Intrastrain recurrent idiotypes among anti-DNA antibodies of (NZB X NZW)F1 hybrid mice

Immunization of NZB and A/J mice against an anti-DNA hybridoma antibody (F227) derived from (NZB x NZW)F₁ (B/W) mice allowed the preparation of two anti-idiotype antisera. These two reagents were shown to recognize different idiotopes of the F227 monoclonal antibody. NZB anti-idiotypic antibodies recognized non-ligand-modifiable idiotypic determinants. These idiotopes were private or present at undetectable level in BW mouse sera since it was found that only two of the 24 B/W mouse sera tested were recognized by these antibodies. Conversely, A/J anti-idiotypic antibodies recognized partially ligand-modifiable idiotopes which were found in all B/W mouse sera tested. These results demonstrate that anti-DNA antibodies share similar idiotypic specificities and suggest that these autoantibodies occur as families of structurally related proteins.     

Molecular and serological diversity of anti-DNA autoantibodies from NZB and (NZB X NZW) F1 mice

We have carried out an analysis of the serological and molecular diversity of a panel of monoclonal anti-DNA autoantibodies and serum autoantibodies from NZB and (NZB X NZW) F1 mice, in an attempt to obtain insights into the mechanisms responsible for the development of systemic autoimmune disease. Our data show that the autoantibodies are quite diverse. A dominant, binding-site idiotope on one of our monoclonal autoantibodies is expressed at variable levels in anti-DNA binding antibodies in the sera of both NZB and (NZB X NZW) F1 mice, but on none of the other monoclonal autoantibodies in our panel. We have cloned and sequenced the heavy chain variable region (VH) gene of one anti-DNA hybridoma and by hybridization have determined the VH and V kappa gene segments expressed by 14 others. All of the autoantibodies express members of known V gene subfamilies. A total of four different VH and at least six V kappa subfamilies are expressed by the hybridomas. Thus, a broad spectrum of the total murine Ig repertoire is represented in the anti-DNA autoantibodies present in these strains.     

Autoantibodies to anti-DNA with anti-allotypic and anti-idiotypic specificities in (NZB X NZW)F1 mice

The monoclonal A52 (IgG2b, kappa) anti-DNA autoantibody represents a major cross-reactive idiotype in the murine and human autoimmune response to DNA. Examination of sera and purified IgG derived from (NZB X NZW)F1 mice showed that these mice develop an age-dependent binding reactivity with the pure anti-DNA IgG. Three monoclonal antibodies possessing this reactivity were prepared from unprimed female (NZB X NZW)F1 mice. One of these monoclonal antibodies appeared to be directed against allotypic determinants present in the NZB IgG2b; the other two antibodies exhibited a marked preference for idiotypic determinants of the A52 IgG. The IgG anti-allotype and anti-idiotype activities in (NZB X NZW)F1 mice may, therefore, represent the products of a deregulated immune system and/or constitute the normal elements of a functional immune regulation system.     

Immunohistochemical characterization of sialadenitis in NZB X NZW F1 mice

The spontaneously developing sialadenitis in female autoimmune NZB X NZW F1 (NZB/W) mice has been studied with the help of immunohistochemistry and monoclonal antibodies to cell surface antigens. Semiquantitative assessment of stained cells within the infiltrates disclosed a progressive focal inflammation most pronounced in submandibular and parotid glands. The majority of cells expressed Ly-1 (all T cells) and L3T4 (T helper) phenotype, whereas only few Lyt-2 (cytotoxic/suppressor) expressing T cells were seen. A large proportion of the infiltrating cells stained for Ia antigens, which was also found on salivary gland ductal epithelium in the proximity of lymphoid infiltrates. The phenotypic pattern in sialadenitis of NZB/W mice thus closely resembles the pattern previously described for human Sj?gren's syndrome (SS). Accordingly, immunomorphological analysis of the NZB/W sialadenitis may be useful in further studies of pathogenesis and therapy of both experimental and human SS.     

Requirement of H-2 heterozygosity for autoimmunity in (NZB X NZW)F1 hybrid mice

In the F1 hybrid of autoimmune New Zealand Black (NZB) and phenotypically normal New Zealand White (NZW) mice, there occurs a severe systemic lupus erythematosus (SLE)-like autoimmune disease more fulminant than that found in the parental NZB mice. To determine the role of the H-2 complex in the pathogenesis of autoimmune disease of the (NZB X NZW)F1 hybrid, we developed H-2-congenic NZB (NZB.H-2z) and NZW (NZW.H-2d) strains, and compared the degree of autoimmune features between congenic H-2d/H-2d and H-2z/H-2z homozygous F1 hybrids and the original H-2d/H-2z heterozygous (NZB X NZW)F1 hybrid. We found that autoimmune features such as productions of IgG class anti-DNA antibodies and retroviral gp70 immune complexes and the development of renal disease were to a great extent reduced in both H-2 homozygous F1 hybrids, as compared with the H-2 heterozygous (NZB X NZW)F1 hybrid. It would thus appear that the heterozygosity of H-2d haplotype derived from NZB and H-2z from NZW is essential for the autoimmune disease characteristic of the (NZB X NZW)F1 hybrid.     

Sequential study of C 1 q-binding substances and of anti-DNA antibodies in (NZB X NZW)F1 mice: evidence for an acute phase of the lupus disease in 2-month-old mice.

Longitudinal studies of C 1 q and DNA-binding substances were performed in sera from 38 (NZB X NZW)F1 female mice between days 39 and 150 of life. Results suggest a two-phase evolution of circulating immune complexes and anti-DNA antibodies in young (NZB X NZW)F1 mice and show the existence of an acute phase of the disease during the second month of life of these mice.     

Variable region primary structures of monoclonal anti-DNA autoantibodies from NZB/NZW F1 mice

VH and VL region primary structures of five NZB/NZW F1 derived monoclonal anti-DNA autoantibodies were determined from cloned cDNA. Comparative analysis of VH genes showed that except for two VH genes that shared complete identity the overall VH gene usage was diverse. Comparison of VH genes with those utilized in a variety of antibody responses showed they were generally unique to the autoanti-DNA response although framework homologies allowed assignment of four of five VH genes to existing murine heavy chain gene families. Only one out of five D segments shared homology to existing germline D segments, and all were rearranged to JH3. V kappa genes showed restriction for four of five light chains to the V kappa 1 subgroup. The V kappa 1 subgroup has been shown previously to be utilized in several anti-DNA autoantibodies as well as a variety of antibodies to exogenous antigens. H and L chain amino acid residues associated with the active site of a ssDNA specific autoantibody, 04-01, are discussed based on recently obtained crystallographic data.     

Effects of the thymic microenvironment on autoantibody production in (NZB X NZW)F1 mice

The effects of the thymic microenvironment on autoantibody production in (NZB X NZW)F1 mice were studied. Neonatally thymectomized male and female F1 mice reconstituted with a parental or F1-irradiated thymic lobe were compared to nonreconstituted and sham-thymectomized controls. While maleness retarded the spontaneous production of ss- and ds-DNA antibodies, thymic grafts did not suppress antibodies to ss-DNA in either sex, but did suppress the production of antibodies to ds-DNA in female mice. A unique property of NZB thymic grafts was the inability to suppress anti-RBC antibodies in male mice. Thus, (i) the gender of the F1 recipient was the most important determinant of production of antibodies to ss-DNA, (ii) either maleness or the thymic microenvironment could retard production of anti-ds-DNA antibodies, and (iii) both gender and the thymic microenvironment were important in the regulation of anti-RBC antibody production. Since the administration of thymosin did not suppress autoantibody production, the effects of the thymic grafts was not solely via thymic hormone production. These studies suggest that sex hormones and/or the thymic microenvironment can exert a suppressive effect on autoantibody production and that autoantibodies differ in their susceptibility to such suppression.     

Hidden anti-nuclear antibodies in sero-negative systemic lupus erythematosus patients and in NZB and (NZB x NZW) F 1 mice

Anti-nuclear antibodies could be demonstrated in sero-negative patients with immune complex glomerulonephritis by means of an acid dissociation method. The antibodies were also found in sero-negative NZB and (NZB × NZW) F₁ mice with complex glomerulonephritis. It is suggested that the antigen is bound to anti-nuclear antibodies, thus forming circulating soluble immune complexes, which cause the glomerular deposits.     

Comparison of natural antibodies to autoantibodies arising during lupus in (NZB x NZW)F1 mice

Autoantibodies arising in (NZB x NZW)F1 (B/W) mice during the lupus-like syndrome were studied and compared to natural antibodies present in normal mice. The antibody activities were tested in sera, circulating immune complexes (CIC) and kidney eluates, using an enzyme immunoassay against a panel of self and non-self antigens: actin, myosin, tubulin, DNA, myoglobin, spectrin and trinitrophenylated bovine serum albumin (TNP/BSA). In the B/M mouse sera, IgM antibodies reacting with all the panel of antigens (PAg) and comparable to those of normal mice, increased moderately from 5 to 9 months and markedly during the last stage preceding death (10 months), when particularly high levels of anti-DNA, anti-tubulin and anti-myoglobin antibodies were noted. Polyreactive IgM antibodies present in CIC were moderately increased while those present in complexes deposited in kidneys were strongly enhanced after the 8th month. IgG antibodies showed an early increase (2 months) in B/W sera for anti-TNP activity, which remained more or less constant until death, while a later (5-6 months) and greater increase of activity, mainly directed against DNA but also against the other antigens of the panel, was observed. In CIC, IgG, mainly anti-DNA but also anti-TNP, were enhanced at the end of the disease while at the same time IgG reacting with all the PAg were found in kidney deposits. Isolation of antibodies from sera on a DNA-immunoadsorbent demonstrated that eluted IgM reacted with all the PAg but mainly with DNA, while IgG reactivity was more restricted to DNA and to a lesser degree to TNP. The D23 idiotype, characteristics of natural polyspecific antibodies, was expressed on IgM and IgG autoantibodies from B/W mice and was enhanced, particularly in kidneys, at the end of the disease. These results demonstrate that natural antibodies are a part of the population of increased autoantibodies in this disease and could participate with IgG anti-DNA antibodies in lupus.     

Proliferation of Ly-1 B cells in autoimmune NZB and (NZB x NZW)F1 mice

Spontaneous autoimmune disease in NZB and (NZB x NZW)F1 (B/W) mice is associated with a spectrum of lymphoproliferative abnormalities, but the relationship between autoimmunity and lymphoproliferation is poorly understood. Lymphomas occur commonly in NZB mice, but they appear to be rare in B/W mice, perhaps because B/W mice die of murine lupus before the lymphomas are evident. We recently reported that autoimmune disease in B/W mice could be reversed by weekly treatment with monoclonal antibodies to the L3T4 antigen on "helper/inducer" T cells. This has enabled us to examine the evolution of lymphoproliferation in B/W mice that survive beyond the usual life span, both in long-term survivors of treatment with anti-L3T4 and in the occasional B/W mouse that spontaneously survives beyond 1 year of age. We find that all of these mice develop marked proliferation of a distinct subpopulation of B cells that express the Ly-1 antigen in low density. These Ly-1+ B cells account for a 2-10-fold increase in the number of splenic, lymph node and peripheral blood lymphocytes. The Ly-1 B cells in individual mice are restricted in their expression of immunoglobulin light chains, suggesting a clonal origin. NZB mice. develop similar proliferation of Ly-1 B cells, suggesting that this is due to underlying genetic and/or viral factors in NZB and B/W mice, and that it is not the result of treatment with anti-L3T4. Although recent studies have implicated Ly-1 B cells in the production of autoantibodies, proliferation of Ly-1 B cells in B/W mice was not associated with production of anti-DNA antibodies or with any paraprotein.     

Effect of an autoreactive T cell clone from (NZB x NZW)F1 mice on the production of anti-DNA antibodies in vivo and in vitro

An autoreactive T lymphocyte clone, designated as F1C4 was established from an autoimmune mouse strain (NZB/NZW)F1. This clone proliferated in the presence of mitomycin C-treated splenic adherent cells (MMC-SAC) from syngeneic mice. This response was dependent on the numbers of MMC-SAC. The specificity of F1C4 for I-A was determined by an inhibition test carried out with monoclonal anti-Ia sera. Furthermore, the F1C4 cells did not exhibit alloreactivity in a proliferation assay and did not react to foreign antigens such as fetal calf serum (FCA) used in the culture medium. When F1C4 cells were cultured with autologous non-T cells in the absence of antigen, they strongly enhanced IgM class anti-ssDNA production from non-T cells of both young and old B/W F1 mice at appropriate cell numbers in vitro. Furthermore, the production of IgG class anti-ssDNA from non-T cells of old B/W F1 mice was also enhanced. The adoptive transfer of F1C4 cells enhanced the levels of both IgM and IgG anti-ssDNA antibodies in the serum of aged B/W F1 mice. Moreover, the serum levels of anti-ssDNA of IgG2a and IgG2b subclasses were readily enhanced by the transfer of F1C4 in vivo.     

Distinct surface phenotypes of B cells responsible for spontaneous production of IgM and IgG anti-DNA antibodies in autoimmune-prone NZB x NZW F1 mice

Autoimmune-prone NZB x NZW F1 (B/W F1) mice produce a high titer of anti-DNA antibodies, In vivo and in vitro studies showed that in the early life of these mice, the immunoglobulin isotype of these antibodies almost exclusively belongs to IgM class, however, IgG anti-DNA antibodies begin to develop when the mice are about 5-6 months old and the titer exceeds that of IgM antibodies from age 7 months on. We asked whether or not the B cell population responsible for IgM and IgG antibody production belongs to the same lineage. The surface phenotypes of B cell populations responsible for the spontaneous production of either IgM or IgG anti-DNA antibodies were examined using panning and sorting methods with several monoclonal antibodies to B cells, including CD5 (Ly-1) and Lp-3; the latter defines a unique B cell differentiation antigen. We obtained evidence that surface phenotypes of B cells secreting IgM anti-DNA antibodies belong to CD5+ Lp-3- and those of B cells secreting IgG anti-DNA antibodies which occur only in old B/W F1 mice belong to CD5- Lp-3+ subpopulations. The majority of peritoneal B cells were CD5+ Lp-3+ throughout the life span of the mice and anti-DNA antibody production was never evidenced. These findings were discussed in relation to age-associated changes of B cell populations in the spleen of this strain of mice.     

Mercury-induced renal immune complex deposits in young (NZB × NZW)F1 mice: characterization of antibodies/autoantibodies

It is well demonstrated that mercury induces a systemic autoimmune disease in susceptible mouse strains. One of the major characteristics of mercury-induced autoimmune disease in mice is the development of renal immune complex deposits. We have previously shown that continual injection of mercury into young autoimmune prone (NZB × NZW)F₁ mice induced an increase in antibody/autoantibody production as well as development of early renal immune complex deposits. In the present study, we characterized the isotype, the specificity and the possible pathogenicity of deposited immunoglobulins in the kidneys of mercury-injected (NZB × NZW)F₁ hybrids. We found that young (NZB × NZW)F₁ mice injected with mercuric chloride (HgCl₂) for 6 weeks developed intense antibody formation of all immunoglobulin isotypes (except for IgG2b) as well as high levels of granular deposits of IgM, IgG1, IgG2a and IgG3 antibodies in the renal mesangium. Increased levels of the same antibody isotypes were also found in the kidney eluate of mercury- but not saline-injected mice. The dominant antibody in the kidney eluate of mercury-injected mice was of IgG1 isotype and found to be directed against double-stranded DNA, collagen, cardiolipin, phosphatidylethanolamine, and the hapten trinitrophenol, but not against nucleolar antigens. Further studies demonstrated that mercury-induced renal immune complex deposits in young (NZB × NZW)F₁ mice did not lead to a severe kidney injury. Thus, in response to mercury, young (NZB × NZW)F₁ mice develop renal immunoglobulin deposits with an isotype and specificity pattern correlating with that seen in the spleen and in the serum.     

Analysis of the genetic encoding of oestradiol suppression of delayed-type hypersensitivity in (NZB x NZW) F1 mice

Oestrogen (E2) has been suggested to be responsible for the female preponderance for systemic lupus erythematosus (SLE) and for exacerbations of disease during pregnancy. In lupus-prone (NZB x NZW) F1 (NZB/W) mice, sex hormones also influence disease progression, thus long-term treatment of NZB/W mice with high doses of oestradiol increases the mortality in immune-complex mediated disease. We have previously demonstrated that E2 suppression of delayed-type hypersensitivity (DTH) to oxazolone (OXA) in NZB/W mice is inherited from the healthy NZW (H-2z) and not from the autoimmune NZB (H-2d) parental strain. In this paper we have analysed the influence of E2 on DTH and antibody responses to OXA in backcrosses of NZB/W mice and the NZB and NZW parental strains. Suppressed DTH was found in 15/16 (94%) of female (NZB/W x NZW) F1 (NZB/W/W) mice treated with E2. In contrast, only 32/36 (51%) of (NZB/W x NZB)F1 (NZB/W/B) mice treated with E2 displayed suppressed DTH reactivity. These two findings are compatible with a single, rather than multiple, dominant gene inherited from the NZW strain encoding for E2-mediated suppression of DTH in NZB/W mice. FACS analysis, using a monoclonal antibody recognizing the H-2z but not the H-2d locus, identified the H-2 expression (H-2dd and H-2dz) of the NZB/W/B backcrosses and revealed that E2 suppression of DTH is not linked to the H-2 haplotype of the backcrosses. Furthermore, E2 treatment of NZB/W/W mice, but not of NZB/W/B mice, significantly enhanced the serum levels of anti-OXA antibodies of both IgG and IgM classes. Based on our results it is tempting to speculate whether similar genetic factors for E2 sensitivity of the immune system may be of importance for the female predominance of human SLE.     

Anti-histone antibodies in the serum of autoimmune MRL and NZB/NZW1 F1 mice

Anti-histone antibodies have been reported in a number of human autoimmune diseases, most notably idiopathic and drug-induced lupus erythematosus. In the current study, anti-histone antibody activity was detected using ELISA and electroblotting techniques in sera from autoimmune NZB/W, MRL-lpr, and MRL-(+)/+ mice. Anti-histone activity increased with age, maturing earlier in females, in both NZB/W and MRL-lpr mice. Testosterone treatment decreased anti-histone activity in NZB/W mice and estrogen treatment from 2 weeks of age increased anti-histone activity in MRL-lpr mice, suggesting that gonadal hormones modified the expression of autoantibodies recognizing these protein antigens. Estrogen also increased serum IgG levels in MRL-lpr mice. Sex hormones affected expression of antibodies recognizing soy milk proteins but not ovalbumin in a similar manner. Nitrocellulose Western blots of SDS gels probed with sera from both types of autoimmune mice most often demonstrated reactivity with histone1. Some mice, usually mature females, also recognized histone4, histone3, and histone2.     

Independent appearance of anti-thymocyte and anti-RNA antibodies in NZB/NZW F1 mice.

Mouse antibodies to soluble bovine skin (type I) collagen react with determinants which are located in the rigid triple-helical portion of the antigen and become destroyed upon unfolding the molecule. Helical antigenic determinants are dependent on the genuine chain assembly, e.g. alpha[1(I)]2alpha2. Artefactual triplehelical structures of the composition [alpha1(I)]3 or [alpha2]3 or a genetically distinct type II collagen from cartilage showed no or only weak cross-reactivity. Pepsin treatment of type I collagen known to remove short, non-helical sequences at both ends of the molecule had virtually no effect on antigenicity and immunogenic activity. A radioimmunoassay failed to detect antibodies in three congenic resistant mouse strains immunized with denatured type I collagen. These strains had been previously classified as high or low responders to native type I collagen. Agglutination titres vs denatured collagen culd already be demonstrated in nonimmune sera. The agglutinating activity was labile against heating at 56 degrees and could not be increased by immunization. Two out of five inbred strains showed a high response against pepsin-dissolved bovine type II collagen with the chain composition [alpha1(II)]3. Lack of correlation in the responder state to both collagen types indicated control by different immune response genes. Antibodies to type II collagen also reacted against triple-helical antigenic determinants and showed neglible cross-reaction with type I collagen.     

The immune response in NZB × NZW F1 hybrid mice

A study was made of the immune response of NZB × NZW F₁ hybrid (BW) mice against `H' antigen of Salmonella, sheep red blood cells, bovine serum albumin and allogeneic tumour cells. The responses of old BW mice having autoimmune glomerulonephritis were compared with those of young healthy mice. It was found that the mice develop immune depression concurrently with the autoimmune process. In old BW mice, the immune response is of the same type as the response observed in neonatally thymectomized mice. Neither age alone, nor functional deficiency of the thymus could account for this immune depression. It is suggested that the ability to respond to foreign antigens was probably depressed by the competition provided by autoantigens.     

Regulation of the immune response in experimental models of autoimmune disorders: resistance of (NZB X NZW)F1 mice to tolerance induction in vivo

Studies were carried out to test whether tolerance to alloantigens and to heterologous proteins could be induced in (NZB X NZW)F1 (B/W) female mice, compared with females of various other mouse strains, including BALB/c, C3H/eb, C57Bl/Ka and (BALB/c X C57Bl/6)F1. Untreated BALB/c and B/W mice were resistant to tolerance induction by deaggregated BSA, while all other strains were susceptible, as indicated by their lack of response to antigen challenge. Tolerance induction to BSA was further potentiated in all mouse strains including BALB/c with the exception of B/W, following prior conditioning of the mice with total lymphoid irradiation (TLI). Similarly, specific and permanent tolerance to H-2 incompatible alloantigens was successfully induced in TLI conditioned BALB/c, C3H/eb, (BALB/c X C57Bl/6)F1 injected with bone marrow cells, however, B/W mice were resistant. Stable chimeras could be established in TLI treated B/W mice only across a semi-allogeneic combination (BALB/c--greater than B/W). No graft vs host disease (GVHD) was observed in any of the chimeras including B/W mice. We conclude that B/W mice are resistant to tolerance induction to heterologous proteins and alloantigens, even after TLI conditioning. We postulate that this phenomenon is a function of both the intrinsic properties of the haemopoietic stem cells, including their differentiated progeny, as well as characteristics of their cellular microenvironment.