Biomedical subjects

P Heeringa

Publications and source records attributed to P Heeringa.

25 records · Page 2Linked to original sources

PubMed1997

Systemic injection of products of activated neutrophils and H2O2 in myeloperoxidase-immunized rats leads to necrotizing vasculitis in the lungs and gut.

The strong association of anti-neutrophil cytoplasmic antibodies with various forms of systemic vasculitis suggests a role for these autoantibodies in the pathophysiology of systemic vasculitis. In the present study, we tested the hypothesis that release of neutrophil lysosomal enzymes in the presence of an anti-myeloperoxidase (anti-MPO) immune response may underlie the development of systemic vasculitis. Brown Norway rats were immunized with MPO in complete Freund's adjuvant or complete Freund's adjuvant alone. Two weeks after immunization, rats bad developed antibodies to human and rat MPO as measured by enzyme-linked immunosorbent assay. Next, rats were intravenously infused with 400 micrograms of a human neutrophil lysosomal extract containing 200 micrograms of MPO followed by 0.5 ml of a 1 mmol/L solution of H2O2 through a cannula inserted into the right jugular vein. Rats were sacrificed at 4 hours, 24 hours, 7 days, or 14 days, and several organs (lungs, heart, liver, spleen, gut, and kidneys) were examined for vasculitic lesions and inflammatory cell infiltrates. Macroscopically, patchy hemorrhagic spots were observed in the lungs and gut of MPO-immunized rats at days 7 and 14 after systemic infection of the neutrophil lysosomal extract and H2O2. Such changes were not observed at earlier time points or in control immunized rats. Histologically, the lungs of MPO-immunized rats sacrificed at days 7 and 14 showed patchy inflammatory cell infiltrates associated with vasculitis, granuloma formation, giant cells, and foci of hemorrhage. At 14 days, early signs of fibrosis were found with deposition of collagen and proliferation of fibroblasts. Furthermore, a prominent leukocytoclastic vasculitis was found in the small intestine of these rats characterized by fibrinoid necrosis and an extensive neutrophilic infiltrate. No inflammatory changes were found in the other organs studied (heart, liver, spleen, and kidneys). Control immunized rats, sacrificed at days 7 and 14 showed only some small foci of inflammatory infiltrates in the lungs whereas no inflammatory changes were found in the gastrointestinal tract. These studies show that release of products from activated neutrophils in the presence of anti-MPO autoantibodies may be relevant to the pathogenesis of anti-MPO-associated vasculitides.

Animals↗

PubMed1996

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 24h 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.

Animals↗

PubMed1996

Autoantibodies to myeloperoxidase aggravate mild anti-glomerular-basement-membrane-mediated glomerular injury in the rat.

Autoantibodies to myeloperoxidase (MPO) are present in sera from patients with various forms of vasculitis-associated glomerulonephritis. Evidence for a pathogenic role of anti-MPO antibodies has been provided mainly by in vitro studies. We studied the pathogenic role of autoantibodies to MPO in a rat model of mild immune-mediated glomerular injury. Brown Norway rats were immunized with human MPO in complete Freund's adjuvant or with complete Freund's adjuvant alone. At 2 weeks after immunization, rats had developed antibodies to human and rat MPO as detected by indirect immunofluorescence, enzyme-linked immunosorbent assay, and immunoprecipitation. At this time point, rats were intravenously injected with a subnephritogenic dose of 150 micrograms of rabbit anti-rat GBM. Rats were sacrificed at 4 hours, 24 hours, 4 days, and 10 days after antibody administration. Control immunized rats developed mild glomerulonephritis characterized by slight proteinuria at day 10 (14.8 +/- 8.1 mg/24 hours) and moderate intraglomerular accumulation of ED1+ macrophages. Crescent formation, tuft necrosis, and tubular atrophy were not observed in those rats. In contrast, rats immunized with MPO developed severe glomerulonephritis characterized by the early occurrence of severe hematuria, marked proteinuria at day 10 (76.2 +/- 18.2 mg/24 hours), and massive glomerular deposition of fibrin. Complement and rat IgG were present in insudative lesions, but no linear pattern along the glomerular capillary wall was observed. By light microscopy, severe glomerular lesions were found at day 10 consisting of crescent formation and fibrinoid necrosis of capillary loops. In the interstitium, tubular necrosis and atrophy and marked interstitial mononuclear infiltration were found in conclusion, autoantibodies to MPO severely aggravate subclinical anti-GBM disease demonstrating their in vivo pathogenic potential.

Animals↗

PubMed1994

Neutrophil activation in vitro and in vivo in Wegener's granulomatosis.

The mechanisms underlying glomerular capillary wall injury in Wegener's granulomatosis (WG) are not well understood. Anti-neutrophil cytoplasmic antibodies (ANCA), present in sera from patients with WG, are known to stimulate respiratory burst and degranulation of primed polymorphonuclear neutrophils (PMN) in vitro. Experimental studies have shown that oxygen radical production and lysosomal enzymes are important mediators of glomerular capillary wall injury. In the present study we investigated the presence of activated PMN and the extracellular localization of lysosomal enzymes in 28 consecutive renal biopsies from patients with WG. The presence of activated PMN within the renal biopsies was compared with the capacity of ANCA, isolated from simultaneously drawn serum samples, to activate primed PMN obtained from a normal donor. Both parameters were also related to renal function. Renal biopsies were obtained from newly diagnosed WG patients before therapy had started. Activation of PMN in the biopsies was assessed by measuring hydrogen peroxide production in situ. The number of activated PMN in the biopsy correlated with the extent of impairment of renal function. Proteinase 3, myeloperoxidase, and elastase, all targets of ANCA, were localized extracellularly in renal tissue and were also found within tubular epithelial cells. All ANCA positive samples were capable of activating primed PMN. The amount of activation correlated with the ANCA titer in those samples. No correlation, however, was found between the in vitro capacity of ANCA-positive IgG fractions to activate primed PMN and the number of activated PMN present in the renal biopsy. We conclude that activated PMN producing toxic oxygen metabolites and releasing lysosomal enzymes, are present in renal biopsies from patients with WG. The amount of activated PMN present within the kidney, and not the capacity of the corresponding ANCA to activate PMN, correlates with renal tissue damage as assessed by serum creatinine levels.

Antibodies, Antineutrophil Cytoplasmic↗

PubMed1994

Activation of granulocytes by anti-neutrophil cytoplasmic antibodies (ANCA): a Fc gamma RII-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 MoAbs showed that anti-Fc gamma RII antibodies were able to turn off the ANCA-induced respiratory burst, whereas anti-Fc gamma RIII antibodies did not. Plasma samples that induced the respiratory burst did not differ from samples that did not induce superoxide production with respect to ANCA titre, but had higher levels of the IgG3 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 Fc gamma RII-dependent, and appears to be facilitated by antibodies of the IgG3 subclass.

Antibodies, Antineutrophil Cytoplasmic↗

PubMed1994

Podocyte expression of MHC class I and II and intercellular adhesion molecule-1 (ICAM-1) in experimental pauci-immune crescentic glomerulonephritis.

We examined immunopathological changes of podocytes in vivo which, based on in vitro studies, are thought to be relevant for the pathogenesis of renal diseases. We investigated the alterations of podocytes in local inflammation in a recently developed model of pauci-immune necrotizing crescentic glomerulonephritis (NCGN) in the rat. Frozen and plastic embedded kidney sections at different time points of the disease were incubated with antibodies directed to MHC class I, MHC class II, ICAM-1 and to relevant cytokines. Strong glomerular expression of MHC class I, II and ICAM-1 was found within 4 days, and plastic embedded sections clearly demonstrated increased cell membrane staining of podocytes. Increased glomerular interferon-gamma (IFN-gamma) was detected within 24 h of induction of NCGN, and IL-1 beta and tumour necrosis factor-alpha (TNF-alpha) were found from day 4. The potency of these cytokines to induce adhesion molecules on podocytes was investigated on rat glomerular epithelial cells in vitro. By using FACS analysis and electron microscopical techniques, we found that the in vivo expression of MHC class I, II and ICAM-1 by podocytes could in vitro be simulated by IFN-gamma. IFN-alpha weakly induced MHC class I, while IL-1 beta and TNF-alpha were ineffective. We hypothesize that podocytes in this in vivo model are important to maintain the local inflammatory process in the glomerulus by expression of relevant adhesion molecules and MHC molecules upon stimulation with specific cytokines.

Animals↗

PubMedDate not supplied

The role of myeloperoxidase in the pathogenesis of systemic vasculitis.

Wegener's granulomatosis, microscopic polyangiitis, Churg-Strauss syndrome and idiopathic pauci-immune necrotizing crescentic glomerulonephritis are strongly associated with the presence of anti-neutrophil cytoplasmic antibodies (ANCA). These ANCA-associated vasculitides can serologically be separated into myeloperoxidase (MPO)-ANCA and proteinase 3 (PR3)-ANCA positive patients. The unique properties of the antigen targeted by the anti-MPO antibodies could help to explain the specific characteristics of MPO-ANCA associated disease. Recently, an animal model has been developed that proves that anti-mouse MPO immunoglobulins alone are capable of causing disease similar to that in humans. Also, the in vitro pathologic effects of binding of MPO-ANCA to MPO are better understood. MPO-ANCA can activate (primed) neutrophils directly causing extensive reactive oxygen species formation and degranulation of neutrophil constituents, including MPO, resulting in a destructive inflammatory response towards the vessel wall. MPO-ANCA can prevent the clearing and inactivation of MPO by ceruloplasmin as well, resulting in increased myeloperoxidase activity. Myeloperoxidase produces not only the strong oxidant bleach (hypochlorous acid) out of hydrogen peroxide and chloride ions but also oxidizes LDL into a macrophage high-uptake form, inactivates protease inhibitors, and consumes nitric oxide. These may contribute to endothelial dysfunction and add to the chronic renal lesions observed in patients with MPO-ANCA. MPO levels are influenced by genetic factors including two, MPO463 and MPO129, single nucleotide polymorphisms. The MPO 463 polymorphism has been associated with an increased risk of development of MPO-ANCA associated disease.

Animals↗