The soluble adhesion molecules are products of activated endothelial cells. They are known for their involvement in processes of inflammation. Ciebiada et al., (2011) declared that concentrations of sICAM-1 are significantly higher in patients with asthma, and are dependent on a seriousness of disease. Our observations of increased adhesion molecules are in agreement with findings of Kristovich who stated that in the context of the pulmonary microenvironment, TNF-α elaborated by particulate-laden alveolar monocytes could act upon proximal septal capillary endothelial cells, inducing their expression of endothelial leukocyte adhesion molecules ICAM-1, vascular cell adhesion molecule (VCAM -1 and E-selectin (Kristovich et al., 2004). Based on this fact we can speculate that levels of sICAM-1 corresponded with inflammation of the airways. Levels of sICAM-1 were increased in the asbestos exposed group. This was not surprising because asbestos fibers are persistent and insoluble in the lungs and are known as causative factor of inflammation. Although in the case of rock wool was a rather disturbing finding for a reason of better elimination of these inhaled synthetic mineral fibers from organism. Usually they have a high solubility and short-term durability in the airways. Among others, there was a shorter duration and lower concentration of fiber in the case of rock wool than asbestos exposure. We noted a statistically significantly higher elevation of sICAM-1 levels in individuals exposed to rock wool compared to the group exposed to asbestos. Glass fibers were not associated with differences in sICAM-1 levels. Adhesion molecules seem to be a sensitive indicator of activation of the immune system and inflammatory response in humans exposed to mineral fibers. Oxidative stress and production of ROS is an important component of the multiple effects of asbestos on human airways (Manning et al., 2002; van Helden et al., 2009). ROS modulate receptor signals and immune responses under physiological conditions, but their overproduction mediates endothelial damage through growth and migration of inflammatory cells, overexpression of inflammatory cytokines and adhesion molecules such as ICAM-1, VCAM-1, and E-selectin (Urso et al., 2011). The elevated production of IL-8, sICAM-1 (rock wool exposure) and sE-selectin (glass fibers exposure) signify immunotoxic effects of synthetic fibers from the airways and increased production of ROS.

#### **6. Summary**

364 Recent Advances in Immunology to Target Cancer, Inflammation and Infections

(Mitschik et al., 2008). In connection with asbestosis, there are cytokines, mainly IL-1β, IL-6, IL-8, which appear to have a role in pathology of this disease (Mossman & Churg, 1998; Tsuda et al., 1997). In spite of the fact that IL-1β is a proinflammatory cytokine required for the synthesis of others cytokines (e.g. IL-8), we did not detect differences in exposed groups in comparison to controls. Our findings were in accordance to observations of Simeonova and Luster (Simeonova & Luster et al., 1996) who noted an enhancement of IL-8 without IL-1β stimuli. IL-6 was previously known as a factor for B-cell differentiation and immunoglobulin production. The increased level of IL-6 may be associated with the increased IgE and IgA levels seen in asbestos exposed individuals. Monitoring of IL-8 in peripheral blood could serve as an early and sensitive marker of developing pulmonary inflammation in consequence of asbestos, glass fibers and rock wool exposure. Across all three exposed groups we observed an increase of cytokine IL-8. Despite the highly significant (p<0.001) differences in IL-8 between exposed workers and human control subjects, these interleukin levels were still in normal reference

The soluble adhesion molecules are products of activated endothelial cells. They are known for their involvement in processes of inflammation. Ciebiada et al., (2011) declared that concentrations of sICAM-1 are significantly higher in patients with asthma, and are dependent on a seriousness of disease. Our observations of increased adhesion molecules are in agreement with findings of Kristovich who stated that in the context of the pulmonary microenvironment, TNF-α elaborated by particulate-laden alveolar monocytes could act upon proximal septal capillary endothelial cells, inducing their expression of endothelial leukocyte adhesion molecules ICAM-1, vascular cell adhesion molecule (VCAM -1 and E-selectin (Kristovich et al., 2004). Based on this fact we can speculate that levels of sICAM-1 corresponded with inflammation of the airways. Levels of sICAM-1 were increased in the asbestos exposed group. This was not surprising because asbestos fibers are persistent and insoluble in the lungs and are known as causative factor of inflammation. Although in the case of rock wool was a rather disturbing finding for a reason of better elimination of these inhaled synthetic mineral fibers from organism. Usually they have a high solubility and short-term durability in the airways. Among others, there was a shorter duration and lower concentration of fiber in the case of rock wool than asbestos exposure. We noted a statistically significantly higher elevation of sICAM-1 levels in individuals exposed to rock wool compared to the group exposed to asbestos. Glass fibers were not associated with differences in sICAM-1 levels. Adhesion molecules seem to be a sensitive indicator of activation of the immune system and inflammatory response in humans exposed to mineral fibers. Oxidative stress and production of ROS is an important component of the multiple effects of asbestos on human airways (Manning et al., 2002; van Helden et al., 2009). ROS modulate receptor signals and immune responses under physiological conditions, but their overproduction mediates endothelial damage through growth and migration of inflammatory cells, overexpression of inflammatory cytokines and adhesion molecules such as ICAM-1, VCAM-1, and E-selectin (Urso et al., 2011). The elevated production of IL-8, sICAM-1 (rock wool exposure) and sE-selectin (glass fibers exposure) signify immunotoxic effects of synthetic

**Soluble adhesion molecules sICAM-1, sVCAM-1 and sE-selectin**

fibers from the airways and increased production of ROS.

range.

This chapter addresses the effects of asbestos, man made mineral fibers (rock wool, glass wool, ceramic fibers) and nickel oxide nanoparticles on the immune system using *in vitro* model, animal model and molecular-epidemiological studies. Data from *in vitro* studies contained results of experiments on alveolar macrophages (AM) and alveolar epithelial type II cells (TII). Stone wool, refractory ceramic fibers (RCF), asbestos (crocidolite) and wollastonite have been tested by lectin histochemistry. Stone wool caused moderate membrane injury of AM and incomplete phagocytosis in a small fraction of AM. RCF caused gaps and reduplicated changes in membranes of both cell types (high dose). Wollastonite caused a decreased reaction in the membranes (high dose). After exposure to the lowest dose of asbestos (crocidolite), the membranes of both cell types were fragmented irregularly and frustrated phagocytosis could be found in AM. Analysis using transmission electron microscopy found severe damage in the organelles and cell death of both cell types exposed to crocidolite. No alterations were found after RCF or stone wool exposure. Analysis of proinflammatory peptides showed that exposure to wollastonite did not change production MCP-1 and MIP-1α in TII cells but in AM the production was significantly enhanced. Different doses of stone wool enhanced production of both peptides in TII cells and AM cells. Crocidolite evoked statistically significant dose dependent enhancement of the production of MCP-1 in AM, for MIP-1α; and both cytokines in TII cells. Comparing the results from different fibers on 2 various primary cell types the following differences are clearly seen: crocidolite (asbestos) evoked the greatest changes, both morphologically and functionally. Increased effects in wollastonite were seen when compared to stone wool. AM cells are more sensitive to the fiber exposure than TII cells.

#### **Intratracheal instillation studies in rat model**

Four types of mineral fibers were administered intratracheally to rats. Four (4w) and 16 weeks (16w) later, immune parameters were examined. Amosite, wollastonite (4w) and rock wool (16w) significantly decreased number of white blood cells; while opposite effect of glass fibers was seen (4w). A consistent increase in percentage of neutrophils was found in animals exposed to all fibers (4w) while decreased percentage of lymphocytes was observed only in rock wool fiber-treated rats (4w). Analysis of lymphocyte subsets in amosite exposed rats showed significantly increased percentage of T-lymphocytes (4w, 16w), mainly cytotoxic cells (4w) and decreased percentage of B-lymphocytes (4w). An increased percentage of T-helper cells was seen in wollastonite group (4w). Exposure to mineral fibers decreased expression of adhesion molecule CD54 (ICAM-1) on peripheral blood leukocytes (amosite, glass fibers and rockwool; all 4w) and CD11b (glass fibers, wollastonite; 4w). Although amosite (4w, 16w) seems to be most potent suppressor of Tand B-lymphocyte proliferation, especially in high-dosed animals, wollastonite (4w) and rock wool (4w, 16w) also interfered with lymphocyte proliferation and suppressed the response of T-lymphocytes. The opposite, stimulative, effect on proliferative capacity of Bcells was found in animals exposed to glass fibers (4w). A highly significant dosedependent suppression of phagocytic activity of neutrophils and monocytes was found mainly in rock wool and glass fiber exposed animals (4w, 16w), but present also in wollastonite and amosite group (4w).

Immune System and Environmental Xenobiotics

workers exposed to mineral fibers.

designed for drug delivery or food components.

**7. Conclusions**

**Molecular epidemiological studies in human population**

In the context of a large-scale molecular epidemiology study, the possible immunomodulatory effects of mineral fibers, in workers occupationally exposed to asbestos, rockwool and glass fibers, were examined. Results of hematological evaluation shown decreased white blood cell count and increased number of lymphocytes and (common) eosinophil and basophil count in glass fiber exposed population. Our findings indicate that exposure to all three types of fibers examined the modulation of immune response to a different degrees. Suppression of T-cell immunity was found in the workers from a former asbestos cement plant, while stimulation of T-cell response was observed in rockwool workers. In addition to an elevated T- lymphocyte response, stimulated T-dependent B-cell response and basal proliferative activity of lymphocytes was seen in workers from glass fiber factory. Changes in lymphocyte subpopulation of CD 16+56 (natural killer cells) in peripheral blood may indicate negative effects of glass fibers on natural cellular immunity. No significant alterations between workers exposed to asbestos, rock wool and glass fibers and controls were found in proportion of CD3+, CD4+, CD8+, CD19+ cells in peripheral blood. Significantly increased serum levels of immunoglobulins IgA (asbestos), IgE (asbestos, rockwool) and decreased levels of IgM (rockwool) were recorded in people exposed to fibers. Increased levels of proinflammatory cytokines (IL-6 asbestos; IL-8 all three fibers), expression of adhesion molecule L-selectin on granulocytes and monocytes (asbestos), levels of soluble adhesion molecules in sera (ICAM-1 asbestos, rockwool; Eselectin glass fibers), increased levels of immunoglobulin E (asbestos and rockwool) and elevated expression of activation markers on eosinophils (CD66b asbestos, glass fibers; CD69 asbestos) may indicate hypersensitivity and an elevated inflammatory status in

With the increasing commercial needs for substitutes of asbestos fibers, a number of manmade and other naturally occurring mineral fibers will appear as a part of living and occupational environments. Fibers discussed in this chapter can enter the human body mainly via the lungs, significance of exposure through digestive tract is less clear. Asbestos has long been recognized as a cause of both benign and malignant lung disease. Man made mineral fibers, once inhaled and displaced to lung tissues, can cause respiratory diseases related to inflammation and fibrosis. Skin diseases have been also reported. In reference to nanoparticles besides the lung and digestive tract, penetration via the skin also occurs (Fig. 1). Knowledge from air pollution showing increased risk of cardiopulmonary, respiratory, hypersensitivity disease and cancer requires specific assessments to be performed for newly produced nanoparticles. The assays currently used to test the safety of materials might be applicable to identify hazards of nanoparticles. Special attention is needed for nanoparticles

To optimize risk assessment for immune system toxicity, it is still necessary to increase our understanding of the underlying immunomodulatory mechanisms which cause negative effects and the quantitative relationships between the immunological tests conducted in the laboratory and manifestation of disease in human populations. There is no universal "consequence of exposure", each type of immunotoxicant should be treated individually when health risks are expected. As mentioned throughout this chapter, the immune system

#### **Inhalation studies in rat model - combined effect of mineral fibers and tobacco smoke on inflammatory response and cytotoxicity**

In rats administered with amosite, weak dose-dependence was seen in simple exposure to fibers without smoking but inflammatory parameters were mostly changed in animals with combined exposure to high dose of fibers and tobacco smoke. In case of wollastonite exposure, no clear dose-dependence in changes of inflammatory parameters was recorded in those administered with fibers alone and very weak in combined exposure groups (fibers and tobacco smoke). Additionally, mild dose dependence of cytotoxic parameters changes in groups without or with tobacco smoke was observed. Tobacco smoke alone induced changes predominantly in inflammatory parameters; alterations in cytotoxic parameters were not explicit.

#### **Combined effect of mineral fibers and tobacco smoke on immune parameters**

Inhalation of high dose of both fibers (amosite and wollastonite) resulted in a significantly increased percentage of B-lymphocytes in peripheral blood of exposed rats. Except the percentage the B-cells, the combined exposure to wollastonite and smoking caused a significant, dose-dependent increase of cytotoxic cells, but total T-lympocytes were decreased. Exposure to amosite and wollastonite increased expression of adhesion molecule CD11b on peripheral blood leukocytes. The proliferative activity of T-lymphocytes and Tdependent B-cell response in animals exposed to amosite in simple or combined exposure with smoking was mostly suppressed. The only exception was combined exposure to amosite fibers and smoking resulting in significant increase of proliferative activity of Bcells. Enhanced proliferative reponse of T-cells was found in animals given high dose of wollastonite. A marked suppressive effect of amosite and wollastonite on phagocytic activity of leukocytes was observed. Moreover, decrease of phagocytosis was recorded in combined exposure to wollastonite and cigarette smoke.

#### **Assessment of immunotoxicity of ceramic fibers and NiO nanoparticles**

Immunophenotypic analysis of leukocytes was examined only 6 months after exposure to fibers and nanoparticles. Analysis revealed statistically significant decreased expression of marker for T-lymphocyte subpopulations (CD4+, CD8+) in rats administered with ceramic fibers. On the other hand, increased expression of CD4+ marker after combined exposure was observed. Exposure to NiO nanoparticles significantly increased expression of MHC II on leukocytes. A similar effect was found on expression of MHC II with combined exposure. A significant decrease of proliferative activity of lymphocytes stimulated with all three mitogens was found in animals exposed to ceramic fibers one month after exposure. To the contrary, 6 months after exposure, opposite effect was seen. Moreover, significant increase of basal proliferative response of spleen cells derived from rats was seen 1 month after exposure to NiO nanoparticles alone and combined exposure to fibers and nanoparticles. Combined exposure to nickel oxide nanoparticles manifested a significant increase of proliferative activity of T-lymphocytes after stimulation with Con A. No alterations in phagocytic activity and respiratory burst were shown one month after exposure of animals to ceramic fibers and/or NiO nanoparticles. However 6 months after exposure, situation was different. Exposure to NiO nanoparticles and combined exposure to ceramic fibers and NiO nanoparticles caused significantly increased phagocytic activity of granulocytes, as well as percentage of cells with respiratory burst.

#### **Molecular epidemiological studies in human population**

In the context of a large-scale molecular epidemiology study, the possible immunomodulatory effects of mineral fibers, in workers occupationally exposed to asbestos, rockwool and glass fibers, were examined. Results of hematological evaluation shown decreased white blood cell count and increased number of lymphocytes and (common) eosinophil and basophil count in glass fiber exposed population. Our findings indicate that exposure to all three types of fibers examined the modulation of immune response to a different degrees. Suppression of T-cell immunity was found in the workers from a former asbestos cement plant, while stimulation of T-cell response was observed in rockwool workers. In addition to an elevated T- lymphocyte response, stimulated T-dependent B-cell response and basal proliferative activity of lymphocytes was seen in workers from glass fiber factory. Changes in lymphocyte subpopulation of CD 16+56 (natural killer cells) in peripheral blood may indicate negative effects of glass fibers on natural cellular immunity. No significant alterations between workers exposed to asbestos, rock wool and glass fibers and controls were found in proportion of CD3+, CD4+, CD8+, CD19+ cells in peripheral blood. Significantly increased serum levels of immunoglobulins IgA (asbestos), IgE (asbestos, rockwool) and decreased levels of IgM (rockwool) were recorded in people exposed to fibers. Increased levels of proinflammatory cytokines (IL-6 asbestos; IL-8 all three fibers), expression of adhesion molecule L-selectin on granulocytes and monocytes (asbestos), levels of soluble adhesion molecules in sera (ICAM-1 asbestos, rockwool; Eselectin glass fibers), increased levels of immunoglobulin E (asbestos and rockwool) and elevated expression of activation markers on eosinophils (CD66b asbestos, glass fibers; CD69 asbestos) may indicate hypersensitivity and an elevated inflammatory status in workers exposed to mineral fibers.

#### **7. Conclusions**

366 Recent Advances in Immunology to Target Cancer, Inflammation and Infections

**Inhalation studies in rat model - combined effect of mineral fibers and tobacco smoke on**

In rats administered with amosite, weak dose-dependence was seen in simple exposure to fibers without smoking but inflammatory parameters were mostly changed in animals with combined exposure to high dose of fibers and tobacco smoke. In case of wollastonite exposure, no clear dose-dependence in changes of inflammatory parameters was recorded in those administered with fibers alone and very weak in combined exposure groups (fibers and tobacco smoke). Additionally, mild dose dependence of cytotoxic parameters changes in groups without or with tobacco smoke was observed. Tobacco smoke alone induced changes predominantly in inflammatory parameters; alterations in cytotoxic parameters

Inhalation of high dose of both fibers (amosite and wollastonite) resulted in a significantly increased percentage of B-lymphocytes in peripheral blood of exposed rats. Except the percentage the B-cells, the combined exposure to wollastonite and smoking caused a significant, dose-dependent increase of cytotoxic cells, but total T-lympocytes were decreased. Exposure to amosite and wollastonite increased expression of adhesion molecule CD11b on peripheral blood leukocytes. The proliferative activity of T-lymphocytes and Tdependent B-cell response in animals exposed to amosite in simple or combined exposure with smoking was mostly suppressed. The only exception was combined exposure to amosite fibers and smoking resulting in significant increase of proliferative activity of Bcells. Enhanced proliferative reponse of T-cells was found in animals given high dose of wollastonite. A marked suppressive effect of amosite and wollastonite on phagocytic activity of leukocytes was observed. Moreover, decrease of phagocytosis was recorded in

Immunophenotypic analysis of leukocytes was examined only 6 months after exposure to fibers and nanoparticles. Analysis revealed statistically significant decreased expression of marker for T-lymphocyte subpopulations (CD4+, CD8+) in rats administered with ceramic fibers. On the other hand, increased expression of CD4+ marker after combined exposure was observed. Exposure to NiO nanoparticles significantly increased expression of MHC II on leukocytes. A similar effect was found on expression of MHC II with combined exposure. A significant decrease of proliferative activity of lymphocytes stimulated with all three mitogens was found in animals exposed to ceramic fibers one month after exposure. To the contrary, 6 months after exposure, opposite effect was seen. Moreover, significant increase of basal proliferative response of spleen cells derived from rats was seen 1 month after exposure to NiO nanoparticles alone and combined exposure to fibers and nanoparticles. Combined exposure to nickel oxide nanoparticles manifested a significant increase of proliferative activity of T-lymphocytes after stimulation with Con A. No alterations in phagocytic activity and respiratory burst were shown one month after exposure of animals to ceramic fibers and/or NiO nanoparticles. However 6 months after exposure, situation was different. Exposure to NiO nanoparticles and combined exposure to ceramic fibers and NiO nanoparticles caused significantly increased phagocytic activity of granulocytes, as well

**Combined effect of mineral fibers and tobacco smoke on immune parameters**

combined exposure to wollastonite and cigarette smoke.

as percentage of cells with respiratory burst.

**Assessment of immunotoxicity of ceramic fibers and NiO nanoparticles**

**inflammatory response and cytotoxicity**

were not explicit.

With the increasing commercial needs for substitutes of asbestos fibers, a number of manmade and other naturally occurring mineral fibers will appear as a part of living and occupational environments. Fibers discussed in this chapter can enter the human body mainly via the lungs, significance of exposure through digestive tract is less clear. Asbestos has long been recognized as a cause of both benign and malignant lung disease. Man made mineral fibers, once inhaled and displaced to lung tissues, can cause respiratory diseases related to inflammation and fibrosis. Skin diseases have been also reported. In reference to nanoparticles besides the lung and digestive tract, penetration via the skin also occurs (Fig. 1). Knowledge from air pollution showing increased risk of cardiopulmonary, respiratory, hypersensitivity disease and cancer requires specific assessments to be performed for newly produced nanoparticles. The assays currently used to test the safety of materials might be applicable to identify hazards of nanoparticles. Special attention is needed for nanoparticles designed for drug delivery or food components.

To optimize risk assessment for immune system toxicity, it is still necessary to increase our understanding of the underlying immunomodulatory mechanisms which cause negative effects and the quantitative relationships between the immunological tests conducted in the laboratory and manifestation of disease in human populations. There is no universal "consequence of exposure", each type of immunotoxicant should be treated individually when health risks are expected. As mentioned throughout this chapter, the immune system

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has been identified as a potential target organ for chemicals including particles and fibers. The immune system plays a critical role in host defense from disease as well as in normal homeostasis; thus identification of immunotoxic risk is important in the protection of human, animal and wildlife health. Clear understanding of normal development of cellular components of the immune system, the means by which they interact, and the known parameters by which their structure and function can be modified is necessary for designing investigations into how environmental agents may affect health through the immune system.

A growth of knowledge in immunology and cell biology connected with an explosion in methodologic and technologic capabilities is very promising for the science of immunotoxicology. There are several challenges yet to be solved within the discipline of immunotoxicology: (1) to improve traditional tests and establish a new tests, which reflect the variety of potential impacts of immunotoxicity; (2) to identify valid, sensitive human biomarkers of immunotoxicity; (3) to interpret minor, moderate, or significant immunotoxic effects in animal models in relation to human risk assessment; (4) better integration of methods of exposure assessment and immunotoxicological risk assessment, especially for simultaneous exposure to multiple agents ; (5) to design better human studies to assess the impact on the immune system in the species of the greatest interest in the context of risk assessment and (6) the better understanding of the role of genetic predisposition and susceptibility in identifying sensitive subpopulations to immune-altering agents (Kaminiski et al., 2008). These challenges are not unique to immunotoxicology, but they are critical, and need to be addressed through intensive and systematic efforts to improve human immune testing strategies.

#### **8. Acknowledgment**

The work was supported by EU grant, contract No. QLK4-CT-1999-01629-FIBRETOX; NIEHS grants of University of Iowa, Iowa, USA: IMUGENFIB, US NIH # 2 D43 TW00621- 006 and NIEHS # 510 20 5240 00000 1 15010 9220; Hungarian-Slovak grants: NKFP-1/B-047/2004, OTKA 046733, ETT 154/2003-6 (Hungarian Medical Research Council), NILU internal grant 106170, EC FP7 [Health-2007-1.3-4, Contract no: 201335].

We would like to express our gratitude to Viera Vachalkova, Helena Turazova, Maria Valentova, Edita Mrvikova, Olga Liskova, Mikulas Krnac, Adriana Paulikova, Darina Cepcova, Jarmila Jantoskova and Lubica Mikloskova and for their excellent technical help.

This article was created by the realization of the project ITMS No.24240120033, based on the supporting Operational research and development program financed from the European Regional Development Fund.

#### **9. References**

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A growth of knowledge in immunology and cell biology connected with an explosion in methodologic and technologic capabilities is very promising for the science of immunotoxicology. There are several challenges yet to be solved within the discipline of immunotoxicology: (1) to improve traditional tests and establish a new tests, which reflect the variety of potential impacts of immunotoxicity; (2) to identify valid, sensitive human biomarkers of immunotoxicity; (3) to interpret minor, moderate, or significant immunotoxic effects in animal models in relation to human risk assessment; (4) better integration of methods of exposure assessment and immunotoxicological risk assessment, especially for simultaneous exposure to multiple agents ; (5) to design better human studies to assess the impact on the immune system in the species of the greatest interest in the context of risk assessment and (6) the better understanding of the role of genetic predisposition and susceptibility in identifying sensitive subpopulations to immune-altering agents (Kaminiski et al., 2008). These challenges are not unique to immunotoxicology, but they are critical, and need to be addressed through

The work was supported by EU grant, contract No. QLK4-CT-1999-01629-FIBRETOX; NIEHS grants of University of Iowa, Iowa, USA: IMUGENFIB, US NIH # 2 D43 TW00621- 006 and NIEHS # 510 20 5240 00000 1 15010 9220; Hungarian-Slovak grants: NKFP-1/B-047/2004, OTKA 046733, ETT 154/2003-6 (Hungarian Medical Research Council), NILU

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**Basic of Immunology and Parasite Immunology**

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**15**

*Iran*

Jalil Mehrzad1,2

*Faculty of Veterinary Medicine,*

**Molecular Aspects of Neutrophils as Pivotal**

**Protect Mammary Gland from Pathogens**

*1Sections Immunology and Biotechnology, Department of Pathobiology,*

*2Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad,*

**Circulating Cellular Innate Immune Systems to**

As a pivotally cellular and molecular arms of circulating innate immune system, polymorphonuclear cells (PMNs) are the most vital primary mobile phagocytes in the body of mammals; their appropriate function is very essential to enhance animals' and humans' health performance. As the first type of innate immune cells arriving at the site of infection, neutrophils play a key role in initiating an innate, inflammatory, and specific immune responses; their importance for protection of organs in the body from pathogens has long been a crucial concern (Burvenich et al., 1994; Paape et al., 1996; Reeves et al., 2002; Burvenich et al., 2003; Mehrzad et al., 2004; 2005a; Letiember, et al., 2005; Liu et al., 2005; Borregaard et al., 2007; Stevens, et al., 2011a; 2011b; Bruhn et al., 2011). The proof of vital roles of neutrophils is that the neutropenic animals/humans are always highly susceptible to many pathogens. Clearly, the complex phenomenon of PMN chemotaxis, diapedesis, phagocytosis, and eventually microbicidal activity each contributes to the ability of PMN to provide an effective first line defense for the body and organs like udder (Burvenich et al., 2003; Mehrzad et al., 2000; 2001a; 2001b; 2002a; 2002b; 2004; 2005a; 2007; 2008a; 2008b; 2009; Mayadas & Cullere, 2005; Borregaard et al., 2008). In this concept many powerful afferent (sensing) and efferent (effector) arms of the neutrophils inside and outside of the cytoplasm are involved; the most common arms are enzymes, granules, free radicals or reactive oxygen species (ROS) and reactive nitrogen species (RNS) and in phagolysosome, into which microbicidal agents are released, neutrophil extracellular traps (NETs), neutrophils' membrane receptors like pattern recognition receptors (PRRs), opsonin receptors etc. that sense, bind and efficiently kill invading microbes, destroy virulence factors, or prevent them

Oscillation and/or impairment of neutrophils' functions, originating from the bone marrow, is a peculiar feature during the physiological and environmental stresses; this impairment might be cumulative upon diapedesis/extravasation of neutrophils (Mehrzad et al., 2001a; 2002a; 2005a, 2004; 2007; 2008a; 2008b; Van Oostveldt et al., 2002a; 2002b; Burvenich et al., 2003). Generalised PMN impairment can be multifactorial, e.g. due to metabolic (Suriyasathaporn et al., 1999) and hormonal (Gray et al., 1982; Alexandrova, 2009; Lai &

**1. Introduction**

from spreading.

### **Molecular Aspects of Neutrophils as Pivotal Circulating Cellular Innate Immune Systems to Protect Mammary Gland from Pathogens**

Jalil Mehrzad1,2

*1Sections Immunology and Biotechnology, Department of Pathobiology, Faculty of Veterinary Medicine, 2Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran*

#### **1. Introduction**

As a pivotally cellular and molecular arms of circulating innate immune system, polymorphonuclear cells (PMNs) are the most vital primary mobile phagocytes in the body of mammals; their appropriate function is very essential to enhance animals' and humans' health performance. As the first type of innate immune cells arriving at the site of infection, neutrophils play a key role in initiating an innate, inflammatory, and specific immune responses; their importance for protection of organs in the body from pathogens has long been a crucial concern (Burvenich et al., 1994; Paape et al., 1996; Reeves et al., 2002; Burvenich et al., 2003; Mehrzad et al., 2004; 2005a; Letiember, et al., 2005; Liu et al., 2005; Borregaard et al., 2007; Stevens, et al., 2011a; 2011b; Bruhn et al., 2011). The proof of vital roles of neutrophils is that the neutropenic animals/humans are always highly susceptible to many pathogens. Clearly, the complex phenomenon of PMN chemotaxis, diapedesis, phagocytosis, and eventually microbicidal activity each contributes to the ability of PMN to provide an effective first line defense for the body and organs like udder (Burvenich et al., 2003; Mehrzad et al., 2000; 2001a; 2001b; 2002a; 2002b; 2004; 2005a; 2007; 2008a; 2008b; 2009; Mayadas & Cullere, 2005; Borregaard et al., 2008). In this concept many powerful afferent (sensing) and efferent (effector) arms of the neutrophils inside and outside of the cytoplasm are involved; the most common arms are enzymes, granules, free radicals or reactive oxygen species (ROS) and reactive nitrogen species (RNS) and in phagolysosome, into which microbicidal agents are released, neutrophil extracellular traps (NETs), neutrophils' membrane receptors like pattern recognition receptors (PRRs), opsonin receptors etc. that sense, bind and efficiently kill invading microbes, destroy virulence factors, or prevent them from spreading.

Oscillation and/or impairment of neutrophils' functions, originating from the bone marrow, is a peculiar feature during the physiological and environmental stresses; this impairment might be cumulative upon diapedesis/extravasation of neutrophils (Mehrzad et al., 2001a; 2002a; 2005a, 2004; 2007; 2008a; 2008b; Van Oostveldt et al., 2002a; 2002b; Burvenich et al., 2003). Generalised PMN impairment can be multifactorial, e.g. due to metabolic (Suriyasathaporn et al., 1999) and hormonal (Gray et al., 1982; Alexandrova, 2009; Lai &

Molecular Aspects of Neutrophils' Pivotal Role in Mammary Gland Immunity 385

Fig. 1. Upper: Whole blood smear (left), immature neutrophil (arrow); milk neutrohils after first step of centrifugation (right), which is almost indistinguishable, with the techniques

phagocytosed microbes by milk neutrophils (far right); these engulfed microbes must be destroyed effectively to limit infection. Lower: Isolated bovine blood neutrohils from

healthy animal (left), which is pure, and postdiapedetic udder (right) neutrohils (B, C and D)

The most prominent characteristic of the PMN is the multilobulated nucleus (see figures 1 and 2). The multilobulated nucleus is important because it allows the PMN to line up its nuclear lobes in a thin line, permitting rapid migration between endothelial cells. Macrophages on the other hand have a large horseshoe shaped nucleus that makes migration between endothelial cells more difficult. Thus, the PMN is the first newly migrated phagocytic cell to arrive at an infection site. Their surface microvilli are also pivotal for their functionality (see figures 2 and 3). Within the cytoplasm there are isles of glycogen that make up 20% of the cell on a dry weight basis and numerous bactericidal granules that are used by the cell for bactericidal activity. Generally, human neutrophils have two predominantly distinct granule populations; azurophilic (primary) granules which are large and appear as electron dense granules on electron microscopy, and specific (secondary) granules which are small and appear as light staining granules on electron microscopy. Azurophilic granules are more abundant in immature lineage of neutrophils than specific granules (Borregaard et al., 2007). Similarly, bovine PMN contains azurophilic and specific granules (figure 2). They also contain a third novel granule that is larger, denser and more numerous than the other two granules. These granules contain lactoferrin, which is also found in secondary granules, but they do not contain constituents common to azurophilic granules. Instead, they contain a group of highly cationic proteins and are the exclusive store of powerful oxygen-independent bactericidal compounds (Gennaro et al., 1983). The most important antibacterial mechanism derived from azurophilic granules is the MPO-H2O2-halide system (Heyneman et al., 1990; Heyneman & Burvenich, 1992; Mehrzad et al., 2001a; 2002a; 2009). MPO in the presence of hydrogen peroxide (H2O2) and halide ions kills bacteria. The functionality of these ROS producing

established in routine laboratories they can easily be distinguishable. A typical

macrophage (A) and lymphocyte (E).

Gallo, 2009) changes. The inevitable occurring of general and local immunocompromised conditions in biologically pivotal organ, mammals' udder, (Burvenich et al., 1994; Hoeben et al., 2000a; Burvenich et al., 2003; Mehrzad et al., 2001a; 2004; 2005a; 2008a; 2009) leads to udder infection and/or inflammation and breast disorders, affecting adult mammals, especially high yielding dairy cows, thereby causing neonatal infections, critical public health damage and economic losses to bovine, meat, dairy and food industries and overall human food chains.

Most researchers see the immunocompromised condition in animals and human as a result of neutrophils' dysfunction; this neutrophils' dysfunctional status can be cumulative upon neutrophils' influx into the udder (Mehrzad et al., 2001a; 2004; 2005a; 2008a; 2009). Despite all progresses and advances in the efferent and afferent branches of molecular aspects of neutrophils, the molecular basis of neutrophils interactions with other immune and nonimmune cells in the udder is incompletely understood.

This chapter presents the cellular and acellular branches of neutrophils-pathogens interactions and factors affecting the effectiveness of particularly neutrophils' efferent arms of innate molecules in bovine. I chose bovine as a model to update our knowledge and to incorporate new observations that broaden our understanding of 1) overall neutrophils' involvement in innate immunity 2) how neutrophils engulf and kill invading pathogens and 3) how some key mechanisms of neutrophil's oscillatory events occur in bovine model. I discuss various aspects of bovine neutrophils that are absolutely relevant to their pivotal roles in an efficient innate immune response against pathogens. Aspects such as cellular and molecular innate immunity and host-pathogen interactions, biology, biochemistry and biophysics of bovine neutrophils as well as immunotoxicology, especially environmental immunotoxins (Mehrzad et al., 2011) and some nutritional immunology (Ibeagha et al., 2009), applying basic techniques like luminometry and flow cytometry will be schematically addressed to gain more insight into the static and dynamics branches of circulating and post-diapedetic neutrophils' functions. Although it is hardly possible in this chapter to address the breadth of information available on efferent arms of neutrophils in healthy and diseased animals and humans, our readers are therefore referred to many more recent detailed references of the many aspects of neutrophils in healthy and diseased hosts and their impacts on udders' health and performance as well.

#### **2. Fine structure of neutrophils**

As the primary and pivotal cells providing innate host defence against pathogens, neutrophils are characterized by their multi-lobed or sometimes picnotic dark-bluish stained nuclei (see figure 1) with the plenty of membrane receptors. The fine structure of bovine PMN has been exclusively demonstrated in classic studies (Paape et al., 2002; Mehrzad et al., 2001a; 2005b). The cell is delineated by a plasma membrane that has a number of functionally important receptors. These include L-selectin and 2-integrin adhesion molecules associated with the binding of PMN to endothelial cells that are important for migration into sites of infection (Stevens et al., 2011a; 2011b; Pezeshki et al., 2011). Membrane receptors for the Fc component of the IgG2 and IgM classes of immunoglobulins and C3b are necessary for mediating effective phagocytosis of invading microbes (Paape et al., 1996). Dying or apoptotic PMN express receptors that mark them for quick disposal by macrophages.

Gallo, 2009) changes. The inevitable occurring of general and local immunocompromised conditions in biologically pivotal organ, mammals' udder, (Burvenich et al., 1994; Hoeben et al., 2000a; Burvenich et al., 2003; Mehrzad et al., 2001a; 2004; 2005a; 2008a; 2009) leads to udder infection and/or inflammation and breast disorders, affecting adult mammals, especially high yielding dairy cows, thereby causing neonatal infections, critical public health damage and economic losses to bovine, meat, dairy and food industries and overall

Most researchers see the immunocompromised condition in animals and human as a result of neutrophils' dysfunction; this neutrophils' dysfunctional status can be cumulative upon neutrophils' influx into the udder (Mehrzad et al., 2001a; 2004; 2005a; 2008a; 2009). Despite all progresses and advances in the efferent and afferent branches of molecular aspects of neutrophils, the molecular basis of neutrophils interactions with other immune and non-

This chapter presents the cellular and acellular branches of neutrophils-pathogens interactions and factors affecting the effectiveness of particularly neutrophils' efferent arms of innate molecules in bovine. I chose bovine as a model to update our knowledge and to incorporate new observations that broaden our understanding of 1) overall neutrophils' involvement in innate immunity 2) how neutrophils engulf and kill invading pathogens and 3) how some key mechanisms of neutrophil's oscillatory events occur in bovine model. I discuss various aspects of bovine neutrophils that are absolutely relevant to their pivotal roles in an efficient innate immune response against pathogens. Aspects such as cellular and molecular innate immunity and host-pathogen interactions, biology, biochemistry and biophysics of bovine neutrophils as well as immunotoxicology, especially environmental immunotoxins (Mehrzad et al., 2011) and some nutritional immunology (Ibeagha et al., 2009), applying basic techniques like luminometry and flow cytometry will be schematically addressed to gain more insight into the static and dynamics branches of circulating and post-diapedetic neutrophils' functions. Although it is hardly possible in this chapter to address the breadth of information available on efferent arms of neutrophils in healthy and diseased animals and humans, our readers are therefore referred to many more recent detailed references of the many aspects of neutrophils in healthy and diseased hosts and

As the primary and pivotal cells providing innate host defence against pathogens, neutrophils are characterized by their multi-lobed or sometimes picnotic dark-bluish stained nuclei (see figure 1) with the plenty of membrane receptors. The fine structure of bovine PMN has been exclusively demonstrated in classic studies (Paape et al., 2002; Mehrzad et al., 2001a; 2005b). The cell is delineated by a plasma membrane that has a number of functionally important receptors. These include L-selectin and 2-integrin adhesion molecules associated with the binding of PMN to endothelial cells that are important for migration into sites of infection (Stevens et al., 2011a; 2011b; Pezeshki et al., 2011). Membrane receptors for the Fc component of the IgG2 and IgM classes of immunoglobulins and C3b are necessary for mediating effective phagocytosis of invading microbes (Paape et al., 1996). Dying or apoptotic PMN express receptors that mark them for quick disposal by

human food chains.

immune cells in the udder is incompletely understood.

their impacts on udders' health and performance as well.

**2. Fine structure of neutrophils**

macrophages.

Fig. 1. Upper: Whole blood smear (left), immature neutrophil (arrow); milk neutrohils after first step of centrifugation (right), which is almost indistinguishable, with the techniques established in routine laboratories they can easily be distinguishable. A typical phagocytosed microbes by milk neutrophils (far right); these engulfed microbes must be destroyed effectively to limit infection. Lower: Isolated bovine blood neutrohils from healthy animal (left), which is pure, and postdiapedetic udder (right) neutrohils (B, C and D) macrophage (A) and lymphocyte (E).

The most prominent characteristic of the PMN is the multilobulated nucleus (see figures 1 and 2). The multilobulated nucleus is important because it allows the PMN to line up its nuclear lobes in a thin line, permitting rapid migration between endothelial cells. Macrophages on the other hand have a large horseshoe shaped nucleus that makes migration between endothelial cells more difficult. Thus, the PMN is the first newly migrated phagocytic cell to arrive at an infection site. Their surface microvilli are also pivotal for their functionality (see figures 2 and 3). Within the cytoplasm there are isles of glycogen that make up 20% of the cell on a dry weight basis and numerous bactericidal granules that are used by the cell for bactericidal activity. Generally, human neutrophils have two predominantly distinct granule populations; azurophilic (primary) granules which are large and appear as electron dense granules on electron microscopy, and specific (secondary) granules which are small and appear as light staining granules on electron microscopy. Azurophilic granules are more abundant in immature lineage of neutrophils than specific granules (Borregaard et al., 2007). Similarly, bovine PMN contains azurophilic and specific granules (figure 2). They also contain a third novel granule that is larger, denser and more numerous than the other two granules. These granules contain lactoferrin, which is also found in secondary granules, but they do not contain constituents common to azurophilic granules. Instead, they contain a group of highly cationic proteins and are the exclusive store of powerful oxygen-independent bactericidal compounds (Gennaro et al., 1983). The most important antibacterial mechanism derived from azurophilic granules is the MPO-H2O2-halide system (Heyneman et al., 1990; Heyneman & Burvenich, 1992; Mehrzad et al., 2001a; 2002a; 2009). MPO in the presence of hydrogen peroxide (H2O2) and halide ions kills bacteria. The functionality of these ROS producing

Molecular Aspects of Neutrophils' Pivotal Role in Mammary Gland Immunity 387

electron micrograph of a neutrohil isolated from post-diapedesis in udder. As a result of the cell ingesting its membrane material during phagocytosis of milk fat globules and casein miscelles, the neutrophils lost the protruding pseudopods required for phagocytosis (×20,000). (B) Scanning electron micrograph of a neutrohil isolated from blood. It has a highly convoluted cell membrane that forms protruding pseudopods (×15,000). Far right: Scanning electron micrograph of a PMN isolated from milk with three Staphylococcus aureus (arrows) are present on the surface of the neutrophil; the bacterium at lower left is partially engulfed by a pseudopod (×20,000). Lower left, transmission electron micrograph of bovine neutrophils isolated from blood. The neutrohil, which is limited by the plasma membrane, contains portions of a multilobed nucleus (N1 to N4), glycogen granules (G), specific granules (S), azurophilic granules (A), and large electron dense granules (D). Azurophilic granules are stained more intensely than the specific and large electron dense granules because the neutrophils were incubated with diaminobenzidine and hydrogen peroxide. As a result, an electron-dense product, indicative of peroxidatic activity, has formed in azurophilic granules (×22,000). Lower right, transmission electron micrograph of bovine neutrophils isolated from lactating udder. Deposition of an electron-dense product performed on neutrophils, which was not stained, electron dense product corresponds to areas that are high in peroxidatic activity.   Partially

**3. Movement of neutrophils from bone marrow to the mammary glands**

The issue of life span, mechanomics and biophysics of this dynamically mobile cell, neutrophil, is very pivotal issue in biomedical research. The neutrophilic PMN leukocytes of the blood circulation are specialized terminally differenciated with a short life-span. All blood and immune cells originate from a self-renewing small population of pluripotent stem cells (CFU-S) that can replicate themselves, or can become committed to a particular development pathway. Neutrophils are the major class of leukocytes in peripheral blood of human and domestic animals. The circulation of a healthy human adult contains 4,500- 10,000 leucocytes/µl with approximately 60% or more being neutrophils (Nathan, 2006). A healthy adult human produces ~1011 neutrophils each day each of which survives about 6-8 hours in the circulation. Almost similar pattern on leukocyte and neutrophils quantities existed in bovine leukocyte and neutrophils (see e.g., Mehrzad et al., 2001a; 200ab; 2001c; 2002a; 2004; 2005a and plenty more). These vital circulating innate immune cells, neutrophils, are formed through the multi-step process of granulopoiesis, from the colonyforming unit of granulocytes (CFU-G) through myeloblasts, promyelocytes, myelocytes, metamyelocytes, and band cells. Precursor cells undergo substantial morphologic, biochemical and functional changes during granulocytic maturation. These changes are associated with significant changes in cell size and nuclear shape, and with the development of stage-specific proteins essential for phagocytosis and microbial killing (Smit et al., 1996; Van Merris et al., 2001a; 2001b; 2002; Burvenich et al., 2003; Mehrzad et al., 2001a; 2001c).

The efficiency of PMN against invading pathogens was previously shown to be highly dependent on the rate of diapedesis into the infection site (Heyneman et al., 1990; Heyneman & Burvenich, 1992; Mehrzad et al., 2001b; 2004; 2005a; 2005b) and on the ability of these PMN to generate ROS (Heyneman & Burvenich, 1992) and Mehrzad et al., 2001a;

adapted from (Paape M., Mehrzad j., et al., 2002).

granules might be altered during physiological and pathological conditions of animals, and should therefore be further investigated.

Fig. 2. Upper panel: upper: Isolated neutrophils from blood (left), in which the presence of immature neutrophil (arrow) is distinguishable and post-diapedetic neutrophils in lactating udder (right), in which phagocytosed bacteria (arrows) are visible. Lower: Scanning electron micrograph of PMN isolated from blood (left) and milk (right). Protruding pseudopods needed for phagocytosis in PMN, which is distinguishable in blood and post-diapedetic neutrophils. The blood PMN has higher convulsed cell membrane that forms protruding pseudopods. This might be due to phagocytosis of milk fat globules and casein miscelles. Samples were taken from cows suffering from *E. coli* mastitis. Lower panel: (A) Scanning

granules might be altered during physiological and pathological conditions of animals, and

Fig. 2. Upper panel: upper: Isolated neutrophils from blood (left), in which the presence of immature neutrophil (arrow) is distinguishable and post-diapedetic neutrophils in lactating udder (right), in which phagocytosed bacteria (arrows) are visible. Lower: Scanning electron micrograph of PMN isolated from blood (left) and milk (right). Protruding pseudopods needed for phagocytosis in PMN, which is distinguishable in blood and post-diapedetic neutrophils. The blood PMN has higher convulsed cell membrane that forms protruding pseudopods. This might be due to phagocytosis of milk fat globules and casein miscelles. Samples were taken from cows suffering from *E. coli* mastitis. Lower panel: (A) Scanning

should therefore be further investigated.

electron micrograph of a neutrohil isolated from post-diapedesis in udder. As a result of the cell ingesting its membrane material during phagocytosis of milk fat globules and casein miscelles, the neutrophils lost the protruding pseudopods required for phagocytosis (×20,000). (B) Scanning electron micrograph of a neutrohil isolated from blood. It has a highly convoluted cell membrane that forms protruding pseudopods (×15,000). Far right: Scanning electron micrograph of a PMN isolated from milk with three Staphylococcus aureus (arrows) are present on the surface of the neutrophil; the bacterium at lower left is partially engulfed by a pseudopod (×20,000). Lower left, transmission electron micrograph of bovine neutrophils isolated from blood. The neutrohil, which is limited by the plasma membrane, contains portions of a multilobed nucleus (N1 to N4), glycogen granules (G), specific granules (S), azurophilic granules (A), and large electron dense granules (D). Azurophilic granules are stained more intensely than the specific and large electron dense granules because the neutrophils were incubated with diaminobenzidine and hydrogen peroxide. As a result, an electron-dense product, indicative of peroxidatic activity, has formed in azurophilic granules (×22,000). Lower right, transmission electron micrograph of bovine neutrophils isolated from lactating udder. Deposition of an electron-dense product performed on neutrophils, which was not stained, electron dense product corresponds to areas that are high in peroxidatic activity.   Partially adapted from (Paape M., Mehrzad j., et al., 2002).

#### **3. Movement of neutrophils from bone marrow to the mammary glands**

The issue of life span, mechanomics and biophysics of this dynamically mobile cell, neutrophil, is very pivotal issue in biomedical research. The neutrophilic PMN leukocytes of the blood circulation are specialized terminally differenciated with a short life-span. All blood and immune cells originate from a self-renewing small population of pluripotent stem cells (CFU-S) that can replicate themselves, or can become committed to a particular development pathway. Neutrophils are the major class of leukocytes in peripheral blood of human and domestic animals. The circulation of a healthy human adult contains 4,500- 10,000 leucocytes/µl with approximately 60% or more being neutrophils (Nathan, 2006). A healthy adult human produces ~1011 neutrophils each day each of which survives about 6-8 hours in the circulation. Almost similar pattern on leukocyte and neutrophils quantities existed in bovine leukocyte and neutrophils (see e.g., Mehrzad et al., 2001a; 200ab; 2001c; 2002a; 2004; 2005a and plenty more). These vital circulating innate immune cells, neutrophils, are formed through the multi-step process of granulopoiesis, from the colonyforming unit of granulocytes (CFU-G) through myeloblasts, promyelocytes, myelocytes, metamyelocytes, and band cells. Precursor cells undergo substantial morphologic, biochemical and functional changes during granulocytic maturation. These changes are associated with significant changes in cell size and nuclear shape, and with the development of stage-specific proteins essential for phagocytosis and microbial killing (Smit et al., 1996; Van Merris et al., 2001a; 2001b; 2002; Burvenich et al., 2003; Mehrzad et al., 2001a; 2001c).

The efficiency of PMN against invading pathogens was previously shown to be highly dependent on the rate of diapedesis into the infection site (Heyneman et al., 1990; Heyneman & Burvenich, 1992; Mehrzad et al., 2001b; 2004; 2005a; 2005b) and on the ability of these PMN to generate ROS (Heyneman & Burvenich, 1992) and Mehrzad et al., 2001a;

Molecular Aspects of Neutrophils' Pivotal Role in Mammary Gland Immunity 389

functionality make this pivotal cell of the circulating innate immune system as one of the cornerstones of the induction and shaping of adaptive immunity in body and organs. Microbicidal activity of neutrophils occurs mainly intracellularly during phagocytosis, with the contribution of many soluble and insoluble proteins, enzymes and ROS produced both

compounds, such as inflammatory mediators, cytokines and ligands for many receptors like pattern recognition receptors (PRRs) (e.g. via Toll-like receptors or TLRs). The activation elicits classic neutrophil functions such as chemotaxis, adherence, ingestion and eventually killing of phagocytosed microbes. Some mediators, cytokines, hormones and metabolites suppress neutrophils' functions throughout the body. Like blood PMN, post-diapedetic PMN functional impairment occurs immediately around parturition; this is coincided with oscillatory events in the cellular and molecular parts of post-diapedetic PMN like viability and killing capacity in the udder. These impairments are more pronounced immediately after parturition and beginning of lactogenesis, which is far more pronounced in older animals. This diagram is based on the author's own studies on bovine PMN functions (see some references appeared in the reference section). The scheme observed in animal model of innate immune system is a fundamental consideration in human, and many molecular aspects of this diagram remains to be further studied in the area of innate immunology in animals and human. Lower panel: Simple scheme of the complex blood-milk barrier

showing enterance of invading microbes, which is almost always ascendingly, via teat canal, in the udder cisterna. When microbes enter the gland the triger of innate immune cells in the gland, mainly macrophages and neutrophils, producing veriety of cytokines, chemokins and plenty more immunogenic molecules, creating cell-cell signaling, activating epithelial and endothelial cells, thereby resulting in a masive recruitment of neutrophils in the mammary gland. When real professional phagocytes, neutrophils, reach the site of infection they capture and ingest microbes by phagocytosis and eventually destroy the pathogenic microbes with their microbicidal arsenal, mainly ROS and proteases and RNS (reactive nitrogen species). This final step of first line defence mechanism is the main focus of the

2001c; 2005a; 2005b; 2009).  The impairment of ROS production was attributed to the absence of membranebound NADPH-oxidase activity, as myeloperoxidase was already present in the rare azurophilic granules at the promyelocytic stage (Van Merris et al., 2002). Thus, when maturation is impaired due to an increased proliferation rate, a higher number of immature neutrophils will appear in the blood circulation. These findings support the hypothesis postulated by (Heyneman & Burvenich, 1992; Mehrzad et al, 2001a; 2005a; 2005b; 2009) namely that the presence of myelocytes, metamyelocytes and band cells (shift to the left) observed during acute inflammation and sepsis may compromise the animals' resistance by supplying more cells that are morphologically immature and functionally insufficient. Van Werven et al. (1997) and Mehrzad et al. (2001a; 2002a; 2004; 2005a; 2007) demonstrated that the increase of neutrophil functionality was a result of increased enzyme activity per neutrophil, rather than an increase of the number of neutrophils. Therefore, the enhanced enzymatic activity in neutrophils after onset of infection/inflammation was believed to be

future resaerch in the area of molecular immunobiology.

neutrophils are the first cells recruited to the site of infection, their capacity and

inside and outside of the neutrophils. Neutrophils are activated by a wide array of

Fig. 3. Upper panel: A schematic representation of the major contributing factors such as some physiological conditions (lactation, parity or age and status of inflammation) to microbicidal capacity of neutrophils in the blood and mammary gland, and the strong link of these conditions to some key immunophysiological parameters of innate host defence in bovine model. This interrelated link happens not only in the blood, but also in the bone marrow, affecting on the most pivotal efferent branches of neutrophil functions in the organs and body, thereby contributing to the outcome or severity of infection. Because

neutrophils are the first cells recruited to the site of infection, their capacity and functionality make this pivotal cell of the circulating innate immune system as one of the cornerstones of the induction and shaping of adaptive immunity in body and organs. Microbicidal activity of neutrophils occurs mainly intracellularly during phagocytosis, with the contribution of many soluble and insoluble proteins, enzymes and ROS produced both inside and outside of the neutrophils. Neutrophils are activated by a wide array of compounds, such as inflammatory mediators, cytokines and ligands for many receptors like pattern recognition receptors (PRRs) (e.g. via Toll-like receptors or TLRs). The activation elicits classic neutrophil functions such as chemotaxis, adherence, ingestion and eventually killing of phagocytosed microbes. Some mediators, cytokines, hormones and metabolites suppress neutrophils' functions throughout the body. Like blood PMN, post-diapedetic PMN functional impairment occurs immediately around parturition; this is coincided with oscillatory events in the cellular and molecular parts of post-diapedetic PMN like viability and killing capacity in the udder. These impairments are more pronounced immediately after parturition and beginning of lactogenesis, which is far more pronounced in older animals. This diagram is based on the author's own studies on bovine PMN functions (see some references appeared in the reference section). The scheme observed in animal model of innate immune system is a fundamental consideration in human, and many molecular aspects of this diagram remains to be further studied in the area of innate immunology in animals and human. Lower panel: Simple scheme of the complex blood-milk barrier showing enterance of invading microbes, which is almost always ascendingly, via teat canal, in the udder cisterna. When microbes enter the gland the triger of innate immune cells in the gland, mainly macrophages and neutrophils, producing veriety of cytokines, chemokins and plenty more immunogenic molecules, creating cell-cell signaling, activating epithelial and endothelial cells, thereby resulting in a masive recruitment of neutrophils in the mammary gland. When real professional phagocytes, neutrophils, reach the site of infection they capture and ingest microbes by phagocytosis and eventually destroy the pathogenic microbes with their microbicidal arsenal, mainly ROS and proteases and RNS (reactive nitrogen species). This final step of first line defence mechanism is the main focus of the future resaerch in the area of molecular immunobiology.

2001c; 2005a; 2005b; 2009).  The impairment of ROS production was attributed to the absence of membranebound NADPH-oxidase activity, as myeloperoxidase was already present in the rare azurophilic granules at the promyelocytic stage (Van Merris et al., 2002). Thus, when maturation is impaired due to an increased proliferation rate, a higher number of immature neutrophils will appear in the blood circulation. These findings support the hypothesis postulated by (Heyneman & Burvenich, 1992; Mehrzad et al, 2001a; 2005a; 2005b; 2009) namely that the presence of myelocytes, metamyelocytes and band cells (shift to the left) observed during acute inflammation and sepsis may compromise the animals' resistance by supplying more cells that are morphologically immature and functionally insufficient. Van Werven et al. (1997) and Mehrzad et al. (2001a; 2002a; 2004; 2005a; 2007) demonstrated that the increase of neutrophil functionality was a result of increased enzyme activity per neutrophil, rather than an increase of the number of neutrophils. Therefore, the enhanced enzymatic activity in neutrophils after onset of infection/inflammation was believed to be

Molecular Aspects of Neutrophils' Pivotal Role in Mammary Gland Immunity 391

production rates in sever responders. This is very important and basic cellular part of innate immunity of mammary gland. This different response is mainly due to the far stronger preinfection blood and post-daipedetic neutrophils' functions, especially, ROS production as

infection/inflammation. So, the capacity of PMN ROS production (especially intracellular) and quality of pos-diapedetic neutrophils both before and during early infection, is crucial for the severity of the diseases, and leads to a faster elimination of pathogens, becuae the

Besides increased expression of selectins, TNF- α and IL-1 also enhance endothelial cell expression of vascular cell adhesion moledule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1) and ICAM-2, which are ligands for the neutrophil integrins very late antigen-4 (VLA-4) and leukocyte function antigen-1 (LFA-1 or CD11a/18 or αLβ2) and macrophage-1 antigen (Mac-1, CD11b/18 or αmβ2), respectively. Interactions of neutrophils with these molecules results in firm attachment to the endothelium, followed by their activation, and spreading. Interactions of neutrophil receptors CXCR1 and CXCR2 down a concentration gradient of their ligand chemokine CXCL8 result in transmigration of neutrophils between endothelial cell spaces to the infectious site (Paulsson et al., 2010). Neutrophil LFA-1-endothelial ICAM-1 and CD177-CD31 (platelet endothelial cell adhesion

A potential role of adhesion molecules like L-selectin in the release of neutrophils from the bone marrow is very critical, because L-selectin is highly expressed on mature PMN in the post-mitotic pool in the bone marrow and in the circulation (Diez-Fraile et al., 2004). The process of granulopoiesis is strictly controlled by regulatory growth factors, comprising cytokines and colony-stimulating factors, which have pleiotropic effects on proliferation, differentiation and functional activation of precursor cells (Burvenich et al., 2003). Using an optimised cell culture assay for the bovine (Smit et al., 1996; Van Merris et al., 2001a; 2001b), it was demonstrated that physiological concentrations of -hydroxybutyric acid and acetoacetic acid induced remarkable suppression on the proliferation of hematopoietic cells (van Werven et al., 1997; Hoeben et al., 1999). Bovine pregnancy-associated glycoprotein also reduced the proliferative activity of bovine progenitor cells (Hoeben et al., 1999). Therefore, the neutrophil circulating pool is largely depending on the proliferative capacity of the bone marrow. After having exerted their role in immune function, PMN die by senescence (Mehrzad et al., 2001a; Van Merris et al., 2002; Burvenich et al., 2003). Aged PMN undergo spontaneous apoptosis in the absence of pro-inflammatory agents prior to their removal by macrophages (Paape et al., 2002; Burvenich et al., 2003), thus preventing the release of their cytotoxic content. Inflammation and infection hugely increase the rate of PMN production, shortening the maturation time, thereby leading to the release of

Advances in mammary gland immunology of recent decades have provided insights into the mechanisms responsible for the defense of the mammary gland against infection. PMN has a pivotal role in the protection of the gland from infections (Paape et al., 2002; Burvenich et al., 2003; Mehrzad et al., 2001a; 2001b; 2001c; 2002; 2004; 2005a; 2005b; 2009). The life cycle of the bovine PMN is short. Formed in the bone marrow, PMN require 10 to 14 days to mature (Bainton et al., 1971; Burvenich et al., 2003). After maturation, PMN may be stored

well as the neutrophils' ROS production during the "early phase" of

fast-strong local response in moderate responders facilitated recovery of

molecule-1 [PECAM-1]) interactions facilitate this migration (Sachs et al., 2007).

immature neutrophils in the circulation pool.

infection/inflammation.

induced by granulocyte-moncyte colony-stimulating factors (GM-CSF), reflecting an increased proliferation and differentiation of bone marrow granulocytes (Heyneman et al., 1990; Heyneman & Burvenich, 1992; Mehrzad et al., 2001a; 2001b; 2004; 2005a). It has been postulated that myeloid stem cells leave the bone marrow almost by a pipeline mechanism, the older cells being released first (Heyneman & Burvenich, et al., 1992). The molecular mechanisms that control the release of mature PMN from the bone marrow into the circulating pool and then extravasation are very complex and poorly understood (see figures 3 and 4).

Neutrophils circulate in the blood until recruited to sites of infection by chemical signals. This migration begins when neutrophils interact with activated endothelial cells. Inflammatory cytokines like tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), and interferon- (IFN-) produced by activated macrophages at sites of infection activate endothelial cells and induce expression of receptors like E-selectin (CD62E) and P-selectin (CD62P) and ligands like Sialyl-Lewis X on glycan-bearing cell adhesion molecule-1 (GlyCAM-1) and P-selectin glycoprotein ligand-1 [PSGL-1]) which interact with neutrophil receptors like L-selectin (CD62L) (Diez-Fraille et al., 2004; Sohn et al., 2007a; 2007b ; Zarbock A & Ley, 2008). Selectin-ligand interactions are of low affinity leading to only "rolling" and slowing of neutrophils on the endothelial cell surface in post capillary venules to enable stronger associations.

Fig. 4. This figure shows the overall disparities of neutrophils' diapedesis rate, postdiapedetic neutrophil ROS production capacity and *E. coli* CFU dynamics in modertate and severe responders of animal infection model; it is based on the study of the kinetics of chemiluminescence and intramammary infection/inflammation (Mehrzad et al., 2001b, 2004; 2005a). Moderate responders' neutrophils are functioning much more appropriately than severs'. The fast increase in neutrophils' diapedesis rate and post-diapedetic neutrophil ROS production capacity during acute infection of mammary gland lags exponential growth of *E. coli* in the gland. Compared with severe responders, the fast-strong local response in moderate responders facilitated recovery of acute infection and inflammation. The bacterial growth in the lactating udder is exceeded to the neutrophils' diapedesis and ROS

induced by granulocyte-moncyte colony-stimulating factors (GM-CSF), reflecting an increased proliferation and differentiation of bone marrow granulocytes (Heyneman et al., 1990; Heyneman & Burvenich, 1992; Mehrzad et al., 2001a; 2001b; 2004; 2005a). It has been postulated that myeloid stem cells leave the bone marrow almost by a pipeline mechanism, the older cells being released first (Heyneman & Burvenich, et al., 1992). The molecular mechanisms that control the release of mature PMN from the bone marrow into the circulating pool and then extravasation are very complex and poorly understood (see

Neutrophils circulate in the blood until recruited to sites of infection by chemical signals. This migration begins when neutrophils interact with activated endothelial cells. Inflammatory cytokines like tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), and interferon- (IFN-) produced by activated macrophages at sites of infection activate endothelial cells and induce expression of receptors like E-selectin (CD62E) and P-selectin (CD62P) and ligands like Sialyl-Lewis X on glycan-bearing cell adhesion molecule-1 (GlyCAM-1) and P-selectin glycoprotein ligand-1 [PSGL-1]) which interact with neutrophil receptors like L-selectin (CD62L) (Diez-Fraille et al., 2004; Sohn et al., 2007a; 2007b ; Zarbock A & Ley, 2008). Selectin-ligand interactions are of low affinity leading to only "rolling" and slowing of neutrophils on the endothelial cell surface in post capillary venules to enable

Fig. 4. This figure shows the overall disparities of neutrophils' diapedesis rate, post-

growth in the lactating udder is exceeded to the neutrophils' diapedesis and ROS

diapedetic neutrophil ROS production capacity and *E. coli* CFU dynamics in modertate and severe responders of animal infection model; it is based on the study of the kinetics of chemiluminescence and intramammary infection/inflammation (Mehrzad et al., 2001b, 2004; 2005a). Moderate responders' neutrophils are functioning much more appropriately than severs'. The fast increase in neutrophils' diapedesis rate and post-diapedetic neutrophil ROS production capacity during acute infection of mammary gland lags exponential growth of *E. coli* in the gland. Compared with severe responders, the fast-strong local response in moderate responders facilitated recovery of acute infection and inflammation. The bacterial

figures 3 and 4).

stronger associations.

production rates in sever responders. This is very important and basic cellular part of innate immunity of mammary gland. This different response is mainly due to the far stronger preinfection blood and post-daipedetic neutrophils' functions, especially, ROS production as well as the neutrophils' ROS production during the "early phase" of infection/inflammation. So, the capacity of PMN ROS production (especially intracellular) and quality of pos-diapedetic neutrophils both before and during early infection, is crucial for the severity of the diseases, and leads to a faster elimination of pathogens, becuae the fast-strong local response in moderate responders facilitated recovery of infection/inflammation.

Besides increased expression of selectins, TNF- α and IL-1 also enhance endothelial cell expression of vascular cell adhesion moledule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1) and ICAM-2, which are ligands for the neutrophil integrins very late antigen-4 (VLA-4) and leukocyte function antigen-1 (LFA-1 or CD11a/18 or αLβ2) and macrophage-1 antigen (Mac-1, CD11b/18 or αmβ2), respectively. Interactions of neutrophils with these molecules results in firm attachment to the endothelium, followed by their activation, and spreading. Interactions of neutrophil receptors CXCR1 and CXCR2 down a concentration gradient of their ligand chemokine CXCL8 result in transmigration of neutrophils between endothelial cell spaces to the infectious site (Paulsson et al., 2010). Neutrophil LFA-1-endothelial ICAM-1 and CD177-CD31 (platelet endothelial cell adhesion molecule-1 [PECAM-1]) interactions facilitate this migration (Sachs et al., 2007).

A potential role of adhesion molecules like L-selectin in the release of neutrophils from the bone marrow is very critical, because L-selectin is highly expressed on mature PMN in the post-mitotic pool in the bone marrow and in the circulation (Diez-Fraile et al., 2004). The process of granulopoiesis is strictly controlled by regulatory growth factors, comprising cytokines and colony-stimulating factors, which have pleiotropic effects on proliferation, differentiation and functional activation of precursor cells (Burvenich et al., 2003). Using an optimised cell culture assay for the bovine (Smit et al., 1996; Van Merris et al., 2001a; 2001b), it was demonstrated that physiological concentrations of -hydroxybutyric acid and acetoacetic acid induced remarkable suppression on the proliferation of hematopoietic cells (van Werven et al., 1997; Hoeben et al., 1999). Bovine pregnancy-associated glycoprotein also reduced the proliferative activity of bovine progenitor cells (Hoeben et al., 1999). Therefore, the neutrophil circulating pool is largely depending on the proliferative capacity of the bone marrow. After having exerted their role in immune function, PMN die by senescence (Mehrzad et al., 2001a; Van Merris et al., 2002; Burvenich et al., 2003). Aged PMN undergo spontaneous apoptosis in the absence of pro-inflammatory agents prior to their removal by macrophages (Paape et al., 2002; Burvenich et al., 2003), thus preventing the release of their cytotoxic content. Inflammation and infection hugely increase the rate of PMN production, shortening the maturation time, thereby leading to the release of immature neutrophils in the circulation pool.

Advances in mammary gland immunology of recent decades have provided insights into the mechanisms responsible for the defense of the mammary gland against infection. PMN has a pivotal role in the protection of the gland from infections (Paape et al., 2002; Burvenich et al., 2003; Mehrzad et al., 2001a; 2001b; 2001c; 2002; 2004; 2005a; 2005b; 2009). The life cycle of the bovine PMN is short. Formed in the bone marrow, PMN require 10 to 14 days to mature (Bainton et al., 1971; Burvenich et al., 2003). After maturation, PMN may be stored

Molecular Aspects of Neutrophils' Pivotal Role in Mammary Gland Immunity 393

and neutrophils' surface receptors, e.g., C5a-C5aR, C5aR-TLR4 interactions. Normally, C5aR is up-regulated in inflammatory environment (Bruhn et al., 2011; Stevens et al., 2011a; 2011b). There is considerable evidence for the participation of C5aR in the harmful consequences of experimental infection, sepsis and cancer in human and animals. Therefore, interception of either C5a or C5aR dramatically improves the exaggerated inflammatory reactions which can be good direction to treat and remodel tissue injuries and damages. This evidence would open a new door to the molecular aspects of controlling and treating of inflammation, infection and sepsis. For example, *in vivo* blockade of C5aR resulted in greatly improved survival of animals after sepsis. Similar phenamena could be observed and would be applied for other key molecules like IL-1-IL-1R interactions as well as TLRs

Extravasation of activated PMN occurs after their adhesion to the endothelial surface. This is accomplished by the expression of specific membrane adhesion molecules. The essential role of the CD11/CD18 family of adhesion molecules in bovine PMN-surface adhesion is welldocumented (Diez-Fraille et al., 2004). These molecules bind to ICAM-1, ICAM-2 and endothelial leukocyte adhesion molecules (ELAM-1) on the endothelial surface. After binding to these molecules, PMN leave the circulation and are ready to function at the infection site. Down-regulated CD11/CD18 in circulating PMN can cause a harder and slower PMN recruitment into the mammary gland (Burvenich et al., 2003; Diez-Fraille et al., 2004). Inflammatory environment of the udder induces adherence of circulating PMN to the endothelium by up-regulation of CD11b/CD18 (Diez-Fraille et al., 2004), of which activity is crucial to bovine PMN diapedesis across the blood/milk barrier (Smits et al., 2000); in such an environment, blood PMN number, and effective adhesion, migration, opsonization, phagocytosis and killing are of crucial importance to the outcome of the infection and the severity of the disease (Gray et al., 1982; Burvenich et al., 1999; 2003; Mehrzad et al., 2001a; 2001b; 2005a; 2005b; 2009). The impact of fast PMN diapedesis during udder infection/inflammation on PMN quality and their ROS production capability could cause dissimilarities between post-diapedetic PMN from inflamed and non-inflamed quarters (Mehrzad et al., 2001b; 2005a; 2005b; 2009) (see later figures of this chapter). The underlying cellular and molecular mechanisms of this disparity would be pivotal for further investigation in the area of animals and human mammary gland immunobiology and

The source of host and/or pathogen-derived cytokines in udder secretions and their impact on udder PMN function has been a subject of investigation. There is evidence of cytokines secretion by mammary macrophages and epithelial cells during both physiological and pathological conditions of the gland (Boudjellab et al., 1998; Mehrzad et al., 2001b; 2004; 2005a; 2005b; Rainard, 2002; Stevens et al., 2011a; 2011b; Pezeshki et al., 2011). These cytokines influence PMN function. For example, the IL-8 is involved in the recruitment of PMN and T lymphocytes into the gland (Barber et al., 1999). Proinflammatory cytokines, like TNF- , IL-1, and LPS suppress the gene expression of cytochrome P-450 1A1 (*cyp1a1*), by activating the transcription nuclear factor B (NF-B) (Notebaert et al., 2005). PMN also play a crucial role in the recruitment of other leukocytes such as CD4+ T lymphocyte and CD8+ T lymphocyte to the inflammation sites (Mehrzad et al., 2008a; 2008b). From bone marrow to the blood stream and the extravasation, the on time PMN influx to the site of inflammation is important in limiting injury and promoting recovery of severe inflammation (Carey et al.,

antagonists/agonists in the inflammatory environment.

1997; Mehrzad et al., 2001b; 2004; 2005a; 2005b).

neoplasia.

for a few additional days. Mature PMN leave the hematopoietic compartment of the bone marrow and enters the vascular sinus by travelling in migration channels through endothelial cells. Normally, mature neutrophils circulate in the blood stream briefly (halflife of ~9 hours), then leave the blood stream by diapedesis and enter tissues where they function as strong phagocytes for 1 to 2 days. In healthy animals, production and destruction of PMN is tightly regulated, which keeps their number in blood, milk, and tissue almost constant (Heyneman et al., 1990; Heyneman & Burvenich, 1992; Paape et al., 2002; Burvenich et al., 2003; Mehrzad et al., 2002a) and this is an essential element of their role in the first line of immune defense. Continuous influx of PMN is orchestrated by the local accumulation of chemotactic factors, which may be of endogenous or exogenous origin. Examples of the former include complement derived factors (e.g., C5a), lipid-derived mediators (e.g., leukotriene B4, platelet-activating factor or PAF) and tissue-derived chemokines (in particular IL-8). The dynamic of PMN diapedesis through blood/milk barrier helps to explain the observed PMN activity fluctuations in milk (Smits et al., 1999; Mayadas & Cullere 2005; Pezeshki et al., 2011).

Transendothelial/epithelial concentrations of neutrophils is very critical to effectively kill invading microbes (Li et al., 2002; Mehrzad et al., 2005a; 2005b). The issue of blood and postdiapedetic PMN functions and concentrations in different physiological and pathological conditions remains the focus of most concern. Although the presence of strong chemotactic factors in non-inflamed udder is the subject of debate, their presence in inflammatory environment of udder and milk is indisputable (Manlongat et al., 1998; Rainard, 2002; Stevens et al., 2011a; 2011b; Pezeshki et al., 2011). Most inflammatory chemoattractants are only induced and released during acute infection. However, a restricted number of chemoattractants can be constitutively present in normal plasma at high concentrations, e.g. Regakine-1 (Struyf et al., 2001). During mastitis, inflammatory chemoattractants simply guide PMN toward infection foci. Potent bovine PMN chemoattractants include C5a, an active cleavage product of the C5 in the complement system, various lipopolysaccharides (LPS), IL-1, IL-2 and IL-8 (Gray et al., 1982). These chemoattractants bind to specific receptors on the PMN plasma membrane.

When the completely equipped neutrophils reach the site of infection/inflammation, they with macrophages capture and ingest microbes by phagocytosis and destroy them with their microbicidal arsenal.  Neutrophils also bind to pathogens coated with various opsonins like IgG, complement components (e.g. iC3b), and lectins. Activated neutrophils express the high affinity Fc-receptor, FcR1, which binds to IgG-Fc of antibody-coated microbes, and β2 integrins on the neutrophil surface can capture pathogens coated with the iC3b. Bound pathogens are then surrounded by neutrophil membrane projections and engulfed in the cellular cytoplasm inside a phagosome. The phagosome fuses with a lysosome to produce a phagolysosome where the captured pathogens are destroyed.

One of the critically hottest topics in the molecular aspects of inflammation, infection and sepsis in relation to neutrophils in animals and human would be the cross-talk between C5a

for a few additional days. Mature PMN leave the hematopoietic compartment of the bone marrow and enters the vascular sinus by travelling in migration channels through endothelial cells. Normally, mature neutrophils circulate in the blood stream briefly (halflife of ~9 hours), then leave the blood stream by diapedesis and enter tissues where they function as strong phagocytes for 1 to 2 days. In healthy animals, production and destruction of PMN is tightly regulated, which keeps their number in blood, milk, and tissue almost constant (Heyneman et al., 1990; Heyneman & Burvenich, 1992; Paape et al., 2002; Burvenich et al., 2003; Mehrzad et al., 2002a) and this is an essential element of their role in the first line of immune defense. Continuous influx of PMN is orchestrated by the local accumulation of chemotactic factors, which may be of endogenous or exogenous origin. Examples of the former include complement derived factors (e.g., C5a), lipid-derived mediators (e.g., leukotriene B4, platelet-activating factor or PAF) and tissue-derived chemokines (in particular IL-8). The dynamic of PMN diapedesis through blood/milk barrier helps to explain the observed PMN activity fluctuations in milk (Smits et al., 1999;