The effect of oxidants on neutrophil-mediated degradation of glomerular basement membrane collagen

The contribution of activated oxygen species to neutrophil-mediated degradation of basement membrane collagen was investigated. In preliminary experiments, pre-exposure of either albumin or glomerular basement membrane to neutrophil myeloperoxidase with H2O2 and chloride increased their susceptibility to proteolysis 2-3-fold. In the basement membrane model, neutrophils are stimulated by trapped immune complexes to adhere, produce oxidants and degranulate. Degradation, measured as the amount of hydroxyproline solubilised, was due to neutral proteinases, particularly elastase, and depended on cell number and the amount of proteinase released. Experiments with oxidant scavengers and inhibitors and with neutrophils from donors with chronic granulomatous disease or myeloperoxidase deficiency showed that oxidants did not affect degradation of the basement membrane when this was measured on a per cell basis. However, oxidative inactivation of the released granule enzymes occurred. Activities of elastase, beta-glucuronidase and lysozyme were 1.5-2-times higher in the presence of catalase, but were unaffected by superoxide dismutase or hydroxyl radical scavengers. Inactivation did not occur with chronic granulomatous disease or myeloperoxidase deficient neutrophils. When related to the activity of released elastase, or to other degranulation markers, collagen degradation was decreased in the presence of catalase, or with chronic granulomatous disease or myeloperoxidase deficient cells. This implies that the basement membrane was made more digestible by myeloperoxidase-derived oxidants, as occurred in the cell-free experiments. Taken together, the results indicate that neutrophil oxidants have two opposing effects. They increase the susceptibility of the collagen to proteolysis and inactivate the proteinases responsible.     

Proteolytic machinery of glomerular epithelial cells against IgG.

Accumulation of immune complexes in the subepithelial region of the glomerular basement membrane results in the lesion of membranous nephropathy. The inefficient handling of immune complexes by the glomerular epithelial cell was investigated by studying the mechanism of IgG proteolysis by the intracellular proteases of cultured epithelial cells. Radiolabelled IgG was incubated with extracts of cells and the digestion of IgG was monitored by SDS-PAGE analysis. Prolonged incubation of IgG with the cell extracts resulted in only partial degradation of the IgG. The enzyme responsible for the breakdown was determined to be the lysosomal cathepsin D based on the pH optimum and the presence of aspartate in the active site of the enzyme. SDS-PAGE analysis of the digestion fragments revealed that a large proportion of the incompletely degraded IgG was the (Fab)2 fragment, which was resistant to further proteolysis. This could be one of the possible explanations for the slow removal of IgG from the subepithelial location of the basement membrane.     

Neutrophil activation by surface bound IgG: pertussis toxin insensitive activation

Surface bound IgG induces neutrophil degranulation and production of superoxide radicals by a mechanism that is not inhibited by either pertussis toxin or cholera toxin, whereas these functions induced by soluble mediators such as FMLP and soluble aggregates of IgG are profoundly inhibited by pertussis toxin. Interaction of neutrophils with surface bound IgG triggers the loss of 32P labeled PIP2 and PIP and the influx of extracellular calcium. Neither of these cellular events when induced by surface bound IgG is inhibited by pertussis toxin. These observations suggest that neutrophil activation induced by surface bound IgG proceeds along a pathway which is not regulated by proteins which are inhibited by either pertussis or cholera toxins.     

Schistosoma japonicum: Immunopathology of nephritis in Macaca fascicularis

Macaca monkeys experimentally infected with Schistosoma japonicum developed a chronic progressive kidney lesion characterized by an increase of mesangial matrix, local glomerular hypercellularity, and local thickening of glomerular basement membrane. Immunofluorescence studies revealed the localization of IgG, IgM, IgA, and IgE immunoglobulins mostly in the mesangial area of the glomeruli accompanied by the deposition of Schistosoma antigens. By electron microscopy, in addition to the local thickening of the glomerular basement membrane, dense homogeneous deposits and those with moth-eaten appearance were detected in the mesangial matrix. These findings suggest that worms in the bloodstream continuously release antigenic materials that stimulate host's antibody response belonging to various immunoglobulin classes including IgE. The produced antibodies and antigens would form immune complexes that deposited in the glomeruli. The increased vascular permeability caused by antigen-IgE antibody interaction may play an important role in the deposition of immune complexes and in the rapid development of kidney injury.     

Increased susceptibility to immunologically mediated glomerulonephritis in IFN-gamma-deficient mice.

It is postulated that IFN-gamma confers susceptibility to immunologically mediated tissue injury. To test this hypothesis, we compared the intensity of accelerated anti-glomerular basement membrane glomerulonephritis between wild-type (IFN-gamma+/+) and IFN-gamma gene knockout (IFN-gamma-/-) mice. This disease model is initiated by binding of heterologous (sheep) anti-glomerular basement membrane Abs to the glomeruli of mice preimmunized with sheep IgG. The secondary cellular and humoral immune responses to the planted Ag then lead to albuminuria and glomerular pathology. We found that IFN-gamma-/- mice or IFN-gamma+/+ mice injected with IFN-gamma-neutralizing Ab develop worse albuminuria and glomerular pathology than IFN-gamma+/+ mice. The humoral response to sheep IgG (serum mouse anti-sheep IgG titers and intraglomerular mouse IgG deposits) was comparable in the IFN-gamma+/+ and IFN-gamma-/- groups. In contrast, IFN-gamma-/- mice mounted a stronger cellular immune response (cutaneous delayed-type hypersensitivity reaction) to sheep IgG than IFN-gamma+/+ mice. These findings provide evidence that endogenous IFN-gamma has a protective role in immunologically mediated glomerulonephritis initiated by foreign Ags.     

Anomalous Raji cell activity related to DNA binding in some patients with autoimmune disease

We examined sera from patients with autoimmune disease that showed a discrepancy between the Raji cell assay and other tests for circulating immune complexes where the Raji cell activity was highly elevated. Using gel filtration chromatography, we found that the Raji cell activity was associated with monomeric IgG and little evidence of aggregates in the samples. Samples elevated for circulating immune complexes by all tests showed aggregates with associated Raji cell activity. The activity in discrepant samples was decreased by up to 40% by absorption of the IgG fraction with DNA-cellulose prior to the Raji cell assay. It is suggested that binding by autoantibodies to the Raji cell membrane is due to a variety of mechanisms.     

Ultramicroscopic localization of cationized antigen in the glomerular basement membrane in the course of active, in situ immune complex glomerulonephritis

The sequence of antigen localization and the interaction of immune deposits with the anionic sites of the glomerular basement membrane (GBM) were investigated in an active model of in situ immune complex glomerulonephritis using a cationized ferritin. Three weeks after immunization with native horse spleen ferritin, the left kidneys of rats were perfused with 500 micrograms of cationized ferritin through the left renal artery. One h after renal perfusion, most of ferritin particles localized subendothelially, corresponding to the anionic sites of the lamina rara interna. In the glomerular capillary loops, infiltrating polymorphonuclear leukocytes and monocytes were seen. Some of these monocytes were in direct contact with immune complexes containing ferritin aggregates associated with anionic sites of the lamina rara interna. At 24 h, numerous ferritin aggregates were present subepithelially, preferentially beneath the slit membrane. The subepithelial location of ferritin did not always correspond to the anionic sites of the lamina rara externa. From days 3 to 7, there was remarkable endocapillary cell proliferation in some loops and pronounced effacement of epithelial foot processes. Focal detachment of epithelium from the GBM was observed occasionally. From days 14 to 28, most of ferritin aggregates were located intramembranously and subepithelially. Membranous transformation has already begun around the subepithelial deposits. This morphological study provides insight into the fate of immune deposits and injury to the GBM in the glomerulonephritis.     

Biochemical studies of neutrophils from male and female rats: a differential response to basement membrane treated with nephrotoxic antiserum

Rat neutrophils added to 3H-labelled glomerular basement membrane (GBM) treated with rabbit anti-rat GBM antiserum degraded the GBM as judged by the release of 3H-labelled peptides. Cells from female animals promoted a more marked degradation than cells from males. This correlated with measurements of higher levels of elastase in granule fractions from the cells. The subcellular distributions of granule marker enzymes was found not to differ between the sexes. Levels of myeloperoxidase, lysozyme, cathepsin G, alkaline phosphatase, gamma-glutamyltranspeptidase and N-acetyl-beta-glucosaminidase showed no sex-based differences. No alpha-mannosidase could be detected in the cells.     

Nuclear pore complexes deposited in the glomerular basement membrane are associated with autoantibodies in a case of membranous nephritis

Massive deposits of organelles, morphologically identical with nuclear pore complexes (NP), were identified in the glomerular basement membrane of an individual with membranous nephritis. Analysis by immunofluorescence demonstrated that the patient's serum contained autoantibodies that became bound within intact cells to cellular components, including centrosomes or centrioles and filaments of the cytoskeleton. Furthermore, these antibodies became bound to numerous small structures at the nuclear periphery. The above evidence suggests that among the autoantibodies were some specifically directed toward the NP, and that the glomerular deposits were NP, possibly in association with IgG.     

Presence of lysosomal enzymes in the normal glomerular basement membrane matrix

Summary The question posed in the present study was: are there hydrolytic enzymes, including proteases, present in the extracellular matrix of the glomerular basement membrane? If these enzymes are present they may play a role in the catabolism of the glomerular basement membrane (GBM) and removal of macromolecular debris resulting from ultrafiltration. Enzymes, acid phosphatase - the marker for lysosomal enzymes - β-galactosidase, β-glucuronidase and acid protease (using albumin as substrate) were biochemically assayed in purified basement membrane preparations. It was found that all enzymes were present in significant amounts in the basement membrane. Compared to other enzymes, acid protease activity was present in much higher amounts. The pH optima of these enzymes were variable but all had significant activity at neutral pH. A method was developed to localize the marker enzyme, acid phosphatase, ultrastructurally in the basement membrane in order to substantiate the biochemical findings. Activity was shown by the presence of dense deposits of lead phosphate. Staining for acid phosphatase could also be shown on isolated, purified basement membrane. The demonstration of acid hydrolases in the GBM matrix argues for their role in (i) the extracellular turnover of basement membrane macromolecules, and (ii) clearance of debris of ultrafiltration which tend to clog the membrane pores.     

Selective inhibition of neutrophil activation by the subendothelial extracellular matrix: Possible role in protection of the vessel wall during diapedesis

Mobilization of circulating neutrophils toward an inflamed area involves adherence of the cells to the vascular endothelium and subsequent penetration through the endothelial cell layer without causing significant damage. To investigate the nature of a possible protective mechanism, granulocytes were incubated with the extracellular matrix (ECM) produced by cultured endothelial cells and tested for release of enzymes, chemoattractants, and free oxygen radicals. In the absence of exogenously added stimuli, the neutrophils adhered to the ECM but there was no detectable release of lysozyme, chemotactic activity, or production of O2-. In contrast, the cells readily released a heparan sulfate-degrading endoglycosidase (heparanase) to an extent comparable with that released in contact with polystyrene surfaces. Neutrophils treated with the calcium ionophore A23187 or with the peptide FMLP produced O2- to a much lesser degree when incubated in contact with ECM-coated surfaces than did those incubated in contact with uncoated polystyrene culture dishes. The ECM itself was devoid of superoxide dismutase activity. Stimulation with opsonized zymosan was not inhibited by the ECM. Experiments with isolated constituents of the ECM revealed that fibronectin but not collagen type IV or laminin could partially inhibit O2- production by Ca2+ ionophore-stimulated neutrophils. Treatment of the ECM with proteolytic enzymes, but not with heparanase, abolished its inhibitory effect on neutrophil activation. These results indicate that the subendothelial basement membrane has the capacity to inhibit release of potentially noxious agents excluding heparanase, suggesting a preferential involvement of this enzyme in neutrophil diapedesis.     

Fate of plasma membrane during endocytosis. II. Evidence for recycling (shuttle) of plasma membrane constituents

Cultured rat embryo fibroblasts were first allowed to store for 24 h fluorescein-labeled goat immunoglobulins directed against rabbit immunoglobulins (F anti-R IgG), and were subsequently exposed for 24 h to [(3)H]acetylated rabbit immunoglobulins known to bind to the cell membrane either specifically (anti-plasma membrane IgG: A anti-PM IgG) or unspecifically (contol IgG: AC IgG). As a result of immunological interaction between the two antibodies (no effect was found if the cells had been preloaded with control goat FC IgG), a substantial portion of the stored F anti-R IgG was unloaded from its intracellular storage site, appearing in the medium in the form of soluble immune complexes with rabbit A IgG. Part of the unloaded F anti-R IgG also was recovered in association with the plasma membrane, but only when A anti-PM IgG was used. In addition, significant reverse translocation of AC IgG from plasma membrane to lysosomes or some related intracellular storage compartment was also observed. With A anti-PM IgG, this translocation was less marked and affecte at the same time the plasma membrane marker 5’- nucleotidase. Cells that had stored horseradish peroxidase (HRP) simultaneously with F anti-R IgG did not unload HRP when exposed to A anti-PM IgG. These results support strongly, though not unequivocally, the concept that plasma membrane patches interiorized by endocytosis are recycled, or shuttled, back to the cell surface. In the framework of this concept, recycling antibody-coated membrane is taken to serve as vehicle for the selective intracellular capture and extracellular discharge of immunologically bound F anti-R IgG. The alternative explanation of regurgitation triggered off by immune complexes is considered less likely in view of the lack of HRP unloading.     

Lupus Nephritis: Enigmas,Conflicting Models and an Emerging Concept

Autoantibodies to components of chromatin, which include double-stranded DNA (dsDNA), histones and nucleosomes, are central in the pathogenesis of lupus nephritis. How anti-chromatin autoantibodies exert their nephritogenic activity, however, is controversial. One model assumes that autoantibodies initiate inflammation when they cross-react with intrinsic glomerular structures such as components of membranes, matrices or exposed nonchromatin ligands released from cells. Another model suggests glomerular deposition of autoantibodies in complex with chromatin, thereby inducing classic immune complex–mediated tissue damage. Recent data suggest acquired error of renal chromatin degradation due to the loss of renal DNaseI enzyme activity is an important contributing factor to the development of lupus nephritis in lupus-prone (NZBxNZW)F1 mice and in patients with lupus nephritis. Down-regulation of DNaseI expression results in reduced chromatin fragmentation and in deposition of extracellular chromatin–IgG complexes in glomerular basement membranes in individuals who produce IgG anti-chromatin autoantibodies. The main focus of the present review is to discuss whether exposed chromatin fragments in glomeruli are targeted by potentially nephritogenic anti-dsDNA autoantibodies or if the nephritogenic activity of these autoantibodies is explained by cross-reaction with intrinsic glomerular constituents or if both models coexist in diseased kidneys. In addition, the role of silencing of the renal DNaseI gene and the biological consequences of reduced chromatin fragmentation in nephritic kidneys are discussed.     

Binding characteristics of dimeric IgG subclass complexes to human neutrophils

Immune complexes were prepared by incubation of human IgG paraproteins with F(ab')2 fragments of the mAb K35 against the kappa-L chain of human IgG. The composition of these complexes was analyzed by centrifugation over sucrose gradients, by gel filtration, by RIA with either IgG Sepharose or K35 Sepharose and by double-labeling studies. The results indicated that the complexes consist of saturated tetramers composed of two IgG molecules cross-linked by two F(ab')2 fragments of the mAb. These complexes were used to study the binding of the different IgG subclasses to human neutrophils at 4 degrees C. Human neutrophils bound IgG3 complexes approximately three times faster than IgG1 complexes. Binding of IgG2 or IgG4 dimers to the neutrophils was undetectable. The same number of IgG1 complexes and IgG3 complexes bound to the neutrophils, but considerable inter-donor variation was found (mean number of Fc gamma R per neutrophil: 190,000, range 120,000 to 400,000). The Ka for the binding of IgG1 complexes to neutrophils (median 11 x 10(7) M-1) was lower than the Ka for the binding of IgG3 complexes (median 47 x 10(7) M-1). Competition studies between labeled IgG1 complexes or IgG3 complexes and unlabeled complexes showed that the Fc gamma R of human neutrophils do not display an IgG subclass specificity. Incubation of neutrophils with a mAb against the FcRIII completely blocked the binding of IgG1 complexes and IgG3 complexes. Incubation with a mAb against the FcRII reduced the affinity of the complexes for the neutrophils but had no effect on the maximum number of complexes bound. This indicates that one complex may bind simultaneously to one FcRIII and to one FcRII.     

Immunoelectron microscopy localization of immunoglobulin G in human placenta

Immunoelectron microscopy of IgG molecules in human mature placenta has shown that IgG bound to microvillar surfaces and the inner wall of endocytotic vesicles of syncytiotrophoblasts. The endocytotic vesicles, containing both bound and unbound IgG molecules, tended to fuse with each other or with other cellular organelles, particularly with lysosomes. The phagolysosomes were more abundant in the basal regions of the cells. Apparently some IgG molecules were not digested by lysosomal enzymes. Vesicles with residual IgG were found to fuse with the basal and basolateral cell membrane and to discharge their contents into the extracellular space by exocytosis. It is suggested that IgG molecules were transported through the trophoblastic basement membrane and the interstitial space by diffusion to the endothelial basement membrane. The IgG molecules then migrated into the fetal vascular lumen via endothelial gaps and interendothelial spaces.     

C3d,g is present in normal human epidermal basement membrane

mAb as well as polyclonal anti-human C3d antibodies were found to specifically bind to the epidermal basement membrane zone of normal human adult and neonatal skin in a linear continuous pattern on direct immunofluorescence microscopy. No such binding was found in dermal microvascular basement membranes. Studies of normal adult human skin using a rat mAb specific for C3g revealed the same pattern of epidermal basement membrane staining. Control polyclonal antibodies directed against C3, C3c, C5, IgG, IgA, or IgM showed no evidence of epidermal basement membrane binding or in situ deposits of immune complexes in samples of normal human skin that were all positive for C3d and C3g. Pre-adsorption of monoclonal or polyclonal anti-human C3d with purified human C3d completely blocked these reagents' epidermal basement membrane reactivity. Anti-human C3d epidermal basement membrane binding was not diminished by pre-treatment of substrate with antibodies directed against C3, C3c, C5, laminin, fibronectin, or type IV collagen as well as bullous pemphigoid, KF-1, or epidermolysis bullosa acquisita Ag. Direct immunofluorescence microscopy studies on 1 M NaCl split human skin showed that C3d and C3g were found in the base of the cleavage plane created within the lamina lucida. By immunoelectron microscopy, C3d was found along the base of the lamina densa and in the sublamina densa region of normal human epidermal basement membrane. Although anti-human C3d epidermal basement membrane binding was not altered by treatment of 6 micron skin sections with buffers of varying pH and ionic concentration, binding was abolished by treating dermal portions of salt split skin with 0.1 M dithiothreitol in 8 M urea. Studies of a patient with congenital C3 deficiency revealed that there was no binding of anti-human C3d or anti-human C3g to this subject's epidermal basement membrane. Moreover, treatment of this patient's skin with aged human serum containing C3d,g or purified human C3 did not restore epidermal basement membrane anti-human C3d binding. These studies demonstrate that C3d,g or a closely related C3 fragment is present in the epidermal basement membrane zone of normal human skin.     

Critical but overlapping role of FcgammaRIII and FcgammaRIV in activation of murine neutrophils by immobilized immune complexes

Immune complex-induced activation of neutrophils through cell surface FcRs plays a central role in the pathogenesis of autoimmune inflammatory diseases. These diseases are often modeled using genetically modified mice. However, in contrast to the number of studies on human cells, the identity of FcRs involved in immune complex activation of murine neutrophils is at present unknown. Furthermore, little is known about the cellular functions mediated by the recently identified murine FcgammaRIV. In this study, we tested the identity of FcRs involved in the activation of neutrophils by plate-bound immune complexes, using various knockout mouse strains, function-blocking mAbs, or the combination of both approaches. Activation of murine neutrophils by immobilized IgG immune complexes was abrogated in FcR gamma-chain-deficient cells, but not by the single or combined deficiency of the gamma-chain-associated FcgammaRI and FcgammaRIII, or by blocking Abs against either FcgammaRIII or FcgammaRIV alone. However, treatment of FcgammaRIII-deficient neutrophils with FcgammaRIV-blocking Abs or simultaneous blocking of FcgammaRIII and FcgammaRIV in wild-type cells completely inhibited the immune complex-induced cellular responses. In parallel studies, activation of human neutrophils by immobilized immune complexes was abrogated by blocking Abs against either FcgammaRIIA or FcgammaRIIIB alone. Taken together, neutrophil activation by immobilized immune complexes requires the murine FcgammaRIII/FcgammaRIV or the human FcgammaRIIA/FcgammaRIIIB molecules. Although both of the two human receptors are required for this response, the two murine receptors play overlapping, redundant roles. These results promote our understanding of autoimmune diseases and identify an IgG-dependent cellular function of FcgammaRIV.     

Neutrophil activation by surface bound IgG is via a pertussis toxin insensitive G protein

Pre-treatment of neutrophils with either pertussis or cholera toxins does not inhibit neutrophil activation by surface bound IgG. In contrast, pretreatment with the phorbol ester, phorbol myristate acetate, results in a dose dependent inhibition of degranulation by surface bound IgG. This inhibition is similar to that seen with soluble ligands where it is thought to be due to interference with the interaction of an activated guanine nucleotide binding protein with phospholipase C (J. Biol. Chem.,262,6121,1987). More directly, GTP binding and GTPase activity are enhanced when human neutrophil membranes are incubated in wells containing surface bound IgG. Neither of these G protein functions were inhibited when membranes were prepared in the presence of pertussis toxin, suggesting that neutrophil activation by surface bound IgG proceeds by a mechanism that involves a pertussis toxin insensitive G protein.     

The role of the neutrophil in inflammatory diseases of the lung

Under certain circumstances, the neutrophil has been implicated in causing disease by damaging normal host tissue. This may occur in the adult respiratory distress syndrome (ARDS). The neutrophil has been implicated since a) substances that activate neutrophils are produced in association with the predisposing risks that lead to ARDS; b) activated neutrophils migrate into the alveolar spaces and their toxic products can be found in lung lavage fluid and in the breath of patients with ARDS; and c) the magnitude of the physiologic alterations correlate with the number of neutrophils in the alveolar space. Additionally, the neutrophils may be primed by substances which are released by activated platelets within the confines of the lung. Both platelet adenine nucleotides and the platelet-derived extracellular matrix protein (ECM), thrombospondin, can prime the neutrophil for subsequent O2- generation following activation of the cells with the chemotactic peptide, F-met-leu-phe (FMLP). Furthermore, neutrophils can be primed or O2- generation by the basement membrane ECM protein, laminin. Since neutrophils express receptors for both laminin and thrombospondin, these constituents may serve to modulate neutrophil behavior for subsequent oxidative metabolism and contribute to exacerbating pulmonary disease.     

Usefulness of grading of neutrophil aggregate size by laser-light scattering technique for characterizing stimulatory and inhibitory effects of agents on aggregation

We attempted to apply the particle counting method that employs laser-light scattering technique to quantify the change in numbers of neutrophil homotypic aggregates of 3 graded-sizes (small, medium and large). Ex vivo activation of human neutrophils by a chemotactic peptide, fMLP, predominantly produced small-sized aggregates (< 15 cells), and also, transiently, medium-sized aggregates (16-130 cells). Co-treatment of neutrophils with fMLP and cytochalasin B mainly produced medium-sized aggregates, with very few large-sized aggregates (> 130 cells). Interestingly, when protein kinase C was activated with phorbol 12-myristate 13-acetate small-, medium- and even large-sized aggregates of neutrophils were formed. Presence of extracellular calcium was required to produce these neutrophil aggregations. Both prostaglandin E2 (PGE2) and wortmannin, an inhibitor of phosphatidyl inositol 3-kinase (PI-3K), inhibited neutrophil aggregation, whereas dbcAMP, a cell permeable analog of cyclic AMP, did not, confirming that PGE2 causes neutrophil aggregation probably through PI-3K inhibition rather than activation of adenylate cyclase. These results suggest that the application of the light scattering technique to characterize human neutrophil aggregates by both size and numbers, has advantages over conventional optical turbulent aggregometry, in that it discriminates neutrophil aggregations produced by different mechanisms of intracellular signaling.