Reactive oxygen species deglycosilate glomerular alpha-dystroglycan.

In the kidney, dystroglycan (DG) has been shown to cover the basolateral and apical membranes of the podocyte. alpha-DG is heavily glycosilated, which is important for its binding to laminin and agrin in the glomerular basement membrane. Furthermore, alpha-DG is negatively charged, which maintains the filtration slit open. Reactive oxygen species (ROS) are known to degrade and depolymerize carbohydrates, and to play a role in several glomerular diseases. Therefore, we evaluated the effect of ROS on the glycosilation of glomerular alpha-DG. By using specific antibodies directed against the core protein or glyco-epitopes of alpha-DG, this was studied in a solid-phase assay, in situ on kidney sections, and in vivo in adriamycin nephropathy. A ligand overlay assay was used to study binding of alpha-DG to its ligands. Exposure to ROS leads to a loss of carbohydrate epitopes on alpha-DG both in vitro and on kidney sections. In the in vitro assays, a decreased binding of deglycosilated alpha-DG to laminin and agrin was found. In adriamycin nephropathy, where radicals play a role, we observed a loss of alpha-DG carbohydrate epitopes. We conclude that deglycosilation of glomerular alpha-DG by ROS leads to disruption of the agrin-DG complex, which in vivo may lead to the detachment of podocytes. Furthermore, loss of negative charge in the filtration slit may lead to foot process effacement of podocytes.

Differential expression of agrin in renal basement membranes as revealed by domain-specific antibodies.

We determined the specificity of two hamster monoclonal antibodies and a sheep polyclonal antiserum against heparan sulfate proteoglycan isolated from rat glomerular basement membrane. The antibodies were characterized by enzyme-linked immunosorbent assay on various basement membrane components and immunoprecipitation with heparan sulfate proteoglycan with or without heparitinase pre-treatment. These experiments showed that the antibodies specifically recognize approximately 150-, 105-, and 70-kDa core proteins of rat glomerular basement membrane heparan sulfate proteoglycan. Recently, we showed that agrin is a major heparan sulfate proteoglycan in the glomerular basement membrane (Groffen, A. J. A., Ruegg, M. A., Dijkman, H. B. P. M., Van der Velden, T. J., Buskens, C. A., van den Born, J., Assmann, K. J. M., Monnens, L. A. H., Veerkamp, J. H., and van den Heuvel, L. P. W. J. (1998) J. Histochem. Cytochem. 46, 19-27). Therefore, we tested whether our antibodies recognize agrin. To this end, we evaluated staining of Chinese hamster ovary cells transfected with constructs encoding full-length or the C-terminal half of rat agrin by analysis on a fluorescence-activated cell sorter. Both hamster monoclonals and the sheep antiserum clearly stained cells transfected with the construct encoding full-length agrin, whereas wild type cells and cells transfected with the construct encoding the C-terminal part of agrin were not recognized. A panel of previously characterized monoclonals, directed against C-terminal agrin, clearly stained cells transfected with either of the constructs but not wild type cells. This indicates that both hamster monoclonals and the sheep antiserum recognize epitopes on the N-terminal half of agrin. By immunohistochemistry on rat renal tissue, we compared distribution of N-terminal agrin with that of C-terminal agrin. The monoclonal antibodies against C-terminal agrin stained almost exclusively the glomerular basement membrane, whereas the anti-N-terminal agrin antibodies recognized all renal basement membranes, including tubular basement membranes. Based on these results, we hypothesize that full-length agrin is predominantly expressed in the glomerular basement membrane, whereas in most other renal basement membranes a truncated isoform of agrin is predominantly found that misses (part of) the C terminus, which might be due to alternative splicing and/or posttranslational processing. The possible significance of this finding is discussed.

Antigen specificity of anti-nuclear antibodies complexed to nucleosomes determines glomerular basement membrane binding in vivo.

Monoclonal anti-nuclear antibodies which are complexed to nucleosomes are able to bind to the glomerular basement membrane (GBM) in vivo, whereas purified antibodies do not bind. The positively charged histone moieties in the nucleosome are-responsible for the binding to anionic determinants in the GBM. We tested the hypothesis that the specificity of the autoantibodies complexed to the nucleosome influences the glomerular binding of the antibody-nucleosome complex. We induced the formation of these immune complexes in vivo, by intraperitoneal inoculation of hybridomas producing monoclonal anti-nuclear antibodies (four anti-histone, three anti-double stranded (ds)DNA and three anti-nucleosome antibodies) into nude BALB/c mice. In ascites and plasma from the mice inoculated with these hybridomas, nucleosome/autoantibody complexes were detected in comparable amounts. Immunofluorescence of kidney sections revealed that about 60% of the mice inoculated with anti-nucleosome or anti-dsDNA hybridomas had immunoglobulin deposits in the GBM, whereas only 15% of the mice with anti-histone hybridomas showed these deposits (p < or = 0.04). In the Matrigel-ELISA (used as a GBM surrogate) ascites from anti-nucleosome or anti-DNA hybridomas displayed significantly higher titers (p < or = 0.002) than ascites from anti-histone hybridomas. In conclusion, nucleosome/immunoglobulin complexes comprising anti-nucleosome or anti-dsDNA auto-antibodies do bind more frequently to the GBM in vivo than nucleosome/immunoglobulin complexes containing anti-histone antibodies. It therefore appears that the specificity of the antibody bound to the nucleosome is a critical determinant for the nephritogenic potential of the nucleosome-autoantibody complex.

Role of polymorphic Fc receptor Fc gammaRIIa in cytokine release and adverse effects of murine IgG1 anti-CD3/T cell receptor antibody (WT31).

Anti-CD3 monoclonal antibody (mAb) OKT3 is immunosuppressive, but causes severe adverse effects during the first administration ("first-dose reaction"). These adverse effects are presumably caused by cytokine release that results from T-cell activation. In vitro, T-cell activation by anti-CD3 mAb requires interaction with monocyte Fc receptors. The Fc receptor for murine IgG1, Fc gammaRIIa, is polymorphic. In some individuals, murine IgG1 anti-CD3 mAb causes T-cell proliferation and cytokine release in vitro (high responders [HR]), whereas in individuals with the low-responder (LR) phenotype it does not. We have now investigated the role of this Fc gammaRIIa polymorphism in the release of cytokines in vivo and the occurrence of adverse effects after the administration of WT31, a murine IgG1 anti-CD3/T cell receptor mAb. WT31 caused an increase of plasma tumor necrosis factor-alpha in all four HR patients and none of the five LR patients. In all HR patients except one, plasma gamma-interferon and interleukin 6 also increased, and a first-dose response was observed, whereas no cytokine release or adverse effects occurred in any of the LR patients. WT31 caused lymphopenia in all HR and none of the LR patients. FACS analysis demonstrated that in HR patients, after the initial disappearance of CD3+ cells from peripheral blood, modulation of CD3 occurred, whereas in LR patients a high degree of coating of the lymphocytes was observed. Surprisingly, WT31 also induced a marked granulocytopenia, as well as a decrease of thrombocytes, in three of the four HR patients (and in none of the LR patients). These data provide direct clinical evidence that Fc receptor interaction determines the release of cytokines and the occurrence of adverse effects after administration of anti-CD3/T cell receptor mAb. Furthermore, these data suggest that tumor necrosis factor-alpha by itself is not sufficient to induce the first-dose reaction.

Analysis at the DNA level of human Fc gamma RII isoforms in relation to the polymorphic binding of murine IgG2b to human B cells.

Three classes of human leucocyte Fc gamma receptors (hFc gamma R) have been identified so far: hFc gamma RI, hFc gamma RII, and hFc gamma RIII. Previous studies have demonstrated that genetically determined differences between individuals exist both with respect to the binding of murine IgG1 (mIgG1) to hFc gamma receptors, and with respect to the binding of murine IgG2b (mIgG2b). The polymorphism in binding of mIgG1 could be ascribed to hFc gamma RIIA, an isoform of hFc gamma RII. The authors have now investigated whether one of the isoforms of hFc gamma RII is also responsible for the polymorphism in binding of mIgG2b. In these studies the authors used EBV-transformed human B cells that demonstrated either binding or no binding of mIgG2b in EA-rosetting assays. mRNA obtained from these cells was amplified by reverse transcriptase and polymerase chain reaction (RT-PCR). Hybridization experiments with the RT-PCR products revealed that the hFc gamma RIIB but not the hFc gamma RIIA isoform was present in these cells. DNA sequencing further demonstrated that the nucleotide sequence of both the extracellular part and the cytoplasmic moiety of hFc gamma RIIB was identical for all individuals tested, regardless of their ability to bind mIgG2b. These findings indicate that the polymorphic binding of mIgG2b cannot be ascribed to one of the isoforms of hFc gamma RII. Since hFc gamma RI and hFc gamma RIII are not present on the cell surface of these cells, the authors conclude that an Fc receptor different from the known hFc gamma receptors must be responsible for the polymorphic binding of mIgG2b. These data further expand the complexity of hFc gamma R.

Removal of monocytes from cell suspensions with anti-CD14 antibody and carbonyl-iron, using Fc gamma R-dependent accessory function as a sensitive measure of monocyte presence.

Human (Fc gamma RI-positive) monocytes are required as accessory cells when T cell proliferation is induced by murine IgG2a anti-CD3 monoclonal antibodies (mAbs). This T cell proliferation assay provides a sensitive method for detecting the presence of monocytes (less than 1% of monocytes can be detected), and we have used it to monitor the effectiveness of different procedures for the removal of monocytes from peripheral blood mononuclear cells. Counterflow centrifugation, phagocytosis of carbonyl-iron, adherence to plastic, monocyte depletion with magnetic beads (Dynabeads M450), and panning with anti-CD14 antibodies each strongly reduced the number of monocytes. However, none of these methods, when used on their own, were capable of completely abolishing the mitogenic response to murine IgG2a anti-CD3 mAb. A virtually complete depletion of monocytes was obtained when the panning procedure using anti-CD14 antibodies was combined with phagocytosis of carbonyl-iron. Importantly, this method could also be used with cryopreserved cells. We have applied this improved method for the removal of monocytes, to study T cell proliferation induced by murine IgG2b anti-CD3 mAb. We were able to demonstrate with this model that cells other than monocytes were able to provide accessory function.

A new radiometric assay for the quantitation of surface-bound IgG on sensitized erythrocytes.

We have developed a sensitive, straightforward method for the quantitation of surface-bound IgG on sensitized erythrocytes. The assay is based on the consumption by sensitized cells of anti-IgG antiserum. The remaining anti-IgG is quantitated in a second incubation by precipitation with 125I-IgG in the presence of polyethylene glycol. Calibration curves for this assay were constructed using known amounts of unlabeled IgG. The method can be performed in microtitre plates and eliminates the use of purified anti-erythrocyte antibodies, or highly purified specific anti-IgG antisera. The results were completely consistent with those of immunofluorescence assays, but our method was much more sensitive, less than 500 molecules of IgG per cell being detected reproducibly. The technique is not laborious and takes much less time than previously described methods with similar sensitivity. As an example of the applicability of this test, the implications of ligand density for the detection by EA rosetting of Fc receptors on human monocytes are shown. The results suggest that a large variation exists in the affinity of the different types of Fc receptors for their ligands.

Complement-dependent and independent mechanisms in acute antibody-mediated rejection of skin xenografts in the mouse.

Complement dependency of acute antibody-mediated rejection (AAR) was studied in a xenogeneic skin graft model in the mouse. PVG/c rat skin grafted to immunosuppressed mice was acutely destroyed by i.v. administered mouse anti-rat serum on day 7 after grafting. Depression of the hemolytic complement titer in the recipients was indicative for complement consumption during the rejection process. Complete and long-lasting complement depletion induced by treatment with cobra venom factor (COVF) decreased the sensitivity of the grafts to AAR. However, complete protection was not achieved, since high doses of antidonor serum again induced destruction. Similar results were obtained in C5-deficient recipients and in COVF-treated C5-deficient recipients. These results indicated that complement-independent rejection mechanisms were operative. This was further substantiated by the finding that purified noncomplement-fixing IgC1 subclass antibodies were able to elicit mice with a normal complement status was caused by intravascular coagulation without primary involvement of polymorphonuclear leukocytes. In complement-depleted animals an Arthus-like reaction was seen, with dense intravascular accumulation of polymorphonuclear leukocytes (PMNs) that, apparently, were attracted to the graft through complement-independent mechanisms.

Passive enhancement of mouse skin allografts by alloantibodies is Fc dependent.

The capacity of F(ab')2 fragments of alloantibodies to enhance mouse allografts was studied in B6AF1 recipients of B10.D2 skin grafts. F(ab')2 obtained by digestion of B6AF1 anti-B10.D2 antibodies was purified by means of affinity chromatography, with anti-subclass antisera and protein A. The degree of contaminating IgG was less than 0.02%. Administration of F(ab')2 with an antigen-binding capacity similar to the IgG from which it originated, inhibited acute antibody-mediated graft rejection but was unable to induce enhancement. Even a dose that was 130 times the molar amount of the minimal enhancing dose of undigested IgG2 was ineffective. We conclude, therefore, that passive enhancement of mouse skin allografts by alloantibodies requires the Fc part.

Isolation of pure IgG subclasses from mouse alloantiserum and their activity in enhancement and hyperacute rejection of skin allografts.

B6AF1 anti-B10.D2 ascites fluid was fractionated by molecular sieving. From the 7S fraction, IgG subclasses were isolated by affinity chromatography with specific antisera against mouse immunoglobulin subclasses coupled to CNRr-activated Sepharose 4B. Thus, 7S IgG1, 7S IgG2, 7S IgG2a, and 7S IgG2b were obtained which were completely pure by criteria of immunodiffusion and immunoelectrophoresis. In the in vitro tests the 7S IgG1 completely lacked cytotoxic activity. All the other subclasses were able to induce lysis of B10.D2 cells in the presence of rabbit complement. All subclasses were tested for their capacity to induce enhancement and hyperacute destruction of B10.DI skin grafts in B6AF1 recipient mice, and for the dose-dependency of these effects. 7S IgG1, although showing clearcut enhancing activity, was completely inactive in inducing hyperacute destruction of the grafts, even if high doses were administered. By contrast, 7S IgG2 (IgG2a as well as IgG2b) was not only able to induce enhancement but also hyperacute destruction of the frafts. The results obtained with 7S IgG2 demonstrate that opposite biologic activities can be present within a single subclass.