Fc gamma receptors differ in their structural requirements for interaction with the tyrosine kinase Syk in the initial steps of signaling for phagocytosis

Receptors for the constant region of IgG, Fc gamma receptors, are expressed on the surface of hematopoietic cells, where they mediate signaling events, such as phagocytosis, essential for host defense. Fc gamma receptors also play a role in the pathophysiology of autoimmune diseases. We have demonstrated that members of each of the three classes of human Fc gamma receptors, Fc gamma RI, Fc gamma RII, and Fc gamma RIII, mediate phagocytosis, but that important differences exist in their requirements for phagocytic signaling. For example, the Fc gamma receptors Fc gamma RI and Fc gamma RIIIA induce signaling largely by association with a gamma subunit containing a conserved cytoplasmic motif (ITAM) whose tyrosines are phosphorylated following receptor stimulation. Fc gamma RIIA contains a similar motif in its own cytoplasmic domain and does not require the gamma chain for phagocytic signaling. The tyrosine kinase Syk associates with the cytoplasmic domain of both the Fc gamma receptor gamma chain and Fc gamma RIIA and is required for phagocytosis by both Fc gamma receptor systems. To elucidate the differences in phagocytic signaling by the gamma chain and Fc gamma RIIA, we investigated the requirements for Fc gamma receptor/Syk co-immunoprecipitation, tyrosine phosphorylation, and phagocytosis. Both Fc gamma RIIA and the human gamma chain contain a tyrosine seven amino acids upstream of the ITAM motif. We observed that the upstream tyrosine plays a role in Fc gamma RIIA phagocytic signaling but is not involved in phagocytic signaling by the human gamma chain. Our data also indicate that the two ITAM tyrosines of the human gamma chain and Fc gamma RIIA do not contribute equally to Fc gamma receptor association with Syk kinase and phagocytic signaling. The data indicate that the carboxy-terminal tyrosine of the receptor cytoplasmic domain is especially important both for the interaction with Syk kinase and for phagocytosis. Elucidating such differences in gamma chain and Fc gamma RIIA signaling may be valuable in designing strategies for therapeutic intervention in hematopoietic and immunological disorders.     

Analysis of FcγRIIA Cytoplasmic Tail Requirements in Signaling for Serotonin Secretion: Evidence for an ITAM‐Dependent,PI3K‐Dependent Pathway

The human Fc receptor, FcγRIIA, is known to mediate phagocytosis and endocytosis, yet the greatest numbers of these receptors are expressed on the surface of non‐phagocytic platelets, where they are involved in serotonin secretion. FcγRIIA harbours three tyrosine (Y) residues within its cytoplasmic domain. Y1 is upstream of both Y2 and Y3, which are contained within an immunoreceptor tyrosine‐based activation motif (ITAM), required for many signaling events. We have demonstrated that the two ITAM tyrosines are required for phagocytic signaling and that mutation of a single ITAM tyrosine decreases but does not abolish phagocytic signaling. Furthermore, we have identified that the YMTL motif is required for endocytosis. These observations suggest that FcγRIIA utilizes different sequences for various signaling events. Therefore, we investigated the sequence requirements for another important FcγRIIA‐mediated signaling event, serotonin secretion, using Rat Basophilic Leukemia (RBL‐2H3) cells transfected with wildtype (WT) FcγRIIA or mutant FcγRIIA. Stimulation of cells expressing WT FcγRIIA induced release of serotonin at a level 7‐fold greater than that in nonstimulated WT FcγRIIA‐transfected cells or nontransfected RBL cells. Mutation of either ITAM tyrosine (Y2 or Y3) to phenylalanine was sufficient to abolish serotonin secretion. Further, while inhibition of Syk with piceatannol blocked phagocytosis as expected, it did not inhibit serotonin secretion. Additionally, inhibition of phosphoinositol‐3‐kinase (PI3K) with wortmannin only had a partial effect on serotonin signaling, despite the fact that the concentrations used completely abolished phagocytic signaling. These data suggest that the requirements for serotonin secretion differ from those for phagocytosis mediated by FcγRIIA.     

FcγRIa–γ-chain complexes trigger antibody-dependent cell-mediated cytotoxicity (ADCC) in CD5+ B cell/macrophage IIA1.6 cells

Most receptors for immunoglobulins exist as multi-subunit complexes, with unique ligand binding α-chains, combined with accessory signalling (γ-, β-, or ζ-) chains. The myeloid class I receptor for IgG (FcγRIa) has been shown to be dependent on the FcR γ-chain for surface expression in vivo. In this study we assess the capacity of FcγRIa–γ-chain complexes expressed in IIA1.6 cells to trigger phagocytosis and ADCC. An intact immunoreceptor tyrosine-based activation motif (ITAM) signalling motif proved essential for triggering of biological function via the FcγRIa receptor complex. Both the FcR γ-chain and the FcγRIIa–ITAM proved active in directing phagocytosis of Staphylococcus aureus and ADCC of erythrocytes, triggered by the FcγRIa complex. The capacity of FcγRIa to trigger phagocytic and cytolytic activity by IIA1.6 cells, both considered ‘professional phagocyte’ functions, motivated us to re-evaluate the cell lineage and developmental stage of IIA1.6 cells. Although originally described as mouse B lymphocytes, the IIA1.6 cells proved positive for non-specific esterase activity and expressed the CD5 antigen. These combined characteristics place the IIA1.6 cells within a unique CD5⁺ B cell/macrophage lineage, optimally suited for cell biological analyses of phagocyte receptors.     

Chimeric Fc receptors identify immunoglobulin-binding regions in human FcγRII and FcϵRI

FcγRII and Fc?RI are functionally distinct cell surface receptors for immunoglobulin (Ig); FcγRII binds IgG with low affinity, whereas Fc?RI binds IgE with high affinity, yet they are homologous in structure and sequence having extracellular regions containing two Ig-like domains with 38% amino acid identity. Chimeric receptors derived from human FcγRII and FcγRI were produced by exchanging homologous regions of the two receptors to define binding region(s) for IgG in FcγRII and IgE in Fc?RI. Firstly, a chimeric form of the Fc?RI α chain was produced by replacing the transmembrane region and cytoplasmic tail with that of FcγRII. This mutant α chain could be expressed on the cell surface independently of associated β and γ subunits, and retained high-affinity IgE binding, indicating that the extracellular region of the FcγRI α chain is sufficient for high-affinity IgE binding. Secondly, to identify the role of the individual domains in Fc binding of both FcγRII and FcγRI, chimeric receptors were generated by exchanging the first extracellular domains between FcγRII and the α chain mutant and used to demonstrate that the second extracellular domain of both receptors contains region(s) directly involved in Ig binding. Additional chimeric receptors were constructed to localize the Ig interactive regions in domain two of FcγRII and FcγRI; these identified a single region of IgG binding in FcγRII located between residues Ser¹³⁶ to Val¹⁶⁹, and at least three independent IgE binding regions in the FcγRI α chain, between residues Trp⁸⁷ to Lys¹²⁸, Tyr¹²⁹ to Asp¹⁴⁵, and Ser¹⁴⁶ to Val¹⁶⁹.     

Lysosomal routing of Fc gamma RI from early endosomes requires recruitment of tyrosine kinases.

The high-affinity receptor for immunoglobulin G (Fc gamma RI) plays a central role in the clearance of immune complexes by mediating their internalization and delivery to lysosomes. In monocytic U937 cells, receptor internalization is independent of tyrosine kinase activity. However, the tyrosine kinase inhibitor, genistein, prevents further progress of the receptor to lysosomes and traps it in a sub-plasma membrane early endosome. Similarly, Fc gamma RI expressed in COS cells is able to internalize immune complexes but is unable to translocate to lysosomes. This suggests that Fc gamma RI, whose cytoplasmic tail is devoid of known signalling motifs, must recruit tyrosine kinases via its gamma-chain to achieve lysosomal delivery. We show that a chimera of the extracellular domain of Fc gamma RI and the cytoplasmic tail of the gamma-chain is both internalized and efficiently trafficked to lysosomes. Our study suggests that a key function of the gamma-chain is recruitment of tyrosine kinases to initiate the intracellular signalling pathways required to target Fc gamma RI following immune complex aggregation to lysosomes and not to initiate endocytosis per se.     

Enhanced phagocytosis in neonatal monocyte-derived macrophages is associated with impaired SHP-1 signaling

Resident macrophages represent a first line of defense through the ingestion of microbial pathogens. Phagocytosis mediated by immunoglobulin-binding Fc receptors is a complex series of events involving tyrosine phosphorylation and dephosphorylation. In the present study we determined that the phagocytic capacity of neonatal monocyte-derived macrophage (MDM) was enhanced in comparison to adult MDM. Cross-linking of surface FcγRIIa receptors enhanced tyrosine phosphorylation of several proteins in both groups, followed by a reduction in tyrosine phosphorylation in adult but not neonatal MDM. Expression of the tyrosine phosphatase SHP-1 was similar in neonatal and adult MDM; however, baseline SHP-1 activity levels were diminished in neonatal MDM. Cross-linking of FcγRIIa receptors induced a greater increase in SHP-1 activity in adult MDM vs. neonatal MDM. The cytoplasmic adapter protein Cbl is a SHP-1 ligand and negatively affects phagocytosis. As determined by co-immunoprecipitation assays, SHP-1 and Cbl did not associate to the same extent in neonatal as compared to adult MDM. Our data suggest that the enhanced phagocytic capacity of neonatal MDM is associated with decreased SHP-1 activity and alteration of downstream signaling pathways.     

Syk-dependent signaling pathways in neutrophils and macrophages are indispensable in the pathogenesis of anti-collagen antibody-induced arthritis

Spleen tyrosine kinase (Syk) is associated with Fcγ receptors (FcγRs) and transmits activation signals through FcγRs in myeloid cells. Thus, application of drugs to inhibit Syk activity can affect the development of immune diseases mediated by autoantibodies, while unexpected systemic effects by the inhibition may be concerned because Syk has multiple physiological functions. We used tamoxifen-inducible systemic conditional Syk knockout (KO) mice to evaluate the role of Syk in the pathogenesis of autoimmune arthritis and to investigate the systemic effects of Syk deletion. In a collagen antibody-induced arthritis model, Syk KO mice were almost completely protected from disease induction and showed significantly attenuated accumulation of neutrophils and macrophages in the joints. Syk-deleted macrophages showed less IL-6 and MCP-1 production upon FcγR ligation and exhibited reduced FcγR-mediated phagocytosis in vitro. Syk-deleted macrophages produce more RANTES upon FcγR ligation, indicating a Syk-independent signaling through the FcγR. We further found that both wild-type and Syk-deleted macrophages induced neutrophil chemotaxis upon FcγR ligation in vitro, and air-pouch model demonstrated that Syk-deleted neutrophils have a potential to infiltrate into local tissues in response to collagen and anti-collagen antibodies. However, Syk-deleted neutrophils exhibited greatly decreased neutrophil extracellular traps formation and FcγR-mediated phagocytosis. Our results indicated that Syk deficiency rendered mice completely unresponsive to immune activation by anti-collagen antibodies with disabling one pathway of FcγR-mediated signaling that was crucial for arthritis induction.     

Anti-inflammatory role of the IgA Fc receptor (CD89): From autoimmunity to therapeutic perspectives

The human immunoglobulin A (IgA) plays a key role in immune protection. IgA is the second most prevalent antibody in the serum. IgA-deficient patients frequently develop autoimmune or inflammatory diseases. The IgA function is mainly mediated through its interaction with the myeloid IgA Fc receptor FcαRI (CD89). FcαRI, an immunoreceptor tyrosine-based activation motif (ITAM)-bearing receptor, has a dual function in immunity. Monovalent targeting of FcαRI, by anti-FcαRI Fab or monomeric IgA, triggers potent inhibitory ITAM (ITAMi) signaling through the associated FcRγ chain. This results in a low-intensity signaling cascade promoting recruitment of the tyrosine phosphatase SHP-1, which induces cell inhibition of multiple types of activation signals. In contrast, cross-linking of FcαRI by multimeric ligand induces a high-intensity signaling pathway that leads to the recruitment of the tyrosine kinase Syk and to cell activation. Thus, FcαRI acts as a regulator, which mediates both anti- and pro-inflammatory functions of IgA depending on the type of interaction. This balance is of great importance to prevent tissue damage in immunopathology and to ensure the return of activated cells to a resting state. The role of the IgA–FcαRI interaction in the activation of different signaling pathways and its multifaceted role in immunity are discussed.     

Stimulation of the intracellular killing of Staphylococcus aureus by human monocytes mediated by Fcγ receptors I and II

Previous studies have shown that intracellular killing of bacteria by monocytes is stimulated by interaction between IgG and Fey receptors (FcγR) in the membrane of these cells. In the present study anti-FcγR monoclonal antibodies (mAb) were used to investigate the relative contributions of the various classes of FcγR to the intracellular killing of Staphylococcus aureus by human monocytes and the biochemical pathways involved. Anti-FcγRI or anti-FcγRII mAb, but not anti-FcγRIII mAb, efficiently stimulated the intracellular killing of bacteria by monocytes. Cross-linking FcγRI or FcγRII, but not FcγRIII, on monocytes with mouse anti-FcγR mAb followed by bridging with F(ab′)₂ fragments of goat anti-mouse IgG enhanced this process. Since the NADPH oxidase inhibitor diphenyleneiodonium blocked the FcγR-mediated intracellular killing of S. aureus, oxygen-dependent bactericidal mechanisms are most probably involved. Cross-linking FcγRI or FcγRII but not binding of the mAb to the FcγR on monocytes activated phospholipase C, as demonstrated by the increase in the intracellular concentration of inositol- (1,4,5)-triphosphate. The enhanced intracellular killing stimulated by cross-linking FcγR on monocytes was completely blocked by U-73122, an inhibitor of phospholipase C-dependent processes. Protein kinase C activity, but not the rise in the cytosolic free Ca⁺⁺ concentration or pertussis toxin-sensitive G proteins, is essential for the FcγR-mediated intracellular killing of bacteria by monocytes. Together, these results demonstrate that cross-linking FcγRI or FcγRII is equally effective in stimulating the intracellular killing of bacteria by monocytes and that this stimulation is a phospholipase C-dependent process.     

Human Fcγ receptors compete for TGN1412 binding that determines the antibody's effector function

The first‐in‐human clinical trial of the CD28‐specific monoclonal antibody (mAb) TGN1412 resulted in a life‐threatening cytokine release syndrome. Although TGN1412 was designed as IgG4, known for weak Fc:Fcγ receptor (FcγR) interactions, these interactions contributed to TGN1412‐induced T‐cell activation. Using cell lines (TFs) expressing human FcγRI, ‐IIa, ‐IIb, or ‐III, we show that TGN1412 and TGN1412 as IgG1 and IgG2 are bound by FcγRs as it can be deduced from literature. However, upon coculture of TGN1412‐decorated T cells with TFs or human primary blood cells, we observed that binding capacities by FcγRs do not correlate with the strength of the mediated effector function. FcγRIIa and FcγRIIb, showing no or very minor binding to TGN1412, mediated strongest T cell proliferation, while high‐affinity FcγRI, exhibiting strong TGN1412 binding, mediated hardly any T‐cell proliferation. These findings are of biological relevance because we show that FcγRI binds TGN1412, thus prevents binding to FcγRIIa or FcγRIIb, and consequently disables T‐cell proliferation. In line with this, FcγRI^?FcγRII⁺ but not FcγRI⁺FcγRII⁺ monocytes mediate TGN1412‐induced T‐cell proliferation. Collectively, by using TGN1412 as example, our results indicate that binding of monomeric IgG subclasses does not predict the FcγR‐mediated effector function, which has major implications for the design of therapeutic mAbs.     

Association of tissue factor with a γ chain homodimer of the IgE receptor type I in cultured human monocytes

Tissue factor (TF) is a high-affinity receptor for coagulation factors VII (F VII) and VIIa. The F VII/VIIa/TF complex is the major cellular initiator of the extrinsic coagulation cascade. We found that the occupancy of TF by its ligand, F VIIa, is involved with signal transduction and that TF is associated with the γ chain homodimer identified as a component of IgE receptor type I (FcϵRI). When 4-day cultured human monocytes were incubated with F VIIa, several polypeptides, especially a 70-kDa polypeptide, were transiently phosphorylated on tyrosine, residues. These phosphorylation events were inhibited by prior binding of anti-TF monoclonal antibody (mAb) HTF-K14, but not anti-TF mAb HTF-K180 to intact cultured monocytes. HTF-K14 blocked the binding of FVII/Vila to cell surface TF, whereas HTF-K180 did not. Anti-TF immunoprecipitates prepared from 1 % digitonin lysates of cultured human monocytes incorporated phosphate in a γ chain homodimer when incubated with [γ-³²P] ATP. The identity of the TF-associated structures as γ chains was established by immunoblot analysis of anti-TF mAb immunoprecipitates with anti-γ chain mAb. In addition, anti-TF immunoblot analysis showed that TF co-precipitated with anti-γ chain mAb. Our data suggest that γ chains may play an important role in signaling via TF in human monocytes/macrophages.     

Modulation of Fcγ receptor-mediated early events by the tyrosine phosphatase CD45 in primary human monocytes. Consequences for interleukin-6 production

Stimulation of primary human monocytes from several donors by cross-linking of Fcγ receptor type I (FcγRI) and FcγRII gave rise to calcium mobilization and protein tyrosine phosphorylation. These early events were not observed without cross-linking. CD45, a transmembrane tyrosine phosphatase, when co-cross-linked with either FcγRI or FcγRII, could prevent FcγRI and FcγRII-mediated calcium mobilization and protein tyrosine phosphorylation. When interleukin (IL)-6 production was measured, we noted a strong IL-6 production after activation of primary human monocytes by cross-linking of FcγRI or FcγRII. In contrast to calcium mobilization and tyrosine phosphorylation of proteins, IL-6 production was not affected by co-cross-linking of CD45 with either FcγRI or FcγRII. Interestingly, cross-linking of the CD45 itself was sufficient to induce IL-6 production. Our results show that the CD45 molecule is important in modulating early events following stimulation of primary human monocytes by cross-linking of FcγRI or FcγRII. However, triggering of CD45 alone can also induce IL-6 production, indicating that CD45 ligation itself can give signals and may have an important role in cytokine induction pathways.     

FcγRI mediates serum amyloid P inhibition of fibrocyte differentiation

Fibrotic diseases, such as cardiac and pulmonary fibrosis, have a poor prognosis with no FDA approved therapies. Monocyte-derived, fibroblast-like cells, called fibrocytes, participate in the formation of fibrotic lesions. The conserved pentraxin protein SAP inhibits fibrocyte differentiation in cell culture, and injections of SAP significantly reduce fibrosis in several animal models. SAP binds to the receptors for the Fc portion of IgG (FcγR) and has been crystallized bound to FcγRIIa (CD32a). The in vivo activity of SAP appears to be dependent on the FcRγ. We find that mutagenesis of the residues critical for SAP binding to FcγRIIa only moderately decreases the ability of SAP to inhibit fibrocyte differentiation. In murine cells, deletion of FcRγ or FcγRI (CD64) significantly reduced sensitivity to SAP. Deletion of the combination of FcγRIIb, FcγRIIIa, and FcγRIV did not significantly affect sensitivity to SAP, whereas deletion of just the inhibitory receptor FcγRIIb (CD32b) increased sensitivity to SAP. In human cells, siRNA-mediated reduction of FcRγ or FcγRI levels significantly decreased sensitivity to SAP, whereas reduction of FcγRIIb levels increased sensitivity to SAP. These observations suggest that SAP, at least in part, uses FcγRI and FcRγ to inhibit fibrocyte differentiation.     

Protein tyrosine kinase Syk is involved in Thy-1 signaling in rat basophilic leukemia cells

Thy-1, a glycosyl-phosphatidylinositol-anchored surface glycoprotein, has been shown to possess transmembrane signaling capacity. In rat mast cells and rat basophilic leukemia cells (RBL) aggregation of surface Thy-1 with antibodies triggers a series of intracellular events, resembling those induced by aggregation of the high-affinity receptor for IgE (FcεRI), including tyrosine phosphorylation of multiple proteins and release of secretory components. Unlike the FcεRI-mediated activation, where both the membrane-associated protein tyrosine kinase (PTK) Lyn and the cytoplasmic PTK Syk are responsible for initiating the signaling cascade, only Lyn has been implicated in Thy-1-mediated activation in RBL cells. Here we report that Syk is also rapidly tyrosine phosphorylated upon Thy-1 cross-linking. Increased Syk tyrosine phosphorylation is observed only in cells in which extensive aggregation of Thy-1 is induced by two layers of cross-linking reagents. RBL-derived mutant cells deficient in the expression of surface Thy-1 and transfectants re-expressing surface Thy-1 were used to exclude the possibility that Syk activation reflects an interaction of the cross-linking reagents with surface molecules other than Thy-1. As FcεRI γ subunits are well known to promote activation of Syk and its recruitment to membrane complexes, we also investigated the role of these subunits in Thy-1-mediated Syk activation, using RBL-derived mutant cells deficient in the expression of FcεRI γ subunits and their revertants. Consistent with the lack of FcεRI expression, no IgE-induced response could be elicited, while Thy-1-inducible Syk phosphorylation was preserved. Our results suggest that Syk might be one of the kinases responsible for signal propagation upon Thy-1 cross-linking in a FcεRI-independent pathway.     

Association of the p56lck protein tyrosine kinase with the FCγRIIIA/CD16 complex in human natural killer cells

The multimeric FcγRIIIA (CD16) complex is expressed on the surface of natural killer (NK) cells and is composed of a 50–70-kDa transmembrane glycoprotein Fcγ receptor (CD16), the T cell receptor (TCR)-ζ chain, and the FcεRIγ chain. Cross-linking FcγRIIIA initiates the rapid tyrosine phosphorylation of multiple substrates including the ζ, subunit and causes subsequent cell activation and antibody-dependent cellular cytotoxicity (ADCC). The subunits of the FcγRIIIA complex lack intrinsic protein tyrosine kinase (PTK) activity, suggesting that receptor-induced tyrosine phosphorylation events are mediated by a nonreceptor PTK. We report here that the human FcγRIIIA is complexed with p56^lck, a src-family PTK previously found associated with the CD4 and CD8 receptors on T cells. Upon engagement of the CD16 receptor, p56^lck is rapidly (within 30 s) and transiently phosphorylated on tyrosine residues. Several FcγRIIIA-associated proteins are identified in immune complex kinase assays including the TCR-ζ, subunit, a p70–90 ζ-associated protein (ZAP), p50a (acidic) and p50b (basic), and p56^lck. We demonstrate that the src-family protein tyrosine kinase inhibitor, herbimycin A, blocks increased intracellular calcium levels and ADCC caused by CD16 cross-linking on NK3.3 cells. Likewise cross-linking CD16 with the protein tyrosine phosphatase CD45, abrogates CD16-induced calcium mobilization. These data suggest that p56^lck is physically associated with FcγRIIIA(CD16) and functions to mediate signaling events related to the control of NK cellular cytotoxicity.     

Protein tyrosine kinase activity is essential for Fc gamma receptor-mediated intracellular killing of Staphylococcus aureus by human monocytes.

Our previous study revealed that the intracellular killing of Staphylococcus aureus by human monocytes after cross-linking Fc gamma receptor I (Fc gamma RI) or Fc gamma RII is a phospholipase C (PLC)-dependent process. The aim of the present study was to investigate whether protein tyrosine kinase (PTK) activity plays a role in the Fc gamma R-mediated intracellular killing of bacteria and activation of PLC in these cells. The results showed that phagocytosis of bacteria by monocytes was not affected by the PTK inhibitors genistein and tyrphostin-47. The intracellular killing of S. aureus by monocytes after cross-linking Fc gamma RII or Fc gamma RII with anti-Fc gamma R monoclonal antibody and a bridging antibody or with human immunoglobulin G (IgG) was inhibited by these compounds in a dose-dependent fashion. The production of O2- by monocytes after stimulation with IgG or IgG-opsonized S. aureus was almost completely blocked by the PTK inhibitor. These results indicate that inhibition of PTK impairs the oxygen-dependent bactericidal mechanisms of monocytes. Genistein and tyrphostin-47, which do not affect the enzymatic activity of purified PLC, prevented activation of PLC after cross-linking Fc gamma RI or Fc gamma RII, measured as an increase in the intracellular inositol 1,4,5-trisphosphate concentration. Cross-linking Fc gamma RI or Fc gamma RII induced rapid tyrosine phosphorylation of several proteins in monocytes, one of which was identified as PLC-gamma 1, and the phosphorylation could be completely blocked by PTK inhibitors, leading to the conclusion that activation of PLC after cross-linking Fc gamma R in monocytes is regulated by PTK activity. Together, these results demonstrate that PTK activity is essential for the activation of PLC which is involved in the Fc gamma R-mediated intracellular killing of S. aureus by human monocytes.     

c‐Jun activating binding protein 1 binds to the IgA receptor and modulates protein levels of FcαRI and FcRγ‐chain

The receptor for IgA, FcαRI or CD89, is expressed on myeloid cells and can trigger phagocytosis, tumor cell lysis, and release of inflammatory mediators. These functions critically depend on the associated FcR γ‐chain; however, some biological functions, like receptor internalization, are solely mediated by FcαRI α‐chain. Little is known as to how FcαRI regulates these processes and the FcαRI intracellular domain does not contain recognized signalling motifs. We searched for associating proteins and identified c‐Jun activating binding protein 1 (JAB1) as a binding partner specifically for FcαRI. We found increased FcαRI surface expression after ectopic expression of JAB1 as well as diminished protein levels of total FcR γ‐chain levels after JAB1 knock‐down. These data functionally link JAB1 with controlling protein expression levels of FcαRI‐FcR γ‐chain protein complex.     

Functional characterization of receptor-type protein tyrosine phosphatase CD148 (HPTPη/DEP-1) in Fcγ receptor IIa signal transduction of human neutrophils

Activation of the 40-kDa low-affinity receptor for IgG (FcγRIIa, CD32) leads to tyrosine phosphorylation, increase of cytosolic free calcium concentration ([Ca²⁺]_i), and production of superoxide anions (O₂^?) in neutrophils (PMN). It has been established that protein tyrosine kinases (PTK) and phosphatases (PTP) are essential for the regulation of intracellular signaling. CD45 is a type I receptor-type protein tyrosine phosphatase (RPTP) with two PTP domains. Recently it has been demonstrated that co-cross-linking of CD45 modulates the signal transduction pathway of FcγRIIa in PMN. In contrast, the functional characteristics of CD148 (HPTPη/DEP-1), a new RPTP with only one PTP domain, is unknown. CD148 is expressed on PMN in slightly lower density than CD45, and in higher density than on lymphocytes. [Ca²⁺]_i measured with fluo-3-loaded PMN by flow cytometry and O₂^? production determined by lucigenin-dependent chemiluminescence were inhibited by co-cross-linking of CD45 with FcγRIIa in comparison to isotype control monoclonal antibody (mAb). In contrast, preincubation with CD148 mAb 143-41 abolished O₂^? generation, but did not inhibit [Ca²⁺]_i rise. In summary, both clustered human RPTP, CD45 and CD148, inhibit FcγRIIa-induced O₂^? production in PMN, but they differ in regulation of [Ca²⁺]_i. Therefore, it is suggested that co-cross-linking of FcγRII with CD45 and CD148 leads to dephosphorylation of different substrates. These distinct functional capacities may be important for differential regulation of FcγR signaling by currently unknown ligands.     

Dectin-1 and Dectin-2 in innate immunity against fungi

Dectin-1 and Dectin-2 are type II transmembrane proteins of the C-type lectin family with single carbohydrate recognition domains (CRDs) in their extracellular region. They are expressed mainly in dendritic cells and macrophages. Dectin-1 recognizes β-glucans with its CRD and transduces signals through its immunoreceptor tyrosine-based activation motif (ITAM)-like motif in the cytoplasmic domain, whereas Dectin-2 recognizes α-mannans and transduces its signal through association with the ITAM-containing Fc receptor γ chain. Upon ligand binding, spleen tyrosine kinase is recruited to the ITAM and activates the caspase recruitment domain family member 9 (CARD9)-nuclear factor-κB axis, resulting in the activation of various genes including those encoding pro-inflammatory cytokines. Both β-glucans and α-mannans are major cell wall components of fungi including Candida albicans and Pneumocystis carinii. Recently, it was reported that Dectin-1 is important in protection against P. carinii by inducing reactive oxygen species, whereas both Dectin-1 and Dectin-2 play important roles in defense against C. albicans by preferentially inducing T(h)17 cell differentiation. In this review, we briefly revisit the structures, ligands, signal transduction and functional roles of Dectin-1 and Dectin-2 in host defense against fungal infection.