**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 antibodymediated 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) [47]. This leads to the formation of different types of immune complexes. Several *in vivo* studies have indeed suggested that different IgG subclasses can activate particular cell responses. For example, in mice, IgG2b was better than IgG1 at eliminating B cell [48] and T cell lymphomas [49]. Also, antierythrocyte antibodies of IgG2a and IgG2b subclasses were better than antibodies of IgG1 and IgG3 subclasses in mediating phagocytosis of opsonized erythrocytes [50]. In humans, it was shown that most FcγRs bind primarily IgG1 and IgG3 over the other subclasses of IgG [6, 7]. Together, these reports confirm that different IgG subclasses mediate different cellular responses *in vivo,* and suggest that different cellular activities result from cross-linking different FcγRs. However, the mechanism used to generate this IgG-FcγR selectivity is not completely understood. Accordingly, a great interest exists for determining which type of IgG binds to which FcγR and what particular receptor is involved in mediating a certain cellular function.

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 [51] and for all human FcγRs [35]. Through these studies, it was found that IgG1 and IgG3 bind to all FcγR. IgG2 binds mainly to FcγRIIa (H131 isoform),

**29**

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

**4.2 The IgG glycosylation pattern**

trials to test their therapeutic potential [59].

**4.3 Polymorphisms of receptors**

responses to different antibodies.

**5. Fcγ receptor signaling**

binding to FcγRs and more binding to other receptors [45].

and FcγRIIIa (V158 isoform), but not to FcγRIIIb [35]. IgG4 binds to many FcγRs [35]. Thus, it is clear that different IgG subclasses engage different Fcγ receptors depend-

All IgG molecules are glycoproteins with an N-glycosylated carbohydrate side chain that is important for antibody function [52]. Deletion of this carbohydrate (sugar) side chain results in poor binding to FcγRs [53]. The N-glycans are heterogeneous in their sugar composition and are attached to asparagine 297 (Asp297) in the Fc portion of the IgG [54]. The carbohydrate side chain may contain sugar residues such as galactose, fucose, and sialic acid in straight or branching patterns [46], and the differences in the glycosylation pattern seem to regulate IgG activity [55].

Many IgG antibodies present a fucose residue linked to an N-acetylglucosamine residue [56]. When this residue is removed, IgG molecules present an increased affinity to the FcγRIIIa [57], 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

Many IgG antibodies also present a carbohydrate side chain that terminates with sialic acid residues [60]. 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 [63]. Therefore, terminal sialic acid can modify IgG activity by promoting less

Another factor influencing the affinity of antibody molecules is the existence of several polymorphisms for the unique FcγRIIa and FcγRIIIb present on human neutrophils [64]. There are two isoforms for FcγRIIa with different amino acids at position 131. These are identified as low-responder (H131) and high-responder (R131) [65]. Similarly, for FcγRIIIb two isoforms exist differing at four positions, NA1 (R36 N65 D82 V106) and NA2 (S36 S65 N82 I106) [66], and with different glycosylation patterns [67]. In addition, another FcγRIIIb isoform named SH is generated by a point mutation (A78D) in the NA2 allele [68]. These multiple FcγR isoforms display diverse binding affinity for different IgG classes [35], creating variable cell

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) [70]. The initial signaling steps for all immunoreceptors are alike and involve first crosslinking 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

ing on the relative affinity of these receptors for a particular IgG class [33].

*Neutrophils*

motif (ITIM) within its cytoplasmic tail (**Figure 3**). The FcγRIIb negatively regulates various cell functions including antibody production by the B cell [37], proliferation, degranulation, and phagocytosis in other leukocytes when it is crosslinked with activating FcγRs [38, 39]. Most leukocytes express both activating and inhibitory FcγRs, hence simultaneous cross-linking establishes a threshold for cell

FcγRIII has two isoforms: FcγRIIIa is a receptor with a transmembrane domain

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 antibodymediated 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

There are four subclasses of IgG (IgG1, IgG2a, IgG2b, and IgG3 in mice; and IgG1, IgG2, IgG3, and IgG4 in humans) [47]. This leads to the formation of different types of immune complexes. Several *in vivo* studies have indeed suggested that different IgG subclasses can activate particular cell responses. For example, in mice, IgG2b was better than IgG1 at eliminating B cell [48] and T cell lymphomas [49]. Also, antierythrocyte antibodies of IgG2a and IgG2b subclasses were better than antibodies of IgG1 and IgG3 subclasses in mediating phagocytosis of opsonized erythrocytes [50]. In humans, it was shown that most FcγRs bind primarily IgG1 and IgG3 over the other subclasses of IgG [6, 7]. Together, these reports confirm that different IgG subclasses mediate different cellular responses *in vivo,* and suggest that different cellular activities result from cross-linking different FcγRs. However, the mechanism used to generate this IgG-FcγR selectivity is not completely understood. Accordingly, a great interest exists for determining which type of IgG binds to which FcγR and what particular receptor is involved in mediating a

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 [51] and for all human FcγRs [35]. Through these studies, it was found that IgG1 and IgG3 bind to all FcγR. IgG2 binds mainly to FcγRIIa (H131 isoform),

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 exclu-

activation [40] that maintains a balanced immune response [41, 42].

sive receptors that are not found in other species [33, 43].

glycosylation pattern [45, 46], and receptor polymorphisms.

**4. IgG binding to Fcγ receptors**

**4.1 The type of IgG subclass**

certain cellular function.

**28**

and FcγRIIIa (V158 isoform), but not to FcγRIIIb [35]. IgG4 binds to many FcγRs [35]. 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 [33].
