Genetic diversity of murine rheumatoid factors

Anti-Ig autoantibodies (rheumatoid factors, RF) have been implicated in the pathogenesis of human and murine rheumatoid arthritis as well as in the regulation of normal immune responses. Their genetic origin, clonal diversity, and inducing agents, and the relatedness between RF associated with disease and those occurring under physiologic conditions are not well understood. In this study, the genetic and clonotypic origin of 34 monoclonal IgM RF-secreting hybridomas from arthritic MRL-lpr/lpr and nonarthritic MRL-+/+ and C57BL/6-lpr/lpr mice was examined by RNA hybridization. For this purpose, we used probes for 10 VH and 13 Vk gene families as well as all JH and Jk gene segments. The majority of hybridomas expressed distinct Ig gene segment patterns and, hence, were clonally unrelated. Overall, a variety of different V and J gene segments were expressed in the hybridoma panel, suggesting that a large number of distinct genetic elements participates in expression of RF-like activity. RF from arthritic mice expressed Vk messages from the overlapping Vk22 and Vk28 gene families more frequently than did those from nonarthritic mice. RF from autoimmune MRL mice, both arthritic MRL-lpr/lpr and nonarthritic MRL-+/+, showed skewed JH4 segment usage, whereas those from C57BL/6-lpr/lpr preferentially expressed JH2.     

VH gene analysis of spontaneously activated B cells in adult MRL-lpr/lpr mice. J558 bias is not limited to classic lupus autoantibodies

To determine the genetic origins of lupus auto-antibodies, we analyzed the relationship between VH gene usage and auto-Ag-binding properties of 352 B cell hybridomas derived from MRL-lpr/lpr mice. The hybridomas were derived from neonatal, 1-month-old, 3-month-old, and 6-month-old mice. The experimental strategy provided that the hybridomas were monoclonal at initial evaluation, so the Ag binding and V gene frequencies of the entire population could be determined. Initially, 1032 Ig-producing hybridomas were evaluated for binding to six Ag; VH gene family use was determined in 119 anti-DNA and anti-rabbit thymus extract (RTE) antibodies (autoantibodies) and in 233 age-matched Ig that did not bind to any of the six Ag (nonbinders). Neonatal B cells, including cross-reactive IgM autoantibodies and nonbinder IgM, used relatively 3' VH genes. The majority of B cells in adult mice used VH genes of the J558 family. Although J558 use was significantly higher among the autoantibodies (anti-DNA and anti-RTE) than among the nonbinder Ig, this difference was due to a higher frequency of J558 use by 1-month-old mice. At 3 months, J558 use by the nonbinder Ig increased to the same frequency of J558 use as in the autoantibody population. J558 use in both groups of antibodies exceeded a previously reported estimation of J558 expression in the functional B cell repertoire of young adult MRL-lpr/lpr mice. Several subgroups of antibodies that share properties with pathogenic Ig, including IgG, cross-reactive Ig, and anti-dsDNA autoantibodies, demonstrated a marked preferential expression of the J558 family. These results suggest that there is an age-related bias in the activation of B cells using J558 VH genes in MRL-lpr/lpr mice that is under the influence of a selective force distinct from, or in addition to, an ssDNA or RTE auto-Ag-driven response.     

The autoimmune mouse MRL/Mp-lpr/lpr contains cells with spontaneous cytotoxic activity against target cells bearing self-determinants

Spontaneously cytotoxic cells possessing specificity and having a complex pattern of reactivity directed at least partly against self-determinants develop by the age of 10 weeks in the autoimmune mouse strain MRL/Mp-lpr/lpr (MRL-lpr) and by the age of 6 months in C57BL/6-lprl/lpr. Similar effector cells do not develop in either MRL/Mp-+/+(MRL-+/+) or normal C57BL/6 mice up to 6 months old. Freshly prepared suspensions of both lymph node and bone marrow cells from individual MRL-lpr mice could kill Con A- or LPS-induced blast cells and fresh thymocytes from MRL-+/+ and other mouse strains with strong preference for strains carrying (self)-H-2k determinants in a 4-hr51Cr release assay. These results imply that self-reactive cells are generated as part of the lpr gene defect.     

Cryoglobulinemia induced by monoclonal immunoglobulin G rheumatoid factors derived from autoimmune MRL/MpJ-lpr/lpr mice

A MRL strain bearing the autosomal recessive mutant gene, lpr (lymphoproliferation), spontaneously develops, in addition to a lupus-like syndrome, unique serological and pathological manifestations. Production of high titers of IgG rheumatoid factors (RF) may be related to the formation of extremely large amounts of cryoglobulins and the development of tissue lesions such as necrotizing polyarteritis, arthritis, and glomerulonephritis. To analyze more directly the relationship of IgG RF to the development of cryoglobulins and tissue injuries, we have established four monoclonal IgG RF secreting hybridomas from unimmunized MRL-lpr/lpr mice and determined their pathogenic effects in normal strains of mice. All the monoclonal IgG RF obtained in this study were of the IgG3 subclass and generated cryoglobulins. However, the fact that not only IgG3 Rf monoclonals but also four of five non-RF IgG3 monoclonals were able to form cryoglobulins, which were composed exclusively of each IgG3 monoclonal, indicates that the IgG3 molecule has a unique physicochemical property to self-associate via nonimmunological interaction and the ability to form cryoglobulins. When the in vivo pathogenic activities of these IgG3 RF and non-RF monoclonals were examined, three of IgG3 RF monoclonals with the specificity to IgG2a were able to induce extensive pathologic manifestations including peripheral vasculitis and glomerulonephritis characteristic of patients with cryoglobulinemia. Our results indicate that the IgG3 itself, independently of its specificity, could be a potential source of cryoglobulins and IgG3 RF, combined with its activity of cryoglobulin formation, may play a significant role in the development of glomerulonephritis and cutaneous vascular lesions of ears and foot pads observed frequently in aged MRL-lpr/lpr mice.     

Influence of the Ig H chain locus on autoantibody production in autoimmune mice.

Autoimmune disease is influenced by multiple genes. In this study, we investigated the role of one genetic locus, Ig H chain. IgG2a antichromatin, anti-ssDNA, and antihistone autoantibodies (autoAb) from (MRL/Mp-lpr/lpr x C57BL/6-lpr/lpr), (Ighj/b); (C57BL/6-lpr/lpr x C57BL/6-lpr/lpr-Igha), (Ighb/a); and (MRL/Mp-lpr/lpr x MRL/Mp-lpr/lpr-Ighb), (Ighj/b) mice were determined using allotype-specific ELISA. Strikingly, antichromatin and antihistone antibodies (Ab) were comprised of significantly more b allotype than either a or j allotype in all cohorts of F1 mice examined. In mice that produced anti-Sm Ab, the b allotype was used preferentially for these autoAb as well. However, no allotype skewing was observed in IgG2a Ab directed against TNP or DNA, or for total IgG2a. An Igh recombinant locus was utilized to examine the genetic control of b allotype skewing in lpr mice and in chronic graft vs host disease. In both models, the VH region did not appear to be responsible for the preferential use of b allotype. These results indicate a contribution to autoimmunity by the Igh locus and raise the possibility that Ig allotype may influence autoimmune disease by its effect on the production of certain autoAb.     

Direct quantitative in situ hybridization studies of Ig VH utilization. A comparison between unstimulated B cells from autoimmune and normal mice

This study examines Ig VH utilization in murine lupus with emphasis on the relative contribution of 3' and 5' gene families. We used in situ hybridization with 35S-labeled ssRNA probes to detect VH expression in individual spleen cells. Cells were taken from unmanipulated animals, and were not stimulated in vitro. This approach allows analysis of VH usage among only those B cells which have undergone activation in vivo, while minimizing the potential for skewing in vitro. We compared usage of the 3' 7183 and Q52 families with the more 5' J558 family in adult NZB, MRL-lpr/lpr, and nonautoimmune NIH Swiss mice. VH utilization in the autoimmune strains was proportionate to VH family size, and was not biased toward the 3' families when compared with the Swiss repertoire. Moreover, 3' skewing did not develop in NZB mice with increasing age. Thus, systemic autoimmunity is not associated with impaired normalization of the adult repertoire away from the 3' bias of early ontogeny. Instead, our data support a stochastic model for VH gene usage in the activated B cells and plasma cells of adult lupus mice.     

Variable region sequence analysis of anti-DNA antibodies: evidence for a family of closely related germ-line VH genes encoding lupus autoantibodies.

Cloning and sequencing of the V regions of the anti-DNA monoclonal antibodies (mAbs), H438 and H130, indicate that H438 is encoded by a J558 VH gene, a single D region nucleotide, and unmutated JH1, V kappa-1C and J kappa 1 genes, and the H130 L chain is encoded by a V kappa-21 subgroup gene J kappa 1 gene. Identification of VH438, which shared VH hybridization pattern with 6% of a panel of 352 MRL/lpr hybridomas, suggests that the frequency of J558 use among spontaneously activated B cells in MRL/lpr mice is greater than previously reported. The VHH438 J558 family gene is identical to VHPAR, which encodes the independently derived MRL/lpr autoantibody, MRP-2, and is highly homologous to the previously reported VHH130, which is identical to a BALB/c germ-line VH gene. Comparison of consensus sequences of homologous autoantibodies and previously reported restriction mapping suggest that a minimum of three highly related J558 germ-line genes encode lupus autoantibodies.     

Idiotypic analysis of a monoclonal anti-Sm antibody. II. Strain distribution of a common idiotypic determinant and its relationship to anti-Sm expression

The idiotypes borne by Y2, a monoclonal anti-Sm antibody of MRL-lpr/lpr mouse strain origin, were investigated to elucidate genetic mechanisms in this autoantibody response. An anti-Y2 anti-idiotypic antiserum was raised in a rabbit and was rendered specific for idiotype by extensive absorption with globulins of the B6 and BALB/c strains as well as the BALB/c myeloma UPC 10. By using a sensitive assay for idiotype by inhibition ELISA, the Y2 determinant was found to be commonly expressed in sera of MRL-lpr/lpr and MRL-+/+ mice. Moreover, sera of several normal strain mice also bore the idiotype and, in mice bearing the lpr gene, idiotype levels were increased 1.5 to fivefold, even in the absence of a serum anti-Sm response. The relationship of this idiotype to anti-Sm expression was further assessed by determining the idiotype content of affinity-purified anti-Sm antibodies from MRL-lpr/lpr mice. Anti-Sm from serum pools or individual animals showed no significant enrichment of the Y2 idiotype in comparison to unselected MRL-lpr/lpr IgG. These results suggest that the Y2 idiotype defines only a minor component of the anti-Sm autoantibody response, and that most antibodies with this determinant express other antigenic specificities.     

Autoimmune CD4+ T cells interfere with immune tolerance to a thymic-independent antigen

The ability of autoimmune T cell subsets to interfere with tolerization of B cells can be studied by using thymic-independent Ag. We have defined an abnormality within the CD4+ T cell compartment in young NZB and MRL-lpr/lpr mice by studying tolerance of spleen and B cells to the thymic independent Ag, fluorescein-Brucella abortus. Tolerization of spleen cells is defective in MRL-lpr/lpr mice, but not MRL-+/+ or C3H.lpr mice, suggesting that the defect requires both the autosomal MRL background and the lpr gene to be present. T enriched cells from NZB mice and from MRL-lpr/lpr mice (but not MRL-+/+ or C3H.lpr mice) reverse tolerance in spleen cells from [NZB X DBA/2]F1 and C3H/HeJ mice, respectively. This interference is removed by treatment with anti-CD4 antibody and C. Supernatants from cultured T cells of NZB and MRL-lpr/lpr mice also prevent tolerance in spleen cells of [NZB X DBA/2]F1 and MRL-+/+ mice, respectively, unless CD4+ cells are removed prior to T cell culture. Removal of T cells from NZB and MRL-lpr/lpr spleen cells allows normal tolerization of B cells, which is abrogated by the addition of syngeneic T cells or cultured T cell supernatants. This effect also depends on the presence of CD4+ T cells. These studies show that in MRL-lpr/lpr mice, through interaction of the lpr and MRL background genes in a T cell subset, and in NZB mice, CD4+ T cells interfere with B cell tolerance to a thymic-independent Ag.     

IgG3 is the major source of cryoglobulins in mice

A total of 20 of 23 IgG3 mAb derived from unmanipulated autoimmune MRL/MpJ-lpr/lpr mice was shown to generate cryoglobulins which were composed exclusively of IgG3. Although three IgG3 mAb failed to develop cryoglobulins, they were able to bind nonspecifically to any IgG3 molecules as efficiently as cryoprecipitable IgG did. The direct role of the gamma 3 constant region for the generation of cryoglobulins was demonstrated by the following findings: 1) the cryoglobulin activity was independent of the specificity of the IgG3 mAb, 2) no mAb other than those of the IgG3 subclass, including IgM rheumatoid factors (RF), generated cryoglobulins, and 3) the cryoglobulin activity was gained after the Ig class switch of mAb from IgM to IgG3. Analysis of Ig components in three different sources of cryoglobulins, either induced by the injection of bacterial LPS or by the infection with Plasmodium yoelii in BALB/c mice or developed spontaneously in MRL/MpJ-lpr/lpr mice, revealed the selective concentration of IgG3 in these cryoglobulins; greater than 99%, 73% and 58% of IgG recoverable from these three cryoglobulins, respectively, were IgG3. This further attests to the major role of IgG3 in the generation of cryoglobulins in mice. In addition, the enhanced formation and even induction of IgG3 cryoglobulins in the presence of IgM anti-IgG3 RF mAb, and the enrichment of IgM RF in LPS- or malaria-induced cryoglobulins indicated that IgM RF can be involved in the generation of cryoglobulins by interacting with noncryoprecipitable IgG3 as well as cryoprecipitable IgG3.     

Organization of the murine immunoglobulin VH complex in the inbred strains.

Deletion mapping analyses have been employed to order the heavy chain variable region (VH) gene families in three inbred murine strains. These nine VH gene families have been positioned with respect to the J558 and 3660 VH families in A/J (Ighe) as follows: 3609-J558-(J606,VGAM3-8,S107)-3660-(X24,Q52,7183 )-DH. Maps generated with respect to J558 in the BALB/c (Igha) and C57BL/6 (Ighb) strains are consistent with these results. The organization of the VH complex produced by deletion mapping is quite different from the accepted map generated by other methods, particularly in that J558 is more DH distal and 3660 is more DH proximal than previously thought. The order presented here is compatible with VH rearrangement frequencies suggesting preferential utilization of DH-proximal VH gene segments. Our data also indicate that interspersion of some VH family members may be a common feature of the murine VH complex since the 3609 VH family is interdigitated in the three strains and a Q52 VH gene segment is interspersed in C57BL/6.     

Abnormal IgG galactosylation and arthritis in MRL-Fas(lpr) or MRL-FasL(gld) mice are under the control of the MRL genetic background.

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.     

Eleven MRL-lpr/lpr anti-DNA autoantibodies are encoded by genes from four VH gene families: a potentially biased usage of VH genes

The genes encoding 11 independently derived anti-DNA autoantibodies from the lupus-prone mouse strain, MRL-lpr/lpr, were examined with VH, D, and JH gene probes. These autoantibodies do not define new VH gene families, since all of the autoantibodies were encoded by VH genes from four of the nine known gene families. A minimum of nine different VH genes encoded this panel of 11 anti-DNA autoantibodies. These results are consistent with the stochastic use of the VH gene repertoire and the expression of multiple VH genes. However, the data is also consistent with a biased usage of the VH gene repertoire. First, two pairs of autoantibodies, one from the J558 family and one from the 7183 family, appear to express identical or closely related VH genes as determined by the position of two restriction enzyme sites 5' of the expressed VH genes. In addition, three autoantibodies that appear to be sister clones might define a third VH gene that is used repeatedly. Secondly, about 45% of the panel is encoded by the Q52 and 7183 families, which are the 3' most families. These families have been shown to be preferentially rearranged early in B cell ontogeny. This suggests that some anti-DNA autoantibodies might originate from a population of B cells that predominate early in ontogeny. An alternative hypothesis is that the potential bias in VH gene and gene family usage could be due to antigen selection. All four JH genes are expressed, although the JH1 gene appears to be underutilized in both expressed and unexpressed rearrangements. Two members of the panel that bind double-stranded DNA were encoded by two different VH gene families, the S107 family and the J558 family.     

Molecular characterization of the A/J J558 family of heavy chain variable region gene segments

The nucleotide sequences of the coding as well as the flanking regions of 11 A/J J558 heavy chain variable region (VH) gene segments are presented. Among these J558 VH segments was the unrearranged germline VH gene segment recruited in the predominant A strain-specific anti-arsonate response. Three other VH gene segments that are greater than 92% related to the p-azophenylarsenate (Ars) A VH gene segment were also isolated. Detailed analysis of the nucleotide sequences of these as well as the remaining seven J558 VH gene segments reveal that the J558 VH gene family is composed of distinct, but related, J558 VH subfamilies. Deletion mapping analyses were used to position the Ars A VH gene segment proximally with respect to the DH-JH clusters within the J558 VH gene family and distally with respect to its own J558 subfamily. The documentation of J558 VH subfamilies is discussed in the context of J558 VH family evolution and diversification.     

Autoreactive T cells in MRL/Mpr-lpr/lpr mice. Characterization of the lymphokines produced and analysis of antigen-presenting cells required

Lymph node cells from 4-wk-old MRL/Mp-lpr/lpr mice, but not from MRL/Mp-+/+ mice, when cultured in vitro for 5 to 7 days, will spontaneously proliferate and produce IL-2. We examined the expression of several cell surface Ag on lymph node cells from MRL/Mp-lpr/lpr mice before and after in vitro culture. There is an increase in the expression of Thy-1, L3T4, IL-2R, T cell activating protein, T cell receptor, and T3 complex on the surface of cultured cells. Cultured cells produced IL-3, IFN-gamma, and small but detectable amounts of IL-1 in addition to IL-2. Gamma irradiation of APC from young MRL/Mp-lpr/lpr mice or treatment of APC with a mAb (J11D) and C, completely abrogated their stimulatory capacity. These experiments suggest that B cells are the predominant APC responsible in the activation of autoreactive T cells in MRL/Mp-lpr/lpr mice. Lymph node cells from C57BL/6-lpr/lpr or C3H-lpr/lpr mice were unable to spontaneously proliferate or produce IL-2. Lymph node cells from (MRL/Mp-lpr/lpr x C57BL/6-lpr/lpr) F1 mice or (C3H-lpr/lpr x MRL/Mp-lpr/lpr) F1 mice did proliferate and produced IL-2 after in vitro culture. Using T cells from these F1 animals and APC from each parental haplotype, we found that APC from MRL/Mp-lpr/lpr mice induced more proliferation and greater amounts of IL-2, when compared to APC from F1 animals. APC from C57BL6-lpr/lpr mice or C3H-lpr/lpr were unable to induce spontaneous proliferation and IL-2 production. Therefore, B cells from MRL/Mp-lpr/lpr mice appear to possess unique features that enable them to activate autoreactive T cells more effectively than B cells from other mice bearing the lpr/lpr gene.     

A pathogenic monoclonal antibody, G8, is characteristic of antierythrocyte autoantibodies from Coombs'-positive NZB mice.

With age, NZB mice develop anti-RBC autoantibodies resulting in the development of autoimmune hemolytic anemia. We now have evidence that this spontaneous autoantibody response consists of antibodies that are similar in specificity and Id expression to a pathogenic autoantibody (G8) that was cloned from an autoimmune NZB mouse. Similar to autoantibodies eluted from Coombs'-positive mouse E (MRBC), the G8 mAb recognizes native (unmodified) MRBC but not RBC from other species. Interestingly, G8 and four additional mAb bind with a higher titer to bromelain-treated MRBC than to native MRBC. Nucleotide sequence analysis reveals, however, that unlike "natural" antibodies that react solely with bromelain-MRBC, G8 is encoded by a J558 VH gene and a V kappa 12,13 L-chain gene. Thus G8 is clearly distinct from antibodies to bromelain-MRBC which are encoded by unrelated V genes. Instead, the sequence of the G8 VH chain was found to be nearly identical to that of an anti-DNA mAb derived from an MRL-lpr/lpr mouse. The results suggest Coombs'-positive autoantibodies from NZB mice are not derived from "natural" antibodies, but rather, consist of a restricted set of autoantibodies expressing the G8 IdX.     

Murine cryoglobulinemia: pathogenic and protective IgG3 self-associating antibodies

A murine IgG3 mAb, 6-19, derived from autoimmune MRL-lpr/lpr mice, is a rheumatoid factor (RF) specific for IgG2a and is able to generate cryoglobulins via nonspecific IgG3 Fc-Fc interaction. Intra-peritoneal passive transfer of ascites containing the 6-19 mAb into BALB/c mice induces skin leukocytoclastic vasculitis and acute glomerulonephritis associated with cryoglobulinemia. Because IgG3 interact with each other, we have determined whether noncryoprecipitating IgG3 mAb were able to inhibit the cryoprecipitation of 6-19 mAb and the development of related tissue lesions. In vitro, the cryoprecipitation of 6-19 mAb was almost completely inhibited by a fourfold excess of a noncryoprecipitating non-RF IgG3 (9-106) mAb derived from MRL-lpr/lpr mice. Cryoprecipitation of five other IgG3 mAb was similarly inhibited by the 9-106 mAb, and two other noncryoprecipitating IgG3 mAb, including the 2-6D antinuclear autoantibody, inhibited the cryoprecipitation of 6-19 mAb. In vivo, pretreatment of BALB/c mice with 9-106 or 2-6D mAb prevented the development of skin vasculitis and glomerulonephritis induced by the 6-19 mAb. The cryoglobulin formation was greatly diminished in 9-106 or 2-6D mAb-treated mice, although their serum levels of 6-19 mAb and RF activity were comparable to those of control mice. This indicated that pretreatment with non-cryoglobulin IgG3 inhibited the cryoglobulin generation and cryoglobulin-associated tissue lesions induced by an IgG3 RF cryoglobulin-generating mAb. These results suggest that the balance of formation of IgG3 autoantibodies with or without the cryoglobulin activity may be critical for the development of IgG3 cryoglobulin-mediated tissue lesions in murine lupus, particularly in MRL-lpr/lpr mice.