Neutrophils, the most abundant leukocytes in blood, are relevant cells of both the innate and the adaptive immune system. Immunoglobulin (Ig) G antibody molecules are crucial activators of neutrophils. IgGs identify many types of pathogens via their two Fab portions and are in turn detected through their Fc portion by specific Fcγ receptors (FcγRs) on the membrane of neutrophils. Thus, antibodies bring the specificity of the adaptive immune response to the potent antimicrobial and inflammatory functions of neutrophils. Two types of FcγRs with several polymorphic variants exist on the human neutrophil. These receptors are considered to be redundant in inducing cell responses. Yet, new evidence presented in recent years on how the particular IgG subclass and the glycosylation pattern of the antibody modulate the IgG–FcγR interaction has suggested that a particular effector function may in fact be activated in response to a specific type of FcγR. In this chapter, we describe the main types of FcγRs on neutrophils and our current view on how particular FcγRs activate various signaling pathways to promote unique effector cell functions, including phagocytosis, activation of integrins, nuclear factor activation, and formation of neutrophil extracellular traps (NETs).
- Fc receptors
Neutrophils are the most abundant cell type in human blood. They are produced in the bone marrow and then released into the circulation. At sites of infection or inflammation, neutrophils migrate to tissues, where they complete their functions. Finally, neutrophils die by apoptosis and are eliminated by macrophages. Neutrophils are an essential part of the innate immune system , with significant antimicrobial functions, including phagocytosis, degranulation, and the formation of neutrophil extracellular traps (NETs). These antimicrobial functions were believed to be the only goal of neutrophils. However, it has recently become clear that neutrophils display many functional responses that go beyond the simple killing of microorganisms. Neutrophils produce cytokines  and other inflammatory factors  that regulate the whole immune system [4, 5]. Consequently, neutrophils are also key effector cells of the adaptive immune system.
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 promote unique effector cell functions.
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 . 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 . 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 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  (Figure 1). Important chemoattractants for neutrophils are activated complement components, such as the anaphylatoxin C5a, bacterial components, such as formyl-methionyl-leucyl-phenylalanine (fMLF) and cytokines, such as interleukin (IL) 8.
2.2. Antimicrobial mechanisms of neutrophils
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 . During maturation, antimicrobial molecules are delivered into the phagosomal lumen, and the vesicle is transformed into a phagolysosome . 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) . Together, these actions form a toxic environment for the microorganism.
During neutrophil formation in the bone marrow, immature neutrophils synthesize proteins that are sorted into different granules . 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 . 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 . 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 . The hierarchical mobilization of neutrophil granules and secretory vesicles depend on intracellular Ca2+-level .
2.2.3. Neutrophil extracellular traps (NETs)
When neutrophils cannot ingest large microorganisms, they can display another antimicrobial strategy . 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) . The process of NETs formation is called NETosis . 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).
3. Fcγ receptors
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 . 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 , 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 functions .
3.1. Human Fcγ receptors
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 , and induce different cellular responses . 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] (Figure 3).
FcγRI is a high affinity receptor, having three Ig-like extracellular domains. It binds mainly monomeric IgG . 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 signaling .
FcγRIIa contains its own ITAM within its cytoplasmic tail. In contrast, the inhibitory receptor FcγRIIb contains an immunoreceptor tyrosine-based inhibition motif (ITIM) within its cytoplasmic tail (Figure 3). The FcγRIIb negatively regulates various cell functions including antibody production by the B cell , proliferation, degranulation, and phagocytosis in other leukocytes when it is cross-linked with activating FcγRs [38, 39]. Most leukocytes express both activating and inhibitory FcγRs, hence simultaneous cross-linking establishes a threshold for cell activation  that maintains a balanced immune response [41, 42].
FcγRIII has two isoforms: FcγRIIIa is a receptor with a transmembrane domain and a cytoplasmic tail, associated with an ITAM-containing homodimer of Fc receptor γ chains (Figure 3). It is expressed mainly on macrophages, natural killer (NK) cells, and dendritic cells [7, 8]. In contrast, FcγRIIIb is expressed exclusively on neutrophils and it is a glycosylphosphatidylinositol (GPI)-linked receptor missing a cytoplasmic tail. Also, no other subunits are known to associate with it (Figure 3). It is important to mention that human FcγRIIa and FcγRIIIb are exclusive receptors that are not found in other species [33, 43].
4. IgG binding to Fcγ receptors
As mentioned before, there is one high-affinity Fcγ receptor, FcγRI (CD64), and two groups of low-affinity Fcγ receptors, FcγRII and FcγRIII (Figure 3). This causes that a single IgG molecule cannot bind to most Fcγ receptors. However, when IgG molecules form antigen-antibody (immune) complexes, they can have many low affinity interactions with Fcγ receptors. Thus, only immune complexes are able to induce the cross-linking of FcγR required for the activation of various antibody-mediated cell functions. It is clear then that depending on the nature of the immune complex, the interaction with various FcγR will change. Several factors have been identified as having an important influence on the affinity of antibody molecules for particular FcγRs. These factors include the type of IgG subclass [7, 44], the IgG glycosylation pattern [45, 46], and receptor polymorphisms.
4.1. The type of IgG subclass
There are four subclasses of IgG (IgG1, IgG2a, IgG2b, and IgG3 in mice; and IgG1, IgG2, IgG3, and IgG4 in humans) . This leads to the formation of different types of immune complexes. Several
Obviously, this selectivity depends mainly on the affinities of different IgG subclasses to particular Fcγ receptors. For this reason, detailed studies to measure the affinities of IgG subclasses to the various Fcγ receptors have been conducted both for mice FcγRs  and for all human FcγRs . Through these studies, it was found that IgG1 and IgG3 bind to all FcγR. IgG2 binds mainly to FcγRIIa (H131 isoform), and FcγRIIIa (V158 isoform), but not to FcγRIIIb . IgG4 binds to many FcγRs . Thus, it is clear that different IgG subclasses engage different Fcγ receptors depending on the relative affinity of these receptors for a particular IgG class .
4.2. The IgG glycosylation pattern
All IgG molecules are glycoproteins with an N-glycosylated carbohydrate side chain that is important for antibody function . Deletion of this carbohydrate (sugar) side chain results in poor binding to FcγRs . The N-glycans are heterogeneous in their sugar composition and are attached to asparagine 297 (Asp297) in the Fc portion of the IgG . The carbohydrate side chain may contain sugar residues such as galactose, fucose, and sialic acid in straight or branching patterns , and the differences in the glycosylation pattern seem to regulate IgG activity .
Many IgG antibodies present a fucose residue linked to an N-acetylglucosamine residue . When this residue is removed, IgG molecules present an increased affinity to the FcγRIIIa , and also an increase in antibody-dependent cell cytotoxicity (ADCC) activity against various tumor cells [51, 57, 58]. Based on these findings, recombinant IgG antibodies with low fucose levels have been produced in order to increase their ADCC activity. Several of these antibodies are now in clinical trials to test their therapeutic potential .
Many IgG antibodies also present a carbohydrate side chain that terminates with sialic acid residues . Contrary to antibodies without fucose, terminal sialic acid usually correlates with low affinity for FcγRs and also with lower ADCC activity [61, 62]. Interestingly, these sialic acid-rich antibodies seem to preferentially bind other receptors different from FcγRs. The receptor dendritic cell specific ICAM-3 grabbing nonintegrin (DC-SIGN) was identified as a receptor for sialic acid-rich IgG . Therefore, terminal sialic acid can modify IgG activity by promoting less binding to FcγRs and more binding to other receptors .
4.3. Polymorphisms of receptors
Another factor influencing the affinity of antibody molecules is the existence of several polymorphisms for the unique FcγRIIa and FcγRIIIb present on human neutrophils . There are two isoforms for FcγRIIa with different amino acids at position 131. These are identified as low-responder (H131) and high-responder (R131) . Similarly, for FcγRIIIb two isoforms exist differing at four positions, NA1 (R36 N65 D82 V106) and NA2 (S36 S65 N82 I106) , and with different glycosylation patterns . In addition, another FcγRIIIb isoform named SH is generated by a point mutation (A78D) in the NA2 allele . These multiple FcγR isoforms display diverse binding affinity for different IgG classes , creating variable cell responses to different antibodies.
5. Fcγ receptor signaling
The human neutrophil expresses two unique activating Fc receptors: FcγRIIa and FcγRIIIb. FcγRIIa is a receptor containing ITAM sequences [36, 69], and it signals similarly to other typical immunoreceptors, such as the antigen receptor of T lymphocytes (TCR) and the antigen receptor of B lymphocytes (BCR) . The initial signaling steps for all immunoreceptors are alike and involve first cross-linking of the receptors on the membrane of the cell, followed by the activation of Src family tyrosine kinases (Figure 4). These kinases lead to activation of spleen tyrosine kinase (Syk), which in turn phosphorylates tyrosines within the ITAM sequence. Phosphorylated ITAM then becomes a binding site for Syk. After binding to the receptor, Syk phosphorylates multiple substrates leading to different cell responses [6, 31, 71] (Figure 4). Syk can phosphorylate and activate phospholipase Cγ (PLCγ), which in turn generates diacylglycerol (DAG) and inositol triphosphate (IP3). DAG also activates protein kinase C (PKC), an important serine/threonine kinase that can lead to the activation of MAP kinases extracellular signal-regulated kinase (ERK) and p38 (Figure 4). IP3 induces release of intracellular calcium from the endoplasmic reticulum. Calcium regulates several proteins such as calmodulin and calcineurin. Syk can also induce activation of phosphatidylinositol-3 kinase (PI3K), which produces phosphatidylinositol 3,4,5-trisphosphate (PIP3). This phospholipid is relevant to the activation of small GTPases, such as Rho and Rac, which are involved in cytoskeleton remodeling for phagocytosis. Rac also leads to activation of the MAPK/ERK kinase (MEK)—ERK pathway, and to activation of c-Jun N-terminal kinases (JNK). These kinases are important for activation of nuclear factors, such as Elk-1, AP-1, and nuclear factor of activated T cells (NFAT) (Figure 4). These nuclear factors induce the expression of cytokines important for inflammation and immune regulation, such as IL-2, IL-6, IL-8, IL-10, tumor necrosis factor α (TNF-α), and IFN-γ [72, 73, 74] (Figure 4).
In contrast, the human FcγRIIIb is a GPI-linked receptor that lacks an intracellular portion. Thus, it is not clear how it can connect to intracellular signaling molecules. However, there is no doubt that FcγRIIIb is an activating receptor inducing several neutrophil responses such as increase in calcium concentration , activation of the respiratory burst , activation of integrins , and induction of NETosis [78, 79]. Despite, the initial signaling mechanism for FcγRIIIb remains unknown, the signaling pathway for this receptor engages Syk and then transforming growth factor-β-activated kinase 1 (TAK1), as well as the MEK/ERK cascade  (Figure 5). One possibility to connect FcγRIIIb with Syk is that the receptor could link with signaling molecules such as Src family tyrosine kinases on the plane of the cell membrane. Because GPI-linked proteins, like the FcγRIIIb, concentrate in lipid rafts on the cell membrane together with Src kinases [81, 82], we can imagine that after cross-linking FcγRIIIb, it associates somehow with these kinases and activates Syk. A possible connection is the binding of the receptor, within the lipid rafts, to a putative ITAM-containing molecule . Many steps are still unknown and future research will help in completely elucidate this signaling pathway.
6. Each FcγR leads to unique cellular responses
The signaling pathways activated by immune complexes binding to Fcγ receptors stimulate different neutrophil responses including phagocytosis, respiratory burst, cytokine and chemokine production, and antibody-dependent cellular cytotoxicity (ADCC) [7, 8, 33]. However, our understanding of what particular function is activated in a cell responding to an individual type of FcγR is still very limited. This lack of knowledge is due, in part, to the fact that each cell expresses several types of FcγRs and all receptors can bind to more than one type of IgG. Thus, it is not clear whether each receptor leads to a particular response or the average signaling from various receptors activates a predetermined cell response. Traditionally, it has been thought that each cell is set to activate a particular cell function after FcγR cross-linking. More recently, however, another interpretation has been considered: each FcγR activates a particular signaling pathway leading to a unique cell response. In the traditional view, each cell is already programmed to perform a particular cell function after FcγR cross-linking, independently of the receptor used. This idea is not really supported by experimental evidence. As indicated above, different IgG subclasses bind particular Fcγ receptors with different affinity, leading to unique cell functions
The idea that particular Fcγ receptors could activate unique cell functions was initially published more than 20 years ago. It was found that the neutrophil FcγRIIIb induced actin polymerization in a Ca2+-dependent manner, while FcγRIIa did not . This initial report was not followed by similar reports and the idea of one receptor one response was forgotten. However, with time, other reports have provided new evidence that supports this idea. Some years later, it was reported that FcγRIIa, but not FcγRIIIb caused shedding of L-selectin expression  (Figure 6). Consequently, it was proposed that binding of antibodies to FcγRIIIb could induce a proadhesive phenotype of neutrophils . More recently, new evidence supporting this idea was found. When each receptor was selectively activated with specific monoclonal antibodies, FcγRIIIb but not FcγRIIa, was able to activate β1 integrins  (Figure 7). This activation resulted from an increase in binding affinity to fibronectin . Thus, after neutrophils leave the circulation, engagement of FcγRIIIb could lead to activation β1 integrins, allowing the cells to adhere to extracellular matrix proteins and migrate into tissues  (Figure 1). In contrast, for antibody-mediated phagocytosis , FcγRIIa was the main Fcγ receptor mediating this response, while FcγRIIIb contribution to phagocytosis was minimal . Therefore, at least in human neutrophils, each Fcγ receptor initiates particular cell functions. FcγRIIa induces phagocytosis (Figure 6), while FcγRIIIb promotes an adhesive phenotype via activation of β1 integrins (Figure 7).
In addition, it was also reported that FcγRIIIb signals to the neutrophil nucleus more efficiently than FcγRIIa. FcγRIIIb, but not FcγRIIa, induced a large increase in phosphorylated ERK in the nucleus, and also efficient phosphorylation of the nuclear factor Elk-1  (Figure 7). Interestingly, FcγRIIa also induced phosphorylation of ERK in the cytosol [84, 90], but this active ERK seems to function mainly in enhancing phagocytosis and not in nuclear signaling  (Figure 4).
A recently discovered antimicrobial function of neutrophils is the formation of neutrophil extracellular traps (NETs) [92, 93]. NETs are induced by several pathogens, including virus, bacteria, fungi, and parasites . Also, pro-inflammatory stimuli such as IL-8, TNF-α, and phorbol-12-myristate-13-acetate (PMA) are efficient inducers of NETs . Because, antigen-antibody complexes are also capable of inducing NET formation ; it was clear that FcγRs were involved in NET formation. Recently, it was found that FcγRIIIb, but not FcγRIIa, is the receptor responsible for NET formation [78, 79, 80] (Figure 8).
Together, all these reports strongly reinforce the modern view that each FcγR induces a particular signaling pathway that activates a single cellular function. Elucidating the conditions that engage a single type of FcγR to activate a particular cellular response would be very helpful in the future for controlling some of cellular functions in clinical settings. For example, in intense infections, it may be important to activate phagocytosis. Because IgG2 binds better to FcγRIIa than to FcγRIIIb [33, 35], it is likely that IgG2 antibodies would activate phagocytosis by neutrophils much better than other IgG subclass antibodies. In consequence, promoting IgG2 antibodies against certain pathogens would result in better phagocytosis against them.
Fcγ receptors expressed in different immune cells are capable of activating different cellular responses important not only for controlling microbial infections but also for regulating immunity [71, 97]. Different subclasses of IgG antibodies bind the various Fcγ receptors with different affinities [33, 35] and can activate various cellular functions of great importance for host defense and for immune regulation. In the human neutrophil, it is clear that a specific Fcγ receptor activates particular cellular responses. FcγRIIa induces efficient phagocytosis , while FcγRIIIb signals to the nucleus for nuclear factor activation  and for NETs formation . Therefore, in principle, a particular cell response could be induced or inhibited by engaging or blocking the corresponding FcγR. Information similar to the one described for neutrophil Fcγ receptors on other immune cells, such as monocytes or dendritic cells, is not available. Future research is needed in this area.
This work was supported by Grant 254434 from Consejo Nacional de Ciencia y Tecnología, México.
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