Immunopathologic aspects of woodchuck hepatitis.

The natural history of infection with woodchuck hepatitis virus (WHV) has been studied in a colony of 38 Marmota monax. Besides serologic assessment for WHV markers, light-microscopic findings of 61 liver biopsies were correlated with the results of immunofluorescence analysis for nucleocapsid (WHcAg) and surface (WHsAg) antigens. Twenty-four chronic WHsAg carriers all featured signs of continuous viral replication. Two major immunomorphologic patterns were observed in their livers: 1) portal hepatitis in which WHcAg accumulated in the cytoplasm and WHsAg was associated with the hepatocyte membrane and 2) periportal hepatitis in which WHcAg shifted toward nuclear localization and WHsAg became mostly intracytoplasmic. Progression from portal to periportal hepatitis, observed in 7 woodchucks, appeared to be induced by a partial recovery of specific immune reactivity to WHV, insufficient, however, to interrupt WHV replication. Deposits of WHsAg and immunoglobulins were present in the kidney and spleen of animals with severe hepatitis.     

Hepadna virus nucleocapsid and surface antigens and the antigen-specific antibodies associated with hepatocyte plasma membranes in experimental woodchuck acute hepatitis

Hepatocyte plasma membranes purified from five woodchucks with distinct serologic and histologic patterns of experimentally induced acute woodchuck hepatitis virus (WHV) infection were studied to determine the virus antigens expression and anti-viral specificity of the bound immunoglobulins. WHV core, e, and surface antigens (WHcAg, WHeAg, and WHsAg, respectively) were analyzed with the use of immunoblotting technique both in the native form of these membranes and in the membranes treated with high molar urea or a nonionic detergent. The eluted material was tested either for the presence of WHV antigens or reactivity of the antibodies directed to the virus antigens. The data revealed that acute WHV infection is accompanied by hepatocyte plasma membrane expression of all three viral antigens tested. In all cases, native membranes displayed both WHeAg and WHsAg, whereas WHcAg presence was detected in hepatocyte plasma membranes after their disruption with urea or a detergent. The data indicated that a part or, in some instances, even the whole detectable WHcAg specificity can be incorporated into plasma membrane structure in such a way that it is not accessible for recognition by the specific antibodies (anti-WHc), suggesting at least a partial functional disability of this antigen as a target for immunologic reactions in in vivo conditions. In contrast, WHeAg specificity was detectable in all native membrane preparations studied and its expression was not evidently influenced by the employed treatments, whereas that of WHsAg tended to decline. Further, anti-WHc reactivity was identified in all membrane eluates tested, but antibodies to WHeAg (anti-WHe) were exclusively found in the material eluted from membranes originating from woodchucks with borderline histologic activity of acute hepatitis, which cleared away e antigen from the serum shortly before liver perfusion. Antibodies to WHsAg (anti-WHs) did not show up in the eluates. The present findings demonstrated that WHeAg specificity is not only exposed on the surface of infected hepatocytes, but is also relatively more easily accessible for serologic recognition than that of WHcAg in acute WHV infection. The above observation suggests that e antigen can serve as a potential plasma membrane target for hepatocytolytic attack in addition to that of WHsAg or WHcAg. Moreover, the results of this study demonstrated an apparent relationship between low histologic activity of liver inflammation, e antigen clearance from the circulation, and detectability of hepatocyte plasma membrane-bound anti-e antibodies in acute hepadna viral hepatitis.     

Purification of woodchuck hepatitis surface antigen using a monoclonal antibody raised against the antigen

Woodchuck hepatitis virus (WHV) is closely related to the human hepatitis B virus (HBV) and infection of woodchucks with WHV creates a useful model for studies of immunity, pathogenesis and therapy of HBV infection. To increase the usefulness of this model, monoclonal antibodies were raised to woodchuck hepatitis surface antigen (WHsAg) and one of these antibodies was used to purify the antigen by affinity chromatography from serum, a simpler and quicker method of purification than the current ultracentrifugation methods. The bands found by SDS-polyacrylamide gel electophoresis of WHsAg were the major 25 and 29 kilodalton (kDa) bands and a triplet of 45, 51 and 55 kDa which are thought to be the glycosylated and unglycosylated middle and large WHsAg. Both the antibody and the antigen are valuable reagents for the study of WHV infection.     

Radioimmunoassay and characterization of woodchuck hepatitis virus core antigen and antibody

Solid-phase radioimmunoassays for woodchuck hepatitis virus core antigen (WHcAg) and antibody (anti-WHc) were developed. WHcAg in woodchuck liver homogenates was characterized by ultracentrifugation in CsCl gradients; both heavy (1.35 g/cm3) and light (1.31 g/cm3) cores were obtained from the liver of an animal during acute WHV infection, which is consistent with observations in hepatitis B virus infection in man. Endpoint titers of anti-WHc were higher in chronic WHV carriers than in animals recovered from acute infections. Both IgM and IgG anti-WHc antibodies were produced by infected woodchucks. A survey of colony woodchucks demonstrated that 88/89 animals having one or more markers of past or ongoing WHV infection were positive for anti-WHc. Thus, serum anti-WHc appears to be a sensitive marker of WHV infection.     

Natural and experimental infection of woodchucks with woodchuck hepatitis virus, as measured by new, specific assays for woodchuck surface antigen and antibody.

Solid-phase radioimmunoassays for woodchuck hepatitis virus (WHV) surface antigen (WHsAg) and antibody to it (anti-WHs) were developed. The test for WHsAg could detect as little as 10 ng/ml. In both tests it was necessary to employ radiolabeled WHsAg instead of anti-WHs as the probe because the latter appeared to be labile to the conditions of labeling. The tests were used to characterize naturally acquired and experimental WHV infections of woodchucks. Forty-three of 72 wild-caught woodchucks had serological evidence of WHV infections; 16 of these resulted in chronic infection, and the remainder were self-limiting. All chronically infected animals were positive for WHsAg and DNA polymerase activity. During 3 years of observation, 11 of the 16 WHsAg-positive animals and 3 of the 27 anti-WHs-positive animals, but none of the 21 uninfected animals developed hepatocellular carcinoma. Seroconversion, possibly resulting from infection with WHV, was documented in a chimpanzee inoculated with WHV. An immune adherence hemagglutination test for WHsAg was also developed by using anti-WHs of chimpanzee origin as a reagent, but the test was not useful for detecting anti-WHs of woodchuck origin because of the lability of the latter.     

Progressive cytomegalovirus glomerulonephritis - An experimental model.

Although acute infection with murine cytomegalovirus (MCMV) resulted in a transient focal glomerulonephritis characterized by mesangial inclusions, infection of HA/ICR mice given antilymphocyte globulin (ALG) led to progressive glomerulonephritis and renal failure. ALG alone without virus failed to produce progressive renal disease. Mice given both MCMV and ALG developed severe proteinuria and azotemia with glomerular crescents by 30 days. By immunofluorescence, viral antigen was limited to mesangial zones and glomerular axial poles. Granular deposits of rabbit IgG from ALG, mouse IgG, and C3 along the peripheral glomerular capillary walls were first observed 12 days after infection. By electron microscopy, virus was found only in glomerular mesangial cells that resembled macrophages. Intramembranous and subepithelial deposits in peripheral capillary walls were associated with accumulations of polymorphonuclear leukocytes dissecting into glomerular basement membranes. These observations best fit a multiphasic mechanism of glomerular injury initiated by persistent virus in the mesangium, followed by deposits of rabbit IgG from ALG, mouse IgG, and C in the peripheral capillary walls, resulting in an amplified immune-complex-mediated injury. Because other viruses localize within the glomerular mesangium, viruses should be considered potential causes of mesangial injury and progressive glomerulonephritis.     

Detection of myeloperoxidase in membranous nephropathy-like deposits in patients with anti-neutrophil cytoplasmic antibody-associated glomerulonephritis

Anti-neutrophil cytoplasmic antibody-associated glomerulonephritis is usually classified as a pauci-immune type. However, it sometimes shows immune complex deposition of unknown origin. We examined the glomerular localization of myeloperoxidase by double immunofluorescence and immunoelectron microscopy in cases of anti-neutrophil cytoplasmic antibody-associated glomerulonephritis with membranous nephropathy-like immunoglobulin G deposition to investigate the immune complex antigens in these cases. Six (35%) of the biopsy samples from 17 cases with anti-neutrophil cytoplasmic antibody-associated glomerulonephritis showed granular deposition of immunoglobulin G along the glomerular capillary walls. Light microscopy revealed necrotizing crescentic glomerulonephritis with segmental thickening of the glomerular basement membrane. Electron microscopy showed electron-dense deposits in intramembranous and mesangial areas. However, the size and distribution of the deposits were irregular and segmental in the examined cases, unlike typical global and subepithelial lesions of membranous nephropathy. Double immunofluorescence using Alexa Fluor 594-labeled anti-myeloperoxidase antibody and fluorescein isothiocyanate-labeled anti-immunoglobulin G antibody, as well as immunoelectron microscopy using anti-myeloperoxidase antibody labeled with 25-nm gold particles revealed partial colocalization of myeloperoxidase and immunoglobulin G within the glomerular basement membrane and mesangium. In some cases of anti-neutrophil cytoplasmic antibody-associated glomerulonephritis, myeloperoxidase may form immune complexes and develop membranous nephropathy-like lesions.     

Use of the cross-reactivity with hepatitis B virus antigens and antibodies for the demonstration of a woodchuck hepatitis virus ‘e’ antigen-antibody system

Woodchuck hepatitis virus (WHV)- associated antigens and antibodies were studied using current sensitive radio- or enzyme immunoassays (RIA, EIA). A significant cross-reactivity was observed between hepatitis B surface antigen (HBsAg) and woodchuck hepatitis surface antigen (WHsAg) using RIA or EIA (Abbott Laboratories, North Chicago, III., U.S.A.) although not with two other commercial EIA tested (Organon Technika, Oss, The Netherlands; Behringwerke AG, Marburg, F.R.G.).A weak but significant reactivity was also found when woodchuck sera positive for WHsAg or anti-WHs by immunodiffusion were tested for HBeAg and anti-HBe by RIA, suggesting the existence of a WHeAganti-WHe system in infected woodchucks. The specificity of this e-anti-e reactivity in the woodchuck was further confirmed by successful absorption experiments.WHsAg and WHeAg could be distinguished serologically by immunodiffusion and separated from each other by ultracentrifugation and ammonium sulphate precipitation. A WHeAg preparation was used to boost the presumed natural antibody activity of an immune woodchuck. The specific anti-HBe response detected by RIA during the immunization experiments demonstrated the existence of a soluble WHeAg cross-reacting with the human HBe-anti-HBe system. This was confirmed in immunodiffusion by a partial identity between the precipitin lines formed by the WHeAg-anti-WHe and HBeAg-anti-HBe reaction.Whether the WHe-Ag-anti-WHe system will mimick HBeAg and anti-HBe in all their clinico-pathological correlations, deserves further study.     

A conserved linear B-cell epitope at the N-terminal region of woodchuck hepatitis virus core protein (WHcAg)

Woodchuck hepatitis virus (WHV) is a member of family Hepadnaviridae and closely related to hepatitis B virus (HBV). The WHV core protein (WHcAg) is a strongly immunogenic protein and forms virus-like particles. WHcAg may represent a suitable carrier system for B- and T-cell epitopes. However, the lack of a high expression system for WHcAg and defined antibodies to detect WHcAg prevents the use of this carrier system. In the present study, vectors expressing WHcAg with carboxyl-terminal truncations were constructed to determine the region of WHcAg required for assembly. The first 144 or 149 amino acid residues of WHcAg were able to efficiently assemble into particulate structures. Both truncated forms of WHcAg were accumulated in E. coli as uniform particles with a diameter of 34nm in large quantities and could be purified in milligram scale. As expected, the particles of truncated WHcAg retained the antigenicity of the full length WHcAg. However, denatured WHcAg remained to be reactive with specific antisera, suggesting that WHcAg may possess additional linear B-cell epitopes. Monoclonal antibodies against denatured WHcAg were generated and tested for their specificity. Five antibodies were found to direct the N-terminal region of WHcAg. Due to the conservation of the amino acid sequence in this region of WHcAg and HBcAg, these antibodies recognized recombinant HBcAg as well. Thus, this linear B-cell epitope is conserved on the core proteins of hepadnaviruses.     

Glomerulonephritis induced by murine chronic graft-versus-host reaction.

Severe glomerulonephritis was induced successfully in (B10 x DBA/2)F1 (BDF1) mice by injection of parental DBA/2 lymphoid cells. The mice manifested typical nephrotic syndrome dying around 10 weeks post injection. Electron microscopical examination demonstrated electron dense deposits first in the mesangial matrix, then in the subepithelium compatible with immune complex glomerulonephritis. Subendothelial deposits were not observed. Immunofluorescent study revealed IgG deposition in the capillary wall and IgM in the mesangium early in the process. As the lesion progressed, both IgG and IgM were present in the mesangial area and along the capillary wall. Some glomeruli showed segmental mesangiolysis, suggesting that altered mesangial cells have a role in the development of glomerular change, which together with rise in serum anti-DNA antibody titer suggest that autoantibodies promote the glomerular lesions in this model system.     

Determination of peripheral blood mononuclear cell responses to mitogens and woodchuck hepatitis virus core antigen in woodchucks by 5-bromo-2′-deoxyuridine or 2[3H]adenine incorporation

Summary.  Characterization of cellular immune response to antigens of woodchuck hepatitis virus (WHV) can contribute to the understanding of acute resolving and chronic outcome of hepadnavirus infection. Studies were limited because peripheral blood mononuclear cells (PBMCs) of woodchucks failed to incorporate [³H]thymidine sufficiently. Therefore, we established a non-radioactive proliferation assay for woodchuck PBMCs using 5-bromo-2′deoxyuridine (BrdU) as thymidine analogue. Mitogen- and WHV core protein(WHcAg) induced PBMC proliferation was detected by BrdU incorporation and compared to an assay using 2[³H]adenine. After stimulation with concanavalin A (ConA) and phytohaemagglutinin (PHA) we observed significant PBMC proliferation with both assays. Mitogen-induced nucleoside uptake of PBMCs into cellular DNA was confirmed by detection of 1′,2′[³H]BrdU and 2[³H]adenine in extracted DNA. PBMCs obtained during the acute phase of WHV infection could be stimulated by WHcAg, whereas no WHcAg-induced proliferation of PBMCs was found in WHV-negative animals. PBMCs of chronic WHV carriers showed only a weak response to WHcAg. The established assays will be useful in determining the kinetics of cellular immune responses to different WHV antigens in the course of WHV infection and may provide an insight into mechanisms responsible for chronic outcome of hepadnavirus infection.     

Glomerulonephritis Induced by Murine Chronic Graft-versus-Host Reaction

Severe glomerulonephritis was induced successfully in (B1OxDBA/2)F₁ (BDF₁) mice by injection of parental DBA/2 lymphoid cells. The mice manifested typical ne-phrotic syndrome dying around 10 weeks post injection. Electron microscopical examination demonstrated electron dense deposits first in the mesangial matrix, then in the subepithelium compatible with immune complex glomerulonephritis. Subendothelial deposits were not ob served. lmmunofluorescent study revealed IgG deposition in the capillary wall and IgM in the mesangium early in the process. As the lesion progressed, both IgG and IgM were present in the mesangial area and along the capillary wall. Some glomeruli showed segmental mesangiolysis, suggest ing that altered mesangial cells have a role in the develop ment of glomerular change, which together with rise in serum anti-DNA antibody titer suggest that autoantibodies promote the glomerular lesions in this model system. Acta Pathol Jpn 42 : 325–332, 1992.     

Glomerular endothelial endocytosis of subendothelial electron dense deposits

In 16 of 52 cases (32.5 per cent) of glomerulonephritis associated with glomerular subendothelial electron dense deposits, we detected endothelial endocytosis of osmiophilic material. Twelve patients had systemic lupus erythematosus, two had poststreptococcal glomerulonephritis, and two had idiopathic immune complex glomerulonephritis. The endocytosis was not observed in 13 patients suffering from membranoproliferative glomerulonephritis, in seven suffering from focal sclerosing glomerulonephritis, and in one suffering from hereditary nephritis. Therefore, the potential for endocytosis appeared to vary according to the illness.By light microscopy the 16 specimens exhibited features of acute proliferative glomerulonephritis. All the 14 with tissue sufficient for immunofluorescence study displayed deposition of immunoglobulins and complement along capillary loops and within the mesangium. By electron microscopy many of the subendothelial deposits were unassociated with endocytosis of osmiophilic matter, yet when endocytosis occurred, it was prominent. Hypertrophied endothelial cells occurred in zones of endocytosis. In nine of 16 cases small vacuoles within endothelial cells contained material of an electron density comparable to that of intramembranous and subendothelial deposits, but lyosomal degradation of the material was not noticed. There was frequent direct communication between osmiophilic material in capillary lumens, the subendothelial aspect of the glomerular basement membrane, and endothelial cytoplasmic processes. These communications suggested movement of osmiophilic material in some fashion. Neutrophils featuring vacuoles that contained osmiophilic material were only rarely observed, indicating that they may not assume a major role in degradation of deposits. Phagocytic activity of mesangial cells and nonresident monocytes was not studied.On the basis of our findings we propose that endothelial endocytosis of osmiophilic material might constitute a component of a transport mechanism or a digestive mechanism of glomerular immune deposits located in the subendothelial region.     

Effect of cyclosporin on immune complex deposition in murine glomerulonephritis.

Chronic glomerulonephritis (GN) was induced in N/M mice by daily injections of human serum albumin (HSA). The glomerular lesion was similar to that observed in human membranous GN and was characterized by intense mesangial and capillary loop immunofluorescent staining for HSA, IgG and C3. Electron microscopic examination revealed numerous electron-dense deposits in the mesangium and along the subepithelial side of the glomerular basement membrane, the latter deposits being associated with membranous spikes. Chronically injected mice that had been treated with cyclosporin (CsA) from Day 1 had different patterns of immune complex deposition. Mesangial deposition was apparently unaltered but no subepithelial deposits or spikes were evident. In addition, only two out of 21 HSA-injected mice which began CsA treatment on Day 21 had subepithelial deposits. There was no significant difference in serum levels of HSA-specific IgG between the three groups of mice. CsA treatment would therefore appear to ameliorate the immunopathology of antigen-induced glomerulonephritis in this model without affecting serum antibody levels, and may be of therapeutic value in the treatment of human membranous GN.     

IgA-IgG nephropathy: a clinicopathologic entity with slow evolution and favorable prognosis.

Renal biopsy specimens were obtained from nine patients with proteinuria and persistent macroscopic or microscopic hematuria. Histologic examination either disclosed no abnormality or showed moderate mesangial thickening and occasionally, evidence of focal segmental glomerulonephritis. Immunofluorescent studies revealed diffuse generalized mesangial deposits of IgA, IgG and betalc in all specimens. Fibrinogen deposits were present in the mesangial space in four specimens only, while IgM was uniformly absent. Serial sections of identical glomeruli allowed the localization of betalc within both IgA and IgG deposits. Ultrastructural studies of the renal biopsy specimens showed accumulation of electron-dense material in the subendothelial region of the capillary loops and the mesangium, with thickening of its matrix. Follow-up data indicated a generally good prognosis.     

Effect of IgA deposits on the glomerular mesangium in Berger's disease

In mesangial IgA glomerulonephritis (Berger's disease), the immunoproteins appeared to gain access from the capillary lumen to the mesangium via endothelial fenestrae or via channels between the endothelial cells. The deposits are transported into the deeper mesangium by a process of inhibition or diffusion, with the matrix acting as the head. There are no true channels or grooves in the mesangial matrix for the transport of the immunoproteins. The contractility of the glomerular myoid fibrils may account for the movement of deposits to the hilus for possible removal. There was partial dissolution of the deposits in the mesangial matrix accompanied by loosening of the matrix. No evidence was found for any significant intracellular phagocytosis and digestion. The mesangial deposits directly or indirectly stimulated the cellular hypertrophy and hyperplasia and increased deposition of mesangial matrix. This was accompanied by formation of collagen fibrils within the thickened matrix and led to atrophy of the mesangial cells and sclerosis of the glomeruli.     

Monoclonal antibodies raised to purified woodchuck hepatitis virus core antigen particles demonstrate X antigen reactivity

Woodchuck hepatitis core antigen (WHcAg) particles purified from the liver of chronically infected animals were used for monoclonal antibody production. Most of the putative clones demonstrated anti-WHc specificity. However, the supernatants from several putative clones bound X antigen sequences from woodchuck hepatitis virus (WHV) and hepatitis B virus (HBV). One monoclonal antibody, designated WC9-85 (an IgM), specifically bound hepatitis B X antigen (HBxAg) residues spanning positions 115-131 (peptide 100). WC9-85 also specifically detected liver-derived WHcAg and duck hepatitis B core antigen (DHBcAg) particles in the same CsCl density gradient fractions as did specific anticore and cross-reactive polyclonal anti-x. WC9-85 did not bind to HBcAg particles made by recombinant DNA techniques, in which only the C-gene sequences are expressed, but did bind to liver-derived HBcAg in identical assays. A second monoclonal anti-x, WC8-62, had similar characteristics. Identification of the immunoreactive species in liver-derived core particles by Western blotting showed that WC9-85 bound the major DHBcAg polypeptide having an apparent molecular weight of 35,000 Da. WC9-85 also bound WHcAg-associated bands at approximately 37,000 and 27,000 Da, but little or no binding at the apparent molecular weight of the major WHcAg polypeptide (about 21,000 Da) was observed. These results are consistent with the conclusions that X determinants are associated with core particles purified from naturally infected livers, that such determinants are associated with the major DHBcAg polypeptide and at least two minor WHcAg-associated polypeptides, and that X reactivity is distinct from core and/or e reactivity in hepadnavirus core particles.     

Glomerular lesions in argyric NZB/NZW mice.

Glomerular basement membranes showed strong silver labelling on electron microscopy in NZB/NZW mice fed 12 mmol AgNO3 for 6 weeks at ages from 1-7 months. Although some silver granules were seen within mesangial immune deposits following their concurrent deposition, silver was usually deposited in electron-lucent areas around pre-existing deposits, or formed a boundary to an expanded mesangium when immune deposits subsequently developed. Immune deposits in capillary loops occurred on the outer or inner aspect of the zone of silver labelling.     

Role of immunoglobulin class in mediation of experimental mesangial glomerulonephritis

The contribution of immunoglobulin class to the histology and ultrastructure of renal lesions were examined in an experimental model of glomerulonephritis in which the glomerular deposits were selected to be predominantly IgM or IgG. The selection was accomplished by immunizing and then injecting rabbits with a carrier preparation plus heat-denatured (HD) DNA (n = 11) or ultraviolet-irradiated (uv) DNA [n = 11). It has been shown previously that HD DNA gives rise to an IgM antibody response and uv DNA to an IgG response. Groups of rabbits immunized with each preparation produced largely IgM (HD DNA) or IgG (uv DNA) anti-DNA antibody. After 10 and 20 weeks of injections, animals receiving both antigens developed diffuse mesangial hypercellularity with either IgM or IgG deposits accompanied by C3; by ultrastructural analysis all deposits were confined to the mesangium. By 28 weeks, heavy mesangial IgM and IgG deposits were noted but no quantitative or qualitative differences in the renal histology was observed. Individual animals developed sporadic hematuria and azotemia but proteinuria was not found. These results show that both IgM and IgG can mediate experimental mesangial proliferative glomerulonephritis and that the immunoglobulin class of the glomerular deposits does not influence the appearance of the renal lesion.     

Immune complex disease. VII. Experimental mesangiopathic glomerulonephritis produced by chronic immunization with thyroglobulin.

Immunohistologic and electron microscipic studies were performed on the kidneys of rabbits given daily intravenous injections of porcine thyroglobulin in amounts adjusted to the immune response of the individual rabbits. Glomerular lesions were restricted to the mesangium, were characterized by varying degrees of proliferation of mesangial cells and increase of mesangial matrix, and were accompanied by accumulations of rabbit immunoglobulins, C3, and porcine thyroglobulin. Electron-dense deposits were localized to the mesangium and the adjacent subendothelial space. Less than 10 per cent of the animals with mesangila lesions developed obvious impairment of glomerular function. Thyroglobulin-containing immune complexes were found to be rapidly removed from the mesangium, so that overloading of the mesangium and consequent accumulation of complexes in the adjacent capillary loops could not occur. Thus, the results provide further evidence that when immune complex deposition is restricted to the mesangium, relatively little interference with glomerular function results. This situation is paralleled in man by the lesions of subclinical lupus nephritis, chance proteinuria and hematuria, and the early lesions of Berger's disease.