FcγR responses to soluble immune complexes of varying size: A scalable cell-based reporter system

Fcγ-receptor (FcγR) activation by antibody derived soluble immune complexes (sICs) is a major contributor to inflammation in autoimmune diseases such as systemic lupus erythematosus (SLE). A robust and scalable test system allowing for the detection and quantification of sICs with regard to receptor activation is missing. We developed a comprehensive cell-based reporter system capable of measuring the sIC-mediated activation of individual human and mouse FcγRs. We show that compared to human FcγRs IIB and III, human FcγRs I and IIA lack sensitivity to sICs. Further, the assay proved to be sensitive to sIC size enabling us to demonstrate for the first time a complete translation of the Heidelberger-Kendall precipitation curve to FcγR responsiveness. The assay also proved useful to quantify sICs-mediated FcγR activation using sera from SLE patients and mouse models of lupus and arthritis. Thus, in clinical practice, it might be employed to measure FcγR activation as a biomarker for disease activity in immune-complex mediated disease.

Interaction with complement proteins and dendritic cells implicates LCCL domain-containing proteins (CCps) of malaria parasites in immunomodulation

The evasion of host immune defense is critical for pathogens to invade, establish infection and perpetuate in the host. The complement system is one of the first lines of innate immune defense in humans that destroys pathogens in the blood circulation. Activation of the complement system through direct encounter with pathogens or some other agents leads to osmolysis of pathogens, clearance of soluble immune complexes and recruitment of lymphocytes at the site of activation. Although malaria parasites are not exposed to the complement system owing to their intracellular development for most part of their life cycle in the human host, the extracellular stages must face the complement system of human or mosquito or both. In a recent issue of the Biochemical Journal, Sharma et al. reported that Plasmodiumfalciparum LCCL domain-containing protein 1 (PfCCp1) inhibited activation of the classical complement pathway and down-regulated effector responses of dendritic cells, which implicate PfCCp1 and related proteins in immunomodulation of the host that likely benefits the parasite. PfCCp1 belongs to a multi-domain protein family that exists as multimeric protein complexes. It needs to be investigated whether PfCCp1 or its multimeric protein complexes have an immunomodulatory effect in vivo and on the mosquito complement system

Restricted immune activation and internalisation of anti-idiotype complexes between drug and antidrug antibodies

ObjectivesTherapeutic antibodies can provoke an antidrug antibody (ADA) response, which can form soluble immune complexes with the drug in potentially high amounts. Nevertheless, ADA-associated adverse events are usually rare, although with notable exceptions including infliximab. The immune activating effects and the eventual fate of these ‘anti-idiotype’ complexes are poorly studied, hampering assessment of ADA-associated risk of adverse events. We investigated the in vitro formation and biological activities of ADA-drug anti-idiotype immune complexes using patient-derived monoclonal anti-infliximab antibodies.MethodsSize distribution and conformation of ADA-drug complexes were characterised by size-exclusion chromatography and electron microscopy. Internalisation of and immune activation by complexes of defined size was visualised with flow imaging, whole blood cell assay and C4b/c ELISA.ResultsSize and conformation of immune complexes depended on the concentrations and ratio of drug and ADA; large complexes (>6 IgGs) formed only with high ADA titres. Macrophages efficiently internalised tetrameric and bigger complexes in vitro, but not dimers. Corroborating these results, ex vivo analysis of patient sera demonstrated only dimeric complexes in circulation.No activation of immune cells by anti-idiotype complexes was observed, and only very large complexes activated complement. Unlike Fc-linked hexamers, anti-idiotype hexamers did not activate complement, demonstrating that besides size, conformation governs immune complex potential for triggering effector functions.ConclusionsAnti-idiotype ADA-drug complexes generally have restricted immune activation capacity. Large, irregularly shaped complexes only form at high concentrations of both drug and ADA, as may be achieved during intravenous infusion of infliximab, explaining the rarity of serious ADA-associated adverse events.

Relationship betweenLeishmaniaIFAT Titer and Clinicopathological Manifestations (Clinical Score) in Dogs

During canine leishmaniasis (CanL) due toLeishmania infantum,high levels of antibodies production are associated with the presence of various clinical signs, because of the deposition of soluble immune complexes in organs and tissues. The immunofluorescence antibody test (IFAT) is one of the most commonly used techniques for detection of anti-Leishmaniaantibodies. The purpose of this study was to assess whether there is a correlation between clinical signs and IFAT titers in dogs naturally infected withLeishmania. A retrospective study was performed on medical records of 49 dogs diagnosed with CanL. Information extracted from the medical records of each dog with CanL was clinical score, IFAT titer, serum total protein (TP), gamma globulin (IgG) and creatinine concentration, and protein creatinine ratio in urine sample (UP/UC) at each follow-up examination. Results show that dogs with highest IFAT titers recorded had higher mean clinical scores indicating a positive relationship (P<0.0001) between anti-Leishmaniaantibodies (IgG) and clinical manifestations, which becomes more evident in severe clinical forms of canine leishmaniasis. Higher TP and IgG serum concentrations were recorded in dogs with higher clinical scores. Significant association was observed between UP/UC and the IFAT titer (P=0.004).

Endocytosis of soluble immune complexes leads to their clearance by FcγRIIIB but induces neutrophil extracellular traps via FcγRIIA in vivo

Soluble immune complexes (ICs) are abundant in autoimmune diseases, yet neutrophil responses to these soluble humoral factors remain uncharacterized. Moreover, the individual role of the uniquely human FcγRIIA and glycophosphatidylinositol (GPI)–linked FcγRIIIB in IC-mediated inflammation is still debated. Here we exploited mice and cell lines expressing these human neutrophil FcγRs to demonstrate that FcγRIIIB alone, in the absence of its known signaling partners FcγRIIA and the integrin Mac-1, internalizes soluble ICs through a mechanism used by GPI-anchored receptors and fluid-phase endocytosis. FcγRIIA also uses this pathway. As shown by intravital microscopy, FcγRIIA but not FcγRIIIB-mediated neutrophil interactions with extravascular soluble ICs results in the formation of neutrophil extracellular traps (NETs) in tissues. Unexpectedly, in wild-type mice, IC-induced NETosis does not rely on the NADPH oxidase, myeloperoxidase, or neutrophil elastase. In the context of soluble ICs present primarily within vessels, FcγRIIIB-mediated neutrophil recruitment requires Mac-1 and is associated with the removal of intravascular IC deposits. Collectively, our studies assign a new role for FcγRIIIB in the removal of soluble ICs within the vasculature that may serve to maintain homeostasis, whereas FcγRIIA engagement of tissue soluble ICs generates NETs, a proinflammatory process linked to autoimmunity.