Signaling through mutants of the IgA receptor CD89 and consequences for Fc receptor gamma-chain interaction

The prototypic receptor for IgA (FcalphaRI, CD89) is expressed on myeloid cells and can trigger phagocytosis, tumor cell lysis, and release of inflammatory mediators. The functions of FcalphaRI and activating receptors for IgG (FcgammaRI and FcgammaRIII) are dependent on the FcR gamma-chain dimer. This study increases our understanding of the molecular basis of the FcalphaRI-FcR gamma-chain transmembrane interaction, which is distinct from that of other activatory FcRs. FcalphaRI is unique in its interaction with the common FcR gamma-chain, because it is based on a positively charged residue at position 209, which associates with a negatively charged amino acid of FcR gamma-chain. We explored the importance of the position of this positive charge within human FcalphaRI for FcR gamma-chain association and FcalphaRI functioning with the use of site-directed mutagenesis. In an FcalphaRI R209L/A213H mutant, which represents a vertical relocation of the positive charge, proximal and distal FcR gamma-chain-dependent functions, such as calcium flux, MAPK phosphorylation, and IL-2 release, were similar to those of wild-type FcalphaRI. A lateral transfer of the positive charge, however, completely abrogated FcR gamma-chain-dependent functions in an FcalphaRI R209L/M210R mutant. By coimmunoprecipitation, we have demonstrated the loss of a physical interaction between FcR gamma-chain and FcalphaRI M210R mutant, thus explaining the loss of FcR gamma-chain-dependent functions. In conclusion, not only the presence of a basic residue in the transmembrane region of FcalphaRI, but also the orientation of FcalphaRI toward the FcR gamma-chain dimer is essential for FcR gamma-chain association. This suggests the involvement of additional amino acids in the FcalphaRI-FcR gamma-chain interaction.     

Immature neutrophils mediate tumor cell killing via IgA but not IgG Fc receptors

Antitumor Abs are promising therapeutics for cancer. Currently, most Ab-based therapies focus on IgG Ab, which interact with IgG FcR (FcgammaR) on effector cells. In this study, we examined human and mouse neutrophil-mediated tumor cell lysis via targeting the IgA FcR, FcalphaRI (CD89), in more detail. FcalphaRI was the most effective FcR in triggering tumor cell killing, and initiated enhanced migration of neutrophils into tumor colonies. Importantly, immature neutrophils that are mobilized from the bone marrow upon G-CSF treatment efficiently triggered tumor cell lysis via FcalphaRI, but proved incapable of initiating tumor cell killing via FcgammaR. This may provide a rationale for the disappointing results observed in some earlier clinical trials in which patients were treated with G-CSF and antitumor Ab-targeting FcgammaR.     

Differential effect of cytokine treatment on Fc alpha receptor I- and Fc gamma receptor I-mediated tumor cytotoxicity by monocyte-derived macrophages

Macrophages represent an important effector cell for Ab-mediated tumor therapy. Previous studies have documented that cytokines can influence Fc receptor (FcR) expression and function. In this study we examined the tumoricidal activities of the type I receptors for IgG (Fc gamma RI) and the IgA FcR (Fc alpha RI) on monocyte-derived macrophages (MDM) cultured in the presence of IFN-gamma, M-CSF, or GM-CSF. Bispecific Abs were used to target a Her2/neu breast carcinoma cell line, SKBR-3, to Fc alpha RI or Fc gamma RI on MDM. Although Fc alpha RI and Fc gamma RI share a common signaling pathway contingent on association with the gamma-chain (FcR gamma subunit), a marked difference in their efficiency in mediating tumoricidal functions was seen in response to specific cytokines. M-CSF- and GM-CSF-treated MDM mediated efficient phagocytosis of SKBR-3 cells by Fc alpha RI and Fc gamma RI; however, IFN-gamma-treated MDM phagocytosed tumor cells only with the Fc gamma RI-directed bispecific Abs. Similarly, IFN-gamma-cultured MDM lysed tumor cells more efficiently via Fc gamma RI then by Fc alpha RI as measured in Ab-dependent cellular cytotoxicity assays. Conversely, GM-CSF-treated MDM mediated more efficient lysis of tumor cells via Fc alpha RI than Fc gamma RI, while M-CSF-cultured MDM were relatively less efficient in mediating Ab-dependent cellular cytotoxicity through either receptor. With the exception of IFN-gamma-mediated enhancement of Fc gamma RI expression and Fc gamma RI gamma-chain complexes, the regulation of Fc gamma RI- or Fc alpha RI-mediated activity occurred without significant change in either receptor expression or total complexes with gamma subunit. These data suggest that the efficiency of Ab-mediated tumor therapy, which depends on FcR effector cell functions, may be modified by the use of specific cytokines.     

Crosslinking of the human Fc receptor for IgA (FcalphaRI/CD89) triggers FcR gamma-chain-dependent shedding of soluble CD89

CD89/FcalphaRI is a 55- to 75-kDa type I receptor glycoprotein, expressed on myeloid cells, with important immune effector functions. At present, no information is available on the existence of soluble forms of this receptor. We developed an ELISA for the detection of soluble CD89 (sCD89) forms and investigated the regulation of sCD89 production. PMA/ionomycin stimulation of monocytic cell lines (U937, THP-1, and MM6), but not of neutrophils, resulted in release of sCD89. Crosslinking of CD89 either via its ligand IgA or with anti-CD89 mAbs similarly resulted in sCD89 release. Using CD89-transfected cells, we showed ligand-induced shedding to be dependent on coexpression of the FcR gamma-chain subunit. Shedding of sCD89 was dependent on signaling via the gamma-chain and prevented by addition of inhibitors of protein kinase C (staurosporine) or protein tyrosine kinases (genistein). Western blotting revealed sCD89 to have an apparent molecular mass of 30 kDa and to bind IgA in a dose-dependent fashion. In conclusion, the present data document a ligand-binding soluble form of CD89 that is released upon activation of CD89-expressing cells. Shedding of CD89 may play a role in fine-tuning CD89 immune effector functions.     

FcalphaRI (CD89) alleles determine the proinflammatory potential of serum IgA

The human IgA FcR (FcalphaRI; CD89) mediates a variety of immune system functions including degranulation, endocytosis, phagocytosis, cytokine synthesis, and cytokine release. We have identified a common, nonsynonymous, single nucleotide polymorphism (SNP) in the coding region of CD89 (844A-->G) (rs16986050), which changes codon 248 from AGC (Ser(248)) to GGC (Gly(248)) in the cytoplasmic domain of the receptor. The two different alleles demonstrate significantly different FcalphaRI-mediated intracellular calcium mobilization and degranulation in rat basophilic leukemia cells and cytokine production (IL-6 and TNF-alpha) in murine macrophage P388D1 cells. In the absence of FcR gamma-chain association in P388D1 cells, the Ser(248)-FcalphaRI allele does not mediate cytokine production, but the Gly(248)-FcalphaRI allele retains the capacity to mediate a robust production of proinflammatory cytokine. This allele-dependent difference is also seen with FcalphaRI-mediated IL-6 cytokine release by human neutrophils ex vivo. These findings and the enrichment of the proinflammatory Gly(248)-FcalphaRI allele in systemic lupus erythematosus populations in two ethnic groups compared with their respective non-systemic lupus erythematosus controls suggest that FcalphaRI (CD89) alpha-chain alleles may affect receptor-mediated signaling and play an important role in the modulation of immune responses in inflammatory diseases.     

Triggering Fc alpha-receptor I (CD89) recruits neutrophils as effector cells for CD20-directed antibody therapy

CD20 Abs induce clinical responses in lymphoma patients, but there are considerable differences between individual patients. In (51)Cr release assays with whole blood as effector source, RAJI cells were effectively killed by a mouse/human chimeric IgG1 construct of CD20 Ab 1F5, whereas ARH-77 proved resistant to killing by this Ab. When whole blood was fractionated into plasma, mononuclear cells, or granulocytic effector cells, RAJI cells were effectively killed in the presence of complement-containing plasma, whereas the mature B cell line ARH-77 proved complement resistant. However, with a bispecific Ab (BsAb) against the myeloid receptor for IgA (CD89; FcalphaRI) and CD20, a broad range of B cell lines were effectively killed. FcalphaRI is expressed on monocytes/macrophages, neutrophils, and eosinophils. As the numbers of these effector cells and their functional activity can be enhanced by application of G-CSF or GM-CSF, lysis via (FcalphaRI x CD20) BsAb was significantly enhanced in blood from patients during therapy with these myeloid growth factors. Interestingly, the major effector cell population for this BsAb were polymorphonuclear neutrophils, which proved ineffective in killing malignant B cells with murine, chimeric IgG1, or FcgammaRI- or FcgammaRIII-directed BsAbs against CD20. Experiments with blood from human FcalphaRI/FcgammaRI double-transgenic mice showed corresponding results, allowing the establishment of relevant syngenic animal models in these mice. In conclusion, the combination of myeloid growth factors and an (FcalphaRI x CD20) BsAb may represent a promising approach to improve effector cell recruitment for CD20-directed lymphoma therapy.     

Destructive arthritis in the absence of both FcgammaRI and FcgammaRIII

Fc receptors for IgG (FcgammaR) have been implicated in the development of arthritis. However, the precise contribution of the individual FcgammaR to joint pathology is unclear. In this study, the role of the different FcgammaR was assessed both in an active and in a passive mouse model of arthritis by analyzing disease development in double and triple knockout (KO) offspring from crosses of FcgammaRI KO, FcgammaRIII KO, FcgammaRI/III double KO, or FcR gamma-chain KO with the FcgammaRII KO on C57BL6 background, which is susceptible for collagen-induced arthritis (CIA). In the active CIA model, onset was significantly delayed in the absence of FcgammaRIII, whereas incidence and maximum severity were significantly decreased in FcgammaRI/II/III triple KO but not in FcgammaRII/III double KO and FcgammaRI/II double KO mice as compared with FcgammaRII KO animals. Remarkably, fully destructive CIA developed in FcgammaRI/II/III triple KO mice. In contrast, FcR gamma/FcgammaRII double KO mice were resistant to CIA. These findings were confirmed with the passive KRN serum-induced arthritis model. These results indicate that all activating FcgammaR play a role in the development of arthritis, mainly in the downstream effector phase. FcgammaRIII is critically required for early arthritis onset, and FcgammaRI can substantially contribute to arthritis pathology. Importantly, FcgammaRI and FcgammaRIII were together dispensable for the development of destructive arthritis but the FcR gamma-chain was not, suggesting a role for another FcR gamma-chain associated receptor, most likely FcgammaRIV. In addition, FcgammaRII plays a negative regulatory role in both the central and effector phase of arthritis.     

Tyrosine phosphorylation of the Fc gamma RIII(CD16): zeta complex in human natural killer cells. Induction by antibody-dependent cytotoxicity but not by natural killing.

NK cells are large granular lymphocytes capable of killing certain tumor cells and virally infected cells in a non-MHC-restricted manner. NK cells can also effect an antibody dependent cytotoxicity that is triggered by CD16, an FcR for IgG. In NK cells, CD16 is expressed in association with zeta, a signal transducing subunit of the TCR complex. Here we show that, just as T cell activation via the TCR complex results in tyrosine phosphorylation of zeta TCR, NK cell activation via CD16 results in tyrosine phosphorylation of zeta NK. Whereas antibody-dependent cytotoxicity also results in tyrosine phosphorylation of zeta, natural cytotoxicity does not. Our results indicate that zeta functions as a transducing element for antibody dependent, but not antibody independent killing by NK cells. Consequently, NK cells are likely to express at least two distinct receptor complexes capable of triggering cytolytic effector function.     

The role of the human Fc receptor Fc gamma RIIA in the immune clearance of platelets: a transgenic mouse model

In humans, the Fc receptor for IgG, FcgammaRIIA, is expressed on macrophages and platelets and may play an important role in the pathophysiology of immune-mediated thrombocytopenia. Mice lack the genetic equivalent of human FcgammaRIIA. To better understand the role of FcgammaRIIA in vivo, FcgammaRIIA transgenic mice were generated and characterized. One transgenic mouse line expressed FcgammaRIIA on platelets and macrophages at levels equivalent to human cells, and cross-linking FcgammaRIIA on these platelets induced platelet aggregation. Immune-mediated thrombocytopenia in this transgenic line was studied using i.v. and i.p. administration of anti-mouse platelet Ab. In comparison with matched wild-type littermates that are negative for the FcgammaRIIA transgene, Ab-mediated thrombocytopenia was significantly more severe in the FcgammaRIIA transgenic mice. In contrast, FcR gamma-chain knockout mice that lack functional expression of the Fc receptors FcgammaRI and FcgammaRIII on splenic macrophages did not demonstrate Ab-mediated thrombocytopenia. We generated FcgammaRIIA transgenic x FcR gamma-chain knockout mice to examine the role of FcgammaRIIA in immune clearance in the absence of functional FcgammaRI and FcgammaRIII. In FcgammaRIIA transgenic x FcR gamma-chain knockout mice, severe immune thrombocytopenia mediated by FcgammaRIIA was observed. These results demonstrate that FcgammaRIIA does not require the FcR gamma-chain for expression or function in vivo. Furthermore, taken together, the data suggest that the human Fc receptor FcgammaRIIA plays a significant role in the immune clearance of platelets in vivo.     

The Fc receptor gamma-chain is necessary and sufficient to initiate signalling through glycoprotein VI in transfected cells by the snake C-type lectin, convulxin.

There is extensive evidence that FcR gamma-chain couples to the collagen receptor glycoprotein VI (GPVI) and becomes phosphorylated on tyrosines upon receptor cross-linking. However, it is not established whether this receptor complex is sufficient to initiate the signalling cascade. We transfected GPVI and the FcR gamma-chain into the human erythroleukaemia cell line K562, which lacks detectable expression of GPVI and the FcR gamma-chain. The results show that GPVI is unable to signal when expressed alone, despite its surface expression, upon stimulation with the snake C-type lectin, convulxin. Coexpression of the FcR gamma-chain confers signalling properties on the receptor. Furthermore, cotransfection of the FcR gamma-chain and two mutant versions of GPVI shows that the transmembrane arginine and cytoplasmic tail of GPVI are necessary for association with the FcR gamma-chain. These results demonstrate that reconstitution of the GPVI-FcR gamma-chain complex in cells expressing the necessary signalling network is sufficient to initiate signalling events in response to convulxin and collagen-related peptide.     

Evidence that phospholipase C-gamma2 interacts with SLP-76, Syk, Lyn, LAT and the Fc receptor gamma-chain after stimulation of the collagen receptor glycoprotein VI in human platelets.

Platelet activation by collagen is mediated by the sequential tyrosine phosphorylation of the Fc receptor gamma-chain (FcR gamma-chain), which is part of the collagen receptor glycoprotein VI, the tyrosine kinase Syk and phospholipase C-gamma2 (PLC-gamma2). In this study tyrosine-phosphorylated proteins that associate with PLC-gamma2 after stimulation by a collagen-related peptide (CRP) were characterized using glutathione S-transferase fusion proteins of PLC-gamma2 Src homology (SH) domains and by immunoprecipitation of endogenous PLC-gamma2. The majority of the tyrosine-phosphorylated proteins that associate with PLC-gamma2 bind to its C-terminal SH2 domain. These were found to include PLC-gamma2, Syk, SH2-domain-containing leucocyte protein of 76 kDa (SLP-76), Lyn, linker for activation of T cells (LAT) and the FcR gamma-chain. Direct association was detected between PLC-gamma2 and SLP-76, and between PLC-gamma2 and LAT upon CRP stimulation of platelets by far-Western blotting. FcR gamma-chain and Lyn were found to co-immunoprecipitate with PLC-gamma2 as well as with unidentified 110-kDa and 75-kDa phosphoproteins. The absence of an in vivo association between Syk and PLC-gamma2 in platelets is in contrast with that for PLC-gamma1 and Syk in B cells. The in vivo function of PLC-gamma2 SH2 domains was examined through measurement of Ca2+ increases in mouse megakaryocytes that had been microinjected with recombinant proteins. This revealed that the C-terminal SH2 domain is involved in the regulation of PLC-gamma2. These data indicate that the C-terminal SH2 domain of PLC-gamma2 is important for PLC-gamma2 regulation through possible interactions with SLP-76, Syk, Lyn, LAT and the FcR gamma-chain.     

Comparison of the TCR ζ-chain with the FcR γ-chain in chimeric TCR constructs for T cell activation and apoptosis

A promising strategy for cancer treatment is adoptive gene therapy/immunotherapy by genetically modifying T cells with a chimeric T cell receptor (cTCR). When transduced T cells (T-bodies) specifically bind to tumor antigens through cTCR, they will become cytotoxic T lymphocytes (CTL) and lyse the tumor cells in a non-major histocompatibility complex (MHC)-restricted manner. Both the FcR gamma-chain and the TCR zeta-chain have been used to construct such cTCR, and both have shown specific cytolytic functions against tumor cells. However, most researchers believe that the zeta-chain generates stronger cytolytic activities against tumor than the gamma-chain and therefore would be a better candidate for cTCR construction. On the other hand, because of the lack of costimulation signaling in such constructs, the T-body might cause activation-induced T cell death (AICD) when bound to tumor antigens. Therefore, one can argue that the gamma-chain might generate less AICD than the zeta-chain because the gamma-chain has only one immunoreceptor tyrosine-based activation motif (ITAM), and the cytolytic activities can be therefore recycled. Two cTCR, GAHgamma and GAHzeta, were constructed and evaluated for cytokine production, specific cytolytic function and AICD in T-bodies after exposure to tumor cells. Using EGP-2-positive LS174T colorectal carcinoma cells as targets, there was no substantial difference observed between a gamma-chain or zeta-chain as the T-body signaling moiety in terms of specific cytolytic functions and induced cytokine production. This paper also demonstrates that, in the absence of a costimulation system, tumor antigen may not trigger apoptosis of T cells transduced with a cTCR carrying either an FcR gamma-chain or a TCR zeta-chain. These observations challenge current ideas about the role of ITAM in T cell activation.     

Crystal structure of the ectodomain of human FcalphaRI

Human FcalphaRI (CD89) is the receptor specific for IgA, an immunoglobulin that is abundant in mucosa and is also found in high concentrations in serum. Although FcalphaRI is an immunoglobulin Fc receptor (FcR), it differs in many ways from FcRs for other immunoglobulin classes. The genes of most FcRs are located on chromosome 1 at 1q21-23, whereas FcalphaRI is on chromosome 19, at 19q13.4, a region called the leukocyte receptor complex, because it is clustered with several leukocyte receptor families including killer cell inhibitory receptors (KIRs) and leukocyte Ig-like receptors (LIRs). The amino acid sequence of FcalphaRI shares only 20% homology with other FcRs but it has around 35% homology with its neighboring LIRs and KIRs. In this work, we analyzed the crystal structure of the ectodomain of FcalphaRI and examined structure similarities between FcalphaRI and KIR2DL1, KIR2DL2 and LIR-1. Our data show that FcalphaRI, KIRs, and LIRs share a common hydrophobic core in their interdomain interface, and FcalphaRI is evolutionally closer to LIR than KIR.     

Delineation of the region in the glycoprotein VI tail required for association with the Fc receptor gamma-chain

The glycoprotein VI (GPVI).Fc receptor gamma-chain (FcRgamma-chain) complex is the major activation receptor for collagen on platelets. GPVI cross-linking mediates activation through tyrosine phosphorylation of an ITAM (immunoreceptor tyrosine-based activation motif) in the FcR gamma-chain by Src family kinases. It has been previously shown that a transmembrane arginine and the cytoplasmic domain of GPVI are required for association with the FcR gamma-chain in immortalized cell lines. In this study, we have delineated the regions in the GPVI tail that promote binding to FcR gamma-chain and mediate functional responses to the snake venom convulxin by reconstitution of mutant forms of GPVI in RBL-2H3 cells. Sequential truncation of the cytoplasmic tail of GPVI revealed a major role for the basic region and a minor role for the juxtamembrane six amino acids in the association with FcR gamma-chain and functional responses to convulxin. Analysis of selective deletions in the GPVI tail supported this conclusion. In addition, we show that the proline-rich domain is required for optimal Ca2+ release, whereas it is dispensable for FcR gamma-chain association.     

Interaction of linker for activation of T cells with multiple adapter proteins in platelets activated by the glycoprotein VI-selective ligand, convulxin

The snake venom toxin convulxin activates platelets through the collagen receptor glycoprotein VI (GPVI)/Fc receptor gamma-chain (FcR gamma-chain) complex leading to tyrosine phosphorylation and activation of the tyrosine Syk and phospholipase Cgamma2 (PLCgamma2). In the present study, we demonstrate that convulxin is a considerably more powerful agonist than collagen or the GPVI-selective collagen-related peptide (CRP). Confirmation that the response to convulxin is mediated solely via Syk was provided by studies on Syk-deficient platelets. The increase in phosphorylation of the FcR gamma-chain is associated with marked increases in tyrosine phosphorylation of downstream proteins including Syk, linker for activation of T cells (LAT), SLP-76, and PLCgamma2. The transmembrane adapter LAT coprecipitates with SLP-76 and PLCgamma2, as well as with a number of other adapter proteins, some of which have not been previously described in platelets, including Cbl, Grb2, Gads, and SKAP-HOM. Gads is constitutively associated with SLP-76 and is probably the protein bridging its association with LAT. There was no detectable association between Grb2 and SLP-76 in control or stimulated cells, suggesting that the interaction of LAT with Grb2 is present in a separate complex to that of LAT-Gads-SLP-76. These results show that the trimeric convulxin stimulates a much greater phosphorylation of the FcR gamma-chain and subsequent downstream responses relative to CRP and collagen, presumably because of its ability to cause a greater degree of cross-linking of GPVI. The adapter LAT appears to play a critical role in recruiting a number of other adapter proteins to the surface membrane in response to activation of GPVI, presumably at sites of glycolipid-enriched microdomains, enabling an organized signaling cascade that leads to platelet activation.     

Signal transduction during human natural killer cell activation: inositol phosphate generation and regulation by cyclic AMP

NK cells mediate both direct cytotoxicity against a variety of tumor cells and indirect (FcR-dependent) cytotoxicity against antibody-coated targets. When cloned human NK cells (CD16+/CD3-) were exposed to NK-sensitive targets for 30 min, the level of inositol phosphates rose two to five times above background. The rise in inositol phosphates induced by NK-sensitive targets was paralleled by an increase in intracellular free calcium concentration ([Ca2+]i). A panel of tumor cells that were resistant to NK cell lysis did not stimulate significant levels of inositol phosphate production and did not induce an elevation of intracellular free calcium. Ligation of the FcR (CD16) with the mAb 3G8 also triggered phosphoinositide turnover. Kinetic experiments demonstrated that stimulation by either susceptible target cells or by FcR ligation led to rapid (less than 1 min) generation of the Ca2+-mobilizing second messenger, inositol trisphosphate, with slower accumulation of inositol bisphosphate and inositol monophosphate. Previous studies have demonstrated that activation of the cAMP-dependent second messenger pathway strongly inhibits NK cell-mediated cytotoxic functions. Treatment of NK effector cells with forskolin to elevate intracellular cAMP levels resulted in a concentration-dependent inhibition of phosphoinositide hydrolysis induced by both NK-sensitive targets and 3G8-mediated FcR ligation. These results suggest that phosphoinositide turnover represents a critical early event in the human NK cell cytolytic process. Moreover, the potent inhibitory effect of cAMP on NK cell cytotoxicity may be explained by the uncoupling of NK receptors from phospholipase C-mediated phosphoinositide hydrolysis.     

Receptor modulation by Fc gamma RI-specific fusion proteins is dependent on receptor number and modified by IgG.

The high-affinity IgG receptor, FcgammaRI (CD64), is constitutively expressed exclusively on professional APCs. Human FcgammaRI binds monomeric IgG with high affinity and is, therefore, saturated in vivo. The binding of IgG to FcgammaRI causes receptor recycling, while Abs that cross-link FcgammaRI cause rapid down-modulation of surface FcgammaRI. Because studies performed in the absence of ligand may not be representative of FcgammaRI modulation in vivo, we investigated the ability of FcgammaRI-cross-linking Abs and non-cross-linking derivatives to modulate FcgammaRI in the presence and absence of ligand. In the absence of ligand mAb H22 and wH22xeGFP, an enhanced green fluorescent protein (eGFP)-labeled fusion protein of H22, cross-linked and rapidly down-modulated surface FcgammaRI on the human myeloid cell line, U937, and its high FcgammaRI-expressing subclone, 10.6. This effect was dependent on the concentration of fusion protein and the level of FcgammaRI expression and correlated with internalization of both wH22xeGFP and FcgammaRI, itself, as assessed by confocal microscopy. A single-chain Fv version, sFv22xeGFP, which does not cross-link FcgammaRI, was unable to modulate FcgammaRI in the absence of IgG. However, if ligand was present, treatment with either monovalent or cross-linking fusion protein led to intracellular receptor accumulation. These findings suggest at least two alternate mechanisms of internalization that are influenced by ligand and demonstrate the physiologic potential of FcgammaRI to transport a large antigenic load into APCs for processing. These studies may lead to the development of better FcgammaRI-targeted vaccines, as well as therapies to down-modulate FcR involved in autoimmune diseases.     

Critical role of the Fc receptor gamma-chain on APCs in the development of allergen-induced airway hyperresponsiveness and inflammation

The FcR common gamma-chain (FcRgamma) is an essential component of the receptors FcepsilonRI, FcgammaRI, and FcgammaRIII, which are expressed on many inflammatory cell types. The role of these receptors in the initiation or maintenance of allergic inflammation has not been well defined. FcRgamma-deficient (FcRgamma(-/-)) and control (wild-type (WT)) mice were sensitized and subsequently challenged with OVA. Following sensitization and challenge to OVA, FcRgamma-deficient (FcRgamma(-/-)) mice developed comparable levels of IgE and IgG1 as WT mice. However, numbers of eosinophils, levels of IL-5, IL-13, and eotaxin in bronchoalveolar lavage fluid, and mononuclear cell (MNC) proliferative responses to OVA were significantly reduced, as was airway hyperresponsiveness (AHR) to inhaled methacholine. Reconstitution of FcRgamma(-/-) mice with whole spleen MNC from WT mice before sensitization restored development of AHR and the numbers of eosinophils in bronchoalveolar lavage fluid; reconstitution after sensitization but before OVA challenge only partially restored these responses. These responses were also restored when FcRgamma(-/-) mice received T cell-depleted MNC, T and B cell-depleted MNC, or bone marrow-derived dendritic cells before sensitization from FcR(+/+) or FcgammaRIII-deficient but not FcRgamma(-/-) mice. The expression levels of FcgammaRIV on bone marrow-derived dendritic cells from FcR(+/+) mice were found to be low. These results demonstrate that expression of FcRgamma, most likely FcgammaRI, on APCs is important during the sensitization phase for the development of allergic airway inflammation and AHR.     

Fc receptor-mediated immunity against Bordetella pertussis.

The relevance of specific Abs for the induction of cellular effector functions against Bordetella pertussis was studied. IgG-opsonized B. pertussis was efficiently phagocytosed by human polymorphonuclear leukocytes (PMN). This process was mediated by the PMN IgG receptors, FcgammaRIIa (CD32) and FcgammaRIIIb (CD16), working synergistically. Furthermore, these FcgammaR triggered efficient PMN respiratory burst activity and mediated transfer of B. pertussis to lysosomal compartments, ultimately resulting in reduced bacterial viability. Bacteria opsonized with IgA triggered similar PMN activation via FcalphaR (CD89). Simultaneous engagement of FcalphaRI and FcgammaR by B. pertussis resulted in increased phagocytosis rates, compared with responses induced by either isotype alone. These data provide new insights into host immune mechanisms against B. pertussis and document a crucial role for Ig-FcR interactions in immunity to this human pathogen.