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MRL mice bearing the lpr (Fas) or gld (Fas ligand) mutation, MRL-Fas(lpr) or MRL-FasL(gld), respectively, develop arthritis similar to rheumatoid arthritis, but C3H and C57BL/6 mice bearing such mutations do not. In MRL-Fas(lpr) mice, agalactosylated oligosaccharides in serum IgG increase significantly in comparison to MRL-+/+ mice without arthritis. In this study, an increased level of agalactosylation in IgG, as compared to MRL-+/+, was found in both MRL-Fas(lpr) and MRL-FasL(gld) mice. In contrast, the incidence of IgG without galactose was comparable among C3H-Fas(lpr), C3H-FasL(gld), and C3H-+/+ mice as well as between C57BL/6-Fas(lpr) and C57BL/6-+/+ mice. These results suggest that the increase in agalactosylated IgG and the development of arthritis in MRL-Fas(lpr) and MRL-FasL(gld) mice are controlled by the MRL genetic background.

A simple method based on the polymerase chain reaction (PCR) was developed for detecting the relative mRNA amounts of the four mouse IgG subclasses in total RNA samples and is described in this report. The main features of this method are, first, cDNA amplification including VH through the constant region(CH2 domain) of each IgG subclass with a set of consensus PCR primers(VH1BACK and 32P-labeled C gamma 32), and secondly, cleavage of the amplified DNA fragments with BamHI and XhoI endonucleases which act at distinct cleavage sites in the constant region of each IgG subclass. The radioactive intensities of the different sized fragments separated on polyacrylamide gel were used to show the relative amounts of IgG subclasses at the RNA level. This method provides clear detection of each IgG subclass using RNA samples from tissues in which Ig-producing cells are rare.

MRL/Mp-lpr/lpr(MRL/lpr) lupus mice develop glomerulonephritis in which the histopathological manifestations of the disease are characterized by diffuse cell-proliferative, crescentic, and/or wire loop-like lesions, resembling those of human lupus nephritis. Although these lesions are thought to be mediated by antibodies, little data is available to explain these regular variations in glomerular lesions induced by antibodies at the monoclonal level. We studied glomerular lesions of normal or severe combined immunodeficient mice injected with nephritogenic immunoglobulin G3-producing hybridoma clones (2B11.3 and 7B6.8), which we previously established from an unmanipulated MRL/lpr mouse. Both clones caused increased serum levels of immunoglobulin G3 with identical patterns over time and both induced glomerular deposits of immunoglobulin G3 and C3. However, 2B11.3 and 7B6.8 induced glomerular lesions that differed in their histopathological manifestations. The 2B11.3 clone generated cell-proliferative lesions associated with marked Mac-2-positive macrophage infiltrates, but the 7B6.8 clone induced lesions characterized by subendothelial hyaline deposits resembling wire loops. The latter was not associated with significant inflammatory cell infiltrates at any point throughout the progression of the lesion. Thus, our findings suggest that the histopathological variation in glomerulonephritis seen in MRL/lpr mice results from clonally expanded B cell clones that produce nephritogenic antibodies with different pathogenic potencies.

Production of IgG3 in MRL/Mp-lpr/lpr (MRL/lpr) lupus mice is one of the major factors to develop glomerulonephritis (GN) in these mice. To examine molecular characteristics of IgG3 responsible for GN in these mice, hybridoma clones producing IgG3 antibodies were prepared from one unmanipulated MRL/lpr mouse. Two clones, 2B11.3 and 7B6.8, were nephritogenic; that is, they caused severe glomerular lesions when injected to normal mice, moreover with a different histopathological manifestation. The 2B11.3 clone generated diffuse cell-proliferative lesions, while those induced by the 7B6.8 clone resembled wire loop lesions in human lupus nephritis. The cDNA sequence analysis of 7B6.8 antibody and the other IgG3 antibody, 1G3, non-nephritogenic, revealed that the C regions of the heavy and light kappa chains were completely the same between them. Furthermore, they were identical in deduced amino acid sequences to those from non-autoimmune BALB/c mice, indicating no allelic difference of Igh-8 between these two strains. The V regions of 2B11.3 and 7B6.8 antibodies were composed of different sets of VH, D, JH, Vk and Jk. Although both of the VH belonged to the J558 family, they seemed to use a different VH germline gene. These findings suggest that GN in MRL/lpr mice is generated by the expansion of clonally different B cells producing particular antibodies possibly with a different pathogenetic potency.

MRL/Mp-lpr/lpr (MRL/lpr) mice spontaneously develop lethal glomerulonephritis (GN) similar to human lupus nephritis, associated with the expression of lymphoproliferation gene lpr. To examine whether a particular IgG subclass is responsible for development of GN in these mice, first quantitative analysis of IgG subclasses in serum and in kidney eluates was performed. Although IgG2a was the dominant subclass in serum throughout the lifespan of mice, the IgG3 level in kidney eluates was three times higher than that of IgG2a at the 16 wk of age, which is the time of onset of development of severe GN. In sera of the 12-wk-old mice, half of the IgG3 was in immune complex form, whereas IgG2a in this form was only 17% of the total amount. Second, cyclosporin A, which ameliorates GN in MRL/lpr mice despite autoantibody production, was found to reduce serum IgG3 and mRNA levels, associated with the revision of cationic shift of the serum IgG3 spectrotype seen in isoelectric focusing. Third, among the hybrid mice with non-autoimmune-prone C3H/HeJ-lpr/lpr (C3H/lpr) mice, MRL/lpr x (MRL/lpr x C3H/lpr) F1, in which the genetic background for GN is likely segregated, the mRNA level for IgG3 correlated well with the degree of glomerular lesion. These findings indicate that production of IgG3 in MRL/lpr mice is one of the major factors responsible for development of GN in these mice, and that this is due to the genetic background of the MRL strain.

The complement (C) dependency of IgG-mediated enhancement of the antibody response was investigated by immunizing mice with trinitrophenyl-coupled keyhole limpet hemocyanin (TNP-KLH) and either a C-activating TNP-specific monoclonal IgG2a antibody (Hy-1.2) or a mutant, non-C-activating variant of Hy-1.2 (M12). Hy-1.2 as well as M12 efficiently enhanced the anti-KLH response, although Hy-1.2 was more active. In addition, also a naturally non-C-activating TNP-specific IgG1 antibody enhanced the response to TNP-coupled bovine serum albumin. Moreover, C-activating IgG could enhance the antibody response in mice depleted of C3 by treatment with cobra venom factor. These findings suggest that the classical pathway of C activation is not required for IgG-mediated enhancement.

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.

The structure of the Fc fragment of human IgG1 immunoglobulin is compared for the native and recombinant proteins. A recombinant human Fc fragment was expressed by an E. coli system [Kitai K., Kudo T., Nakamura S., Masegi T., Ichikawa Y. and Horikoshi K. (1988) Appl. Microbiol. Biotechnol. 28, 52-56]. The recombinant protein, which presumably lacks oligosaccharides, was used along with the native human Fc fragment obtained by proteolytic digestion of a myeloma IgG1 protein. 1H NMR has been employed along with circular dichroism and fluorescence spectroscopy to discuss the structure of these two types of proteins. It has been concluded that (1) the overall structure of the recombinant protein is quite similar to that of the native protein, which possesses asparagine-linked oligosaccharides, but (2) a significant difference in structure exists in the neighborhood of the glycosylation site. The difference in the effector functions for the two kinds of the Fc proteins has been briefly discussed in terms of the structural change detected by 1H NMR.

The Fc portion of IgG is recognized by macrophages via Fc receptors on their cell surface. The recognition of human IgG1-Fc by macrophages was compared for the native and recombinant proteins by a novel assay method using competitive inhibition of the aggregated IgG-mediated chemiluminescence reaction of human macrophage cell line THP-1. Recombinant Fc proteins, prepared in an Escherichia coli system, which lack oligosaccharides and retain normal staphylococcal protein A reactivity and, to a lesser extent, C1q binding capacity, showed inhibitory ability greater than 500 times lower than that of native Fc fragments obtained by proteolytic digestion of myeloma proteins. This suggests that the oligosaccharide chains on IgG1-Fc molecules may be critical for recognition by Fc receptors on macrophages.

Feedback suppression of the antibody response by IgG is known to be dependent on intact Fc regions. However, it is not clear which of the Fc-mediated effector functions is required. In the present report we have studied whether ability or inability of the IgG antibodies to activate the complement system was of consequence for their immunosuppressive effect. First, a monoclonal IgG1-anti-2,4,6-trinitrophenyl (TNP) antibody, unable to activate complement via the classical or alternate pathway, was shown to be able to inhibit more than 90% of the in vivo sheep erythrocyte-specific antibody response in mice when TNP coupled to sheep erythrocytes was used as antigen. Second, we investigated the immunosuppressive ability of a non-complement-activating mutant IgG2a-anti-TNP monoclonal antibody. The mutant differs from the wild type by a single amino acid substitution in the CH2 domain leading to inability to fix complement factor C1q. However, the mutant has the same affinity for antigen and the same Fc receptor-binding capacity as the wild type antibody. It is demonstrated that the mutant was as efficient as the wild type antibody in inhibiting an in vitro antibody response to TNP-coupled sheep erythrocytes. These findings confirm the non-determinant specificity and Fc dependence of IgG-mediated suppression, and show that the Fc-mediated effector mechanism is independent of complement activation. The results instead suggest binding to Fc receptors as a necessary step in feedback immunosuppression and favor inactivation of B cells by cross-linking of Fc and antigen receptors on their surface rather than elimination of antigen by complement-dependent phagocytosis as the effector mechanism.

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.