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Murine lupus is a useful animal model for the study of disease based on some defect in the immune system including the so-called collagen diseases. Seven strains are available in Japan; NZB, NZB/WF1, MRL/Mp-lpr/lpr, BxSB, SL/Ni, NC and C3H/HeJ-gld/gld. Characteristics of these strains of mice are outlined. Care should be taken about applying the knowledge or information obtained from animal models to the analysis of related human disease because there are various genetic or species barriers which cannot be overcome. Therefore, it is rather dangerous to conclude that the pathogenesis of some human disease is similar to that of an animal model from a study of the latter. Therefore, one should divide the data obtained from animal experiments into "factors" and insert each factor in the "formula" of a human disease in order to better understand it. Recent studies by the authors on two strains of lupus mouse; SL/Ni and MRL/Mp-lpr/lpr from such a standpoint are presented and discussed in detail.

Using c-Ha-, c-Ki-, and c-N-ras-specific probes in a RNA-RNA hybridization assay we found enhanced expression of c-Ha-ras protooncogene in stomach adenocarcinomas relative to nonneoplastic epithelium, whereas little or no transcription of either c-Ki- or c-N-ras was detected. Enhanced levels of c-Ha-ras RNA expression were detected in all of the adenocarcinomas examined. Hybridization with c-Ha-ras was also detected in nonneoplastic gastric epithelium adjacent to carcinoma, although the labeling was less intense than that of carcinoma cells. More extensive analysis of the c-Ha-ras p21 expression was then carried out in formalin-fixed, paraffin-embedded tissue sections and extracts from surgically resected stomach tissues using monoclonal antibodies (MAbs) RAP-5 and Y13-259. The data obtained from the immunohistochemical studies were consistent with the results of in situ hybridization assay. Adenocarcinomas were much more reactive with MAb RAP-5 than benign and normal tissues, and the majority of carcinomas demonstrated increased expression of c-Ha-ras p21. Quantitative liquid competition radioimmunoassays using MAb Y13-259 also demonstrated significantly higher levels of c-Ha-ras p21 in extracts from stomach adenocarcinomas than those from normal mucosae. No strict correlation was found between ras p21 expression and the degree of tumor differentiation or histological type. Although advanced carcinomas generally demonstrated higher levels of ras p21 than early carcinomas, no correlation among advanced carcinomas and ras p21 levels was observed in relation to depth of tumor invasion to the muscularis propria, subserosa, or serosa. Benign lesions, in comparison, were much less reactive with MAb RAP-5 than carcinomas. Among the benign lesions tested, dysplastic lesions were more reactive than nondysplastic lesions. Normal stomach mucosa was generally nonreactive with the exception of parietal cells. Our results indicate that transformation of the stomach mucosa from benign to malignant phenotype is associated with an increase in c-Ha-ras p21 expression.

MRL/Mp mice bearing the lymphoproliferation gene (lpr) spontaneously develop systemic granulomatous arteritis coincident with glomerulonephritis (GNP). Although the association of lpr-dependent lymphoproliferation in these mice seems to be a prerequisite for the development of granulomatous arteritis, the genetic basis is poorly understood. The first approach to this problem was to study the ability of another, nonallelic, lymphoproliferative gene, gld (generalized lymphoproliferative disease), inducing arteritis in MRL/Mp mice. The gld gene was placed on an MRL/Mp background by producing reciprocal (MRL/Mp-+/+ X C3H/Hej-gld/gld)F2 hybrid mice. Seventeen percent of these mice with lymphoproliferation had arteritis and GNP, suggesting that more than one lymphoproliferative gene could induce GNP and arteritis in an MRL/Mp background. Next, the effect of rearrangements in the genetic background of MRL/Mp-lpr/lpr mice by hybridization with non-autoimmune lpr-bearing mice was examined. This was done by making MRL/Mp-lpr/lpr X reciprocal (MRL/Mp-lpr/lpr X C57BL/6-lpr/lpr)F1 mice. Thirty-three percent of these mice developed arteritis, but one third of these did not get GNP, thus showing that susceptibility to arteritis was separate from GNP. The histopathologic features of the arteritis in both the F2 hybrids and the backcross mice were granulomatous and were identical to those seen in MRL/Mp-lpr/lpr mice. These findings suggested that it might be possible to dissociated two components (arteritis and GNP) of a severe autoimmune disease of MRL/Mp mice and to study their pathogenesis separately.

The in vitro immunosuppressive ability of a monoclonal IgG2b-anti-trinitrophenyl antibody lacking carbohydrate chains (GORK-Tm) has been compared with the immunosuppressive ability of the intact antibody (GORK). The carbohydrate depletion of GORK-Tm was achieved by culturing the GORK hybridoma cells in media containing tunicamycin, an inhibitor of glycosylation. Both antibodies have been shown to have the same antigen and protein A-binding capacity, although GORK-Tm is deficient in complement fixation, antibody-dependent cellular cytotoxicity, binding to Fc receptors on macrophages, and rapid elimination of antigen-antibody complexes from the circulation. It is shown in these studies that although GORK antibodies are efficient immunosuppressors, suppressing up to 95% of the plaque-forming cell response, GORK-Tm antibody preparations with identical hemagglutinating and antigen-binding capacity are highly deficient in suppressing the immune response. These findings are of interest in two ways. First, it shows that the carbohydrate chains are of great importance for the immunosuppressive effects of IgG. Secondly, it also shows that the isotype IgG2b, in addition to all other murine isotypes, can efficiently suppress the humoral immune response.

Mutagenesis of mAb is a useful means for studying the biologic and pathologic functions of immune complexes. Treatment of the Hy-1.2 hybridoma-producing IgG2a-anti-TNP antibodies with ethylmethanesulfonate provided us with a mutant clone, producing antibodies with reduced capacity for C activation. The antibodies retained normal Ag-binding capacity, staphylococcal protein A reactivity, and association to FcR for IgG on murine macrophages. No significant polypeptide deletion or class-switch was observed, but a significant change in clonotype was revealed by IEF. Intravenous injection of the mutant antibodies in immune complex form induced different tissue distributions of Ag in mice; i.e., more in kidneys and less in spleen, and developed more mesangial deposits in renal glomeruli compared with those of the wild type. Moreover, the production of granulomatous lesions in vivo caused by immune complexes of TNP-Sepharose was augmented by using mutant antibodies. These lesions demonstrated an enhanced accumulation of macrophages with multinucleated giant cells. Availability of this kind of mutant mAb is thus helpful in the elucidation of the biologic functions and consequences of immune complexes.

Removal of asparagine (Asn)-linked carbohydrate chains from IgG antibody molecules reduces their antibody effector functions such as C activation and FcR binding. We have prepared IgG2a mAb with modified structure of carbohydrate chains by treating the hybridoma cells with swainsonine, which inhibits the processing of Asn-linked carbohydrate chains at the site of action of mannosidase II. These antibodies have obtained the capacity to bind lentil lectin and have become sensitive to endoglycosidase H digestion, indicating the structural changes of oligosaccharides from complex type to hybrid type. They behaved in an identical manner to the normal IgG2a antibodies with regards to extracellular secretion, Ag-binding capacity, C-mediated hemolysis and FcR-mediated functions. Critical moieties of Asn-linked carbohydrate chains on IgG molecules to retain their antibody effector functions were discussed.