Importance of Fc Receptor γ-Chain ITAM Tyrosines in Neutrophil Activation and in vivo Autoimmune Arthritis.

Activating Fcγ receptors associated with Fc receptor γ-chain (FcRγ) are critical for mediating neutrophil effector functions in immune complex-mediated autoimmune diseases. FcRγ contains ITAM tyrosines and the in vivo role of these tyrosines has not been defined in neutrophils and arthritis. In this study, the in vivo functions of FcRγ ITAM tyrosines were characterized using wild type and ITAM tyrosine mutant (Y65F/Y76F) transgenic mice crossed to an FcRγ-deficient genetic background. FcRγ-deficient neutrophils showed undetectable cell surface expression of the activating Fcγ receptor IV, defective immune complex-induced superoxide production, degranulation and spreading. Although the re-expression of both the wild type and the ITAM tyrosine mutant (Y65F/Y76F) FcRγ could restore activating Fcγ receptor expression of FcRγ-deficient neutrophils, only the wild type transgenic form could mediate Fcγ receptor-dependent effector functions. In contrast, neutrophils carrying ITAM tyrosine mutant FcRγ were unable to produce superoxide, mediate degranulation and perform active spreading. In addition, our results confirmed the protection of FcRγ-deficient mice from autoimmune arthritis. Importantly, the presence of the wild type FcRγ transgene, in contrast to the ITAM tyrosine mutant transgene, partially reversed autoimmune arthritis development. The reversing effect of the wild type transgene was even more robust when animals carried the wild type transgene in a homozygous form. Collectively, FcRγ ITAM tyrosines play a critical role in the induction of neutrophil effector responses, the initiation and progression of an autoantibody-induced experimental arthritis in vivo, indicating a signaling, rather than just a receptor stabilizing function of the molecule.

Sphingosine 1-phosphate enhances Fc gamma receptor-mediated neutrophil activation and recruitment under flow conditions.

Sphingosine 1-phosphate (S1P) is a bioactive phospholipid that is released by platelets and endothelial cells and has been implicated in diverse biological functions. We hypothesized that S1P may influence immune complex-mediated polymorphonuclear neutrophil activation. Using flow cytometry and fluorescence spectrometry, we found that exogenous addition of S1P led to an enhanced polymorphonuclear neutrophil Fcgamma receptor-mediated rise in intracellular Ca(2+) and reactive oxygen species generation in a pertussis toxin-independent manner, while having only a small effect by itself. Thus, S1P amplifies a positive feedback loop where Fcgamma receptor-mediated rises in Ca(2+) and reactive oxygen species are interdependent, with reactive oxygen species acting to increase tyrosine phosphorylation and activity of upstream signaling intermediates. S1P augmentation of Fcgamma receptor signaling translates to downstream functional consequences, including shape change and recruitment to endothelial surfaces coated with suboptimal levels of immune complexes. Taken together, S1P from activated platelets or endothelial cells may serve to amplify leukocyte recruitment and tissue injury at sites of immune complex deposition in vasculitis.

A promoter haplotype of the immunoreceptor tyrosine-based inhibitory motif-bearing FcgammaRIIb alters receptor expression and associates with autoimmunity. II. Differential binding of GATA4 and Yin-Yang1 transcription factors and correlated receptor expression and function.

The immunoreceptor tyrosine-based inhibitory motif-containing FcgammaRIIb modulates immune function on multiple cell types including B cells, monocytes/macrophages, and dendritic cells. The promoter for the human FCGR2B is polymorphic, and the less frequent 2B.4 promoter haplotype is associated with the autoimmune phenotype of systemic lupus erythematosus. In the present study, we demonstrate that the 2B.4 promoter haplotype of FCGR2B has increased binding capacity for GATA4 and Yin-Yang1 (YY1) transcription factors in both B lymphocytes and monocytes, and that overexpression of GATA4 or YY1 enhances the FCGR2B promoter activity. The 2B.4 haplotype leads to elevated expression of the endogenous receptor in heterozygous donors by approximately 1.5-fold as assessed on EBV-transformed cells, primary B lymphocytes, and CD14(+) monocytes. This increased expression accentuates the inhibitory effect of FcgammaRIIb on B cell Ag receptor signaling, measured by Ca(2+) influx and cell viability in B cells. Our results indicate that transcription factors GATA4 and YY1 are involved in the regulation of FcgammaRIIb expression, and that the expression variants of FcgammaRIIb lead to altered cell signaling, which may contribute to autoimmune pathogenesis in humans.

Complementary roles of glycoprotein VI and alpha2beta1 integrin in collagen-induced thrombus formation in flowing whole blood ex vivo.

Platelets interact vigorously with subendothelial collagens that are exposed by injury or pathological damage of a vessel wall. The collagen-bound platelets trap other platelets to form aggregates, and they expose phosphatidylserine (PS) required for coagulation. Both processes are implicated in the formation of vaso-occlusive thrombi. We previously demonstrated that the immunoglobulin receptor glycoprotein VI (GPVI), but not integrin alpha2beta1, is essential in priming platelet-collagen interaction and subsequent aggregation. Here, we report that these receptors have yet a complementary function in ex vivo thrombus formation during perfusion of whole blood over collagen. With mice deficient in GPVI or blocking antibodies, we found that GPVI was indispensable for collagen-dependent Ca2+ mobilization, exposure of PS, and aggregation of platelets. Deficiency of integrin beta1 reduces the GPVI-evoked responses but still allows the formation of loose platelet aggregates. By using mice deficient in G(alpha)q or specific thromboxane A2 and ADP antagonists, we show that these autocrine agents mediated aggregation but not collagen-induced Ca2+ mobilization or PS exposure. Collectively, these data indicate that integrin alpha2beta1 facilitates the central function of GPVI in the platelet activation processes that lead to thrombus formation, whereas the autocrine thromboxane A2 and ADP serve mainly to trigger aggregate formation.

FDF03, a novel inhibitory receptor of the immunoglobulin superfamily, is expressed by human dendritic and myeloid cells.

In this study, we describe human FDF03, a novel member of the Ig superfamily expressed as a monomeric 44-kDa transmembrane glycoprotein and containing a single extracellular V-set Ig-like domain. Two potential secreted isoforms were also identified. The gene encoding FDF03 mapped to chromosome 7q22. FDF03 was mostly detected in hemopoietic tissues and was expressed by monocytes, macrophages, and granulocytes, but not by lymphocytes (B, T, and NK cells), indicating an expression restricted to cells of the myelomonocytic lineage. FDF03 was also strongly expressed by monocyte-derived dendritic cells (DC) and preferentially by CD14+/CD1a- DC derived from CD34+ progenitors. Moreover, flow cytometric analysis showed FDF03 expression by CD11c+ blood and tonsil DC, but not by CD11c- DC precursors. The FDF03 cytoplasmic tail contained two immunoreceptor tyrosine-based inhibitory motif (ITIM)-like sequences. When overexpressed in pervanadate-treated U937 cells, FDF03 was tyrosine-phosphorylated and recruited Src homology-2 (SH2) domain-containing protein tyrosine phosphatase (SHP)-2 and to a lesser extent SHP-1. Like engagement of the ITIM-bearing receptor LAIR-1/p40, cross-linking of FDF03 inhibited calcium mobilization in response to CD32/FcgammaRII aggregation in transfected U937 cells, thus demonstrating that FDF03 can function as an inhibitory receptor. However, in contrast to LAIR-1/p40, cross-linking of FDF03 did not inhibit GM-CSF-induced monocyte differentiation into DC. Thus, FDF03 is a novel ITIM-bearing receptor selectively expressed by cells of myeloid origin, including DC, that may regulate functions other than that of the broadly distributed LAIR-1/p40 molecule.

IgG-mediated enhancement of antibody responses is low in Fc receptor gamma chain-deficient mice and increased in Fc gamma RII-deficient mice.

Immunization with IgG/Ag or IgE/Ag complexes leads to a higher production of specific Abs than immunization with Ag alone. The enhancing effect of IgE is exclusively dependent upon the low-affinity receptor for IgE, Fc epsilon RII, whereas the mechanism behind IgG-mediated enhancement is unknown. We have investigated whether receptors for the Fc part of IgG are required for responses to IgG/Ag. Mice lacking the gamma subunit of Fc receptors (FcRs) (FcR gamma-/-), Fc gamma RII (Fc gamma RII-/-), or Fc gamma RIII (Fc gamma RIII-/-) were immunized with BSA-2,4,6-trinitrophenyl (TNP) alone or BSA-TNP complexed to monoclonal TNP-specific IgG1, IgG2a, or IgG2b. As expected, all subclasses enhanced the Ab-response to BSA in wild-type mice. Enhancement was in the same order of magnitude in Fc gamma RIII-/- mice (

Reactive oxygen intermediates enhance Fc gamma receptor signaling and amplify phagocytic capacity.

Receptors for the Fc region of IgG (Fc gamma R) mediate internalization of opsonized particles by human neutrophils (PMN) and mononuclear phagocytes. Cross-linking of Fc gamma R leads to activation of protein tyrosine kinases and phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) within Fc gamma R subunits, both obligatory early signals for phagocytosis. Human PMN constitutively express two structurally distinct Fc gamma R, Fc gamma RIIa and Fc gamma RIIIb, and can be induced to express Fc gamma RI by IFN-gamma. We have previously shown that stimulation of PMN through Fc gamma RIIIb results in enhanced Fc gamma RIIa-mediated phagocytic activity that is inhibited by catalase. In the present study, we have tested the hypothesis that reactive oxygen intermediates (ROI) have the capacity to regulate Fc gamma R responses and defined a mechanism for this effect. We show that H2O2 augmented phagocytosis mediated by Fc gamma RIIa and Fc gamma RI in PMN and amplified receptor-triggered tyrosine phosphorylation of Fc gamma R-associated ITAMs and signaling elements. Generation of endogenous oxidants in PMN by cross-linking Fc gamma RIIIb similarly enhanced phosphorylation of Fc gamma RIIa and Syk, a tyrosine kinase required for phagocytic function, in a catalase-sensitive manner. Our results provide a mechanism for priming phagocytes for enhanced responses to receptor-driven effects. ROI generated in an inflammatory milieu may stimulate quiescent cells to rapidly increase the magnitude of their effector function. Indeed, human monocytes incubated in the presence of stimulated PMN showed oxidant-induced increases in Fc gamma RIIa-mediated phagocytosis. Definition of the role of oxidants as amplifiers of Fc gamma R signaling identifies a target for therapeutic intervention in immune complex-mediated tissue injury.

Stimulation of intracellular Ca2+ levels in human neutrophils by soluble immune complexes. Functional activation of FcgammaRIIIb during priming.

Soluble immune complexes bind to unprimed neutrophils and generate intracellular Ca2+ transients but fail to activate the NADPH oxidase. Following priming of the neutrophils with either tumor necrosis factor alpha or granulocyte-macrophage colony-stimulating factor, stimulation of the cells with the soluble immune complexes leads to an enhanced Ca2+ signal and significant secretion of reactive oxidants. The enhanced Ca2+ signal observed in primed neutrophils results from the influx of Ca2+ from the external environment and is partly sensitive to tyrosine kinase inhibitors. This is in contrast to the Ca2+ signal observed in unprimed neutrophils, which arises from the mobilization of intracellular stores. When the surface expression of FcgammaRIIIb on primed neutrophils was decreased either through incubation with Pronase or phosphoinositide-specific phospholipase C, the extra enhanced Ca2+ mobilization seen in primed cells was significantly lowered, while the initial rise in intracellular Ca2+ was unaffected. Depletion of FcgammaRIIIb had no significant effect on the Ca2+ transients in unprimed neutrophils. Cross-linking FcgammaRII, but not FcgammaRIIIb, induced increases in intracellular Ca2+ in unprimed neutrophils, while cross-linking either of these receptors increased Ca2+ levels in primed neutrophils. The FcgammaRII-dependent intracellular Ca2+ rise in primed cells was unaffected by incubation in Ca2+-free medium, whereas the FcgammaRIIIb-dependent transient was significantly decreased when Ca2+ influx was prevented in Ca2+-free medium supplemented with EGTA. Cross-linking either FcgammaRII or FcgammaRIIIb in primed or unprimed cells failed to stimulate substantial levels of inositol 1,4,5-trisphosphate production. These results indicate that following stimulation of primed neutrophils with soluble immune complexes the enhanced Ca2+ mobilization observed is the result of a functional activation of the glycosylphosphatidylinositol-linked FcgammaRIIIb.

ITIMs and ITAMs. The Yin and Yang of antigen and Fc receptor-linked signaling machinery.

The initial stages of an immune response are regulated at the level of the cell-surface antigen and Fc receptors. The extracellular portions of these receptors provide immune specificity and determine the nature of the responding effector cells, whereas the intracellular portion transduces signals into the cell and determines the intensity and duration of the immune response. Recent studies led to the identification of two types of modules within the cytoplasmic region of receptor subunits that are critical for the activation and termination of signal transduction pathways. Phosphorylation of the conserved tyrosine residues within the two modules, the immunoreceptor tyrosine-based activation motif (ITAM) and the immunoreceptor tyrosine-based inhibition motif (ITIM), is followed by the recruitment of different sets of SH2-containing molecules to the receptor site. These proteins regulate the receptor-linked signal transduction pathways in a positive or a negative fashion, which is a reminiscent of the ancestral Yin-Yang principle.

Na+/H+ exchange activity during phagocytosis in human neutrophils: role of Fcgamma receptors and tyrosine kinases.

In neutrophils, binding and phagocytosis facilitate subsequent intracellular killing of microorganisms. Activity of Na+/H+ exchangers (NHEs) participates in these events, especially in regulation of intracellular pH (pHi) by compensating for the H+ load generated by the respiratory burst. Despite the importance of these functions, comparatively little is known regarding the nature and regulation of NHE(s) in neutrophils. The purpose of this study was to identify which NHE(s) are expressed in neutrophils and to elucidate the mechanisms regulating their activity during phagocytosis. Exposure of cells to the phagocytic stimulus opsonized zymosan (OpZ) induced a transient cytosolic acidification followed by a prolonged alkalinization. The latter was inhibited in Na+-free medium and by amiloride analogues and therefore was due to activation of Na+/H+ exchange. Reverse transcriptase PCR and cDNA sequencing demonstrated that mRNA for the NHE-1 but not for NHE-2, 3, or 4 isoforms of the exchanger was expressed. Immunoblotting of purified plasma membranes with isoform-specific antibodies confirmed the presence of NHE-1 protein in neutrophils. Since phagocytosis involves Fcgamma (FcgammaR) and complement receptors such as CR3 (a beta2 integrin) which are linked to pathways involving alterations in intracellular [Ca2+]i and tyrosine phosphorylation, we studied these pathways in relation to activation of NHE-1. Cross-linking of surface bound antibodies (mAb) directed against FcgammaRs (FcgammaRII > FcgammaRIII) but not beta2 integrins induced an amiloride-sensitive cytosolic alkalinization. However, anti-beta2 integrin mAb diminished OpZ-induced alkalinization suggesting that NHE-1 activation involved cooperation between integrins and FcgammaRs. The tyrosine kinase inhibitors genistein and herbimycin blocked cytosolic alkalinization after OpZ or FcgammaR cross-linking suggesting that tyrosine phosphorylation was involved in NHE-I activation. An increase in [Ca2+]i was not required for NHE-1 activation because neither removal of extracellular Ca2+ nor buffering of changes in [Ca2+]i inhibited alkalinization after OpZ or Fc-gammaR cross-linking. In summary, Fc-gammaRs and beta2 integrins cooperate in activation of NHE-1 in neutrophils during phagocytosis by a signaling pathway involving tyrosine phosphorylation.

Increased capacity for store regulated calcium influx in U937 cells differentiated by treatment with dibutyryl cAMP.

We have investigated the mechanisms underlying calcium signalling evoked by cross-linking of the high affinity IgG receptor (Fc gamma RI) in populations of the human monocyte-like cell line, U937, following activation of the cells by cytokine treatment, or differentiation to a more macrophage-like state by treatment with dibutyryl cAMP (Bt2cAMP). We have shown previously that a larger and more prolonged entry of external calcium occurs in Bt2cAMP-differentiated cells, although there is a smaller initial release from internal stores in these cells than in those activated by IFN-gamma treatment. In this paper we demonstrate, by use of the endomembrane Ca(2+)-ATPase inhibitor, thapsigargin, that this effect is explained (at least in part) by an enhanced capacity for store regulated entry of calcium in Bt2cAMP-differentiated cells.

Intravenous immunoglobulins suppress immunoglobulin productions by suppressing Ca(2+)-dependent signal transduction through Fc gamma receptors in B lymphocytes.

A high dose intravenous immunoglobulin (IVIG) therapy is used in the treatment of a wide range of autoimmune disorders. However, the mechanisms of the action of IVIGs remain poorly understood. To analyse the mechanisms of effects of IVIGs on immunoglobulin (Ig) production of B cells, the effects of IVIGs on B lymphoblastoid cell lines transformed by Epstein-Barr virus (LCLs) were investigated. The productions of IgG or IgM of LCLs were dose-dependently suppressed by polyethylene glycol (PEG)-treated IVIG or pH 4-treated IVIG though the productions were not or only slightly suppressed by pepsin-treated IVIG. The suppression by IVIGs was blocked by anti-human IgG Fc or anti-Fc gamma RII. C mu gene expression and mu s C terminal gene expression of LCLs were suppressed by PEG-treated IVIG, whereas neither C mu gene expression nor mu s C terminal gene expression of LCLs were suppressed by pepsin-treated IVIG. Although the increase in intracellular calcium concentration in LCLs was not suppressed by pepsin-treated IVIG, the increase was suppressed by PEG-treated IVIG. This suppressing effect of PEG-treated IVIG on intracellular calcium concentration of LCLs was blocked by anti-human IgG Fc or anti- Fc gamma RII. Our results suggest that IVIGs suppressed the Ca(2+)-dependent signal transduction through Fc gamma R on B-cell membrane, consequently, the transcription of C mu mRNA, especially secreted mu mRNA was suppressed in the B cells.