et al., 2007). Ablation of SATB1 causes dysregulation of genes required for the development of T cells and the development is stalled at the DP stage (Alvarez et al., 2000). Thymocytes from SATB1 knockout mice revealed ectopic expression of genes such as *IL-2R* and *IL-7R*. SATB1 is known to regulate genes by selectively tethering their regulatory regions and via formation of a characteristic cage-like structure around the heterochromatic regions in Thymocytes (Cai et al., 2004), presumably demarcating the active and inactive domains (Galande et al., 2007). SATB1 also acts as a docking site for chromatin remodeling/modifying factors such as ISWI, ASF1 and NURD complex containing HDAC1, leading to the repression of genes (Yasui et al., 2000). Post-translational modifications of SATB1 such as acetylation and phosphorylation act as molecular switches regulating its ability to govern gene expression. The PDZ-like domain of SATB1 undergoes phosphorylation by PKC and acetylation by PCAF acetyltransferase in signal-dependent manner (Kumar et al., 2006). Acetylation of SATB1 negatively influences the DNA binding activity of SATB1 whereas phosphorylated form of SATB1 is shown to bind tightly to the *Il-2* promoter and repress *Il-2*. Interaction of SATB1 with the CtBP1 corepressor via its Nterminal PDZ-like domain represses transcription. Upon inhibition of Wnt signaling by LiCl treatment SATB1 is acetylated, loses its interaction with CtBP1 and thus leads to activation of *Il-2* (Purbey et al., 2009). Further, SATB1 is also known to regulate chromatin loop domain organization ('loopscape') in a cell type-specific manner. In Jurkat T cells, SATB1 organizes the MHC class I locus into a 'loopscape' comprising six loops. However, CHO cells which express comparatively less SATB1 exhibit a different 'loopscape' of the MHC locus. Intersstingly, overexpression of SATB1 in CHO cells rendered the 'loopscape' similar to that in Jurkat cells underscoring the importantance of SATB1 in cell-type specific higher-order chromatin organization (Kumar et al., 2007; Galande et al., 2007). In TH2 cells, SATB1 organizes the loop domain architecture of the TH2 cytokine locus and governs the coordinated expression of *IL-4, IL-5* and *IL-13* and thus regulate TH2 differentiation (Cai et al., 2006). Thus, SATB1 has emerged as an important factor orchestrating gene expression by modulating the higher-order chromatin architecture in a cell-type specific and signaldependent manner.

Number of studies in the past few years have demonstrated the role of SATB1 in cancer. It has been shown that siRNA-mediated knockdown of SATB1 in higly aggressive breast cancer cells reversed the tumorogenic capability of cells and also inhibited the tumor growth (Han et al., 2008). Downregulation of SATB1 in cancerous cells resulted in alteration in the expression of about thousand genes. Furthermore, overexpression of SATB1 in a nonaggressive tumor cell line resulted in augmenting the tumorigenic and metastatic capacity of these cells indicating its direct role in coordinated regulation of multiple genes. SATB1 presumably reprogrammes gene expression by inducing specific epigenetic modifications at target gene loci, leading to upregulation of metastasis-associated genes and simultaneously causing downregulation of tumor suppressor genes (Han et al., 2008). These studies point to a coordinated mechanism of tumor progression.

### **3. SATB1 in T cell development and differentiation**

#### **3.1 Overview of T cell development**

T cells arise from the hematopoietic stem cell precursors that migrate to the thymus. Early stage T cell precursors (ETPs) that migrate to the thymus lose the capability to give rise to B

SATB1: Key Regulator of T Cell Development and Differentiation 199

assays revealed that SATB1:β-catenin interaction regulates the expression of Wnt target genes in TCF-independent manner (Notani et al., 2010). The recruitment of β-catenin to SATB1 target genes is preceded by deacetylation of SATB1 upon Wnt/β-catenin signaling in thymocytes and CD4+ T cells. SATB1 directly binds to cis regulatory elements at the CD8 enhancer and required for the CD8 SP thymocyte development from the DP thymocytes (Yao et al., 2010). Thus, SATB1 which is highly expressed in thymocytes acts as a global

CD4+ SP thymocytes from the thymus migrate to peripheral lymphoid organs, where they encounter antigen presented by the antigens presenting cells (APCs) and further differentiate into T helper (TH) effector phenotypes. TH1 population is involved in cellular immunity wherein they assist macrophages and cytotoxic T cells (Tc) for clearance of infected cells while TH2 cells help B cells in generating humoral response by increasing production of neutralising antibodies against the pathogen (Zhu and Paul, 2008). TH2 population is characterized by the effector cytokines it secretes viz., IL-5, IL-13 and IL-4. Strikingly, SATB1 which is known to have a important role during thymocyte development is upregulated during TH2 differentiation (Lund et al., 2005; Notani et al., 2010). SATB1 was shown to regulate the expression of TH2 cytokines by remodeling the chromatin in an actively transcribed loop form (Cai et al., 2006). T cell activation along with IL-4 cytokine stimulus showed that SATB1 forms higher-order chromatin structure of the 200 Kb TH2 cytokine locus and regulates *Il-5, Il-13* and *Il-4.* SATB1 induces expression of these cytokines by recruiting chromatin modifying enzyme Brg1 and RNA Pol II converting the locus into transcriptionally active region (Cai et al., 2006). Furthermore, induction of SATB1 expression in CD4+ cells during TH2 differentiation is STAT-6 dependent (Lund et al., 2005 and Ahlfors et al., 2010). Transcriptome profiling of differentiating CD4+ cells into TH1/ TH2 subtypes revealed that SATB1 is involved in regulation of over 300 genes indicating its crucial role

An important insight into the role of SATB1 in TH differentiation was obtained when the gene expression profiles of various subsets of TH cells were compared with the TCRactivated CD4+ T cells, a condition referred to as TH0. To ascertain whether SATB1 regulated genes are involved in TH cell differentiation, Ahlfors et al., (2010) silenced expression of SATB1 using siRNAs in TH cells. Their studies revealed that expression of multiple genes was altered upon SATB1 knockdown in TH1, TH2 and TH0 population. The RNA expression profile revealed that in differentiating CD4+ T cells, expression of 319 genes was altered. Out of these, 70 genes were selectively affected in TH2 population while 43 genes had altered expression in TH1 population. Thus total of 40% (43+14+43=127) genes showed altered expression upon cytokine treatment suggesting SATB1 targets were partly specific to TH subsets. Notably, 48% of SATB1 target genes were regulated by IL-4. Furthermore, TCR stimulation alone regulated one third of SATB1 targets and only 18% of SATB1 target genes were not regulated by TCR or combination of TH1/TH2 polarizing cytokines. The gene expression profiling clearly indicated that SATB1 is likely to play an essential role in the development or function of various TH subtypes (Ahlfors et al., 2010). Another important contribution of this study was the finding that IL-5 which is predominantly secreted by TH2 cells is repressed by SATB1 during early stages of polarization. The repression of *Il-5*

regulator in their development.

**3.3 Role of SATB1 in TH2 differentiation**

during TH cell differentiation (Ahlfors et al., 2010).

cells, however they have the propensity to develop into lineages other than T cells such as macrophages, dendritic cells and NKT cells (Yui et al., 2010). The ETPs also called DN1 phenotypically are CD4- , CD8- , CD3- , CD25- , CD44+ cells. These cells undergo extensive proliferation and are not yet completely committed to T lineage (Rothenberg et al., 2010). The next stage of development is characterized by upregulation of CD25 and is called DN2 stage at which cells are CD4-, CD8-, CD3-, CD25+, CD44+. Further, CD44 is downregulated and such cells are referred to as DN3 stage (CD4- , CD8- , CD3- , CD25+, CD44- ) and at this stage they are committed to the T cell lineage. The DN3 cells stop dividing and undergo rearrangement of TCRβ chain. Successful assembly of the β chain facilitates the movement of cells and this process is known as β-selection (Michie & Zuniga-Pflucker, 2002). Subsequently, these cells downregulate both CD25 (IL-2R) and CD44, the stage is called DN4 and these are fully committed towards T lineage and start proliferation. Following the successful rearrangement of αβ TCR, thymocytes start expressing CD4 and CD8 coreceptors on the cell surface. The DP thymocytes undergo a stringent selection process, where the TCRs that cannot bind to self antigens undergo death by neglect, whereas those which bind to self MHC with intermediate affinity undergo positive selection (Marrack & Kappler, 1997). Further, these thymocytes either develop into CD4+ or CD8+ SP thymocytes dependent on the TCR signals and the expression of specific transcription factor(s) (Singer et al., 2008). In the periphery, the mature T cells differentiate into effector T cells depending on the antigen encountered and cytokine signals.

#### **3.2 Role of SATB1 in thymocyte development**

SATB1 knockout mice exhibit a severe defect in T cell development. SATB1-null mice have disproportionately small thymi and spleens as compared to the wild-type mice. At the cellular level, these mice exhibit multiple defects in T-cell development. The population of immature CD3- ,CD4- ,CD8- triple negative (TN) thymocytes is greatly reduced. Most strikingly, the thymocyte development is blocked at the double positive stage and the CD4+ or CD8+ SP thymocytes fail to develop (Alvarez et al., 2000). Ablation of SATB1 also results in dysregulation of multiple genes such as *Il-2R* and *Il-7R* involved in T cell development and differentiation (Alvarez et al., 2000). Within the thymus majority of the DP thymocytes are eliminated via apoptosis during positive and negative selection process (Surh & Sprent, 1994). Dexamethasone-induced apoptosis of thymocytes resulted in rapid dissociation of SATB1 from chromatin. Furthermore, SATB1 is specifically cleaved by caspase-6 after the aspartate residue at position 254 which led to the identification of the PDZ-like domain in the N-terminal region of SATB1. In vitro analysis revealed that caspase-6 cleavage also abolished the DNA-binding ability of SATB1 (Galande et al., 2001). The cleavage of SATB1 during T cell apoptosis might be required for the initiation of DNA fragmentation. In SATB1- null mice peripheral CD4+ T cells fail to respond to activation stimulus and undergo apoptosis demonstrating indispensible role of SATB1 during proper T cell development (Alvarez et al., 2000). Comparison of the wild-type mice with the SATB1-/- mice indicated that repression of *Il-2R* gene was caused specifically by recruitment of histone deacetylases by SATB1 (Yasui et al., 2002). Immunostaining of SATB1 in mouse thymocytes revealed that it forms a unique cage-like structure differentiating euchromatin from heterochromatin (Cai et al., 2003; Notani et al., 2010). In thymocytes, SATB1 is also known to cooperate with other regulatory factors such as β-catenin and CtBP-1 in signal-dependent manner and regulate gene expression (Purbey et al., 2009; Notani et al., 2010). SATB1-binding site-driven reporter assays revealed that SATB1:β-catenin interaction regulates the expression of Wnt target genes in TCF-independent manner (Notani et al., 2010). The recruitment of β-catenin to SATB1 target genes is preceded by deacetylation of SATB1 upon Wnt/β-catenin signaling in thymocytes and CD4+ T cells. SATB1 directly binds to cis regulatory elements at the CD8 enhancer and required for the CD8 SP thymocyte development from the DP thymocytes (Yao et al., 2010). Thus, SATB1 which is highly expressed in thymocytes acts as a global regulator in their development.

### **3.3 Role of SATB1 in TH2 differentiation**

198 Hematology – Science and Practice

cells, however they have the propensity to develop into lineages other than T cells such as macrophages, dendritic cells and NKT cells (Yui et al., 2010). The ETPs also called DN1

proliferation and are not yet completely committed to T lineage (Rothenberg et al., 2010). The next stage of development is characterized by upregulation of CD25 and is called DN2 stage at which cells are CD4-, CD8-, CD3-, CD25+, CD44+. Further, CD44 is downregulated

they are committed to the T cell lineage. The DN3 cells stop dividing and undergo rearrangement of TCRβ chain. Successful assembly of the β chain facilitates the movement of cells and this process is known as β-selection (Michie & Zuniga-Pflucker, 2002). Subsequently, these cells downregulate both CD25 (IL-2R) and CD44, the stage is called DN4 and these are fully committed towards T lineage and start proliferation. Following the successful rearrangement of αβ TCR, thymocytes start expressing CD4 and CD8 coreceptors on the cell surface. The DP thymocytes undergo a stringent selection process, where the TCRs that cannot bind to self antigens undergo death by neglect, whereas those which bind to self MHC with intermediate affinity undergo positive selection (Marrack & Kappler, 1997). Further, these thymocytes either develop into CD4+ or CD8+ SP thymocytes dependent on the TCR signals and the expression of specific transcription factor(s) (Singer et al., 2008). In the periphery, the mature T cells differentiate into effector T cells depending on

SATB1 knockout mice exhibit a severe defect in T cell development. SATB1-null mice have disproportionately small thymi and spleens as compared to the wild-type mice. At the cellular level, these mice exhibit multiple defects in T-cell development. The population of

strikingly, the thymocyte development is blocked at the double positive stage and the CD4+ or CD8+ SP thymocytes fail to develop (Alvarez et al., 2000). Ablation of SATB1 also results in dysregulation of multiple genes such as *Il-2R* and *Il-7R* involved in T cell development and differentiation (Alvarez et al., 2000). Within the thymus majority of the DP thymocytes are eliminated via apoptosis during positive and negative selection process (Surh & Sprent, 1994). Dexamethasone-induced apoptosis of thymocytes resulted in rapid dissociation of SATB1 from chromatin. Furthermore, SATB1 is specifically cleaved by caspase-6 after the aspartate residue at position 254 which led to the identification of the PDZ-like domain in the N-terminal region of SATB1. In vitro analysis revealed that caspase-6 cleavage also abolished the DNA-binding ability of SATB1 (Galande et al., 2001). The cleavage of SATB1 during T cell apoptosis might be required for the initiation of DNA fragmentation. In SATB1- null mice peripheral CD4+ T cells fail to respond to activation stimulus and undergo apoptosis demonstrating indispensible role of SATB1 during proper T cell development (Alvarez et al., 2000). Comparison of the wild-type mice with the SATB1-/- mice indicated that repression of *Il-2R* gene was caused specifically by recruitment of histone deacetylases by SATB1 (Yasui et al., 2002). Immunostaining of SATB1 in mouse thymocytes revealed that it forms a unique cage-like structure differentiating euchromatin from heterochromatin (Cai et al., 2003; Notani et al., 2010). In thymocytes, SATB1 is also known to cooperate with other regulatory factors such as β-catenin and CtBP-1 in signal-dependent manner and regulate gene expression (Purbey et al., 2009; Notani et al., 2010). SATB1-binding site-driven reporter

,CD4-,CD8- triple negative (TN) thymocytes is greatly reduced. Most

, CD8-

, CD3-

, CD44+ cells. These cells undergo extensive

, CD25+, CD44-

) and at this stage

, CD25-

phenotypically are CD4-

immature CD3-

, CD8-

and such cells are referred to as DN3 stage (CD4-

the antigen encountered and cytokine signals.

**3.2 Role of SATB1 in thymocyte development**

, CD3-

CD4+ SP thymocytes from the thymus migrate to peripheral lymphoid organs, where they encounter antigen presented by the antigens presenting cells (APCs) and further differentiate into T helper (TH) effector phenotypes. TH1 population is involved in cellular immunity wherein they assist macrophages and cytotoxic T cells (Tc) for clearance of infected cells while TH2 cells help B cells in generating humoral response by increasing production of neutralising antibodies against the pathogen (Zhu and Paul, 2008). TH2 population is characterized by the effector cytokines it secretes viz., IL-5, IL-13 and IL-4. Strikingly, SATB1 which is known to have a important role during thymocyte development is upregulated during TH2 differentiation (Lund et al., 2005; Notani et al., 2010). SATB1 was shown to regulate the expression of TH2 cytokines by remodeling the chromatin in an actively transcribed loop form (Cai et al., 2006). T cell activation along with IL-4 cytokine stimulus showed that SATB1 forms higher-order chromatin structure of the 200 Kb TH2 cytokine locus and regulates *Il-5, Il-13* and *Il-4.* SATB1 induces expression of these cytokines by recruiting chromatin modifying enzyme Brg1 and RNA Pol II converting the locus into transcriptionally active region (Cai et al., 2006). Furthermore, induction of SATB1 expression in CD4+ cells during TH2 differentiation is STAT-6 dependent (Lund et al., 2005 and Ahlfors et al., 2010). Transcriptome profiling of differentiating CD4+ cells into TH1/ TH2 subtypes revealed that SATB1 is involved in regulation of over 300 genes indicating its crucial role during TH cell differentiation (Ahlfors et al., 2010).

An important insight into the role of SATB1 in TH differentiation was obtained when the gene expression profiles of various subsets of TH cells were compared with the TCRactivated CD4+ T cells, a condition referred to as TH0. To ascertain whether SATB1 regulated genes are involved in TH cell differentiation, Ahlfors et al., (2010) silenced expression of SATB1 using siRNAs in TH cells. Their studies revealed that expression of multiple genes was altered upon SATB1 knockdown in TH1, TH2 and TH0 population. The RNA expression profile revealed that in differentiating CD4+ T cells, expression of 319 genes was altered. Out of these, 70 genes were selectively affected in TH2 population while 43 genes had altered expression in TH1 population. Thus total of 40% (43+14+43=127) genes showed altered expression upon cytokine treatment suggesting SATB1 targets were partly specific to TH subsets. Notably, 48% of SATB1 target genes were regulated by IL-4. Furthermore, TCR stimulation alone regulated one third of SATB1 targets and only 18% of SATB1 target genes were not regulated by TCR or combination of TH1/TH2 polarizing cytokines. The gene expression profiling clearly indicated that SATB1 is likely to play an essential role in the development or function of various TH subtypes (Ahlfors et al., 2010). Another important contribution of this study was the finding that IL-5 which is predominantly secreted by TH2 cells is repressed by SATB1 during early stages of polarization. The repression of *Il-5*

SATB1: Key Regulator of T Cell Development and Differentiation 201

signalling in the polarized cells, SATB1 interacts with β-catenin, recruits it to *Gata-3* promoter and derepresses it leading to TH2 commitment (Figure 2). Several SATB1 regulated genes are activated by SATB1:β-catenin complex in Wnt-dependent manner. Posttranslational modifications of SATB1 act as molecular switches regulating its DNA-binding activity and ability to interact with multiple partner proteins (Kumar et al., 2006). Upon Wnt signaling SATB1 is deacetylated and directly interacts with β-catenin through its PDZ-like domain. The physical interaction between SATB1 and β-catenin is required for TH2 differentiation. The two prominent factors TCF and SATB1 compete for β-catenin interaction. SATB1 competitively recruits β-catenin and hence also affects the transcription of TCF regulated genes. However, TCF and SATB1 do not interact with each other suggesting that they have non-overlapping effects (Notani et al., 2010). Thus, these two mediators of Wnt signaling presumably bind to their genomic targets independent of each other. LEF/TCF family proteins were the only known β-catenin partners for number of years. Another β-catenin partner known to be involved in pituitary gland development and lineage determination is the homeodomain protein Prop-1 (Olson et al., 2006). The report by Notani et al. (2010), demonstrated that homeodomain-containing transciption regulator

SATB1 is also a β-catenin-binding factor and is involved in TH2 differentiation.

Fig. 2. **SATB1: β-catenin complex regulates** *Gata-3* **expression during TH2 differentiation.**  Upon Wnt signaling β-catenin translocates into the nucleus. SATB1 interacts with β-catenin

Role of transcription factor GATA-3 in TH2 polarization by upregulating IL-4 secretion and inhibiting IFN-γ expression is very well established (Avni et al., 2002; Spilianakis et al., 2004). SATB1 positively regulates GATA-3 expression in TH2 cells by recruiting p300 acetyltransferase and β-catenin to *Gata-3* promoter upon Wnt signal (Figure 2). The role of Wnt signaling in TH2 cell differentiation was further demonstrated by using DKK1, an inhibitor of Wnt signaling. Upon DKK1 treatment in TH cells, GATA-3 expression was suppressed and also TH2 cytokines were downregulated. Quantitative transcript profiling revealed that expression of GATA-3 was suppressed upon Dkk1 treatment in TH2 subset, suggesting that Wnt signaling is necessary for the upregulation of GATA-3 during differentiation of TH2 cells. Overexpression and siRNA mediated silencing of SATB1 and -catenin provided the conclusive evidence for their direct roles in the differentiation of CD4+ cells. Upon siRNA-

differentiation. In differentiating TH2 cells, SATB1: β-catenin complex binds to the *Gata-3* promoter and upregulates Gata-3 expression by recruiting the p300 activator complex. SATB1: β-catenin complex regulates *Gata-3* expression in Wnt-dependent manner and thus

and regulates multiple genes. GATA-3 is known to be a master regulator of TH2

regulates TH2 differentiation (Notani et al., 2010).

promoter by SATB1 was during brought about by recruiting HDAC1 corepressor to the *Il-5* locus (Figure 1). Later the course of differentiation, the competition between binding of SATB1 and GATA-3 results in binding of GATA-3 to the *Il-5* promoter which derepresses *Il-5* locus and IL-5 is produced (Ahlfors et al., 2010). IL-5 plays important role in differentiation and activation of eosinophils and dysregulation of *Il-5* results into eosinophila (Mosmann & Coffman 1987; Campbell et al., 1988; Sanderson, 1988). Hence regulation of IL-5 is not only important in proper TH differentiation but also in understanding its role in diseases such as eosinophila.

TH2 differentiation is also regulated by the downstream transcription factors like GATA-3 and STAT-6. GATA-3 is a transcription factor predominantly expressed in T cells and brain (Oosterwegel et al., 1992). GATA-3 has been shown to play an important role in thymocyte development and also during TH2 differentiation (Ho et al., 2010). The essential role of GATA-3 was demonstrated by creating mice lacking GATA-3 expression. GATA-3-deficient CD4+ T cells cannot differentiate into TH2 phenotype and they produce IFNγ under TH2 polarizing conditions (Zhu J et. al., 2004). IL-4-STAT6 signaling pathway is known to cause the upregulation of GATA-3 in TH2 differentiating cells. However, a recent report provided an alternative view by demonstrating that CD4+ T cells can differentiate to TH2 phenotype in absence of STAT6 via notch signaling although with a reduced efficiency (Amsen et al., 2004). In this review we have focused on the newly dicovered mechanism for regulation of GATA-3 expression by SATB1 in Wnt-dependent manner.

Fig. 1. **SATB1 mediated regulation of** *Il-5* **during TH2 differentiation.** IL-5 is a late TH2 cytokine**.** SATB1 directly binds to *Il-5* promoter and inhibits its expression by recruiting HDAC repressor complex. During allergic conditions GATA-3 displaces SATB1 bound to the *Il-5* promoter and upregulates IL-5 cytokine expression (Ahlfors et al., 2010).

#### **3.4 SATB1 as a mediator of Wnt signaling**

Recently, a new role for SATB1 has been discovered as a mediator of Wnt-signaling pathway during TH differentiation (Notani et al., 2010). Wnt signaling is one of the well studied and highly conserved pathways responsible for various developmental processes and cell fate decisions (Logan and Nusse, 2004). β-catenin is the key transducer of canonical Wnt signaling cascade which upon Wnt signaling is stabilized in the cytoplasm, then translocates to the nucleus and interacts with T cell factor (TCF) family transcriptional factors. Asociation of β-catenin with the TCF family proteins alters the expression of Wntresponsive genes (Logan and Nusse, 2004). SATB1 brings about TH2 cell differentiation via Wnt signaling by recruiting β-catenin to its genomic targets (Notani et al., 2010). This study demonstrated that SATB1 represses target genes in undifferentiated cells. Upon Wnt

promoter by SATB1 was during brought about by recruiting HDAC1 corepressor to the *Il-5* locus (Figure 1). Later the course of differentiation, the competition between binding of SATB1 and GATA-3 results in binding of GATA-3 to the *Il-5* promoter which derepresses *Il-5* locus and IL-5 is produced (Ahlfors et al., 2010). IL-5 plays important role in differentiation and activation of eosinophils and dysregulation of *Il-5* results into eosinophila (Mosmann & Coffman 1987; Campbell et al., 1988; Sanderson, 1988). Hence regulation of IL-5 is not only important in proper TH differentiation but also in understanding its role in diseases such as

TH2 differentiation is also regulated by the downstream transcription factors like GATA-3 and STAT-6. GATA-3 is a transcription factor predominantly expressed in T cells and brain (Oosterwegel et al., 1992). GATA-3 has been shown to play an important role in thymocyte development and also during TH2 differentiation (Ho et al., 2010). The essential role of GATA-3 was demonstrated by creating mice lacking GATA-3 expression. GATA-3-deficient CD4+ T cells cannot differentiate into TH2 phenotype and they produce IFNγ under TH2 polarizing conditions (Zhu J et. al., 2004). IL-4-STAT6 signaling pathway is known to cause the upregulation of GATA-3 in TH2 differentiating cells. However, a recent report provided an alternative view by demonstrating that CD4+ T cells can differentiate to TH2 phenotype in absence of STAT6 via notch signaling although with a reduced efficiency (Amsen et al., 2004). In this review we have focused on the newly dicovered mechanism for regulation of

Fig. 1. **SATB1 mediated regulation of** *Il-5* **during TH2 differentiation.** IL-5 is a late TH2 cytokine**.** SATB1 directly binds to *Il-5* promoter and inhibits its expression by recruiting HDAC repressor complex. During allergic conditions GATA-3 displaces SATB1 bound to

Recently, a new role for SATB1 has been discovered as a mediator of Wnt-signaling pathway during TH differentiation (Notani et al., 2010). Wnt signaling is one of the well studied and highly conserved pathways responsible for various developmental processes and cell fate decisions (Logan and Nusse, 2004). β-catenin is the key transducer of canonical Wnt signaling cascade which upon Wnt signaling is stabilized in the cytoplasm, then translocates to the nucleus and interacts with T cell factor (TCF) family transcriptional factors. Asociation of β-catenin with the TCF family proteins alters the expression of Wntresponsive genes (Logan and Nusse, 2004). SATB1 brings about TH2 cell differentiation via Wnt signaling by recruiting β-catenin to its genomic targets (Notani et al., 2010). This study demonstrated that SATB1 represses target genes in undifferentiated cells. Upon Wnt

the *Il-5* promoter and upregulates IL-5 cytokine expression (Ahlfors et al., 2010).

GATA-3 expression by SATB1 in Wnt-dependent manner.

**3.4 SATB1 as a mediator of Wnt signaling**

eosinophila.

signalling in the polarized cells, SATB1 interacts with β-catenin, recruits it to *Gata-3* promoter and derepresses it leading to TH2 commitment (Figure 2). Several SATB1 regulated genes are activated by SATB1:β-catenin complex in Wnt-dependent manner. Posttranslational modifications of SATB1 act as molecular switches regulating its DNA-binding activity and ability to interact with multiple partner proteins (Kumar et al., 2006). Upon Wnt signaling SATB1 is deacetylated and directly interacts with β-catenin through its PDZ-like domain. The physical interaction between SATB1 and β-catenin is required for TH2 differentiation. The two prominent factors TCF and SATB1 compete for β-catenin interaction. SATB1 competitively recruits β-catenin and hence also affects the transcription of TCF regulated genes. However, TCF and SATB1 do not interact with each other suggesting that they have non-overlapping effects (Notani et al., 2010). Thus, these two mediators of Wnt signaling presumably bind to their genomic targets independent of each other. LEF/TCF family proteins were the only known β-catenin partners for number of years. Another β-catenin partner known to be involved in pituitary gland development and lineage determination is the homeodomain protein Prop-1 (Olson et al., 2006). The report by Notani et al. (2010), demonstrated that homeodomain-containing transciption regulator SATB1 is also a β-catenin-binding factor and is involved in TH2 differentiation.

Fig. 2. **SATB1: β-catenin complex regulates** *Gata-3* **expression during TH2 differentiation.**  Upon Wnt signaling β-catenin translocates into the nucleus. SATB1 interacts with β-catenin and regulates multiple genes. GATA-3 is known to be a master regulator of TH2 differentiation. In differentiating TH2 cells, SATB1: β-catenin complex binds to the *Gata-3* promoter and upregulates Gata-3 expression by recruiting the p300 activator complex. SATB1: β-catenin complex regulates *Gata-3* expression in Wnt-dependent manner and thus regulates TH2 differentiation (Notani et al., 2010).

Role of transcription factor GATA-3 in TH2 polarization by upregulating IL-4 secretion and inhibiting IFN-γ expression is very well established (Avni et al., 2002; Spilianakis et al., 2004). SATB1 positively regulates GATA-3 expression in TH2 cells by recruiting p300 acetyltransferase and β-catenin to *Gata-3* promoter upon Wnt signal (Figure 2). The role of Wnt signaling in TH2 cell differentiation was further demonstrated by using DKK1, an inhibitor of Wnt signaling. Upon DKK1 treatment in TH cells, GATA-3 expression was suppressed and also TH2 cytokines were downregulated. Quantitative transcript profiling revealed that expression of GATA-3 was suppressed upon Dkk1 treatment in TH2 subset, suggesting that Wnt signaling is necessary for the upregulation of GATA-3 during differentiation of TH2 cells. Overexpression and siRNA mediated silencing of SATB1 and -catenin provided the conclusive evidence for their direct roles in the differentiation of CD4+ cells. Upon siRNA-

SATB1: Key Regulator of T Cell Development and Differentiation 203

Fig. 3. Model depicting the early events occurring upon Wnt signaling in polarized TH2 cell and role of SATB1 in this process. In the complex paradigm of TH2 polarization model there have been several studies suggesting role(s) of different mechanisms and it is now evident that SATB1 plays a major role during this process of TH2 commitment. **A**, In naïve cells when IL-4 signalling is absent, expression of SATB1 is low and GATA-3 is not upregulated. **B,** Under TH2 conditions, when a peptide antigen is presented by an antigen presenting cell (APC) to the TCR on T cell surface and IL-4 secreted by the APCs causes the activation of Jak Kinases which phosphorylate STAT-6, which in turn upregulates SATB1 and GATA-3. SATB1 interacts with β-catenin which is translocated to the nucleus in Wnt-dependent manner and this complex regulates *Gata-3* expression. STAT-6, SATB1 and GATA-3 coordinatively regulate *Il-4* expression which is a characteristic cytokine of TH 2 differentiation. However, the role of

Wnt signaling, β-catenin is phosporylated by destruction complex and targeted for degradation to proteosomal complex. SATB1 is acetylated and has low DNA-binding affinity in the absence of Wnt signal. Also, in the absence of nuclear β-catenin TCF does not regulate Wnt responsive genes and hence their transcription is suppressed (Figure 3A). Upon Wnt signaling, the destruction complex that sequesters -catenin does not form and βcatenin is stabilized, which then translocates to nucleus. SATB1 is deacetylated upon Wnt signaling and it then competes with TCF for interaction with β-catenin. Deacetylated SATB1 recruits β-catenin to genomic targets and regulates Wnt-responsive genes resulting into TH2 differentiation (Notani et al., 2010). SATB1 also binds to TH2 cytokine locus and upregulates

transcription of *Il-4*, *Il-5* and *Il-13* resulting into TH2 commitment (Figure 3B).

STAT-6 in regulation of SATB1 as depicted here is speculative.

mediated silencing of SATB1 the expression of GATA-3 was downregulated in TH2 cells. Overexpression of SATB1 led to a significant increase in the expression of GATA-3 in TH2, suggesting that SATB1 positively regulates GATA-3 expression (Notani et al., 2010). In summary, Wnt signaling is essential for TH2 differentiation whereby SATB1 upregulates GATA-3 expression which further enhances IL-4 secretion. CD4+ T cells are receptive to Wnt signals because they produce different Wnts themselves (Notani et al., 2010). The differential sensitivity of TH cell subtypes to Wnt signaling could be due to the fact that the downstream processes such as stabilization of -catenin occur prominently in the TH2 subtype and not TH1 (Notani et al., 2010). Thus, these evidences clearly argue in favor of requirement of SATB1 and Wnt/-catenin signaling during TH cell differentiation.

GATA3 facilitates chromatin remodeling of TH2 cytokine locus leading to conversion of the *Il4*–*Il5*–*Il13* locus to an open conformation, allowing transcription of this locus by transcription factors involved in TH2-cell differentiation (Avni et al., 2002). The associated specific epigenetic changes include histone modifications upon binding of GATA-3 to its DNA targets were found to be mainly H3K4 and H3K27 methylation (Wei et al., 2011). Another chromatin protein CTCF binds to TH2 cytokine locus and assists GATA-3 and SATB1 mediated TH2 commitment (Almeida et al., 2009). Thus, collectively the three regulators namely SATB1, GATA-3 and CTCF could be responsible for orchestrating the coordinated regulation of TH cell differentiation.

A model for regulation of TH2 differentiation by SATB1 is illustrated in Figure 3. TH0 cell is activated and polarized by TCR docking and IL-4 cytokine respectively. In the absence of

mediated silencing of SATB1 the expression of GATA-3 was downregulated in TH2 cells. Overexpression of SATB1 led to a significant increase in the expression of GATA-3 in TH2, suggesting that SATB1 positively regulates GATA-3 expression (Notani et al., 2010). In summary, Wnt signaling is essential for TH2 differentiation whereby SATB1 upregulates GATA-3 expression which further enhances IL-4 secretion. CD4+ T cells are receptive to Wnt signals because they produce different Wnts themselves (Notani et al., 2010). The differential sensitivity of TH cell subtypes to Wnt signaling could be due to the fact that the downstream processes such as stabilization of -catenin occur prominently in the TH2 subtype and not TH1 (Notani et al., 2010). Thus, these evidences clearly argue in favor of requirement of SATB1 and

GATA3 facilitates chromatin remodeling of TH2 cytokine locus leading to conversion of the *Il4*–*Il5*–*Il13* locus to an open conformation, allowing transcription of this locus by transcription factors involved in TH2-cell differentiation (Avni et al., 2002). The associated specific epigenetic changes include histone modifications upon binding of GATA-3 to its DNA targets were found to be mainly H3K4 and H3K27 methylation (Wei et al., 2011). Another chromatin protein CTCF binds to TH2 cytokine locus and assists GATA-3 and SATB1 mediated TH2 commitment (Almeida et al., 2009). Thus, collectively the three regulators namely SATB1, GATA-3 and CTCF could be responsible for orchestrating the

A model for regulation of TH2 differentiation by SATB1 is illustrated in Figure 3. TH0 cell is activated and polarized by TCR docking and IL-4 cytokine respectively. In the absence of

Wnt/-catenin signaling during TH cell differentiation.

coordinated regulation of TH cell differentiation.

Fig. 3. Model depicting the early events occurring upon Wnt signaling in polarized TH2 cell and role of SATB1 in this process. In the complex paradigm of TH2 polarization model there have been several studies suggesting role(s) of different mechanisms and it is now evident that SATB1 plays a major role during this process of TH2 commitment. **A**, In naïve cells when IL-4 signalling is absent, expression of SATB1 is low and GATA-3 is not upregulated. **B,** Under TH2 conditions, when a peptide antigen is presented by an antigen presenting cell (APC) to the TCR on T cell surface and IL-4 secreted by the APCs causes the activation of Jak Kinases which phosphorylate STAT-6, which in turn upregulates SATB1 and GATA-3. SATB1 interacts with β-catenin which is translocated to the nucleus in Wnt-dependent manner and this complex regulates *Gata-3* expression. STAT-6, SATB1 and GATA-3 coordinatively regulate *Il-4* expression which is a characteristic cytokine of TH 2 differentiation. However, the role of STAT-6 in regulation of SATB1 as depicted here is speculative.

Wnt signaling, β-catenin is phosporylated by destruction complex and targeted for degradation to proteosomal complex. SATB1 is acetylated and has low DNA-binding affinity in the absence of Wnt signal. Also, in the absence of nuclear β-catenin TCF does not regulate Wnt responsive genes and hence their transcription is suppressed (Figure 3A). Upon Wnt signaling, the destruction complex that sequesters -catenin does not form and βcatenin is stabilized, which then translocates to nucleus. SATB1 is deacetylated upon Wnt signaling and it then competes with TCF for interaction with β-catenin. Deacetylated SATB1 recruits β-catenin to genomic targets and regulates Wnt-responsive genes resulting into TH2 differentiation (Notani et al., 2010). SATB1 also binds to TH2 cytokine locus and upregulates transcription of *Il-4*, *Il-5* and *Il-13* resulting into TH2 commitment (Figure 3B).

SATB1: Key Regulator of T Cell Development and Differentiation 205

Adaptive immune response raised against pathogen includes clonal expansion of antigenspecific T cells which are then cleared from the system mainly by activation-induced cell death (AICD), a type of apoptosis (Krammer et al., 2007). Sézary syndrome which is a variant of cutaneous T cell lymphoma results by clonal accumulation of mature T cells originating from skin (Willemze et al., 2005). This accumulation of cells occurs as a result of resistance of cells to AICD (Klemke et al., 2006). The pathogenesis of Sézary Syndrome (SS) is still not very clear. A recent study by Wang et al. (2011) revealed that the deficiency of SATB1 leads to SS. Sézary cells obtained from patients are CD4+ CD7- mature memory T cells and show a TH2 cytokine profile with loss of expression of CD7. Transcription profiling of the Sézary cells from patients and Hut78 (Sézary-derived cell line) revealed that SATB1 was drastically downregulated in these cells as compared to non-Sézary control cells such as Jurkat T cells. Additionally, immunofluorescence staining showed a lowered nuclear localization of SATB1 in of primary Sézary cells as well as in Hut98 cells (Wang et al., 2011). Retroviral transduction mediated restoration of SATB1 in Hut98 cells increased apoptosis in these cells within 4 days without changing their proliferation rate. Subsequently, it was demonstrated that the SATB1 restored cells were sensitized to AICD. The transcriptome analysis of these SATB1 restored cells showed remarkable up-regulation of FASL/CD95L which is a death receptor ligand. Further, 32 out of total 153 (12%) dysregulated genes in Sézary cells were normalized upon SATB1 restoration in these cells (Wang et al., 2011). The increased AICD in SATB1 restored Sézary cells was shown to be induced by FASL via caspase 8-dependent pathway. These studies strongly suggested that SATB1 plays a very important role in pathogenesis of Sézary syndrome and it plays a vital role in regulation of homeostasis of T cells. Sézary cells are known not to respond to radiation therapy as these cells do not have increased proliferation but rather possess resistance to apoptosis. Currently the therapies for SS include upregulation of FASL to sensitize these cells for apoptosis. Restoration of SATB1 in Sézary cells could be a promising new strategy for the treatment of Sézary syndrome. The SS cells would also serve as a knockout model for

In the field of T cell biology, TH differentiation is itself a complex phenomenon, one reason being that TH cell fate is not pre-decided during development in thymus, it is primarily executed upon the encounter of undifferentiated T cell with the antigen in the peripheral immune system. Hence TH cell polarization leading to final differentiation is a multi-cascade process with several epigenetic changes invoked in response to various signals. In this Chapter we focused on role of SATB1 which is an important global regulator involved in T cell development, maturation and differentiation. We elaborated on the role of SATB1 during TH cell differentiation which is an important pool of cells for humoral as well as cell mediated immunity. To summarize the findings of various studies, it can be concluded that SATB1 plays an important role at the very early stages of TH cell differentiation. The studies discussed here suggest that SATB1 represses the chromatin in undifferentiated cells by recruiting repressors to the gene loci. Upon early events of cell polarization such as TCR signal and cytokine secretion by cells, SATB1 immediately responds to even lower level of cytokine signal such as IL-4 by changing the chromatin 'loopscape' of specific loci in TH2

**5. Loss of SATB1 function: Sézary syndrome**

studying role of SATB1 in human T cell functions.

**6. Conclusions**

#### **4. Regulation of SATB1 via STAT-6**

Signal transducer and activator of transcription (STATs) are important in various biological processes such as development, programed cell death, organogenesis, cell growth regulation and adaptive immunity (Horvath, 2000). Upon appropriate cytokine signaling STAT molecules are phosphorylated by Janus kinases and they form homodimers. The phosphorylated STATs translocate to the nucleus and affect the transcription of their target genes (Schindler and Darnell, 1995). Cytokine signaling mediates the activation of specific STAT molecules and plays an important role during T helper cell differentiation. During the TH differentiation STAT-4 and STAT-6 play seminal roles during TH1 and TH2 differentiation process respectively. IL-12 signaling initiates from binding of IL-12 to the IL-12 receptor, which further associates with protein tyrosine kinases and Jak2. The Jak2 kinase specifically causes the phosphorylation of STAT-4 (Waltford et al., 2004). STAT-4 causes the expression of Interferon and transcription factor Tbet during TH1 differentiation (Thieu et al., 2008, Robertson et al., 2005). IL-4 secreted by the APCs engages to the IL-4 receptor on CD4+ T cells which then recruits Jak 3 kinases and causes the activation of STAT-6 (Witthuhn et al., 1994). STAT-6 regulates the expression of IL-4 and GATA-3 during the TH2 differentiation (Zhu and Paul, 2008). The knockout models of STAT-4 and STAT-6 have revealed that T cells cannot differentiate into their respective effector phenotypes (Wuster et al., 2000). Genome-wide analysis of occupancy of STAT factors have shown that they preferentially bind to the promoters and intergenic regions in the genome. STAT proteins have a palindromic GAA consensus binding site. STAT molecules generally colocalize with the active histone marks, and it is shown that both proteins SAT4 and SAT6 colocalize with H3K4 trimethylation marks in the genome (Wei et al., 2010). Gene expression studies along with elucidation of the epigenetic marks at key loci using STAT knockout mice have revealed that STAT are important for the maintenance of epigenetic marks on such genes and thus regulation of gene expression.

STAT-6 knockdown caused the downregulation of CRTH2 expression in cells polarised to TH2 phenotype (Elo et al., 2010). Another study also demonstrated that STAT-6 knockdown resulted in downregulation of SATB1 expression at both RNA and protein level (Ahlfors et al., 2010). Microarray-based gene expression profiling data from different groups using mouse and human models depicted similar results showing downregulation of SATB1 (Wei et al., 2010; Elo et al., 2010). Bsed on these finding, we hypothesize that STAT-6 may directly bind to the SATB1 promoter and mediate activating epigenetic histone modifications leading to the upregulation of SATB1 during TH2 differentiation. SATB1 in turn causes positive regulation of *Il-4* expression.

Interestingly, two recent studies have implicated Foxp3 in the regulation of SATB1 (Beyer et al. 2011; McInnes et al., 2011). Foxp3 tumor suppressor regulates SATB1 expression in breast epithelial cells and downregulates its expression in miRNA-dependent manner (McInnes et al., 2011). Repression of SATB1 has been also identified as a crucial mechanism for the phenotype and function of T(reg) cells. Foxp3 acts as a transcriptional repressor for the SATB1 locus and indirectly suppresses it through the induction of microRNAs that bound the SATB1 3' untranslated region (Beyer et al., 2011). Thus, elucidation of such regulatory loops will be important steps towards understanding the regulation and in vivo functions of SATB1.

### **5. Loss of SATB1 function: Sézary syndrome**

Adaptive immune response raised against pathogen includes clonal expansion of antigenspecific T cells which are then cleared from the system mainly by activation-induced cell death (AICD), a type of apoptosis (Krammer et al., 2007). Sézary syndrome which is a variant of cutaneous T cell lymphoma results by clonal accumulation of mature T cells originating from skin (Willemze et al., 2005). This accumulation of cells occurs as a result of resistance of cells to AICD (Klemke et al., 2006). The pathogenesis of Sézary Syndrome (SS) is still not very clear. A recent study by Wang et al. (2011) revealed that the deficiency of SATB1 leads to SS. Sézary cells obtained from patients are CD4+ CD7 mature memory T cells and show a TH2 cytokine profile with loss of expression of CD7. Transcription profiling of the Sézary cells from patients and Hut78 (Sézary-derived cell line) revealed that SATB1 was drastically downregulated in these cells as compared to non-Sézary control cells such as Jurkat T cells. Additionally, immunofluorescence staining showed a lowered nuclear localization of SATB1 in of primary Sézary cells as well as in Hut98 cells (Wang et al., 2011). Retroviral transduction mediated restoration of SATB1 in Hut98 cells increased apoptosis in these cells within 4 days without changing their proliferation rate. Subsequently, it was demonstrated that the SATB1 restored cells were sensitized to AICD. The transcriptome analysis of these SATB1 restored cells showed remarkable up-regulation of FASL/CD95L which is a death receptor ligand. Further, 32 out of total 153 (12%) dysregulated genes in Sézary cells were normalized upon SATB1 restoration in these cells (Wang et al., 2011). The increased AICD in SATB1 restored Sézary cells was shown to be induced by FASL via caspase 8-dependent pathway. These studies strongly suggested that SATB1 plays a very important role in pathogenesis of Sézary syndrome and it plays a vital role in regulation of homeostasis of T cells. Sézary cells are known not to respond to radiation therapy as these cells do not have increased proliferation but rather possess resistance to apoptosis. Currently the therapies for SS include upregulation of FASL to sensitize these cells for apoptosis. Restoration of SATB1 in Sézary cells could be a promising new strategy for the treatment of Sézary syndrome. The SS cells would also serve as a knockout model for studying role of SATB1 in human T cell functions.

#### **6. Conclusions**

204 Hematology – Science and Practice

Signal transducer and activator of transcription (STATs) are important in various biological processes such as development, programed cell death, organogenesis, cell growth regulation and adaptive immunity (Horvath, 2000). Upon appropriate cytokine signaling STAT molecules are phosphorylated by Janus kinases and they form homodimers. The phosphorylated STATs translocate to the nucleus and affect the transcription of their target genes (Schindler and Darnell, 1995). Cytokine signaling mediates the activation of specific STAT molecules and plays an important role during T helper cell differentiation. During the TH differentiation STAT-4 and STAT-6 play seminal roles during TH1 and TH2 differentiation process respectively. IL-12 signaling initiates from binding of IL-12 to the IL-12 receptor, which further associates with protein tyrosine kinases and Jak2. The Jak2 kinase specifically causes the phosphorylation of STAT-4 (Waltford et al., 2004). STAT-4 causes the expression of Interferon and transcription factor Tbet during TH1 differentiation (Thieu et al., 2008, Robertson et al., 2005). IL-4 secreted by the APCs engages to the IL-4 receptor on CD4+ T cells which then recruits Jak 3 kinases and causes the activation of STAT-6 (Witthuhn et al., 1994). STAT-6 regulates the expression of IL-4 and GATA-3 during the TH2 differentiation (Zhu and Paul, 2008). The knockout models of STAT-4 and STAT-6 have revealed that T cells cannot differentiate into their respective effector phenotypes (Wuster et al., 2000). Genome-wide analysis of occupancy of STAT factors have shown that they preferentially bind to the promoters and intergenic regions in the genome. STAT proteins have a palindromic GAA consensus binding site. STAT molecules generally colocalize with the active histone marks, and it is shown that both proteins SAT4 and SAT6 colocalize with H3K4 trimethylation marks in the genome (Wei et al., 2010). Gene expression studies along with elucidation of the epigenetic marks at key loci using STAT knockout mice have revealed that STAT are important for the maintenance of epigenetic marks on such genes

STAT-6 knockdown caused the downregulation of CRTH2 expression in cells polarised to TH2 phenotype (Elo et al., 2010). Another study also demonstrated that STAT-6 knockdown resulted in downregulation of SATB1 expression at both RNA and protein level (Ahlfors et al., 2010). Microarray-based gene expression profiling data from different groups using mouse and human models depicted similar results showing downregulation of SATB1 (Wei et al., 2010; Elo et al., 2010). Bsed on these finding, we hypothesize that STAT-6 may directly bind to the SATB1 promoter and mediate activating epigenetic histone modifications leading to the upregulation of SATB1 during TH2 differentiation. SATB1 in turn causes

Interestingly, two recent studies have implicated Foxp3 in the regulation of SATB1 (Beyer et al. 2011; McInnes et al., 2011). Foxp3 tumor suppressor regulates SATB1 expression in breast epithelial cells and downregulates its expression in miRNA-dependent manner (McInnes et al., 2011). Repression of SATB1 has been also identified as a crucial mechanism for the phenotype and function of T(reg) cells. Foxp3 acts as a transcriptional repressor for the SATB1 locus and indirectly suppresses it through the induction of microRNAs that bound the SATB1 3' untranslated region (Beyer et al., 2011). Thus, elucidation of such regulatory loops will be important steps towards understanding the regulation and in vivo functions of

**4. Regulation of SATB1 via STAT-6**

and thus regulation of gene expression.

positive regulation of *Il-4* expression.

SATB1.

In the field of T cell biology, TH differentiation is itself a complex phenomenon, one reason being that TH cell fate is not pre-decided during development in thymus, it is primarily executed upon the encounter of undifferentiated T cell with the antigen in the peripheral immune system. Hence TH cell polarization leading to final differentiation is a multi-cascade process with several epigenetic changes invoked in response to various signals. In this Chapter we focused on role of SATB1 which is an important global regulator involved in T cell development, maturation and differentiation. We elaborated on the role of SATB1 during TH cell differentiation which is an important pool of cells for humoral as well as cell mediated immunity. To summarize the findings of various studies, it can be concluded that SATB1 plays an important role at the very early stages of TH cell differentiation. The studies discussed here suggest that SATB1 represses the chromatin in undifferentiated cells by recruiting repressors to the gene loci. Upon early events of cell polarization such as TCR signal and cytokine secretion by cells, SATB1 immediately responds to even lower level of cytokine signal such as IL-4 by changing the chromatin 'loopscape' of specific loci in TH2

SATB1: Key Regulator of T Cell Development and Differentiation 207

Alvarez JD, Yasui DH, Niida H, Joh T, Loh DY & Kohwi-Shigematsu T. (2000) The MAR-

Amsen D, Blander JM, Lee GR, Tanigaki K, Honjo T & Flavell RA. (2004) Instruction of

Avni O, Lee D, Macian F, Szabo SJ, Glimcher LH & Rao A. (2002) T(H) cell differentiation is

Beyer M, Thabet Y, Müller RU, Sadlon T, Classen S, Lahl K, Basu S, Zhou X, Bailey-

Cai S, Han HJ & Kohwi-Shigematsu T. (2003) Tissue-specific nuclear architecture and gene

Cai S, Lee CC & Kohwi-Shigematsu T. (2006) SATB1 packages densely looped,

Campbell HD, Tucker WQ, Hort Y, Martinson ME, Mayo G, Clutterbuck EJ, Sanderson CJ &

DiPaolo RJ, Brinster C, Davidson TS, Andersson J, Glass D, Shevach EM. (2007)

Dong C. (2008) TH17 cells in development: an updated view of their molecular identity and

Elo LL, Järvenpää H, Tuomela S, Raghav S, Ahlfors H, Laurila K, Gupta B, Lund RJ,

Galande S, Dickinson LA, Mian IS, Sikorska M & Kohwi-Shigematsu T. (2001)

Galande S, Purbey PK, Notani D & Kumar PP. (2007) The third dimension of gene

Han HJ, Russo J, Kohwi Y, Kohwi-Shigematsu T. (2008) SATB1 reprogrammes gene expression to promote breast tumour growth and metastasis. Nature. 452:187-93.

transcriptionally active chromatin for coordinated expression of cytokine genes.

Young IG. (1987) Molecular cloning, nucleotide sequence, and expression of the gene encoding human eosinophil differentiation factor (interleukin 5). *Proc Natl*

Autoantigen-specific TGFbeta-induced Foxp3+ regulatory T cells prevent autoimmunity by inhibiting dendritic cells from activating autoreactive T cells. *J*

Tahvanainen J, Hawkins RD, Oresic M, Lähdesmäki H, Rasool O, Rao KV, Aittokallio T & Lahesmaa R. (2010) Genome-wide profiling of interleukin-4 and STAT6 transcription factor regulation of human Th2 cell programming.

SATB1 cleavage by caspase 6 disrupts PDZ domain-mediated dimerization, causing detachment from chromatin early in T-cell apoptosis. *Mol Cell Biol.* 21:5591-

regulation: organization of dynamic chromatin loopscape by SATB1. *Curr Opin*

genes during T-cell development. *Genes Dev.* 14:521-535.

expression regulated by SATB1. *Nat Genet.* 34:42-51.

genetic programming. *Nat Rev Immunol.* 8:337-348.

cells. Cell. 117:515-526.

*Immunol.* 3:643-651

*Immunol.* 12:898-907.

*Nat Genet.* 38:1278:1288.

*Immunol.* 179:4685-4693.

*Immunity.* 32:852-862.

*Genet Dev.* 17:408-414.

5604.

*Acad Sci U S A.* 84:6629–6633.

binding protein SATB1 orchestrates temporal and spatial expression of multiple

distinct CD4 T helper cell fates by different notch ligands on antigen-presenting

accompanied by dynamic changes in histone acetylation of cytokine genes. *Nature*

Bucktrout SL, Krebs W, Schönfeld EA, Böttcher J, Golovina T, Mayer CT, Hofmann A, Sommer D, Debey-Pascher S, Endl E, Limmer A, Hippen KL, Blazar BR, Balderas R, Quast T, Waha A, Mayer G, Famulok M, Knolle PA, Wickenhauser C, Kolanus W, Schermer B, Bluestone JA, Barry SC, Sparwasser T, Riley JL & Schultze JL. (2011) Repression of the genome organizer SATB1 in regulatory T cells is required for suppressive function and inhibition of effector differentiation. *Nat*

cells which culminates into synthesis of downstream transcription factors required for further differentiation such as GATA-3. Wnt signaling acts as a booster for the differentiation signal in these cells which brings about changes in chromatin organization via SATB1 as a mediator of Wnt signaling and promotes GATA-3 transcription. In the later stages of differentiation, TH subtype specific factors such as GATA-3 take over and competitively overcome the SATB1 mediated repression of TH2 cytokines and in turn upregulate the TH2 signature cytokines such as IL-5. Thus, SATB1 presumably acts as a regulatory switch at the very early stages of cell polarization and differentiation by repressing various cell type specific genes, however it specifically responds to polarization signal by changing its acetylation status. The indispensible role of SATB1 in TH cell differentiation is exemplified by diseases such as Eosinophila and Sézary syndrome, the later manifests as a result of SATB1 deficiency.

### **7. Future perspectives**

The role of SATB1 in differentiation of CD4+ T cells has come into the limelight as described in this review. However, the role of SATB1 during earlier events such as thymocyte maturation are not studied in detail and requires further investigation. Since SATB1 is known to regulate genes such as *Thpok* which are important for the lineage commitment process, it is essential to evaluate whether SATB1 plays a direct role during the thymocyte lineage commitment. Findings from recent studies have highlighted the requirement for delineation of molecular mechanisms governing the expression of SATB1 during the process of thymocyte maturation. In the CD4+ T cells, it would be important to study the regulation of SATB1 which might be regulated by an IL-4:STAT6-dependent mechanism as seen during the differentiation of TH2 cells. It would be also interesting to investigate whether SATB1 plays any role(s) in the differentiation of the other subtypes of CD4+ T cells. Studies elucidating role of miRNAs in the regulation of SATB1 in these various subtypes of T cells would also shed light on the signaling pathways and associated mechanisms regulating the development and differentiation of various subtypes of T cells.

### **8. Acknowledgements**

Work was supported by grants from the Centre of Excellence in Epigenetics program of the Department of Biotechnology, Government of India and IISER Pune. KG is supported by fellowship from the Council of Scientific and Industrial Research, India.

### **9. References**

cells which culminates into synthesis of downstream transcription factors required for further differentiation such as GATA-3. Wnt signaling acts as a booster for the differentiation signal in these cells which brings about changes in chromatin organization via SATB1 as a mediator of Wnt signaling and promotes GATA-3 transcription. In the later stages of differentiation, TH subtype specific factors such as GATA-3 take over and competitively overcome the SATB1 mediated repression of TH2 cytokines and in turn upregulate the TH2 signature cytokines such as IL-5. Thus, SATB1 presumably acts as a regulatory switch at the very early stages of cell polarization and differentiation by repressing various cell type specific genes, however it specifically responds to polarization signal by changing its acetylation status. The indispensible role of SATB1 in TH cell differentiation is exemplified by diseases such as Eosinophila and Sézary syndrome, the

The role of SATB1 in differentiation of CD4+ T cells has come into the limelight as described in this review. However, the role of SATB1 during earlier events such as thymocyte maturation are not studied in detail and requires further investigation. Since SATB1 is known to regulate genes such as *Thpok* which are important for the lineage commitment process, it is essential to evaluate whether SATB1 plays a direct role during the thymocyte lineage commitment. Findings from recent studies have highlighted the requirement for delineation of molecular mechanisms governing the expression of SATB1 during the process of thymocyte maturation. In the CD4+ T cells, it would be important to study the regulation of SATB1 which might be regulated by an IL-4:STAT6-dependent mechanism as seen during the differentiation of TH2 cells. It would be also interesting to investigate whether SATB1 plays any role(s) in the differentiation of the other subtypes of CD4+ T cells. Studies elucidating role of miRNAs in the regulation of SATB1 in these various subtypes of T cells would also shed light on the signaling pathways and associated mechanisms regulating the

Work was supported by grants from the Centre of Excellence in Epigenetics program of the Department of Biotechnology, Government of India and IISER Pune. KG is supported by

Ahlfors A, Limaye A, Elo LL, Tuomela S, Burute M, Notani D, Gottimukkala K, Rasool O,

Almeida C, Heath H, Krpic S, Dingjan G, Hamburg J, Bergen I, Nbelen S, Sleutels F,

Galande S & Lahesmaa R. (2010) SATB1 dictates expression of multiple genes including IL-5 involved in human T helper cell differentiation. *Blood.* 116:1443-1453.

Grosveld F, Galjart N & Hendriks R (2009) Critical role for the transcription regulator CCCTC-Binding factor in the control of Th2 cytokine expression. *J.*

later manifests as a result of SATB1 deficiency.

development and differentiation of various subtypes of T cells.

fellowship from the Council of Scientific and Industrial Research, India.

**7. Future perspectives**

**8. Acknowledgements**

*Immunol.* 182:999-1010.

**9. References**

SATB1: Key Regulator of T Cell Development and Differentiation 209

Oosterwegel M, Timmerman J, Leiden J & Clevers H. (1992) Expression of GATA-3 during lymphocyte differentiation and mouse embryogenesis. *Dev Immunol.* 3:1-11. Pancer Z & Cooper MD. (2006) The evolution of adaptive immunity. *Annu Rev Immunol.*

Pavan Kumar P, Purbey PK, Sinha CK, Notani D, Limaye A, Jayani RS & Galande S. (2006)

Pavan Kumar P, Purbey PK, Sinha CK, Notani D, Limaye A, Jayani RS, Galande S. (2006)

Purbey PK, Singh S, Notani D, Kumar PP, Limaye AS & Galande S. (2009) Acetylation-

Rappl G, Muche JM, Abken H & et al. (2001) CD4+CD7- T cells compose the dominant T-cell

Robertson MJ, Chang HC, Pelloso D & Kaplan MH (2005) Impaired interferon-gamma

Rothenberg EV, Zhang J & Li L. (2010) Multilayered specification of the T-cell lineage fate.

Sakaguchi S, Yamaguchi T, Nomura T & Ono M. (2008) Regulatory T cells and immune

Sanderson CJ. (1988) Interleukin-5: an eosinophil growth and activation factor. *Dev Biol*

Schindler C & Darnell JE Jr. (1995) Transcriptional responses to polypeptide ligands: the

Singer A, Adoro S & Park JH. (2008) Lineage fate and intense debate: myths, models and mechanisms of CD4- versus CD8-lineage choice. *Nat Rev Immunol.* 8:788-801. Singer A. (2002) New prespectives on a developmental dilemma: the kinetic signaling model

Sokolowska-Wojdylo M, Wenzel J, Gaffal E, Steitz J, Roszkiewicz J, Bieber T &Tüting T.

monitoring of patients with Sezary syndrome. *Clin Exp Dermatol*. 30:702-706. Staudt V, Bothur E, Klein M, Lingnau K, Reuter S, Grebe N, Gerlitzki B, Hoffmann M, Ulges

Surh CD & Sprent J. (1994) T-cell apoptosis detected in situ during positive and negative

Thieu VT, Yu Q, Chang HC, Yeh N, Nguyen ET, Sehra S & Kaplan MH. (2008) Signal

bet to promote T helper 1 cell-fate determination. *Immunity.* 29: 679-670.

developmental program of T helper 9 cells. *Immunity.* 33:192-202.

selection in the thymus. *Nature.* 372:100-103.

and the importance of signal duration for the CD4/CD8 lineage decision. *Curr Opin*

(2005) Absence of CD26 expression onskin-homing CLA+ CD4+ T lymphocytes in peripheral blood is a highly sensitive marker for early diagnosis and therapeutic

A, Taube C, Dehzad N, Becker M, Stassen M, Steinborn A, Lohoff M, Schild H, Schmitt E & Bopp T. (2010) Interferon-regulatory factor 4 is essential for the

transducer and activator of transcription 4 is required for the transcription factor T-

regulating its transcriptional activity in vivo. *Mol cell.* 22:231-243.

regulating its transcriptional activity in vivo. *Mol Cell*. 22:231-243.

stem cell transplantation for lymphoma. *Blood.* 106:963-970.

JAK-STAT pathway. *Annu Rev Biochem.* 64:621-651.

by SATB1. *Mol Cell Biol.* 29:1321-1337.

*Dermatol.* 44:456-461.

*Immunol Rev.* 238:150-168.

tolerance. *Cell.* 133:775-787.

*Stand.*69:23–29.

*Immunol.* 14:207-215.

Phosphorylation of SATB1, a global gene regulator, acts as amolecular switch

Phosphorylation of SATB1, a global gene regulator, acts as a molecular switch

dependent interaction of SATB1 and CtBP1 mediates transcriptional repression

clone in the peripheral blood of patients with Sezary syndrome. *J Am Acad*

production as a consequence of STAT4 deficiency after autologous hematopoietic

24:497-518.

Hirota K, Duarte JH, Veldhoen M, Hornsby E, Li Y, Cua DJ, Ahlfors H, Wilhelm C, Tolaini

17 producing T cells in inflammatory responses. *Nat Immunol.* 12:255-263. Ho IC, Tai TS, Pai SY. (2009) GATA3 and the T-cell lineage: essential functions before and

Horvath CM. (2000) STAT proteins and transcriptional responses to extracellular signals.

King C, Tangye SG, Mackay CR. (2008) T follicular helper (TFH) cells in normal and

Kitano M, Moriyama S, Ando Y, Hikida M, Mori Y, Kurosaki T & Okada T. (2011)

Klemke CD, Brenner D, Weiss EM, Schmidt M, Leverkus M, Gülow K & Krammer PH.

Krammer PH, Arnold R & Lavrik IN. (2007) Life and death in peripheral T cells. *Nat Rev*

Kreslavsky T, Gleimer M, Garbe AI & Von Boehmer H. (2010) αβ versus ϒδ fate choice: counting the T-cell lineages at the branch point. *Immunol Rev.* 238:169-181. Kumar PP, Bischof O, Purbey PK, Notani D, Urlaub H, Dejean A & Galande S. (2007)

Lund R, Aittokallio T, Nevalainen O & Lahesmaa R. (2003) Identification of novel genes

Lund R, Ahlfors H, Kainonen E, Lahesmaa AM, Dixon C & Lahesmaa R. (2005)

Marrack P & Kappler J. (1997) Positive selection of thymocytes bearing alpha beta T cell

McInnes N, Sadlon TJ, Brown CY, Pederson S, Beyer M, Schultze JL, McColl S, Goodall GJ,

Michie AM & Zúñiga-Pflücker JC. (2002) Regulation of thymocyte differentiation: pre-TCR

Mosmann TR & Coffman RL. (1987) TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. *Annu Rev Immunol.* 7:145–173. Mosmann TR, Cherwinski H, Bond MW, Giedlin MA & Coffman RL. (1986) Two types of

Notani D, Gottimukkala KP, Jayani RS, Limaye AS, Damle MV, Mehta S, Purbey PK, Joseph

T(H)2 differentiation in Wnt-dependent manner. *PLoS Biol.* 8(1):e1000296.

Bcl6 protein expression shapes pre-germinal center B cell dynamics and follicular

(2009) Lack of T-cell receptor-induced signaling is crucial for CD95 ligand upregulation and protects cutaneous T-cell lymphoma cells from activation-induced

Functional interaction between PML and SATB1 regulates chromatin-loop architecture and transcription of the MHC class I locus. *Nat Cell Biol.* 9:45-56. Logan CY & Nusse R (2004) The Wnt signaling pathway in development and disease. *Annu*

regulated by IL-12, IL-4 or TGF- during the early polarization of CD4+

Identification of genes involved in the initiation of human Th1 or Th2 cell

Barry SC. (2011) FOXP3 and FOXP3-regulated microRNAs suppress SATB1 in

murine helper T cell clone. I. Definition according to profiles of lymphokine

J & Galande S. (2010) Global regulator SATB1 recruits beta-catenin and regulates

after T-helper-2-cell differentiation. *Nat Rev Immunol.* 9:125-135.

dysregulated immune responses. *Annu Rev Immunol.* 26:741-766.

helper T cell heterogeneity. *Immunity.* 34:961-972.

*Trends Biochem Sci.* 10:496-502.

cell death. *Cancer Res*. 69:4175-4183.

*Rev Cell Dev Biol*. 20: 781-810.

lymphocytes. *J Immunol.* 171:5428-5336.

commitment. *Eur J Immunol.* 35: 3307-3319.

breast cancer cells. Oncogene. doi: 10.1038/onc.2011.293.

activities and secreted proteins. *J. Immunol.* 136:2348–574.

signals and beta-selection. *Semin Immunol.* 14:311-323.

receptors. *Curr Opin Immunol.* 9:250-255.

*Immunol. 7*:532-542.

M, Menzel U, Garefalaki A, Potocnik AJ & Stockinger B. (2011) Fate mapping of IL-

**8**

Doris Cerecedo

*México*

**Neutrophil Chemotaxis and Polarization:**

*Laboratorio de Hematobiología, Escuela Nacional de Medicina y Homeopatía,*

Neutrophils (also known as polymorphonuclear leukocytes or PMN), the first line of defense against intruding microorganisms, are produced in the bone marrow from stem cells that in turn proliferate and differentiate into mature neutrophils. They play an important role in host defense and contribute to inflammation-related tissue injuries. During inflammation, neutrophils extravasate across the endothelium that lines the blood vessel wall through a multistep process [1, 2], which includes rolling on and subsequent firm

Neutrophil migration through the vascular endothelial layer into lymphoid or inflamed tissues involves a dynamic regulation of cell adhesion in which new adhesions are formed at the cell's leading edge, [3] while filipodia and lamellipodia are generated as exploratory and motile projections and, coordinately, adhesions are released from the trailing edge [4].

For these events, the supply of adhesion molecules to the site of pseudopodial protrusion must be necessarily replenished in order to enable the cell to move forward. There is evidence that the membrane trafficking pathways that recycle adhesion receptors contribute to cell migration [5], which is crucial for polarization and migration in various cell types [6]. Preferential targeting of proteins to the leading or lagging edge of migrating cells is important for polarity and chemotaxis. Asymmetric distribution of proteins has implications beyond polarity and chemotaxis because these same proteins display characteristic localization patterns when cells undergo morphological changes in general. Several proteins have been identified as contributing to cell polarity organization and subsequent inflammatory-cell migration by regulating membrane trafficking. Ly49Q directs the organization of neutrophil polarization as well as neutrophil migration to inflammation sites by regulating membrane raft functions, reorganizing neutrophils in the presence of inflammatory signals, and maintaining neutrophil homeostasis in the absence of such signals [7]. In addition, regulated exocytosis plays a crucial role in conversion of inactive, circulating neutrophils into fully activated cells capable of chemotaxis, phagocytosis, and

Polarity gives cells morphologically and functionally distinct spatial restriction to leading and/or lagging edges by relocating certain proteins or their activities selectively to the

**1. Introduction**

adhesion to endothelial cells.

bacterial killing [8].

**When Asymmetry Means Movement**

*Instituto Politécnico Nacional (IPN), México DF,*

## **Neutrophil Chemotaxis and Polarization: When Asymmetry Means Movement**

Doris Cerecedo

*Laboratorio de Hematobiología, Escuela Nacional de Medicina y Homeopatía, Instituto Politécnico Nacional (IPN), México DF, México*

### **1. Introduction**

210 Hematology – Science and Practice

Wang Y, Su M, Zhou LL, Tu P, Zhang X, Jiang X, Zhou Y. (2011) Deficiency of SATB1

Watford WT, Hissong BD, Bream JH, Kanno Y, Muul L & O'Shea JJ. (2004) Signaling by IL-

Wei L, Vahedi G, Sun HW, Watford WT, Takatori H, Ramos HL, Takahashi H, Liang J,

Witthuhn BA, Silvennoinen O, Miura O, Lai KS, Cwik C, Liu ET & Ihle JN. (1994)

Yang Q, Jeremiah Bell J & Bhandoola A. (2010) T-cell lineage determination. *Immunol Rev.*

Yao X, Nie H, Rojas IC, Harriss JV, Maika SD, Gottlieb PD, Rathbun G & Tucker PW. (2010)

Yasui D, Miyano M, Cai S, Varga-Weisz P & Kohwi-Shigematsu T. (2002) SATB1 targets chromatin remodelling to regulate genes over long distances. *Nature.* 419:641-645. Yu D, Rao S, Tsai LM, Lee SK, He Y, Sutcliffe EL, Srivastava M, Linterman M, Zheng L,

Yui MA, Feng N & Rothenberg EV. (2010) Fine-scale staging of T cell lineage commitment in

Zhu J, Min B, Hu-Li J, Watson CJ, Grinberg A, Wang Q, Killeen N, Urban JF Jr, Guo L & Paul

Zhu J, Yamane H & Paul WE. (2010) Differentiation of effector CD4 T cell populations. *Annu*

Zlotoff DA & Bhandoola A. (2011) Hematopoietic progenitor migration to the adult thymus.

Zhu J & Paul WE. (2008) CD4 T cells: fates, functions and faults. *Blood.* 112:1557-1569

transcription during T helper cell differentiation. *Immunity.* 32:840-851. Willemze R, Jaffe ES, Burg G, Cerroni L, Berti E, Swerdlow SH, Ralfkiaer E, Chimenti S,

transcription. *Blood*. 117: 3826-3835.

lymphomas. *Blood*. 105:3768-3785.

commitment. *Immunity.* 31:457-468.

responses. *Nat Immunol.* 5:1157-1165.

*Rev Immunol.* 28:445-489.

*Ann N Y Acad Sci.* 1217:122-138.

adult mouse thymus. *J Immunol.* 185:284-293.

238:12-22.

lymphoid and myeloid cells. *Nature*. 370: 153–157.

proteins SATB1 and CDP. *Mol Immunol.* 48:153-163.

expression in Sezary cells causes apoptosis resistance by regulating FasL/CD95L

12 and IL-23 and the immunoregulatory roles of STAT4. *Immunol Rev.* 202:139-156.

Gutierrez-Cruz G, Zang C, Peng W, O'Shea JJ & Kanno Y. (2010) Discrete roles of STAT4 and STAT6 transcription factors in tuning epigenetic modifications and

Diaz-Perez JL, Duncan LM, Grange F, Harris NL, Kempf W, Kerl H, Kurrer M, Knobler R, Pimpinelli N, Sander C, Santucci M, Sterry W, Vermeer MH, Wechsler J, Whittaker S & Meijer CJ. (2005) WHO-EORTC classification for cutaneous

Involvement of the Jak-3 Janus kinase in signaling by interleukins 2 and 4 in

The L2a element is a mouse CD8 silencer that interacts with MAR-binding

Simpson N, Ellyard JI, Parish IA, Ma CS, Li QJ, Parish CR, Mackay CR & Vinuesa CG. (2009) The transcriptional repressor Bcl-6 directs T follicular helper cell lineage

WE. (2004) Conditional deletion of Gata3 shows its essential function in TH1-TH2

Neutrophils (also known as polymorphonuclear leukocytes or PMN), the first line of defense against intruding microorganisms, are produced in the bone marrow from stem cells that in turn proliferate and differentiate into mature neutrophils. They play an important role in host defense and contribute to inflammation-related tissue injuries. During inflammation, neutrophils extravasate across the endothelium that lines the blood vessel wall through a multistep process [1, 2], which includes rolling on and subsequent firm adhesion to endothelial cells.

Neutrophil migration through the vascular endothelial layer into lymphoid or inflamed tissues involves a dynamic regulation of cell adhesion in which new adhesions are formed at the cell's leading edge, [3] while filipodia and lamellipodia are generated as exploratory and motile projections and, coordinately, adhesions are released from the trailing edge [4].

For these events, the supply of adhesion molecules to the site of pseudopodial protrusion must be necessarily replenished in order to enable the cell to move forward. There is evidence that the membrane trafficking pathways that recycle adhesion receptors contribute to cell migration [5], which is crucial for polarization and migration in various cell types [6]. Preferential targeting of proteins to the leading or lagging edge of migrating cells is important for polarity and chemotaxis. Asymmetric distribution of proteins has implications beyond polarity and chemotaxis because these same proteins display characteristic localization patterns when cells undergo morphological changes in general. Several proteins have been identified as contributing to cell polarity organization and subsequent inflammatory-cell migration by regulating membrane trafficking. Ly49Q directs the organization of neutrophil polarization as well as neutrophil migration to inflammation sites by regulating membrane raft functions, reorganizing neutrophils in the presence of inflammatory signals, and maintaining neutrophil homeostasis in the absence of such signals [7]. In addition, regulated exocytosis plays a crucial role in conversion of inactive, circulating neutrophils into fully activated cells capable of chemotaxis, phagocytosis, and bacterial killing [8].

Polarity gives cells morphologically and functionally distinct spatial restriction to leading and/or lagging edges by relocating certain proteins or their activities selectively to the

Neutrophil Chemotaxis and Polarization: When Asymmetry Means Movement 213

Trafficking leukocytes often reduce their migration speed, pause, and polarize toward the bound cell or the tissue structure to execute crucial functions including phagocytosis, cell-tocell signaling, activation, and the release of cytokines or toxic factors toward an encountered

At least three basic kinetic states govern leukocyte positioning in tissues, including fast migration (5 to 25 μm/min), slow and often locally confined movement (2 to 5 μm), and adhesive arrest, and these rapidly interconvert. Based on these kinetic states, leukocyte accumulation in tissues occurs by means of at least three distinct mechanisms: 1) local engagement of adhesion receptors causes individual leukocytes to stick and become immobilized at a specific spot; 2) degradation of promigratory signals causes cell populations to slow down or stop movement, and 3) loss of exit signals confines cells to a

Complete migration arrest is mediated by activation of adhesion receptors on the moving cell followed by attachment to counter-receptors on other cells or on Endothelial cell migration (ECM) structures, leading to an immobilized cell. Within seconds, adhesion overrides ongoing promigratory signals; this is followed by cytoskeletal polarization toward

Endothelial cells (EC) are the critical substrate for leukocyte attachment and motility within the vascular lumen via adhesion molecules such as integrin, ligands whose expression is enhanced on activated ECs, which in turn react to molecules generated during infection and inflammation such as Tumor necrosis factor alpha (TNF-), interleukin 1 (IL-1), and interleukin 17 (IL-17). Expression of these molecules can be further regulated through the cross-talk between EC and leucocytes; binding of PSGL-1 to P-selectin and E-selectin establishes the initial contact between neutrophils and activated ECs. Interaction of EC adhesion molecules (ICAM-1 and VCAM-1) with leukocyte ligands triggers the formation of docking structures or transmigrating cups [16, 17], which embrace adherent leucocytes [18]. Additionally, formation of pro-adhesive sites termed "endothelial adhesive platforms" (EAP) is determined by the existence of pre-formed, tetraspanin-enriched microdomains

Adherent leukocytes may transmigrate at the point of initial arrest, but sometimes rather locomote laterally to preferred sites of Transendothelial cell migration (TECM) [20, 21]; *in vitro* and *in vivo* luminal crawling is dependent on β2 integrins and its blockade appears to increase the incidence of trans- as opposed to paracellular cell migration [21]. The junctional adhesion molecule A (JAM-A), an adhesion molecule expressed on both EC and leukocytes

There are other molecules and mechanisms that have been recently implicated in leukocyte motility; for example, it has been demonstrated both *in vivo* and *in vitro* that platelets enhance neutrophil TECM in inflammation, which is consistent with a mechanistic role for

**2. Trafficking requirements**

local microenvironment despite ongoing migration [14].

the bound cell or the ECM structure [15].

**3. Ensuring tightened adhesion**

such as CD9, CD151, and CD81 [19].

PSGL-1 for this response [24].

[22], regulate integrin internalization and re-cycling [23].

cell.

poles. Polarization provides cells with morphological, functional, and sensitivity differences to the chemoattractant, altering the way the cell responds to a gradient. Thus, polarization generates a bipolar mechanosensory state with a dynamic leading edge for acquiring new contacts and signals, a stiff mid-body, and a sticky uropod that is dragged along the substrate and stabilizes the cell position in complex environments [9, 10]. Hence, integration of signals generated in both cellular poles leads to a coordinated movement of the leukocyte.

Chemotaxis is conceptually divided into motility, directional sensing, and polarity; however, chemotaxis typically incorporates these features. Many molecules involved in chemotaxis include both lipids and proteins and are localized on the membrane or in the cortex, specifically at either the leading or the lagging edge of polarized cells.

Freely diffusing chemoattractant or soluble molecular cues, known as Damage-associated molecular patterns (DAMP), are liberated from damaged tissue in high abundance. DAMP include Adenosine triphosphate (ATP), bacterial peptides, heat-shock proteins, chromatin, and galectins [11], providing short-lived or pulsatile directional information, in addition to longer-lived cues provided by constitutive or induced tissue-bound chemoattractants [11]. Beyond adhesive migration arrest, local reduction of promigratory signals is achieved by down-modulation of chemoattractant receptors, receptor desensitization, and ligand competition, whereas termination of chemoattractant activity occurs through uptake by neutralizing chemoattractant receptors and/or proteolytic degradation. After ligation, chemoattractant receptors become internalized and are either recycled to the leading edge or stored in vesicles in the uropod, thus limiting the availability of both the chemoattractant and its receptor [12]. The end result is a cascade of activation and adhesion events designed to uptake leukocytes along vessel walls, activate these for them to make stable adhesions, allow them to locomote along the endothelial surface, and to transmigrate across endothelial junctions and through the subendothelial basal lamina, guiding them onto the damage site (Figure 1) [13].

Fig. 1. Type chemotaxis in neutrophils. A) Chemotaxis triggered by soluble diffusing compounds leading to formation of the leading edge. B) Directed-mediated migration toward chemoattractants trapped on tissue structures.

### **2. Trafficking requirements**

212 Hematology – Science and Practice

poles. Polarization provides cells with morphological, functional, and sensitivity differences to the chemoattractant, altering the way the cell responds to a gradient. Thus, polarization generates a bipolar mechanosensory state with a dynamic leading edge for acquiring new contacts and signals, a stiff mid-body, and a sticky uropod that is dragged along the substrate and stabilizes the cell position in complex environments [9, 10]. Hence, integration of signals generated in both cellular poles leads to a coordinated movement of the leukocyte. Chemotaxis is conceptually divided into motility, directional sensing, and polarity; however, chemotaxis typically incorporates these features. Many molecules involved in chemotaxis include both lipids and proteins and are localized on the membrane or in the

Freely diffusing chemoattractant or soluble molecular cues, known as Damage-associated molecular patterns (DAMP), are liberated from damaged tissue in high abundance. DAMP include Adenosine triphosphate (ATP), bacterial peptides, heat-shock proteins, chromatin, and galectins [11], providing short-lived or pulsatile directional information, in addition to longer-lived cues provided by constitutive or induced tissue-bound chemoattractants [11]. Beyond adhesive migration arrest, local reduction of promigratory signals is achieved by down-modulation of chemoattractant receptors, receptor desensitization, and ligand competition, whereas termination of chemoattractant activity occurs through uptake by neutralizing chemoattractant receptors and/or proteolytic degradation. After ligation, chemoattractant receptors become internalized and are either recycled to the leading edge or stored in vesicles in the uropod, thus limiting the availability of both the chemoattractant and its receptor [12]. The end result is a cascade of activation and adhesion events designed to uptake leukocytes along vessel walls, activate these for them to make stable adhesions, allow them to locomote along the endothelial surface, and to transmigrate across endothelial junctions and through the subendothelial basal lamina, guiding them onto the damage site

Fig. 1. Type chemotaxis in neutrophils. A) Chemotaxis triggered by soluble diffusing compounds leading to formation of the leading edge. B) Directed-mediated migration

toward chemoattractants trapped on tissue structures.

cortex, specifically at either the leading or the lagging edge of polarized cells.

(Figure 1) [13].

Trafficking leukocytes often reduce their migration speed, pause, and polarize toward the bound cell or the tissue structure to execute crucial functions including phagocytosis, cell-tocell signaling, activation, and the release of cytokines or toxic factors toward an encountered cell.

At least three basic kinetic states govern leukocyte positioning in tissues, including fast migration (5 to 25 μm/min), slow and often locally confined movement (2 to 5 μm), and adhesive arrest, and these rapidly interconvert. Based on these kinetic states, leukocyte accumulation in tissues occurs by means of at least three distinct mechanisms: 1) local engagement of adhesion receptors causes individual leukocytes to stick and become immobilized at a specific spot; 2) degradation of promigratory signals causes cell populations to slow down or stop movement, and 3) loss of exit signals confines cells to a local microenvironment despite ongoing migration [14].

Complete migration arrest is mediated by activation of adhesion receptors on the moving cell followed by attachment to counter-receptors on other cells or on Endothelial cell migration (ECM) structures, leading to an immobilized cell. Within seconds, adhesion overrides ongoing promigratory signals; this is followed by cytoskeletal polarization toward the bound cell or the ECM structure [15].

### **3. Ensuring tightened adhesion**

Endothelial cells (EC) are the critical substrate for leukocyte attachment and motility within the vascular lumen via adhesion molecules such as integrin, ligands whose expression is enhanced on activated ECs, which in turn react to molecules generated during infection and inflammation such as Tumor necrosis factor alpha (TNF-), interleukin 1 (IL-1), and interleukin 17 (IL-17). Expression of these molecules can be further regulated through the cross-talk between EC and leucocytes; binding of PSGL-1 to P-selectin and E-selectin establishes the initial contact between neutrophils and activated ECs. Interaction of EC adhesion molecules (ICAM-1 and VCAM-1) with leukocyte ligands triggers the formation of docking structures or transmigrating cups [16, 17], which embrace adherent leucocytes [18]. Additionally, formation of pro-adhesive sites termed "endothelial adhesive platforms" (EAP) is determined by the existence of pre-formed, tetraspanin-enriched microdomains such as CD9, CD151, and CD81 [19].

Adherent leukocytes may transmigrate at the point of initial arrest, but sometimes rather locomote laterally to preferred sites of Transendothelial cell migration (TECM) [20, 21]; *in vitro* and *in vivo* luminal crawling is dependent on β2 integrins and its blockade appears to increase the incidence of trans- as opposed to paracellular cell migration [21]. The junctional adhesion molecule A (JAM-A), an adhesion molecule expressed on both EC and leukocytes [22], regulate integrin internalization and re-cycling [23].

There are other molecules and mechanisms that have been recently implicated in leukocyte motility; for example, it has been demonstrated both *in vivo* and *in vitro* that platelets enhance neutrophil TECM in inflammation, which is consistent with a mechanistic role for PSGL-1 for this response [24].

Neutrophil Chemotaxis and Polarization: When Asymmetry Means Movement 215

Beyond the endothelium, migrating cells face two further barriers; the pericyte sheath, and the tough venular Basement membrane (BM) [36, 37]. Neutrophils have the ability to

Fig. 2. Hypothetical sequence of events during neutrophil transmigration. Neutrophils are tethered by P- and E-selectin on endothelial cells and PSGL, L-selectin, and CD44 on neutrophils simultaneously participate in neutrophil rolling and activation. Endothelium activation by stimuli such as IL-1, IL17, TNF- promote transmigration dependent of molecules such as PECAM-1, ICAM-1, and JAM-A, thus unzipping the tight junctions and restoring themselves while TNF- promote transmigration via ESAM. Neutrophils take trans- or paracellular routes. Postendothelial cleavage of structural proteins occurs by means

of secreted or membrane-anchored matrix metalloproteases (MMPs). Abbreviations: Basement membrane (BM), Endothelial cells (EC), Platelet endothelial cell adhesion

adhesion molecule (ESAM), Tumor necrosis factor- (TNF-) [39].

**6. Neutrophil polarization and migration structures**

basement membrane and homing into sites of infection or inflammation.

molecule (PECAM-1), Intracellular adhesion molecule-1 (ICAM-1), Endothelial cell-selective

On the other hand, leukocyte penetration of the vascular BM depends on the vascular bed. Additionally, it has recently been shown that the venular BM contains pre-formed regions with low expression of certain BM components, denominated Low expression regions (LER), which are preferentially utilized by transmigrating neutrophils and monocytes [29, 40]. Alignment of these regions with gaps between adjacent pericytes suggests a key role for these cells in vascular BM generation *in vivo*. Vascular BM architecture depends on the migration of neutrophils, but not monocytes, through the LER remodeling these regions and increasing their size [41, 42], suggesting the involvement of proteases in this response.

Neutrophils present in the blood are able to tissue-injury or infection signals by adhering to vascular endothelial cells, then transmigrating across the endothelium through the

migrate through the pericyte sheath via both para- [38] and transcellular pathways

**5. Mobilization beyond the endothelium**

### **4. Neutrophil mobilization**

Leukocyte interactions with the endothelial surface trigger cellular and sub-cellular events that initiate and/or facilitate leukocyte passage through the endothelium by interaction of docking structures with cytoskeleton via adaptor proteins such as vinculin, paxilin, and Ezrin, radixin, and moesin (ERM) proteins [18, 25], although Guanosine triphosphate (GTP)ases (RhoG and RhoA) induce actin polymerization leading to the formation of small membrane protrusions called apical cups or docking structures.

Once firm adhesion is established, two routes can be taken for transendothelial migration: the transcellular road, whereby neutrophils penetrate the individual EC, or the paracellular road, by which neutrophils squeeze between EC Figure 2.

A number of molecules at EC junctions actively facilitate leukocyte transmigration via a paracellular route such as Platelet endothelial adhesion molecular-1 (PECAM-1), Intracellular adhesion molecule-2 (ICAM-2), CD99, Endothelial cell-selective adhesion molecules (ESAM), and junctional adhesion molecules (JAM) [22, 26] and, according to *in vivo* and *in vitro* evidence, a sequence of events has been suggested that regulate neutrophil transmigration to EC walls and that include the following: (i) ICAM-1 and ICAM-2 on the luminal surface of EC and within the junction may provide a haptotactic gradient to guide neutrophils to EC junctions via their 2 partners (LFA-1 and MAC-1) [27]; (ii) once within junctions, endothelial-cell JAM-A (through interaction, possibly with LFA-1) [28], facilitates completion of neutrophil passage through the EC layer, and (iii) within the EC junction, homophilic interactions between endothelial and leukocyte PECAM-1 stimulates neutrophils to express the key leukocyte laminin receptor, integrin α6β1, on their surface, which facilitates neutrophil passage through the vascular basement membrane [29-31]. It is also noteworthy that signals from ICAM-1 activate Src and Pyk-2 tyrosine kinases, which phosphorylate VE-Cadherin, destabilizing its bonds and loosening endothelial cell-cell junctions [32].

The transcellular route is taken by some 20% of neutrophils and has been observed in a broad range of tissues including bone marrow, thymus, lymph nodes, pancreas, and the blood brain barrier [33]. Apparently, there is clear evidence for the formation of a transcellular pore requiring membrane fusion and displacement of cytoplasmic organelles during transcellular migration. Vesicular vacuolar organelles (VVO) are enriched at poreformation sites, apparently providing additional membrane to the area and facilitating the fusion of apical and basal membranes in a process dependent on SNARE-containing membrane fusion complexes [34], and there is increasing evidence for a role for caveolin-1 in determining transendothelial migration route [35].

Carman et al. (2008) [34] have identified *in vitro* and *in vivo* the existence of protrusive podosomes on the basal side of crawling lymphocytes ; these protrusive podosomes appear to identify the cell's thinner peripheral areas rather than the perinuclear region in order to identify a pore formation-permissive site. These dynamic investigatory podosomes can then extend to form invasive podosomes, resembling invadopodia of metastatic tumor cells, which extend down into the EC, bringing the apical and basal membranes into close apposition. U

### **5. Mobilization beyond the endothelium**

214 Hematology – Science and Practice

Leukocyte interactions with the endothelial surface trigger cellular and sub-cellular events that initiate and/or facilitate leukocyte passage through the endothelium by interaction of docking structures with cytoskeleton via adaptor proteins such as vinculin, paxilin, and Ezrin, radixin, and moesin (ERM) proteins [18, 25], although Guanosine triphosphate (GTP)ases (RhoG and RhoA) induce actin polymerization leading to the formation of small

Once firm adhesion is established, two routes can be taken for transendothelial migration: the transcellular road, whereby neutrophils penetrate the individual EC, or the paracellular

A number of molecules at EC junctions actively facilitate leukocyte transmigration via a paracellular route such as Platelet endothelial adhesion molecular-1 (PECAM-1), Intracellular adhesion molecule-2 (ICAM-2), CD99, Endothelial cell-selective adhesion molecules (ESAM), and junctional adhesion molecules (JAM) [22, 26] and, according to *in vivo* and *in vitro* evidence, a sequence of events has been suggested that regulate neutrophil transmigration to EC walls and that include the following: (i) ICAM-1 and ICAM-2 on the luminal surface of EC and within the junction may provide a haptotactic gradient to guide neutrophils to EC junctions via their 2 partners (LFA-1 and MAC-1) [27]; (ii) once within junctions, endothelial-cell JAM-A (through interaction, possibly with LFA-1) [28], facilitates completion of neutrophil passage through the EC layer, and (iii) within the EC junction, homophilic interactions between endothelial and leukocyte PECAM-1 stimulates neutrophils to express the key leukocyte laminin receptor, integrin α6β1, on their surface, which facilitates neutrophil passage through the vascular basement membrane [29-31]. It is also noteworthy that signals from ICAM-1 activate Src and Pyk-2 tyrosine kinases, which phosphorylate VE-Cadherin, destabilizing its bonds and loosening endothelial cell-cell

The transcellular route is taken by some 20% of neutrophils and has been observed in a broad range of tissues including bone marrow, thymus, lymph nodes, pancreas, and the blood brain barrier [33]. Apparently, there is clear evidence for the formation of a transcellular pore requiring membrane fusion and displacement of cytoplasmic organelles during transcellular migration. Vesicular vacuolar organelles (VVO) are enriched at poreformation sites, apparently providing additional membrane to the area and facilitating the fusion of apical and basal membranes in a process dependent on SNARE-containing membrane fusion complexes [34], and there is increasing evidence for a role for caveolin-1 in

Carman et al. (2008) [34] have identified *in vitro* and *in vivo* the existence of protrusive podosomes on the basal side of crawling lymphocytes ; these protrusive podosomes appear to identify the cell's thinner peripheral areas rather than the perinuclear region in order to identify a pore formation-permissive site. These dynamic investigatory podosomes can then extend to form invasive podosomes, resembling invadopodia of metastatic tumor cells, which extend down into the EC, bringing the apical and basal membranes into close

membrane protrusions called apical cups or docking structures.

road, by which neutrophils squeeze between EC Figure 2.

determining transendothelial migration route [35].

**4. Neutrophil mobilization**

junctions [32].

apposition. U

Beyond the endothelium, migrating cells face two further barriers; the pericyte sheath, and the tough venular Basement membrane (BM) [36, 37]. Neutrophils have the ability to migrate through the pericyte sheath via both para- [38] and transcellular pathways

Fig. 2. Hypothetical sequence of events during neutrophil transmigration. Neutrophils are tethered by P- and E-selectin on endothelial cells and PSGL, L-selectin, and CD44 on neutrophils simultaneously participate in neutrophil rolling and activation. Endothelium activation by stimuli such as IL-1, IL17, TNF- promote transmigration dependent of molecules such as PECAM-1, ICAM-1, and JAM-A, thus unzipping the tight junctions and restoring themselves while TNF- promote transmigration via ESAM. Neutrophils take trans- or paracellular routes. Postendothelial cleavage of structural proteins occurs by means of secreted or membrane-anchored matrix metalloproteases (MMPs). Abbreviations: Basement membrane (BM), Endothelial cells (EC), Platelet endothelial cell adhesion molecule (PECAM-1), Intracellular adhesion molecule-1 (ICAM-1), Endothelial cell-selective adhesion molecule (ESAM), Tumor necrosis factor- (TNF-) [39].

On the other hand, leukocyte penetration of the vascular BM depends on the vascular bed. Additionally, it has recently been shown that the venular BM contains pre-formed regions with low expression of certain BM components, denominated Low expression regions (LER), which are preferentially utilized by transmigrating neutrophils and monocytes [29, 40]. Alignment of these regions with gaps between adjacent pericytes suggests a key role for these cells in vascular BM generation *in vivo*. Vascular BM architecture depends on the migration of neutrophils, but not monocytes, through the LER remodeling these regions and increasing their size [41, 42], suggesting the involvement of proteases in this response.

### **6. Neutrophil polarization and migration structures**

Neutrophils present in the blood are able to tissue-injury or infection signals by adhering to vascular endothelial cells, then transmigrating across the endothelium through the basement membrane and homing into sites of infection or inflammation.

Neutrophil Chemotaxis and Polarization: When Asymmetry Means Movement 217

induces actin polymerization through WAVE (Scar) and Arp2/3. WAVE, a member of the WASP family of actin-binding proteins, mediates actin filament formation [56], while Arp2/3 causes sideward branching of actin filaments. Together, these activities generate interconnected, branched networks [57]. Thus, promigratory signals received at the leading edge generate local Rac activation and actin network protrusion, pushing the plasma membrane outward. Preferential receptor-sensitivity mechanisms at the leading edge are likely diverse and may include local signal- amplification mechanisms [58] and exclusion of counter-regulatory proteins. The mid-region generates actomyosin-based stiffness and contractility, limits lateral protrusions, and thereby maintains a stable, bipolar cortex. The cytoskeletal motor protein myosin II, located in the central and rear regions of leukocytes, promotes actin-filament contraction and limits lateral protrusions. Myosin II cross-links actin filaments in parallel, forming the contractile shell required to hold the extending cell

together and propelling the cell nucleus, the most rigid part of the cell, forward [59].

**8. Leukocyte movement in different environments and initial migration**

resistance, a process known as contact guidance (Figure 2).

capability of leukocytes is considerable, especially for neutrophils [63].

Neutrophils are able to migrate along or through 2- or 3- dimensional (2-D or 3-D) surfaces. 2-D Surfaces, such as inner vessel walls, peritoneum, and pleura, require integrin-mediated attachment known as haptokinesis and polarized adhesion through binding of integrins α4β1 and LFA-1 (L2) to their counterparts (VCAM-1 and ICAM-1)(Figure 3A). In contrast, migration in 3-D, ECM environments, which are composed mainly of cellular (lymph node) or fibrillar ECM components, is integrin-independent and cells use weakly adhesive-tononadhesive interaction and traction mechanisms that are mediated by actin flow along the confining ECM scaffold structure, contributing to shape change and squeezing [9, 44, 60] (Figure 3B). It is likely that neutrophils adapt to tissue geometry and follow paths of least

For passage, the first postendothelial tissue structure and barrier to cells undergoing diapedesis, locally confined cleavage of the structural proteins laminin-10 and type IV collagen, occurs by secreted or membrane-anchored Matrix metalloproteases (MMPs) and serine proteases [61, 62]. Cell-body deformation is coupled with cytoplasmic propulsion and streaming through preexisting or newly formed pores; the deformation and constriction

Interestingly, a recent study showed the existence of venule-wall regions in which laminin-10, collagen IV, and nidogen-2 expression is considerably diminished; neutrophil transmigration enlarges the size of these regions, and their protein content is further reduced, an effect that appears to involve neutrophil-derived serine proteases [40]. Location of proteases at the leukocyte cell surface takes place through two different mechanisms: either by endogenous expression as transmembrane proteins or by binding of extracellular proteases to integral membrane receptors. Integrins are shown to act as anchoring receptors for several proteases including MMPs; such interactions have been detected in caveolae, invadopodia, and at the leading edge of migrating cells, where directed proteolytic activity is required [64]. In this regard, pro-MMP-2 and pro-MMP-9 are bound to L2 and M2 on the surface of activated leukemic cells, and inhibition of these complexes blocks 2 integrindependent leukocyte migration [65]. Pro-MMP-9–M2 complexes are primarily localized into intracellular granules of resting neutrophils, but after cellular activation, they are

The following four steps mediate the multiple cycles of attachment and detachment generating neutrophil forward movement during migration: the leading edge protrudes one or several pseudopods by actin flow; protruding membrane and surface receptors interact with the substrate; actomyosin-mediated contraction of the cell body occurs in mid-region, thus the rear of the cell moves forward. Neutrophil migration moves at up to 30 μm/min, lacks strong adhesive interactions to the tissue, and commonly preserves tissue integrity [9].

Receptors such as β2 integrins in neutrophils show discrete relocation toward the tips of ruffles [43]. The mid-region of amoeboid cells contains the nucleus and a relatively immobile cell region that maintains the front-rear axis. The trailing edge contains the highly glycosylated surface receptors CD43 and CD44, adhesion receptors including Intercellular adhesion molecule (ICAM)-1, ICAM-3, β1 integrins, and Ezrin-radixin-moesin adaptor proteins (ERM), as well as GM-1-type cholesterol-rich microdomains [44]. The uropod mediates cell–matrix and cell–cell interactions during migration and has a putative anchoring function [45]. The uropod extends rearward from the nucleus and contains the microtubule-organizing center and rearward-polarized microtubules, the Golgi, and abundant actin-binding ERM proteins. In association with microtubules, mitochondria localize to the rear of the cell that, presumably, due to local ATP delivery to the region of ATP-dependent actomyosin contraction, is required for proper polarization, uropod retraction, and migration [10, 46].

### **7. Polarization of cytoskeletal and signaling scaffolds**

In neutrophils, polarization and migration to chemoattractant gradients such as chemokines and cytokines, lipid mediators, bacterial factors, and Extracellular matrix (ECM) degradation products including collagen, fibronectin, and elastin fragments [47, 48], is known as chemotaxis. After chemokines and chemoattractants bind to the extracellular domains of their cognate G protein-coupled receptor (GPCRs) pseudo- and lamellipodia protrusion are induced. In leukocytes, the majority of GPCRs transmit through the α subunit of Giα. These GPCR include the following: the fMLP (N-formyl-Met-Leu-Phe) receptor and the C5a receptor; chemokine receptors including CCR7, CXCR4, CXCR5, and CCR3; the leukotriene B4 receptor BLT1; sphingosine-1-phosphate receptors 1–4 (S1P1–4), and Lysophosphatidic acid (LPA) receptors 1–3 [49]. All these GPCR mediate promigratory signals but also enhance cell activation. A key GPCR-mediated pathway is signaling through the Phosphatidylinositol-3-kinase (PI(3)K), which contains the p110γ catalytic subunit). PI(3)K-γ is recruited into the inner leaflet of the plasma membrane by the G protein βγ subunit, where it becomes activated and subsequently phosphorylates Phosphatidylinositol phosphates (PIP) and other effectors [50]. PIP serve as docking sites for pleckstrin-homology domain-containing proteins, notably Akt (also known as protein kinase B), which is implicated in inducing actin polymerization and pseudopod protrusion by phosphorylating downstream effectors [51] such as the actin-binding protein girdin [52]. A second pathway linked with PI(3)K activation is induced by ζ-chain-associated receptors, including T cell receptors (TCRs) and receptors FC (FcRs). These receptors signal through tyrosine kinases Lck and Zap-70 to class Ia PI(3)Ks (consisting of p110δ) and activate downstream Akt, as well as the GTPases Rac and Cdc42 [53]. A third, PI(3)K-independent pathway induced by the fMLP receptor in neutrophils leads to the activation of p38 mitogen-associated protein kinase and downstream Rac activation [54, 55]. Ultimately, Rac

The following four steps mediate the multiple cycles of attachment and detachment generating neutrophil forward movement during migration: the leading edge protrudes one or several pseudopods by actin flow; protruding membrane and surface receptors interact with the substrate; actomyosin-mediated contraction of the cell body occurs in mid-region, thus the rear of the cell moves forward. Neutrophil migration moves at up to 30 μm/min, lacks strong adhesive interactions to the tissue, and commonly preserves tissue integrity [9]. Receptors such as β2 integrins in neutrophils show discrete relocation toward the tips of ruffles [43]. The mid-region of amoeboid cells contains the nucleus and a relatively immobile cell region that maintains the front-rear axis. The trailing edge contains the highly glycosylated surface receptors CD43 and CD44, adhesion receptors including Intercellular adhesion molecule (ICAM)-1, ICAM-3, β1 integrins, and Ezrin-radixin-moesin adaptor proteins (ERM), as well as GM-1-type cholesterol-rich microdomains [44]. The uropod mediates cell–matrix and cell–cell interactions during migration and has a putative anchoring function [45]. The uropod extends rearward from the nucleus and contains the microtubule-organizing center and rearward-polarized microtubules, the Golgi, and abundant actin-binding ERM proteins. In association with microtubules, mitochondria localize to the rear of the cell that, presumably, due to local ATP delivery to the region of ATP-dependent actomyosin contraction, is required for proper polarization, uropod

In neutrophils, polarization and migration to chemoattractant gradients such as chemokines and cytokines, lipid mediators, bacterial factors, and Extracellular matrix (ECM) degradation products including collagen, fibronectin, and elastin fragments [47, 48], is known as chemotaxis. After chemokines and chemoattractants bind to the extracellular domains of their cognate G protein-coupled receptor (GPCRs) pseudo- and lamellipodia protrusion are induced. In leukocytes, the majority of GPCRs transmit through the α subunit of Giα. These GPCR include the following: the fMLP (N-formyl-Met-Leu-Phe) receptor and the C5a receptor; chemokine receptors including CCR7, CXCR4, CXCR5, and CCR3; the leukotriene B4 receptor BLT1; sphingosine-1-phosphate receptors 1–4 (S1P1–4), and Lysophosphatidic acid (LPA) receptors 1–3 [49]. All these GPCR mediate promigratory signals but also enhance cell activation. A key GPCR-mediated pathway is signaling through the Phosphatidylinositol-3-kinase (PI(3)K), which contains the p110γ catalytic subunit). PI(3)K-γ is recruited into the inner leaflet of the plasma membrane by the G protein βγ subunit, where it becomes activated and subsequently phosphorylates Phosphatidylinositol phosphates (PIP) and other effectors [50]. PIP serve as docking sites for pleckstrin-homology domain-containing proteins, notably Akt (also known as protein kinase B), which is implicated in inducing actin polymerization and pseudopod protrusion by phosphorylating downstream effectors [51] such as the actin-binding protein girdin [52]. A second pathway linked with PI(3)K activation is induced by ζ-chain-associated receptors, including T cell receptors (TCRs) and receptors FC (FcRs). These receptors signal through tyrosine kinases Lck and Zap-70 to class Ia PI(3)Ks (consisting of p110δ) and activate downstream Akt, as well as the GTPases Rac and Cdc42 [53]. A third, PI(3)K-independent pathway induced by the fMLP receptor in neutrophils leads to the activation of p38 mitogen-associated protein kinase and downstream Rac activation [54, 55]. Ultimately, Rac

retraction, and migration [10, 46].

**7. Polarization of cytoskeletal and signaling scaffolds**

induces actin polymerization through WAVE (Scar) and Arp2/3. WAVE, a member of the WASP family of actin-binding proteins, mediates actin filament formation [56], while Arp2/3 causes sideward branching of actin filaments. Together, these activities generate interconnected, branched networks [57]. Thus, promigratory signals received at the leading edge generate local Rac activation and actin network protrusion, pushing the plasma membrane outward. Preferential receptor-sensitivity mechanisms at the leading edge are likely diverse and may include local signal- amplification mechanisms [58] and exclusion of counter-regulatory proteins. The mid-region generates actomyosin-based stiffness and contractility, limits lateral protrusions, and thereby maintains a stable, bipolar cortex. The cytoskeletal motor protein myosin II, located in the central and rear regions of leukocytes, promotes actin-filament contraction and limits lateral protrusions. Myosin II cross-links actin filaments in parallel, forming the contractile shell required to hold the extending cell together and propelling the cell nucleus, the most rigid part of the cell, forward [59].

### **8. Leukocyte movement in different environments and initial migration**

Neutrophils are able to migrate along or through 2- or 3- dimensional (2-D or 3-D) surfaces. 2-D Surfaces, such as inner vessel walls, peritoneum, and pleura, require integrin-mediated attachment known as haptokinesis and polarized adhesion through binding of integrins α4β1 and LFA-1 (L2) to their counterparts (VCAM-1 and ICAM-1)(Figure 3A). In contrast, migration in 3-D, ECM environments, which are composed mainly of cellular (lymph node) or fibrillar ECM components, is integrin-independent and cells use weakly adhesive-tononadhesive interaction and traction mechanisms that are mediated by actin flow along the confining ECM scaffold structure, contributing to shape change and squeezing [9, 44, 60] (Figure 3B). It is likely that neutrophils adapt to tissue geometry and follow paths of least resistance, a process known as contact guidance (Figure 2).

For passage, the first postendothelial tissue structure and barrier to cells undergoing diapedesis, locally confined cleavage of the structural proteins laminin-10 and type IV collagen, occurs by secreted or membrane-anchored Matrix metalloproteases (MMPs) and serine proteases [61, 62]. Cell-body deformation is coupled with cytoplasmic propulsion and streaming through preexisting or newly formed pores; the deformation and constriction capability of leukocytes is considerable, especially for neutrophils [63].

Interestingly, a recent study showed the existence of venule-wall regions in which laminin-10, collagen IV, and nidogen-2 expression is considerably diminished; neutrophil transmigration enlarges the size of these regions, and their protein content is further reduced, an effect that appears to involve neutrophil-derived serine proteases [40]. Location of proteases at the leukocyte cell surface takes place through two different mechanisms: either by endogenous expression as transmembrane proteins or by binding of extracellular proteases to integral membrane receptors. Integrins are shown to act as anchoring receptors for several proteases including MMPs; such interactions have been detected in caveolae, invadopodia, and at the leading edge of migrating cells, where directed proteolytic activity is required [64]. In this regard, pro-MMP-2 and pro-MMP-9 are bound to L2 and M2 on the surface of activated leukemic cells, and inhibition of these complexes blocks 2 integrindependent leukocyte migration [65]. Pro-MMP-9–M2 complexes are primarily localized into intracellular granules of resting neutrophils, but after cellular activation, they are

Neutrophil Chemotaxis and Polarization: When Asymmetry Means Movement 219

*Co1,Lm* ND *EC, SMC, TC, Monos*

*Col, Epiligrin, Fn, Lm, Invasin RGD EC, TC, EPC, Fb*

*Col, Fn, Lm, Echovirus 1 DGEA Plt, EC, Fb, SMC, TC, EPC*

Fn, Invasin, VCAM-1 EILDV (Fn) TC, Monos, Eos, LC, ER

Fn, Invasin RGD Fb, EC, Monos, TC, Plt

Lm, Invasin ND Plts, TC, EC, EPC

Lm ND Myocytes

Fn,Vn RGD Fb

Fn, Vn RGD Fb

Fn, Tenascin RGD

Col, Fib, Fn, Lm Opn, Pn,

 Col, Fib, Fn, TSP, Vn, vWf, *Borrelia*

 Fib, Fn, Factor X, ICAM-1, iC3b

vWf, HIV Tat, Tenascin,

Col, Lm, Tenascin RGD EPC, Myocytes

Vn, HIV Tat, Adenovirus RGD EC, EPC, Fb, Tumors

Vn RGD Melanoma

Fib, Fn, Vn, vWf RGD PMN

*<sup>2</sup>* ICAMs (1-3) ND TC, BC, LGL, Monos,

Fib, iC3b GPRP Monos, Macros, PMN

Lm ND EC, EPC, Schwann cells

Fn, MAdCAM, VCAM-1 EILDV (Fn) Gut homing, TC

E-Cadherin ND Epithelial TC

Abbreviations: BC = B cells; Col = Collagen; EC = Endothelial cells; Eos = Eosinophils; EPC = Epithelial cells; Fb = Fibroblasts; Fib = Fibrinogen; Fn = Fibronectin; iC3b = inactivated

Macros = Macrophages; Mega = Megakaryocytes; Monos = Monocytes; OPN = Osteopontin; Plt = Platelets; PMN = Neutrophils or Polymorphonuclear leukocytes; SMC = Smooth muscle cells; TC = T cells; TSP = Thrombospondin; Vn = Vitronectin; vWf = von Willebrand

Table 1. Classification of integrins according to ligand motifs and distribution.

component of complement; Lm = Laminin; LGL = Large granular lymphocytes;

ND TC. Macros

**integrin**

**integrins**

**2 integrin**

**Other integrins**

disease. (Modified from [71]).

**v and 3**

*v*

*v*

*v*

*v*

*v*

*IIb*

> *R*

*L*

**Ligands Motifs Distribution**

*QIDSPL(VCAM-1)*

ND *SMC*

TSP, Vn RGD EC, FB, Monos, SMC, OC

KQAGDV Plt, Mega

PMN, Eos

PMN, Monos, Macros, LGL

Adenovirus Plt, Tumors

relocalized to the cell surface [66]. Neutrophils secrete laminin, suggesting that leukocytederived matrix proteins might also contribute to the transmigration process [67].

Fig. 3. Type-substrate interaction with neutrophils. A) Two-dimensional integrin-mediated neutrophil migration*. In vivo* 2-D haptokinetic migration is present during crawling on Endothelial cell (EC) or through Extracellular matrix (EM). B) Three-dimensional integrinindependent neutrophil migration. *In vivo*, this occurs through organized tissue structures.

### **9. Role of cytoskeleton in regulating integrin adhesiveness**

Integrins are a superfamily of heterodimeric cell-surface receptors that are found in a broad range of animal species [68]; their main role, as their name implies, is to integrate the cell cytoskeleton with adhesion points of extracellular matrix and cell-surface ligands in order to mediate essential cellular processes such as cell-cell and cell-extracellular matrix interactions, polarization in response to extracellular cues, cell migration, differentiation, survival, and cell-pathogen interactions [69].

In vertebrates, 19 different integrin subunits and eight different integrin β subunits have been reported, in combination forming about 25 β heterodimers [70]. The majority of /βsubunit combinations can be organized into three fundamental groups based on subunit type (β1, β2, and β3, or v chains, on the extracellular matrix protein-type recognized, or on the specific adhesion motifs [71] (Table 1).

β1 integrins form the first and largest group of integrins and are ubiquitously distributed in nucleated cells as well as in platelets. β1 Integrins are expressed in bone marrow-derived cells (except for neutrophils), in certain tumor cells, and in muscle development. A second major group of integrins shares either the β3 or the v subunit (Table 1) and recognizes different ligands from a broad gamma of cell and tissue sources. Integrins with the v subunit may form dimers with at least five different β chains, including the β1 chain. Subunits v and β3 recognize Arg-Gly-Asp (RGD) domains present in extracellular matrix proteins.

The third group of integrins shares the β2 integrin chain, whose expression is restricted to leukocytes [72] (Table 1). Receptors such as 4β2, also known as the LFA-1 integrin, determine the capability of leukocytes in endothelial epithelium transmigration and recognize members of the Intercellular adhesion molecule (ICAM) family of adhesion proteins. In contrast, expression of Mβ2 is restricted to monocytes, macrophages, and granulocytes; it recognizes

relocalized to the cell surface [66]. Neutrophils secrete laminin, suggesting that leukocyte-

Fig. 3. Type-substrate interaction with neutrophils. A) Two-dimensional integrin-mediated neutrophil migration*. In vivo* 2-D haptokinetic migration is present during crawling on Endothelial cell (EC) or through Extracellular matrix (EM). B) Three-dimensional integrinindependent neutrophil migration. *In vivo*, this occurs through organized tissue structures.

Integrins are a superfamily of heterodimeric cell-surface receptors that are found in a broad range of animal species [68]; their main role, as their name implies, is to integrate the cell cytoskeleton with adhesion points of extracellular matrix and cell-surface ligands in order to mediate essential cellular processes such as cell-cell and cell-extracellular matrix interactions, polarization in response to extracellular cues, cell migration, differentiation,

In vertebrates, 19 different integrin subunits and eight different integrin β subunits have been reported, in combination forming about 25 β heterodimers [70]. The majority of /βsubunit combinations can be organized into three fundamental groups based on subunit type (β1, β2, and β3, or v chains, on the extracellular matrix protein-type recognized, or on

β1 integrins form the first and largest group of integrins and are ubiquitously distributed in nucleated cells as well as in platelets. β1 Integrins are expressed in bone marrow-derived cells (except for neutrophils), in certain tumor cells, and in muscle development. A second major group of integrins shares either the β3 or the v subunit (Table 1) and recognizes different ligands from a broad gamma of cell and tissue sources. Integrins with the v subunit may form dimers with at least five different β chains, including the β1 chain. Subunits v and β3

The third group of integrins shares the β2 integrin chain, whose expression is restricted to leukocytes [72] (Table 1). Receptors such as 4β2, also known as the LFA-1 integrin, determine the capability of leukocytes in endothelial epithelium transmigration and recognize members of the Intercellular adhesion molecule (ICAM) family of adhesion proteins. In contrast, expression of Mβ2 is restricted to monocytes, macrophages, and granulocytes; it recognizes

recognize Arg-Gly-Asp (RGD) domains present in extracellular matrix proteins.

**9. Role of cytoskeleton in regulating integrin adhesiveness**

survival, and cell-pathogen interactions [69].

the specific adhesion motifs [71] (Table 1).

derived matrix proteins might also contribute to the transmigration process [67].

Table 1. Classification of integrins according to ligand motifs and distribution. Abbreviations: BC = B cells; Col = Collagen; EC = Endothelial cells; Eos = Eosinophils; EPC = Epithelial cells; Fb = Fibroblasts; Fib = Fibrinogen; Fn = Fibronectin; iC3b = inactivated component of complement; Lm = Laminin; LGL = Large granular lymphocytes; Macros = Macrophages; Mega = Megakaryocytes; Monos = Monocytes; OPN = Osteopontin; Plt = Platelets; PMN = Neutrophils or Polymorphonuclear leukocytes; SMC = Smooth muscle cells; TC = T cells; TSP = Thrombospondin; Vn = Vitronectin; vWf = von Willebrand disease. (Modified from [71]).

Neutrophil Chemotaxis and Polarization: When Asymmetry Means Movement 221

Recently, dystrophin protein-adhesion complex proteins such as short dystrophins, utrophins, and the dystrophin-associated protein complex -dystroglycan,syntrophin and dystrobrevins) form part of actin-based structures such as lamellipodia and uropod, in which their polarized distribution is evident and their feasible role in chemotaxis and

Other proteins with differential distribution appear in Figure 4 and the list is increasing.

Fig. 4. Neutrophil regions observed after triggered activation and differential protein

For exiting the vasculature, leukocytes follow a consecutive sequence of events that starts with the first contact of free-flowing neutrophil to the vascular endothelium followed by leukocyte rolling along the vessel wall. Both events are mediated by specialized receptor ligand pairs consisting of a member of the selectin family of adhesion molecules and specific carbohydrate determinants on selectin ligands. During rolling, leukocytes are in intimate contact with the vascular endothelium, enabling endothelial-bound chemokines to interact with their respective chemokine receptors on the neutrophil surface. Upon binding to the receptor, chemokine receptor-mediated signaling events trigger the activation of β2 integrins. Activated integrins subsequently interact with endothelium-expressed ligands, which lead to a reduction in leukocyte rolling velocity and eventually, to mediate stable adhesion and migration across the blood vessel wall. Following neutrophil spreading and intravascular crawling along the endothelium, tethered neutrophils reach the correct spot for exiting into tissue. Upon neutrophil stimulation, actin, which is one of the major components of the cytoskeleton in neutrophils, is reorganized through reversible cycles of polymerization and depolymerization, thereby comprising the driving motor for the

migration is strongly suggested [86].

distribution. Adapted from [87].

**10. Conclusions**

fibrinogen and inactivated C3b, playing an important role in the phagocytosis of opsonized particles and bacteria [73]. The fourth group of integrins includes three integrins (6β4, 4β7, and Eβ7); these integrins recognize extracellular matrix components as well as adhesion molecules of the Immunoglobulin superfamily (IgSF). Common integrins expressed on leukocytes and their counterparts are summarized in Table 1.

Association of extended forms of integrins with the cortical cytoskeleton is required to integrate mechanical forces from shear flow and F-actin and to undergo ligand-induced strengthening at endothelial contacts. Key differences between 4 and 2 integrins regarding their increase in cytoskeleton-mediated avidity may occur. The 4 integrins can bind paxillin upon dephosphorylation of Ser988 in their cytoplasmic domain at the sides and rear pole of the cell, whereas PKA-mediated phosphorylation of these integrins is confined to the cell's leading edge. Paxillin regulates 4 integrin function (tethering and firm adhesion) [74], enhancing their migration rate and reducing their spreading, and paxillin–4 interaction downregulates the formation of focal adhesions, stress fibers, and lamellipodia by triggering activation of different tyrosine kinases, such as Focal adhesion kinase (FAK), Pyk2, Src, and Abl [75, 76]. The 4–paxillin complex inhibits stable lamellipodia by recruiting an ADP-ribosylation factor (Arf)-GTPase-activating protein that decreases Arf activity, thereby inhibiting Rac, and limiting lamellipodia formation to the cell front [77]. Recently, it was discovered that integrins can induce PIP5K1C-90 polarization independently of chemoattractants. This integrin-induced PIP5K1C-90 polarization works together with chemoattractant signaling in regulating neutrophil polarization and directionality *in vitro* and infiltration *in vivo* [78].