A prominent response during the Drosophila host defence is the induction of proteolytic cascades, some of which lead to localized melanization of pathogen surfaces, while others activate one of the major players in the systemic antimicrobial response, the Toll pathway. Despite the fact that gain‐of‐function mutations in the Toll receptor gene result in melanization, a clear link between Toll activation and the melanization reaction has not been firmly established. Here, we present evidence for the coordination of hemolymph‐borne melanization with activation of the Toll pathway in the Drosophila host defence. The melanization reaction requires Toll pathway activation and depends on the removal of the Drosophila serine protease inhibitor Serpin27A. Flies deficient for this serpin exhibit spontaneous melanization in larvae and adults. Microbial challenge induces its removal from the hemolymph through Toll‐dependent transcription of an acute phase immune reaction component.

A serine proteinase cascade in insect hemolymph mediates prophenoloxidase activation, a defense mechanism against pathogen or parasite infection. Little is known regarding its initiating proteinase or how this enzyme is activated in response to invading microorganisms. We have isolated from the tobacco hornworm, Manduca sexta, a cDNA encoding a modular protein designated hemolymph proteinase 14 (HP14). It contains five low density lipoprotein receptor class A repeats, a Sushi domain, a unique Cys-rich region, and a proteinase-catalytic domain. The HP14 mRNA exists in fat body and hemocytes of the naive larvae, and its level increases significantly at 24 h after a bacterial challenge. We expressed proHP14 with a carboxyl-terminal hexahistidine tag in a baculovirus/insect cell system and detected the recombinant protein in two forms. The 87-kDa protein was primarily intracellular, whereas the 75-kDa form was present in the medium. Interaction with peptidoglycan resulted in proteolytic processing of the purified zymogen and generation of an amidase activity. Supplementation of hemolymph with proHP14 greatly enhanced prophenoloxidase activation in response to Micrococcus luteus. These data suggest that proHP14 is a pattern recognition protein that binds to bacteria and autoactivates and triggers the prophenoloxidase activation system in the hemolymph of M. sexta.Similar to other invertebrates, insects lack an adaptive immune system and rely solely on their innate immune mechanisms to fight against invading microorganisms (1-3). These defense mechanisms are mediated by hemocytes (e.g. phagocytosis and encapsulation), fat body (e.g. induced synthesis of antimicrobial peptides), and plasma factors (e.g. hemolymph coagulation and melanization). Accumulating evidence indicates that a complex serine proteinase network in insect hemolymph coordinates some of these responses (4). The prophenoloxidase (proPO) 1 activation cascade is probably composed of several serine proteinases that are sequentially activated in response to microbial infection and lead to the proteolytic activation of proPO to phenoloxidase (PO). PO catalyzes the formation of quinones that are reactive intermediates for melanin synthesis (5, 6). Quinones are also involved in cuticle sclerotization, wound healing, and sequestration of parasites or pathogens.Although the physiological importance of the proPO activation in insect immune system has been appreciated for many years, the molecular characterization of the cascade components was reported only recently. We isolated three proPOactivating proteinases from cuticular extract or hemolymph of M. sexta prepupae, and they all required a protein cofactor for proPO activation (7-10). Similar results were obtained from the beetles Holotrichia diomphalia and Tenebrio molitor (11-13). In contrast, the silkworm proPO-activating enzyme does not appear to need any auxiliary factor for proPO activation (14). Limited by substrate availability, current biochemical research is focused on the components at the end of th...

Granulomatosis with polyangiitis (Wegener's) is a rare autoimmune neutrophil-mediated vasculitis that can cause renal disease and mucosal manifestations. Antineutrophil cytoplasmic antibodies (ANCA) are present in many patients, vary in level over time, and induce neutrophil activation through engagement with Fc receptors (FcRs). Given roles for FcRs in ANCA-mediated neutrophil activation and IgA antibodies in mucosal immunity, we hypothesized that FcR genetics and previously unappreciated IgA ANCA affect clinical presentation. We assembled a total of 673 patients and 413 controls from two multicenter cohorts, performed ELISA and immunofluorescence assays to determine IgA and IgG ANCA positivity, and used Illumina, TaqMan, or Pyrosequencing to genotype eight haplotype-tagging SNPs in the IgA FcR ( FCAR ) and to determine NA1/NA2 genotype of FCGR3B, the most prevalent neutrophil IgG FcR. We evaluated neutrophil activation by measuring degranulation marker CD11b with flow cytometry or neutrophil extracellcular trap formation with confocal microscopy. Functional polymorphisms in FCGR3B and FCAR differed between patient groups stratified by renal involvement. IgA ANCA were found in ∼30% of patients and were less common in patients with severe renal disease. Neutrophil stimulation by IgA or IgG ANCA led to degranulation and neutrophil extracellcular trap formation in a FcR allele-specific manner (IgA:FCAR P = 0.008; IgG:FCGR3B P = 0.003). When stimulated with IgA and IgG ANCA together, IgG ANCA induced neutrophil activation was reduced ( P = 0.0001). FcR genotypes, IgA ANCA, and IgG ANCA are potential prognostic and therapeutic targets for understanding the pathogenesis and presentation of granulomatosis with polyangiitis (Wegener's).

The human IgA FcR (FcαRI; 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 (Ser248) to GGC (Gly248) in the cytoplasmic domain of the receptor. The two different alleles demonstrate significantly different FcαRI-mediated intracellular calcium mobilization and degranulation in rat basophilic leukemia cells and cytokine production (IL-6 and TNF-α) in murine macrophage P388D1 cells. In the absence of FcR γ-chain association in P388D1 cells, the Ser248-FcαRI allele does not mediate cytokine production, but the Gly248-FcαRI allele retains the capacity to mediate a robust production of proinflammatory cytokine. This allele-dependent difference is also seen with FcαRI-mediated IL-6 cytokine release by human neutrophils ex vivo. These findings and the enrichment of the proinflammatory Gly248-FcαRI allele in systemic lupus erythematosus populations in two ethnic groups compared with their respective non-systemic lupus erythematosus controls suggest that FcαRI (CD89) α-chain alleles may affect receptor-mediated signaling and play an important role in the modulation of immune responses in inflammatory diseases.

No abstract

Ligand specificity characterizes receptors for antibody and many other immune receptors, but the common use of the FcR-γ-chain as their signaling subunit challenges the concept that these receptors are functionally distinct. We hypothesized that elements for specificity might be determined by the unique cytoplasmic domain (CY) sequences of the ligand-binding α-chains of γ-chain associated receptors. Among Fcγ receptors (FcRs), a protein kinase C (PKC) phosphorylation consensus motif, [RSSTR], identified within the FcγRIIIa (CD16A) CY by in silico analysis, is specifically phosphorylated by PKCs, unlike other FcRs. Phosphorylated CD16A mediates a more robust calcium flux, tyrosine phosphorylation of Syk and pro-inflammatory cytokine production while non-phosphorylatable CD16A is more effective at activation of the Gab2/PI3K pathway, leading to enhanced degranulation. S100A4, a specific protein binding partner for CD16A-CY newly identified by yeast two-hybrid analysis, inhibits phosphorylation of CD16A-CY by PKC in vitro, and reduction of S100A4 levels in vivo enhances receptor phosphorylation upon cross-linking. Taken together, PKC-mediated phosphorylation of CD16A modulates distinct signaling pathways engaged by the receptor. Calcium activated binding of S100A4 to CD16A, promoted by the initial calcium flux, attenuates the phosphorylation of CY, and acting as a molecular switch, may both serve as a negative feedback on cytokine production pathways during sustained receptor engagement and favor a shift to degranulation, consistent with the importance of granule release following conjugate formation between CD16A+ effector cells and target cells. This switch mechanism points to new therapeutic targets and provides a frame for understanding novel receptor polymorphisms.

TNF ligand superfamily member 13B (B-lymphocyte stimulator (BLyS), B cell activating factor (BAFF)) promotes primary B cell proliferation and immunoglobulin production. While the soluble form of BLyS/BAFF is thought to be the primary biologically active form, little is known about the regulation of its cleavage and processing. We provide evidence that Fcγ receptor cross-linking triggers a rapid release of soluble, biologically active BLyS/BAFF from myeloid cells. Surprisingly, this function is primarily mediated by FcγRI, but not FcγRIIa as defined by specific mAb, and can be initiated by both IgG and C reactive protein (CRP) as ligands. The generation of a B cell proliferation and survival factor by both innate and adaptive immune opsonins through engagement of an Fcγ receptor, which can also enhance antigen uptake and presentation, provides a unique opportunity to facilitate antibody production. These results provide a mechanism by which Fcγ receptors can elevate circulating BLyS levels and promote autoantibody production in immune complex mediated autoimmune diseases.

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