humans are confronted with a number of microorganisms , viruses , and parasites that require continued surveillance and defense against [ 1 , 2 ] .
polymorphonuclear leukocytes ( pmns ) , monocytes , and tissue macrophages are important cells in the innate immune response to microorganisms and parasites [ 1 , 3 ] . all derived from the same myeloid progenitor cell in the bone marrow .
blood monocytes are an intermediate stage of development which then further differentiates in tissues to various macrophage populations .
tissue macrophages , as well as , other cells of the innate immune system , are critical for these surveillance and defense activities .
upon stimulation by microbial particles or other endogenous factors such as cytokines , macrophages can de novo synthesize and release a large variety of cytokines ( il-1 , il-6 , il-8 , il-10 , il-12 , tnf , ifn , ifn , mcp - l , mcp-3 , mif , m - csf , g - csf , gm - csf , mip - l , mip-2 , lif , osm , and tgf- ) .
some cytokines can stimulate the production of cytokines by macrophages ( il-3 , gm - csf , ifn ) , while others can suppress it ( il-4 , il-10 , il-13 , tgf ) .
in addition , in many organs , tissue macrophages serve as important regulatory cells , through their synthesis and secretion of a spectrum of cytokines and growth factors [ 57 ] .
toll - like receptors ( tlrs ) of mammalian innate immune defense systems play a crucial role in the initiation of adaptive immune responses to bacteria invasion [ 811 ] .
a critical feature of macrophages required for specific recognition and response includes the expression of optimum levels of different tlrs [ 811 ] . upon the interaction with specific pathogen - associated molecular patterns ( pamps ) ,
tlrs trigger the signal transduction cascades that lead to cellular responses including the engulfment of invasive microbes , induction of inflammatory cytokines , and generation of ros to kill the invasive pathogens .
several mammalian tlrs genes have been identified , and at least ten human tlrs have been cloned .
tlr1 , predominantly distributed in monocytes / macrophages and pmns , is implicated with trl2 in the recognition of the native configuration of mycobacterial lipoprotein and several other lipopeptides [ 10 , 12 ] .
tlr4 and cd14 , on the other hand , are required for the recognition of the gram - negative bacteria - derived lipopolysaccharide ( lps ) . during sepsis ,
the binding of lps to extracellular domain of tlr4 receptors on phagocytic cells , macrophages , and neutrophils elicits activation of the tlr4/irak-2/nf-b complex to signal production and release of proinflammatory mediators ( cytokines ) phagocytic cells .
although tlrs play an important role in the adequate response of immune cells , the deregulation of their expression and function causes defective immune responses [ 1416 ] and the occurrence of chronic diseases [ 1719 ] . to understand the molecular mechanisms underlying macrophage differentiation , investigators have turned to human myeloid cell lines such as hl-60 , u937 , thp-1 , and ml-1 [ 2023 ] . in vitro ,
these myeloid cell lines continuously proliferate in suspension culture and can be induced to differentiate into macrophages by 12-o - tetradecanoylphorbol-13-acetate ( tpa ) or 1,25-dihydroxyvitamin d3 .
these cell lines have been widely used as cell models for studying the molecular and cellular aspects of myeloid differentiation .
ml-1 cells were originally isolated from a patient with acute myeloblastic leukemia [ 25 , 26 ] .
these cells readily differentiate into macrophages in response to a low concentration of tpa ( 0.3  ng / ml or 0.5  nm ) [ 25 , 26 ] . in this process
, the differentiation signal induced by tpa is mediated via a redox - regulated extracellular receptor kinase ( erk1/2 ) .
differentiated ml-1 cells demonstrate a number of biochemical and functional endpoint indicators of macrophage differentiation including : nonspecific esterase ( nse ) phagocytosis , plasma membrane nadph oxidase and accompanying superoxide generation , and cell surface markers [ 2831 ] . in the present study
, we have characterized the temporal upregulation of tlr1 , tlr2 , tlr4 , and irak2 expression during the differentiation of ml-1 cells leading to increased responsiveness to lps and lta .
our data also show that the increased expression of tlr1 and tlr4 induced by tpa is at least in part mediated through activation of erks .
lipoteichoic acid ( lta ) ( major constituent of the gram - positive bacteria cell wall ) and lipopolysaccharide ( lps ) ( escherichia coli ) were purchased from sigma - aldrich .
the human cytokines il-1 alpha , il-1 beta , il-6 , il-8 , il-10 , il-12(p40 ) , gmcsf , ifn - gamma , tnf- and ip-10 cytokine multiplex immunoassay reagents were obtained from upstate biotechnology , lake placid , ny , usa . the alexa fluor 488-labeled mouse anti - human abs against tlr1 , tlr4 , cd14 , and
human myeloid leukemia ml-1 cells , originally isolated from a patient with acute myeloblastic leukemia , were kindly provided by dr .
ruth craig , dartmouth school of medicine , nh . all cell culture media and supplements were obtained from invitrogen ( carlsband , ca ) .
cells were cultured in rpmi 1640 medium supplemented with 10% heat - inactivated fetal bovine serum , 2  mm l - glutamine , antibiotics ( 50 u / ml penicillin and 50  g / ml streptomycin ) , and maintained at 37c in a humidified 5% co2 atmosphere as previously described [ 25 , 26 , 32 ] .
the differentiation of ml-1 cells to macrophages entails a 6-day protocol : three days exposure to tpa ( 5  nm ) and followed by three days of culture in the absence of tpa .
both the concentration of tpa used and the time of its presence are critical for optimal macrophage differentiation , particularly mitochondrial maturation .
louis , mo ) was dissolved in dmso to obtain a 100  m stock solution and further diluted before use .
for all experiments , cells were cultured at an initial density of 5  10 cells / ml . for negative controls ,
the cells were incubated in the absence of tpa , in medium containing an equivalent 1% ( v / v ) dmso .
ml-1 cells were grown at density of 10  cells / ml in a 150  ml dish and treated with vehicle ( dmso ) or 5  nm tpa ( n = 3 ) .
the purified rna was subjected to affymetrix oligonucleotide microarray analysis using human genome u133 2.0 plus array chip .
six replicates , including three controls and three tpa treated ml-1 cells samples , were examined .
the levels of tlr1 , tlr4 , and cd14 were determined using facs - scan analysis .
cells ( 1  10 ) resuspended in pbs containing 0.1% sodium azide and 5% fbs were incubated on ice for 30  min and then incubated with ( alexa - fluor 488 ) conjugated anti - tlr1 , tlr4 , or cd14 antibodies for 1 hour .
the cells were washed twice , fixed in 2% formaldehyde in pbs , and analyzed by facs - scan analysis .
negative controls were stained with isotype - matched ( alexa - fluor 488)-conjugated igg and compensation was adjusted using the single - stained cell samples .
the fluorescence intensities were determined using cellquest software ( becton dickinson , bedford , ma ) .   selected panel of inflammatory mediators ( il-1 alpha , il-1 beta , il-6 , il-8 , il-10 , il-12(p40 ) , gmcsf , ifn - gamma , tnf - a , and ip-10 ) in cell culture medium were analyzed following stimulation of ml-1 cells or differentiated ml-1 cells ( dml-1 ) with lps or lta for 16 hours , using cytokine multiplex immunoassay reagents ( upstate biotechnology , lake placid , new york , united states ) analyzed by luminex 100 ( luminex corporation , austin , texas , united states ) .
the generation of cellular ros , in particularextracellular h2o2 , was assessed using horseradish peroxidase - dependent luminol - derived chemiluminescence(cl ) as previously described .
one million cells were incubated with luminol ( 10  m ) and horseradish peroxidase ( 10  g / ml ) in complete pbs , 500  l ( pbs with 0.5  mm mgcl2 , 0.7  mm cacl2 , 0.1% glucose ) .
cellular cl was measured continuously in a berthold lb9505 luminometer ( pforzheim , germany ) for 1.5 hours .
in our previous studies , we have demonstrated that human myeloid ml-1 cells differentiate to macrophages upon stimulation with the phorbol ester tpa . to further confirm that tpa - treated ml-1 cells exhibit macrophage characteristics , cd14 gene expression , a key macrophage marker , was determined at the mrna and protein level .
examination of the microarray analysis ( figure 1(a ) ) , and flow cytometry analysis ( figure 1(b ) ) revealed a strong induction of cd14 gene expression upon stimulation with tpa and in a time - dependent fashion .
toll - like receptors ( tlrs ) of mammalian innate immune defense systems play a crucial role in the initiation of adaptive immune responses to bacteria invasion [ 811 ] .
accordingly , we assessed the temporal expression of different tlr gene products in response to tpa - induced macrophage differentiation of ml-1 cells .
analysis of microarray data revealed significant increases in the mrna levels of tlr1 , tlr2 , tlr4 , and tlr8 expression also that of irak-2 in ml1 cells compared to control ( dmso - treated cells ) ( figure 2 ) .
interestingly , only the gene expression of the tlr members indicated above were changed in response to tpa .
as shown in figure 2 , the mrna for each of the tlrs showed different expression patterns , with tlr4 showing maximum mrna as early as 6 hours of tpa addition , after which it decreased .
interestingly , irak-2 mrna exhibited the same temporal pattern as tlr4 ( figure 2(b ) ) .
on the other hand , the mrna for tlr2 and tlr8 was apparent at 6 days of differentiation .  
flow cytometry analysis confirmed enhanced tlr1 and tlr4 protein expression by ml-1cells ( figure 3(a ) ) .
immunocytochemistry data revealed a basal level of expression of tlr4 but not tlr1 in the undifferentiated ml-1 cells ( figure 3(b ) ) , which increased with macrophage differentiation . in our previous studies [ 23 , 27 , 35 ]
, we reported that the mitogen - activated protein kinase ( mapk ) cascade plays a crucial role in the initiation of the macrophage differentiation signal induced by tpa . using u0126 , a mek inhibitor
, we examined whether mapk - derived differentiation signal was required for tlr-1 and tlr-4 expression - induced by tpa in ml-1 cells .
pretreatment with 10  m u0126 effectively blocked both tlr1 and tlr4 protein expression induced by tpa in ml-1 cells ( figure 4 ) .
since ml-1 cells already expressed a basal level of tlr4 protein , the observed increase in lps and lta response may be due in part to the upregulation of irak-2 gene expression in response to tpa .
together , these observations suggested that tlr1 , tlr2 tlr4 , and cd14 and irak-2 gene expression is induced in ml-1 cells upon stimulation with tpa .
this observation is supported by a previous report that tpa induces tlr2 expression in u937 cells . to determine whether tpa - differentiated ml-1 cells exhibit macrophage - like responses following stimulation with bacterial particles lps and lta , cytokine multiplex immunoassay was used to assess the levels of selected cytokine production and release .
incubation with lta ( 10  g / ml ) and lps ( 100  ng / ml ) resulted in robust production of various cytokines by the differentiated ml-1cells ( figure 5 ) .
for example , lps either induced il-1 alpha and beta , il-6 , tnf alpha and ip-10 , or to a much greater extent than lta .
the presence of il-10 , il-12p40 , and interferon gamma was similar in response to lps and lta .
these observations support our previous findings that tpa treatment induces ml-1 cell differentiation to functional macrophages . to determine whether tpa - differentiated ml-1 cells exhibit a change in ros levels in response to lps and lta , luminol - derived cl was used .
horseradish peroxidase - dependent luminol - derived cl indicates the presence of extracellular hydrogen peroxide .
as shown in figure 6 , ml-1 cells exhibit a significant luminol - derived cl , indicative of the presence of extracellular hydrogen peroxide .
we have previously determined that in unstimulated macrophages that this basal ros is mitochondrial in origin .
the differentiated ml-1 cells exhibited a delayed but a more sustained production of hydrogen peroxide in the presence of lps or lta .
addition of dpi ( 10  m ) , a potent inhibitor of nad(p)h - dependent enzymes , rapidly suppressed the luminol - derived cl suggesting a role of nadph oxidase and/or mitochondria complex i in the observed ros production by ml-1 cells ( figure 6 ) .
the basal unstimulated ros production by differentiated ml-1 cells was also blocked by dpi , as previously shown ( figure 6 ) .
interestingly , the sustained , increased ros with lps or lta treatment was not apparent until 3040 minutes after their addition . in this
regard , ros from nadph oxidase in alveolar macrophages is apparent almost immediately after tpa addition to activate pkc .
macrophages are key members of the innate immune system and are known to play crucial roles in initiating and maintaining the immune response [ 38 , 39 ] .
our immunocytochemistry data show a basal expression of human myeloid ml-1 cells differentiate into macrophage - like cells when incubated with tpa .
we have demonstrated that the tpa - induced differentiation signal is mediated via a redox - mediated activation of the extracellular - regulated kinase ( erk1/2 ) , a family member of mitogen - activated protein kinases , mapks , to signal cell growth arrest and cell attachment during the initiation of the differentiation process [ 23 , 27 ] . in the present study , we investigated the mechanism by which tpa treatment may lead to increase in ml-1 cell response to stimulation by bacterial particle lps and lta .
we have shown that tpa treatments result in the upregulation of tlr1 , tlr2 , tlr4 , and cd14 in ml-1 cells , which lead in part to the potentiation of their responses to lps and lta .
this observation correlates with a previous study by shuto et al . in 2007 , which has suggested the existence of a direct correlation between the levels of tlr2 proteins expression and the cellular response to tlr2 ligands in differentiated hl-60 cells .
has also shown an induction of tlr2 gene expression in response to tpa in u937 cells .
our immunocytochemistry data show a basal expression of tlr4 but not tlr1 in undifferentiated ml-1 cells .
however , upon tpa treatment , both tlr1 and tlr4 expressions were upregulated not only at the mrna but also at the protein level .
an interesting finding is that interleukin-1 receptor - associated kinases ( irak-2 ) mrna were significantly upregulated in response to tpa treatment in ml-1 cells .
the induction of irak-2 expression in ml-1 cells correlates with the observed increase responsiveness to tlr2 and tlr4 ligands ( lps and lta ) .
irak-2 has been implicated to participate in multiple toll - like receptor signaling pathways that lead to nf-b activation and inflammatory cytokine production [ 41 , 42 ] .
irak-2 loss - of - function mutants or knock - down of irak-2 expression by small interfering rna suppresses tlr3 , tlr4 , and tlr8 signaling to nf-b in human cell lines , and importantly , tlr4-mediated chemokine production in primary human cells [ 41 , 42 ] .
together , these observations strongly suggest that tpa treatment not only induces tlr1 , tlr2 , and tlr4 genes expression , but also that of the irak-2 in ml-1 cells .
irak-2 participates in multiple toll - like receptor signaling pathways that lead to nf-b via activation and inflammatory cytokine production .
the regulation of irak-2 gene expression is known to play a role in the control mechanism of inflammatory responses .
since ml-1 cells express basal tlr4 level , the upregulation of irak-2 gene expression in response to tpa appears to be at least in part a determinant factor in the increased nfb - mediated cytokines production induced by lps and lta .
tlrs expression on macrophages is essential for recognition and response to bacterial particle [ 811 , 43 ] . as such , impaired expression of tlrs
for example , macrophages from tlr1 mice and tlr2 mice were shown to be hyporesponsive to borrelia burgdorferi outer - surface lipoprotein ( ospa ) vaccination .
therefore , defects in the tlr1/2 signaling pathway may account for human hyporesponsiveness to ospa vaccination .
interestingly , recent studies have proposed that the overexpression of tlr4 may play roles in the occurrence of inflammatory diseases [ 1719 ] .
our suggestion that tlr1 and tlr4 expression required mapk activation is based on ( i ) our previous observations that tpa induces a ros - mediated erk1/2 activation in ml-1 cells [ 23 , 27 ] and ( ii ) the tlr1 and tlr4 protein expression in ml-1 induced by tpa was blocked by u0126 , an agent that is known to effectively suppress mek activity .
this observation is consistent with previous reports by jang et al .   that demonstrated that tpa - induced tlr2 expression in u937 cells
ros generation in response to bacterial lps and lta is crucial for pathogen killing by immune cells [ 8 , 44 ] .
however , sustained production of ros during immune responses and sepsis can cause damage to macromolecules , cell death and tissue injury [ 45 , 46 ] . in tpa - differentiated ml-1 cells we have identified two sources of ros , nad(p)h oxidase , and the mitochondrial electron transport chain . here , we demonstrated that tpa - differentiated ml-1 cells exhibit a prolonged generation of ros upon stimulation with lps and lta .
our conclusion that the mitochondrial electron transport chain and/or nadph oxidase may be the source(s ) of this ros is based on the observation that dpi ( 10  m ) , an agent that we have shown effective in inhibiting both nadph oxidase and mitochondria complex iin ml-1 cells , immediately and completely suppressed the prolonged ros generation induced by lps and lta . while dpi inhibition does not allow us to conclude the source of ros elicited by lps and lta , there are several studies that have demonstrated interactions between tlr and mitochondria .
for example , a recent study has shown that a subset of toll - like receptors ( tlr1 , tlr2 , and tlr4 ) promote recruitment of mitochondria to macrophage phagosomes and induce the generation of mitochondrial ros .
these results implicate mros as an important component of antibacterial responses and establish mitochondria as hubs for innate immune signaling .
similarly , toll - like receptor 4 mediates ros - mediated mitochondrial dna damage and biogenic responses in the liver of mice exposed to heat - inactivated e. coli . collectively , the data presented in this study demonstrate that tpa - induced differentiation of ml-1 cells to macrophages is accompanied by the expression of members of the tlr / irak-2/nf-b complex .
this leads to a robust downstream tlr / irak-2/nfb - mediated cytokine production and an increase in sustained cellular oxidative metabolism and ros upon cellular interaction with bacterial lps and lta .
 12-o - tetradecanoylphorbol 13-acetate ( tpa ) induces the differentiation of human myeloid ml-1 cells to macrophages . in the current study , 
 the expression , responsiveness , and regulation of toll - like receptors ( tlrs ) in tpa - induced ml-1-derived macrophages were investigated . 
 we have found that tpa - induced differentiation of ml-1 cells was accompanied by the upregulation of tlr1 , tlr2 , tlr4 , and cd14 expression at both the mrna and protein levels . 
 interestingly , tlr1 and tlr4 protein expression on ml-1 cells could be blocked by pretreatment with u0126 , suggesting the role of an erk1/2-induced differentiation signal in this process . 
 in addition , the expression of irak-2 , a key member of the tlr / irak-2/nf-b - dependent signaling cascade was also induced in response to tpa . 
 accordingly , we demonstrated an increased cellular release of inflammatory cytokines ( tnf- and various interleukins ) upon stimulation with lps and lta ligands for tlr4 and tlr2 , respectively . 
 furthermore , using luminol - dependent chemiluminescence , addition of lps and lta induces a sustained dpi - inhibitable generation of reactive oxygen species ( ros ) by the differentiated ml-1 cells . 
 together , these data suggest that the increase in the responsiveness of tpa - treated ml-1 cells to lps and lta occurs in response to the upregulation of their respective receptors as well as an induction of the irak-2 gene expression . 
1. Introduction
2. Material and Methods
3. Results
4. Discussion