Antibodies represent an important bridge between the specificity of the adaptive immune system, and the highly destructive mechanisms of cells of the innate immune system. Antibodies bind to microorganisms via their antigen-binding sites, and to Fc receptors on the surface of leukocytes, via their carboxyl terminal Fc portion. Receptors for the Fc portion of various immunoglobulin (Ig) classes have been described [20]. Fc Receptors for IgG (FcγR), for IgE (FcεR), and for IgA (FcαR) are known [20]. Crosslinking of Fcγ receptors with their IgG antibody ligands triggers various functions in many cells of the immune system. These cell functions include phagocytosis, cell degranulation, production of various cytokines and chemokines, ADCC, and activation of genes [10,21].

Fcγ receptors are a family of glycoproteins, part of the IgG superfamily. They consist of an IgG binding α-subunit, that usually pairs with accessory γ chains, which are important for receptor signaling (Figure 1).

In humans, three classes of FcγR have been identified, FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16) (Figure 1A) [22]. FcγR are coded for by different genes and differ in their relative avidity for IgG, molecular structure, and cellular distribution. FcγRI α-subunit presents three Ig-like extracellular domains, and binds monomeric IgG [23]. In contrast, FcγRII and FcγRIII present two Ig-like extracellular domains, and bind only multimeric immune complexes. FcγRI, expressed on monocytes, macrophages, and interferon-γ (IFN-γ)-stimulated neu-

trophils, is associated with a dimer of the common Fc receptor (FcR) gamma-chain (also named FcRγ chain). Each γ chain contains tyrosine residues that are phosphorylated upon receptor activation and become docking sites for other signaling molecules. These tyrosine residues are found within a common motif known as ITAM, for immunoreceptor tyrosine-based activation motif [24, 25]. There are several isoforms of FcγRII, derived from its three genes and from alternative splicing. FcγRII isoforms are distributed differently on hematopoietic cells. FcγRIIA and FcγRIIC are found mainly in phagocytic cells (neutrophils, monocytes, and macrophages), whereas FcγRIIB is expressed mainly in B lymphocytes [22]. FcγRIIB expression is inducible in phagocytic leukocytes, for the negative regulation of cell functions, such as phagocytosis [8,26]. The human FcγRIIA is a particular receptor that does not have associated FcRγ chains. FcγRIIA contains an ITAM in its cytoplasmic portion, while FcγRIIB has a different tyrosine-containing motif involved in negative signaling. This motif is known as ITIM, for immunoreceptor tyrosine-based inhibition motif [27]. FcγRIII has two isoforms: FcγRIIIA is a receptor with a transmembrane portion and a cytoplasmic tail, associated with an ITAM-containing homodimer of FcRγ chains on macrophages, natural killer (NK) cells, and dendritic cells. FcγRIIIA expressed on basophils and mast cells associates with a heterodimer of γ/ζ chains and an extra β chain (Figure 1A) [22,23]. FcγRIIIB is present exclusively on neutrophils and it is a glycosylphosphatidylinositol (GPI)-linked receptor, lacking a cytoplasmic tail. No other subunits are known to associate with it, and its signaling mechanism remains unidentified (Figure 1A). It is also worth noting that human FcγRIIA and FcγRIIIB are exclusive receptors that are not found in other species [28].

In mice, several Fcγ receptors have also been described (Figure 1B). These receptors are very similar, yet not identical, to the human FcγR [8]. FcγRI is expressed on monocytes and macrophages, and it is also associated with a dimer of FcRγ chains, which contain the ITAM motifs involved in receptor signaling. FcγRIII is a receptor with a transmembrane portion and a cytoplasmic tail, associated with a dimer of FcRγ chains, containing ITAMs. This receptor is closer to the human FcγRIIA, as revealed by the genetic structure of FcRs in various species [13]. FcγRIV is also an activating receptor expressed together with a dimer of FcRγ chains [29], and it is closer to the human FcγRIIIA (Figure 1B). FcγRIV may be the most relevant activating FcγR in mice, due to its ability to bind IgG2a and IgG2b with higher affinity [13]. FcγRIIB is the negative receptor containing an ITIM motif in its cytoplasmic tail. It is expressed mainly in B lymphocytes but also in phagocytic leukocytes and dendritic cells. FcγRIIB, described first in B lymphocytes, down regulates the activation signals from the B cell antigen receptor (BCR) to inhibit antibody production by the B cell [30]. This inhibitory receptor also helps to regulate initiation of other cell functions in phagocytic leukocytes and dendritic cells by creating, together with activating Fcγ receptors, a threshold for cell activation [13,31].

Fcγ receptors are found on many cells of the immune system, including granulocytes such as neutrophils and eosinophils; phagocytes such as neutrophils, monocytes and macrophages; and lymphocytes such as natural killer cells and B cells [23]. The wide variety of cellular responses regulated by Fcγ receptors is consequently not surprising. Monocytes and macrophages express all types of activating Fcγ receptors, FcγRI, FcγRII, and FcγRIII in humans, and FcγRI, FcγRIII, and FcγRIV in mice (Table 1). Murine neutrophils express FcγRIII, and FcγRIV, whereas human neutrophils express FcγRIIA and FcγRIIIB. It is noteworthy to mention that human neutrophils have the two unique FcγR, not present in neutrophils of other species. Thus, care should be taken when analyzing data derived from mouse studies of neutrophil FcγR function, since conclusions may not necessarily apply to human neutrophils. NK cells exclusively express FcγRIIIA in humans and FcγRIII in mice. Dendritic cells (DCs) also express various FcγRs, while B lymphocytes express mainly the inhibitory FcγRIIB (Table 1). Some T lymphocyte populations have also been reported to express FcγRs [32-34], but the role for these receptors in T cell function or development remains unclear and requires further studies.

Crosslinking of activating Fcγ receptors with their IgG antibody ligands triggers various functions in many cells of the immune system. As mentioned before, all activateing receptors contain ITAM motifs involved in receptor signaling. The exact activation mechanism is not completely clear, but at the initial molecular events involve activation of Src family kinases followed by activation of Syk (spleen tyrosine kinase) family kinases.

The model for the initial steps of activating FcγR signaling is as follows: Upon crosslinking, the receptor associates with lipid rafts. Lipid rafts are small regions of the plasma membrane that are enriched in cholesterol and sphingolipids [35]. There, the receptor co-localizes with Src kinases. These kinases phosphorylate tyrosines within the ITAM. Phosphorylated tyrosines then become docking sites for Syk. This kinase then phosphorylates multiple substrates, including phosphatidylinositol 3- kinase (PI 3-K), phospholipase Cγ (PLCγ), and the adap

Table 1. Fc gamma receptors.

^aData from [64,65]; ^bData from [29,61].

or molecules SLP76 (SH2-domain-containing leukocyte protein of 76 kDa) and LAT (linker for activation of T cells) [36] (Figure 2). These molecules organize and activate several signaling pathways, depending on the cell type, leading to particular cellular responses and to transcriptional changes (Figure 2). For example, SLP76 seems to be important for FcγR signaling in neutrophils [37], while it seems to be dispensable in macrophages and NK cells [38,39]. LAT is another adaptor that seems to participate in many FcγR-mediated functions (Figure 2). LAT was reported to be constitutively associated with the common FcRγ chain in monocytes [40], and to be important for efficient phagocytosis in macrophages [40]. Clearly, further studies are needed to identify the particular adaptor used by each type of Fc receptor in the various leukocytes, and to connect this to a unique cell response.

The inhibitory FcγRIIB is the negative receptor containing an ITIM motif in its cytoplasmic tail instead of an ITAM sequence [41]. It was described first in B lymphocytes, where it down regulates the activation signals from the B cell antigen receptor (BCR) (Figure 3A) to inhibit antibody production by the B cell [30]. Contrary to the activating receptors that engage several kinases, this inhibitory receptor signals by activation of phosphatases. Inositol 5-phosphatase (SHIP1) is the main enzyme activated upon crosslinking of FcγRIIB and BCR (Figure 3B) [27]. This phosphatase binds via its SH2 domain, to the phosphorylated tyrosines within the ITIM sequence of FcγRIIB. SHIP1 transforms phosphatidylinositol-3,4,5-triphosphate (PIP₃) the main product of PI 3-K, into phosphatidylinositol-3,4-biphosphate (PIP₂); pre-

venting in this way the stimulation of key activating enzymes such as Akt, Btk, and PLCγ (Figure 3B).

As indicated above, different leukocytes express more than one activating FcγR (Table 1), and most of these cells also express at the same time the inhibitory FcγRIIB. NK cells are particular leukocytes in this respect, because they only express FcγRIIIA in humans and FcγRIII in mice. The coexpression of both activating and inhibitory FcγR results in simultaneous triggering of activating and inhibitory signal transduction pathways (Figure 4A). Thus, a particular cell will respond when the sum of activating and inhibiting signals reach a threshold of activation that is determined by the relative expression of both types of FcγR (Figure 4B). The importance of the inhibitory FcγRIIB in regulating many IgG-mediated responses in different leukocytes was made evident in FcγRIIB-deficient mice, which showed enhanced activity of many IgG-mediated cell responses including: phagocytosis, immune complex-mediated inflammation, IgG-

mediated passive and active anaphylaxis, and IgE-mediated anaphylaxis [42,43]. They also showed enhanced dendritic cell maturation, and antigen presentation [44-46]. These results thus confirmed that FcγRIIB regulates initiation of cell functions by generating, together with activating Fcγ receptors, a threshold for cell activation [31, 47].

Analysis of FcγR genes in different species has identified

orthologous receptors between mice and humans. The similarities come from gene localization and also from sequence homology of the extracellular portion of the receptors [8,13]. Therefore, the high affinity receptors FcγRIA and FcRI, the low affinity receptors FcγRIIA and FcγRIII, and also the low affinity receptors FcγRIIIA and FcγRIV cluster in the same area of chromosome 1 (Figure 5). Similarly, the inhibitory FcγRIIB gene maps to the same chromosome region, both in mice and humans (Figure 5). In addition, the human FcγRIIA and mouse FcγRIII, as well as the human FcγRIIIA and mouse FcγRIV, present high sequence homology in their extracellular domains [13]. Despite this similarity, the receptors are not equivalent, since important differences have been detected between mice and human responses to IgG. For example, the human FcγRI binds the IgG1 and IgG3 subclasses with high affinity, while the mouse FcγRI only binds IgG2a with high affinity (Table 1). In addition, the mouse FcγRIV is also able to bind IgE, while the human FcγRIIIA is not [48,49].

In addition, there are several polymorphisms in the human FcγRII and FcγRIII. For FcγRIIA two allelic variants exist expressing either arginine or histidine at position 131 [50,51]. For FcγRIIIA also allelic variants exist expressing either valine or phenylalanine at position 158 [52,53]. Similarly, for FcγRIIIB on neutrophils, two isoforms exist, the NA1 and NA2 allotypes [54]. These isoforms differ by five nucleotides and four amino acids, with NA2 containing two additional N-linked glycosylation sites. These differences affect the capacity of FcγRIIIB

to interact with human IgG. Therefore, neutrophils from individuals who are homozygous for the NA1 allele have better phagocytosis of IgG-opsonized targets than do neutrophils from NA2-homozygous individuals [55,56]. These multiple FcγR and their allelic variants vary greatly in their affinity for different IgG classes [57]. Thus, a great interest exists to understand how different IgG molecules engage different FcγR to activate the multiple cell responses associated with antibodies Fcγ receptor signaling.

A single antibody molecule does not bind to Fcγ recaptors. However, antigen-antibody complexes promote many low affinity interactions between FcγR on the surface of leukocytes and antibody complexes. The low affinity of antibodies for individual leukocyte FcγR prevents recaptors from binding antibodies in the absence of antigen, thus reducing the chance of immune cell activation when there is not an infection. Immune complexes induce the crosslinking of FcγR to activate the many different antibody-mediated cell responses. Immune complexes are clearly of different kinds, since they are formed by different classes of antibodies, and in vivo studies have suggested that the different IgG classes have different activities. For example, IgG2b/c was better in eliminating B cells [58] and T cell lymphomas [59] than IgG1. Likewise, using class-switch variants of anti-erythrocyte antibodies it was found that IgG2a and IgG2b were better in mediating phagocytosis of opsonized erythrocytes than IgG1 and IgG3 [60,61]. Also, polioencephalomyelitis induced by infection with lactate dehydrogenaseelevating virus (LDV) was delayed much better by IgG2a anti-LDV antibodies than any other IgG class of antiLDV antibodies [62]. In addition, the severity of glomerular inflammation was greater for IgG2a, followed by IgG2b and finally IgG1 [63]. All these reports confirmed that different IgG classes mediate different cellular responses in vivo through Fcγ receptors.

Because, immune complexes of different kinds induce different cell responses, there has been a great interest in determining which type of IgG binds to which FcγR and what particular receptor is involved in mediating the activity of particular IgG classes. Early studies showed that there is a high affinity receptor for IgG (FcγRI), which binds preferably to IgG1 in humans and IgG2a in mice [64,65]. This receptor is saturated with serum IgG on leukocytes in the blood. As mentioned before, the other receptors have only low affinity and can bind to IgG only in the form of immune complexes (Table 1) [64,65]. In addition, it was clearly established that most FcγR have a binding preference for IgG1 and IgG3 over the other classes of IgG (Table 1). Similarly, in mice it was found that IgG1 binds only to FcγRIII, while IgG2a binds to all types of activating FcγR, and IgG2b binds to FcγRIII and FcγRIV. IgG3 does not seem to bind significantly to any of the FcγR (Table 1) [29,61]. In agreement with these data, IgG1 activity was lost in mice deficient in FcγRIII [61,66]. For IgG2a and IgG2b, however the correlation with particular Fcγ receptors is not as simple. In some model systems the activity of these IgG classes was lost in FcγRIII-deficient mice, while it was not in others [13]. In a model of autoimmune hemolytic anemia, IgG2amediated response was highly dependent on FcγRIII, but also FcγRI and FcγRIV contributed to the development of severe anemia [67]. In another model of arthritis, mice deficient in FcγRI showed reduced cartilage destruction, and impaired protection from a bacterial infection, indicating the prominent role of FcγRI in IgG2a-dependent immune functions [68]. Thus, IgG2a used all activating Fcγ receptors with important contribution from FcγRI and FcγRIII. Similarly, for IgG2b a particular interaction with a particular Fcγ receptor cannot be clearly established. In models of IgG2b-dependent B cell depletion [69], and nephrotoxic nephritis [70], inhibition of FcγRIV prevented B cell destruction and kidney inflammation, suggesting a central role for FcγRIV in IgG2b-mediated functions. However, in models of IgG2b-dependent autoimmune hemolytic anemia [67], acute glomerular inflammation [63], or acute lung injury [71], FcγRIV and also FcγRIII were important for the activity of this IgG class. Thus, an order of activity is observed among the different IgG classes, and a preference of engagement with particular Fcγ receptors.

Part of the mechanism used to create this IgG-FcγR selectivity is revealed by studies that measured the affinities of IgG classes toward both activating Fcγ recaptors and the inhibitory FcγRIIB [13]. In this way, it was found that IgG1 has higher affinity for the inhibitory FcγRIIB than for the activating FcγRIII (Figure 6A)generating a high threshold for activation. In contrast, IgG2a and IgG2b have higher affinity for the activating FcγRIV than for the inhibitory FcγRIIB (Figure 6B), generating a lower threshold for activation. Thus, certain classes of IgG, such as IgG1, are more dependent on the signaling from the inhibitory receptor. In agreement with this view, deletion of the inhibitory FcγRIIB increased IgG1 activity in models of platelet depletion and tumor cell killing [61]. It is important to note that this model for IgG-FcγR selectivity is not static and can be modulated by other factors, such as the pattern of FcγR expression on the different leukocytes (Table 1), and cytokines that can modify FcγR expression (Figure 7). Thus, Th1-type cytokines such as interferon-γ, and the anaphylatoxin C5a upregulate activating FcγRs expression and downregulate FcγRIIB expression [72,73], whereas Th2-type cytokines, such as interleukin (IL)-4, IL-10, and transforming growth factor-beta (TGF-β) upregulate FcγRIIB expression [8,74].

All the reports previously described have confirmed that different IgG classes mediate different cellular responses in vivo by engaging particular Fcγ receptors depending on the relative affinity of these receptors for a particular

IgG class. However, these findings do not explain what cell response is activated in response to a particular type of Fcγ receptor. One possibility is that each leukocyte is already programmed to respond in a certain way when immune complexes crosslink its Fcγ receptors. Another possibility is that each FcγR signals differently to initiate a particular cell response. The first possibility is not very likely because a particular leukocyte can respond with various cell functions, and also because the same class of IgG induces different responses in different leukocytes. The idea that each FcγR is able to initiate a particular cell response is supported by recent reports where individual FcγR were crosslinked on human neutrophils. As described above, human neutrophils express only two FcγR, FcγRIIA and FcγRIIIB [28]. These receptors are different in the way they are anchored to the cell membrane. FcγRIIA has a typical transmembrane and cytoplasmic tail containing an ITAM for signaling. In contrast, FcγRIIIB is a GPI-linked receptor, lacking a cytoplasmic tail, and its signaling mechanism remains unidentified. However, early reports indicated that both FcγR are capable of signaling and inducing particular responses [75]. More recently, it was found that FcγRIIA, but not FcγRIIIB could induce an increase in L-selectin expression [76]. Opposite to this, FcγRIIIB, but not FcγRIIA, was able to activate β1 integrins [77]. In addition, when the major cell response of neutrophils, arguably phagocytosis, was examined, FcγRIIA was the predominant FcγR mediating this response. FcγRIIIB contribution to phagocytosis was minimal [78]. In complete contrast, FcγRIIIB signaling to the neutrophil nucleus was much more efficient than FcγRIIA signaling. FcγRIIIB, but not FcγRIIA, promoted a robust increase in phosphorylated ERK in the nucleus, and also efficient phosphorylation of the nuclear factor Elk-1 [79]. Interestingly, the FcγRIIA signaling pathway resembles the classical ITAM-mediated pathway (Figure 2) [5], while FcγRIIIB signaling pathway remains a mystery (Figure 8). Taken together, these reports strongly support the hypothesis that each FcγR is capable of initiating particular signaling pathways that lead to unique cell responses.