**2. Neutrophils**

Neutrophils are the most abundant leukocytes in blood and because they are the first cells to appear at sites of inflammation and infection; they are regarded as the first line of defense of the innate immune system [10]. Neutrophils can rapidly move from the blood into affected sites through a process known as the leukocyte adhesion cascade. Once in the tissues, they perform important antimicrobial functions, including phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs) [11, 12].

#### **2.1 Leukocyte adhesion cascade**

Neutrophils leave the blood circulation at sites of infection or inflammation by binding to the endothelial cells and then transmigrating into the tissues [13]. This process known, as the leukocyte adhesion cascade (**Figure 1**), begins with the activation of endothelial cells at the affected site. Activated endothelial cells upregulate the expression of adhesion receptors such as E- and P-selectins. Neutrophils bind to these selectins via glycoprotein ligands on their membrane. As a consequence, neutrophils can then roll on endothelial cells. Next, neutrophils get activated by chemokines, which induce a high affinity state on integrins, another group of adhesion receptors. Binding of integrins with their corresponding ligands, such as intercellular adhesion molecule-1 (ICAM-1) and ICAM-2 on endothelial cells, results in slower neutrophil rolling and then firm adhesion that makes neutrophils stop. Finally, neutrophils transmigrate the endothelium into the tissues. Engagement of endothelial-cell adhesion molecules seems to provoke the opening of endothelial-cell contacts by redistributing junctional molecules in a way that promotes transmigration of neutrophils. Molecules that do not help neutrophil migration, such as vascular endothelial cadherin (VE-cadherin), are moved away from junctional regions. Other endothelial junctional molecules for which neutrophils express ligands concentrate on the endothelial cell luminal surface creating an adhesive environment for the neutrophil. Platelet/endothelial-cell adhesion molecule 1 (PECAM1) and CD99 support homophilic interactions between endothelial cells and neutrophils. While, junctional adhesion molecule (JAM)-1 and JAM-2 on the endothelial cell bind to the β1 integrin VLA4, and the β2 integrins LFA-1 and Mac-1 on the neutrophil, respectively. The endothelial cell-selective adhesion molecule (ESAM) is also involved in transmigration by binding to an unknown

**25**

*Neutrophil Activation by Antibody Receptors DOI: http://dx.doi.org/10.5772/intechopen.80666*

ligand on neutrophils [12, 14]. Once neutrophils move into tissues, they follow chemoattractant gradients to reach affected sites using now adhesion of β1 integrins to proteins of the extracellular matrix, such as collagen and fibronectin [15] (**Figure 1**). Important chemoattractants for neutrophils are activated complement components, such as the anaphylatoxin C5a, bacterial components, such as formylmethionyl-leucyl-phenylalanine (fMLF) and cytokines, such as interleukin (IL) 8.

*Leukocyte adhesion cascade. Neutrophils, also known as polymorphonuclear (PMN) cells, move to sites of inflammation via a leukocyte adhesion cascade that includes activation of endothelial cells with upregulation of E- and P-selectins. Neutrophils bind to these selectins via glycoprotein ligands, such as P-selectin glycoprotein ligand-1 (PSGL-1), and begin rolling on endothelial cells. Next, neutrophils get stimulated by chemokines and activate their β2 integrins, which bind to their corresponding ligands, such as intercellular adhesion molecule-1 (ICAM-1). Integrin binding induces firm adhesion and transmigration of neutrophils into tissues. Once in tissues, neutrophils move following chemoattractant gradients to reach affected sites using now adhesion of β1 integrins to proteins of the extracellular matrix, such as collagen and fibronectin. Antibodies (IgG) bind to microorganisms (bacteria) and are in turn recognized by Fcγ receptors (FcγR) on the membrane of neutrophils.*

Neutrophils recruited from the circulation into infected tissues can eliminate microorganisms by phagocytosis, by releasing antimicrobial substances or by form-

Phagocytosis is the process by which particles larger than 5 μm get internalized by the cell into a vacuole called the phagosome. Neutrophils recognize pathogens directly through pattern-recognition receptors (PAMPs), or indirectly through opsonin receptors. Opsonins are host proteins, such as antibody molecules or complement components, that bind to microorganisms and facilitate their detection and destruction by leukocytes [16, 17]. After internalization, the nascent phagosome matures by fusing with lysosomes [18]. During maturation, antimicrobial

**2.2 Antimicrobial mechanisms of neutrophils**

ing NETs [11, 12] (**Figure 2**).

*2.2.1 Phagocytosis*

**Figure 1.**

*Neutrophil Activation by Antibody Receptors DOI: http://dx.doi.org/10.5772/intechopen.80666*

#### **Figure 1.**

*Neutrophils*

promote unique effector cell functions.

**2. Neutrophils**

lular traps (NETs) [11, 12].

**2.1 Leukocyte adhesion cascade**

Immunoglobulin (Ig) G antibody molecules are an essential part of the adaptive immune system. IgGs recognize antigens via their two Fab portions and are in turn linked through their Fc portion to specific Fcγ receptors (FcγRs) on the membrane of leukocytes [6, 7]. In this way, antibodies function as a bridge between the specific adaptive immune response and the potent innate immune functions of leukocytes. In the human neutrophil, two types of FcγR exist. Thus, antibodies are important activators of neutrophils. The Fcγ receptors on the neutrophil are considered to be redundant in inducing cell responses [8, 9]. However, recent findings on how a particular IgG subclass and the glycosylation pattern of the antibody regulate the IgG–FcγR interaction suggest that a particular effector function may in fact be activated in response to a specific type of FcγR. It is the purpose of this chapter to describe the FcγRs on human neutrophils and present our current view of how particular FcγRs activate various signaling pathways to

Neutrophils are the most abundant leukocytes in blood and because they are the first cells to appear at sites of inflammation and infection; they are regarded as the first line of defense of the innate immune system [10]. Neutrophils can rapidly move from the blood into affected sites through a process known as the leukocyte adhesion cascade. Once in the tissues, they perform important antimicrobial functions, including phagocytosis, degranulation, and formation of neutrophil extracel-

Neutrophils leave the blood circulation at sites of infection or inflammation by binding to the endothelial cells and then transmigrating into the tissues [13]. This process known, as the leukocyte adhesion cascade (**Figure 1**), begins with the activation of endothelial cells at the affected site. Activated endothelial cells upregulate the expression of adhesion receptors such as E- and P-selectins. Neutrophils bind to these selectins via glycoprotein ligands on their membrane. As a consequence, neutrophils can then roll on endothelial cells. Next, neutrophils get activated by chemokines, which induce a high affinity state on integrins, another group of adhesion receptors. Binding of integrins with their corresponding ligands, such as intercellular adhesion molecule-1 (ICAM-1) and ICAM-2 on endothelial cells, results in slower neutrophil rolling and then firm adhesion that makes neutrophils stop. Finally, neutrophils transmigrate the endothelium into the tissues. Engagement of endothelial-cell adhesion molecules seems to provoke the opening of endothelial-cell contacts by redistributing junctional molecules in a way that promotes transmigration of neutrophils. Molecules that do not help neutrophil migration, such as vascular endothelial cadherin (VE-cadherin), are moved away from junctional regions. Other endothelial junctional molecules for which neutrophils express ligands concentrate on the endothelial cell luminal surface creating an adhesive environment for the neutrophil. Platelet/endothelial-cell adhesion molecule 1 (PECAM1) and CD99 support homophilic interactions between endothelial cells and neutrophils. While, junctional adhesion molecule (JAM)-1 and JAM-2 on the endothelial cell bind to the β1 integrin VLA4, and the β2 integrins LFA-1 and Mac-1 on the neutrophil, respectively. The endothelial cell-selective adhesion molecule (ESAM) is also involved in transmigration by binding to an unknown

**24**

*Leukocyte adhesion cascade. Neutrophils, also known as polymorphonuclear (PMN) cells, move to sites of inflammation via a leukocyte adhesion cascade that includes activation of endothelial cells with upregulation of E- and P-selectins. Neutrophils bind to these selectins via glycoprotein ligands, such as P-selectin glycoprotein ligand-1 (PSGL-1), and begin rolling on endothelial cells. Next, neutrophils get stimulated by chemokines and activate their β2 integrins, which bind to their corresponding ligands, such as intercellular adhesion molecule-1 (ICAM-1). Integrin binding induces firm adhesion and transmigration of neutrophils into tissues. Once in tissues, neutrophils move following chemoattractant gradients to reach affected sites using now adhesion of β1 integrins to proteins of the extracellular matrix, such as collagen and fibronectin. Antibodies (IgG) bind to microorganisms (bacteria) and are in turn recognized by Fcγ receptors (FcγR) on the membrane of neutrophils.*

ligand on neutrophils [12, 14]. Once neutrophils move into tissues, they follow chemoattractant gradients to reach affected sites using now adhesion of β1 integrins to proteins of the extracellular matrix, such as collagen and fibronectin [15] (**Figure 1**). Important chemoattractants for neutrophils are activated complement components, such as the anaphylatoxin C5a, bacterial components, such as formylmethionyl-leucyl-phenylalanine (fMLF) and cytokines, such as interleukin (IL) 8.

#### **2.2 Antimicrobial mechanisms of neutrophils**

Neutrophils recruited from the circulation into infected tissues can eliminate microorganisms by phagocytosis, by releasing antimicrobial substances or by forming NETs [11, 12] (**Figure 2**).

#### *2.2.1 Phagocytosis*

Phagocytosis is the process by which particles larger than 5 μm get internalized by the cell into a vacuole called the phagosome. Neutrophils recognize pathogens directly through pattern-recognition receptors (PAMPs), or indirectly through opsonin receptors. Opsonins are host proteins, such as antibody molecules or complement components, that bind to microorganisms and facilitate their detection and destruction by leukocytes [16, 17]. After internalization, the nascent phagosome matures by fusing with lysosomes [18]. During maturation, antimicrobial

#### **Figure 2.**

*Antimicrobial mechanisms of neutrophils. Neutrophils can destroy microbial pathogens, such as bacteria by (a) degranulation, (b) phagocytosis, and (c) NETosis. During degranulation, antimicrobial proteins are released outside the neutrophil. In phagocytosis, the pathogen is ingested in a vacuole named phagosome, which then fuses to lysosomes and becomes a phagolysosome, where the pathogen is destroyed. During NETosis, DNA fibers decorated with histones and granular proteins, such as elastase and myeloperoxidase are released in structures known as neutrophil extracellular traps (NETs).*

molecules are delivered into the phagosomal lumen, and the vesicle is transformed into a phagolysosome [19]. In the phagolysosome, reactive oxygen species (ROS) are produced by the NADPH oxidase on the phagosomal membrane, and the pH inside drops to 4.5–5. Also, hydrogen peroxide (H2O2) is converted to hypochlorous acid (HOCl) in a reaction catalyzed by myeloperoxidase (MPO) [20]. Together, these actions form a toxic environment for the microorganism.

#### *2.2.2 Degranulation*

During neutrophil formation in the bone marrow, immature neutrophils synthesize proteins that are sorted into different granules [10]. Granules are classified into three different types based on their content. Azurophilic granules contain mainly myeloperoxidase, elastase, and cathepsin G. Specific granules contain mainly collagenase, lactoferrin, and lysozyme. Gelatinase granules contain mainly gelatinase, lysozyme, and cytochrome b558 [21]. Neutrophils also form secretory vesicles at the last step of their differentiation. These secretory vesicles contain several important receptors on their membrane, including complement receptors (CR1), Fc receptors (CD16), lipopolysaccharide (LPS) receptors (CD-14), and fMLF receptors. Granule heterogeneity is due to the controlled expression of the granule protein genes [22]. Mature neutrophils are released into the circulation and when they reach sites of infection, neutrophils can degranulate in order to deliver their antimicrobial proteins. Secretory vesicles present the greatest predisposition for extracellular release, followed by gelatinase granules, specific granules, and azurophil granules [23]. The hierarchical mobilization of neutrophil granules and secretory vesicles depend on intracellular Ca2+-level [24].

#### *2.2.3 Neutrophil extracellular traps (NETs)*

When neutrophils cannot ingest large microorganisms, they can display another antimicrobial strategy [25]. Neutrophils can release long chromatin fibers that are decorated with proteins from their granules. These fibers can trap microorganisms, and therefore, they have been called neutrophil extracellular traps (NETs) [26]. The process of NETs formation is called NETosis [27]. NETosis has been described as a special form of programmed cell death. The complete mechanisms of NETs formation are still unknown; it seems that NETosis requires NADPH oxidase activation, reactive oxygen species (ROS) production, myeloperoxidase (MPO), and neutrophil elastase (NE) release [28, 29] (**Figure 2**).

**27**

**Figure 3.**

*[39] (red oval).*

*Neutrophil Activation by Antibody Receptors DOI: http://dx.doi.org/10.5772/intechopen.80666*

Antibodies produced by the adaptive immune response are mainly of the IgG class. These antibodies present higher affinity and greater specificity for their particular antigen. Thus, IgG antibodies are key for controlling infections from all types of pathogens, including viruses, bacteria, fungi, and protozoa [30]. However, IgG molecules do not directly damage the microorganisms they recognize. It is in fact, the cells of the innate immune system, which are responsible for the antimicrobial functions of these antibodies. Although, some antibodies can activate complement, which is then deposited on microorganisms to promote phagocytosis via complement receptors [17, 31], or to induce bacterial lysis via the formation of the membrane attack complex [32], most IgG antibodies bind to specific receptors on the membrane of leukocytes [7, 8]. These receptors recognize the fragment crystallizable (Fc) portion of IgG molecules and are therefore known as Fcγ receptors (FcγR). Cross-linking of FcγR on the surface of cells activates several antimicrobial

Human Fcγ receptors comprise a family of glycoproteins expressed on the membrane of immune cells [7, 8]. These receptors can bind to the various IgG subclasses with different affinities [7], and induce different cellular responses [6]. FcγR can be classified as activating receptors (FcγRI/CD64, FcγRIIa/CD32a, FcγRIIIa/CD16a, and FcγRIIIb/CD16b), and one inhibitory receptor (FcγRIIb/CD32b) [7, 9, 33, 34]

FcγRI is a high affinity receptor, having three Ig-like extracellular domains. It binds mainly monomeric IgG [9]. In contrast, FcγRII and FcγRIII are low-affinity receptors, having two Ig-like extracellular domains. They bind only multimeric immune complexes [9, 35]. FcγRI is associated with a dimer of the common Fc receptor γ chain, which contains an immunoreceptor tyrosine-based activation motif (ITAM) sequence (**Figure 3**). The ITAM sequence is important for receptor

FcγRIIa contains its own ITAM within its cytoplasmic tail. In contrast, the inhibitory receptor FcγRIIb contains an immunoreceptor tyrosine-based inhibition

*Human Fcγ receptors. Schematic illustration of human receptors for IgG. Fcγ receptors are shown relative to the cell membrane (brown line). The IgG-binding chain (α) is expressed together with their respective γ2 signaling subunits. FcγRI is a high affinity receptor, having three Ig-like extracellular domains. FcγRII and FcγRIII are low-affinity receptors, having two Ig-like extracellular domains. FcγRIIIb is expressed exclusively on neutrophils, and it is a glycosylphosphatidylinositol (GPI)-linked receptor missing a cytoplasmic tail. ITAM, immunoreceptor tyrosine-based activation motif with consensus sequence YxxI/Lx (6–12)YxxI/L [36] (green oval); ITIM, immunoreceptor tyrosine-based inhibitory motif with the consensus sequence I/V/L/SxYxxL/V* 

**3. Fcγ receptors**

functions [6].

(**Figure 3**).

signaling [36].

**3.1 Human Fcγ receptors**
