Stephanie Konrad

Cell-derived anaphylatoxins as key mediators of antibody-dependent type II autoimmunity in mice

Complement C5a, a potent anaphylatoxin, is a candidate target molecule for the treatment of inflammatory diseases, such as myocardial ischemia/reperfusion injury, RA, and the antiphospholipid syndrome. In contrast, up until now, no specific contribution of C5a and its receptor, C5aR, was recognized in diseases of antibody-dependent type II autoimmunity. Here we identify C5a as a novel key mediator of autoimmune hemolytic anemia (AIHA) and show that mice lacking C5aR are partially resistant to this IgG autoantibody-induced disease model. Upon administration of anti-erythrocyte antibodies, upregulation of activating Fcgamma receptors (FcgammaRs) on Kupffer cells, as observed in WT mice, was absent in C5aR-deficient mice, and FcgammaR-mediated in vivo erythrophagocytosis was impaired. Surprisingly, in mice deficient in FcgammaRI and FcgammaRIII, anti-erythrocyte antibody-induced C5 and C5a production was abolished, demonstrating the existence of a previously unidentified FcgammaR-mediated C5a-generating pathway. These results show that the development of a full-blown antibody-dependent autoimmune disease requires C5a--produced by and acting on FcgammaR--and may suggest therapeutic benefits of C5 and/or C5a/C5aR blockade in AIHA and other diseases closely related to type II autoimmune injury.

Macrophages Induce the Inflammatory Response in the Pulmonary Arthus Reaction through Gαi2 Activation That Controls C5aR and Fc Receptor Cooperation

Complement and FcγR effector pathways are central triggers of immune inflammation; however, the exact mechanisms for their cooperation with effector cells and their nature remain elusive. In this study we show that in the lung Arthus reaction, the initial contact between immune complexes and alveolar macrophages (AM) results in plasma complement-independent C5a production that causes decreased levels of inhibitory FcγRIIB, increased levels of activating FcγRIII, and highly induced FcγR-mediated TNF-α and CXCR2 ligand production. Blockade of C5aR completely reversed such changes. Strikingly, studies of pertussis toxin inhibition show the essential role of Gi-type G protein signaling in C5aR-mediated control of the regulatory FcγR system in vitro, and analysis of the various C5aR-, FcγR-, and Gi-deficient mice verifies the importance of Gαi2-associated C5aR and the FcγRIII-FcγRIIB receptor pair in lung inflammation in vivo. Moreover, adoptive transfer experiments of C5aR- and FcγRIII-positive cells into C5aR- and FcγRIII-deficient mice establish AM as responsible effector cells. AM lacking either C5aR or FcγRIII do not possess any such inducibility of immune complex disease, whereas reconstitution with FcγRIIB-negative AM results in an enhanced pathology. These data suggest that AM function as a cellular link of C5a production and C5aR activation that uses a Gαi2-dependent signal for modulating the two opposing FcγR, FcγRIIB and FcγRIII, in the initiation of the inflammatory cascade in the lung Arthus reaction.

STIM1 is essential for Fcγ receptor activation and autoimmune inflammation

Fcgamma receptors (FcgammaRs) on mononuclear phagocytes trigger autoantibody and immune complex-induced diseases through coupling the self-reactive immunoglobulin G (IgG) response to innate effector pathways, such as phagocytosis, and the recruitment of inflammatory cells. FcRgamma-based activation is critical in the pathogenesis of these diseases, although the contribution of FcgammaR-mediated calcium signaling in autoimmune injury is unclear. Here we show that macrophages lacking the endoplasmic reticulum-resident calcium sensor, STIM1, cannot activate FcgammaR-induced Ca(2+) entry and phagocytosis. As a direct consequence, STIM1 deficiency results in resistance to experimental immune thrombocytopenia and anaphylaxis, autoimmune hemolytic anemia, and acute pneumonitis. These results establish STIM1 as a novel and essential component of FcgammaR activation and also indicate that inhibition of STIM1-dependent signaling might become a new strategy to prevent or treat IgG-dependent immunologic diseases.

Both FcγRIV and FcγRIII are essential receptors mediating type II and type III autoimmune responses via FcRγ-LAT-dependent generation of C5a

FcγRIV is a relatively new IgG Fc receptor (FcγR) that is reported to contribute to the pathogenesis of autoimmune diseases, although its specific role in relation to FcγRIII, complement and IgG2 subclasses remains uncertain. Here we define FcγRIV on macrophages as a receptor for soluble IgG2a/b complexes but not for cellular bound IgG2a and show that simultaneous activation of FcγRIV and FcγRIII is critical to mediate certain type II/III autoimmune responses. FcγRIII‐deficient mice display compensatory enhanced FcγRIV expression, are protected from lung inflammation after deposition of IgG complexes, and show reduced sensitivity to IgG2a/b‐mediated hemolytic anemia, indicating that increased FcγRIV alone is not sufficient to trigger these diseases in the absence of FcγRIII. Importantly, however, blockade of FcγRIV is also effective in inhibiting phagocytosis and cytokine production in IgG2b‐induced anemia and acute lung injury, processes that display a further dependence on C5a anaphylatoxin receptor. Using gene deletion and functional inhibition studies, we found that FcγRIII and FcγRIV are each essential to trigger an FcRγ‐linker for activation of T‐cell‐dependent signal that drives C5a production in the Arthus reaction. Together, the results demonstrate a combined requirement for FcγRIII and FcγRIV in autoimmune injury, and identify the linker for activation of T cells adaptor as an integral component of linked FcγR and C5a anaphylatoxin receptor activation to generate inflammation.

Phosphoinositide 3-Kinases γ and δ, Linkers of Coordinate C5a Receptor-Fcγ Receptor Activation and Immune Complex-induced Inflammation

Fcγ receptors (FcγR) and the C5a receptor (C5aR) are key effectors of the acute inflammatory response to IgG immune complexes (IC). Their coordinated activation is critical in IC-induced diseases, although the significance of combined signaling by these two different receptor classes in tissue injury is unclear. Here we used the mouse model of the passive reverse lung Arthus reaction to define their requirements for distinct phosphoinositide 3-kinase (PI3K) activities in vivo. We show that genetic deletion of class IB PI3Kγ abrogates C5aR signaling that is crucial for FcγR-mediated activation of lung macrophages. Thus, in PI3Kγ-/- mice, IgG IC-induced FcγR regulation, cytokine release, and neutrophil recruitment were blunted. Notably, however, C5a production occurred normally in PI3Kγ-/- mice but was impaired in PI3Kδ-/- mice. Consequently, class IA PI3Kδ deficiency caused resistance to acute IC lung injury. These results demonstrate that PI3Kγ and PI3Kδ coordinate the inflammatory effects of C5aR and FcγR and define PI3Kδ as a novel and essential element of FcγR signaling in the generation of C5a in IC disease.

PI3Kgamma and PI3Kdelta: Linkers of coordinate C5aR-FcgammaR activation and immune complex-induced inflammation

Fcγ receptors (FcγR) and the C5a receptor (C5aR) are key effectors of the acute inflammatory response to IgG immune complexes (IC). Their coordinated activation is critical in IC-induced diseases, although the significance of combined signaling by these two different receptor classes in tissue injury is unclear. Here we used the mouse model of the passive reverse lung Arthus reaction to define their requirements for distinct phosphoinositide 3-kinase (PI3K) activities in vivo. We show that genetic deletion of class IB PI3Kγ abrogates C5aR signaling that is crucial for FcγR-mediated activation of lung macrophages. Thus, in PI3Kγ-/- mice, IgG IC-induced FcγR regulation, cytokine release, and neutrophil recruitment were blunted. Notably, however, C5a production occurred normally in PI3Kγ-/- mice but was impaired in PI3Kδ-/- mice. Consequently, class IA PI3Kδ deficiency caused resistance to acute IC lung injury. These results demonstrate that PI3Kγ and PI3Kδ coordinate the inflammatory effects of C5aR and FcγR and define PI3Kδ as a novel and essential element of FcγR signaling in the generation of C5a in IC disease.

Protective Role of Complement C5a in an Experimental Model of Staphylococcus aureus Bacteremia

The complement system is a key component of the innate immune response. Here, we have examined the role of complement anaphylatoxin C5a in an experimental model of Staphylococcus aureus bacteremia. Our data provide compelling evidence for a protective role of C5a during staphylococcal bloodstream infection.

Intravenous immunoglobulin (IVIG)-mediated neutralisation of C5a: a direct mechanism of IVIG in the maintenance of a high FcγRIIB to FcγRIII expression ratio on macrophages

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Characterization of the Murine IgG Fc Receptor III and IIB Gene Promoters A SINGLE TWO-NUCLEOTIDE DIFFERENCE DETERMINES THEIR INVERSE RESPONSIVENESS TO C5a

The two low affinity IgG Fc receptors (FcγR), FcγRIII and FcγRIIB, are coexpressed on myeloid effector cells, and their genes, as reported here, are positively and negatively regulated by both C5a and interferon-γ through different signaling mechanisms. Two 48- and 43-bp sequences (C5a-inductive region (CIR) and C5a-suppressive region (CSR)) in the FcγRIII and FcγRIIB 5′-flanking regions that are necessary for C5a induction and suppression, respectively, are defined. Sequence analysis of the CIR and CSR, which localize apart from the interferon-γ-responsive regions in each gene, revealed the presence of a novel element that differs by two nucleotides between FcγRIII and FcγRIIB. Mutation analysis of the CIR and CSR showed that this small difference determines inverse responsiveness in an FcγR gene context-dependent manner. Our study suggests that C5a uses similar DNA motifs (defined as GTGAXXTCCA) in both pathways of transcriptional induction and suppression of FcγRIII and FcγRIIB.