P Heeringa

Activation of granulocytes by anti‐neutrophil cytoplasmic antibodies (ANCA): a FcγRII‐dependent process

ANCA have been demonstrated to induce the respiratory burst in primed neutrophils. In this study we have extended the investigations on neutrophil activation by ANCA directed against proteinase 3 (PR3). Myeloperoxidase (MPO) and lactoferrin (LF), and we have analysed the underlying mechanisms. All three ANCA antigens were expressed on the cell surface of primed neutrophils. Superoxide production assayed by both cytochrome c reduction and oxidation of dihydrorhodamine 123, was induced by heterologous polyclonal anti‐MPO and anti‐LF antibodies, and ANCA‐positive plasma samples. Induction of superoxide production was dose‐dependent. F(ab)2 fragments did not induce the respiratory burst. Blockade of Fc receptors by specific oAbs showed that anti‐FcγRII antibodies were able to turn off the ANCA‐induced respiratory burst, whereas anti‐FcγRII antibodies did not. Plasma samples that induced the respiratory bursl did not differ from samples that did not induce superoxide production with respect to ANCA titre, but had higher levels of the lgG3 subclass of ANCA. Levels of the other subclasses of ANCA were comparable between those samples. We conclude that ANCA‐induced activation of primed neutrophils is FcγRII‐dependent, and appears to be facilitated by antibodies of the IgG3 subclass.

Elastase, but not proteinase 3 (PR3), induces proteinuria associated with loss of glomerular basement membrane heparan sulphate after in vivo renal perfusion in rats

Elastase, but not PR3, induces proteinuria associated with loss of glomerular basement membrane (GBM) heparan sulphate after in vivo renal perfusion in rats. PR3 and elastase are cationic neutral serine proteinases present in the azurophilic granules of polymorphonuclear leucocytes. Release of these proteolytic enzymes along the glomerular capillary wall may induce glomerular injury. Here, we investigated the effects of PR3 and elastase on the induction of proteinuria and glomerular injury after renal perfusion of these enzymes in Brown–Norway rats. Perfusion of active elastase induced a dose‐dependent proteinuria 24u2003h after perfusion, while inactivated elastase did not. Perfusion of comparable amounts of active PR3 did not induce proteinuria. Light and electron microscopy showed no morphological abnormalities in any experimental group. However, immunohistology revealed that proteinuria occurring after perfusion of active elastase was associated with a strong reduction in intraglomerular expression of the heparan sulphate side chain and, to a lesser extent, of the protein core of heparan sulphate proteoglycans (HSPG). In vitro, both elastase and PR3 digested HSPG. However, PR3 bound to a lesser extent to HSPG than elastase. We conclude that elastase, but not PR3, induces proteinuria after in vivo renal perfusion. This differential effect probably relates to different binding to the GBM of those enzymes due to differences in their isoelectric points. Degradation of heparan sulfate proteoglycans, leading to the disappearance of their side chains that contribute to the polyanionic structure of the GBM, appears to be involved in the induction of proteinuria after perfusion of elastase.

CONTEMPORARY CHALLENGES IN AUTOIMMUNITY

Introduction: It is hypothesized that IgM antibodies to oxidized LDL are anti‐atherogenic. Myeloperoxidase from plaque‐infiltrating neutrophils catalyzes the production of hypochlorite (HOCl), which oxidizes LDL. Here we study the IgM response to HOCl‐modified LDL in comparison to titers of T15 clonotypic natural antibodies. Methods: Plasma of LDLR−/− mice fed a normal chow or high‐fat diet was obtained after 6 and 16 weeks. The IgM responses to HOCl‐modified LDL and T15 clonotypic natural IgM antibodies were measured by ELISA. Results: The IgM levels in response to HOCl‐modified LDL increased dramatically in the atherosclerotic group after introduction of the high‐fat diet, but not in mice on normal chow. The natural IgM T15 clonotypic antibody titers revealed a more moderate increase during atherogenesis. Conclusion: Our results show that during atherogenesis there is a strong induction of IgM antibodies to HOCl‐modified LDL particles. Whether these induced IgM antibodies are pro‐ or anti‐atherogenic remains to be established.