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.     

Lupus nephritis in autoimmune-prone NZB x NZW F1 mice and mechanisms of transition of the glomerular lesions

The pattern of renal glomerular lesions in systemic lupus erythematosus (SLE)-prone NZB x NZW (B/W) F1 mice shows an age-associated transition, as is often seen in human lupus nephritis during the clinical course of the disease. Observations revealed that the earliest lesions were confined to the mesangium associated mainly with IgM deposits, and to a lesser degree with IgG. In mice over 5 months of age, the lesions extended gradually to the capillary wall with fine granular subepithelial deposits of IgG, but not of IgM. The ultimate pattern of the glomerular lesion was one of diffuse proliferation with diffusely distributed deposits of both IgG and IgM in the mesangium and along the capillary wall. Even at this stage, subepithelial deposits were composed of IgG, but not of IgM. The pattern of glomerular deposits of endogenous retroviral envelope glycoprotein gp70, which is highly anionic, virtually coincided with that of IgG. Taking these findings collectively, it is suggested that the progression of glomerular lesions in B/W F1 mice depends largely on the age-associated appearance of retroviral gp70-IgG anti-gp70 immune complexes in the circulation and their deposition along peripheral subepithelial, and eventually subendothelial areas.     

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.     

Are Ly1 B cells responsible for the IL2-hyperresponsiveness of B cells in autoimmune-prone NZB x NZW F1 mice?

Unlike splenic B cells from normal BALB/c mice, those from autoimmune-prone NZB x NZW (B/W) F1 young mice were hyperresponsive to interleukin 2 (IL2) and proliferated even in the absence of prior stimulation signals. We found that the B cell subpopulation responsible for this event belongs to that with 6B2 (B220)-dull, Ly1(CD5)-positive phenotypes. Cell cycle analysis revealed that the 6B2-dull, Ly1 B cell subpopulation contains a significant number of cells at stages of G1A and G1B, while the majority of 6B2-bright B cells belongs to GO. Therefore, it appears that most of the 6B2-dull, Ly1 B cells in young B/W F1 mice are already stimulated in vivo to the stage of early activation, so that they respond to IL2 without prior stimuli. Unlike other strains of mice, including parental NZB and NZW, the B/W F1 mice showed a rapid decrease in the proportion of splenic Ly1 B cells, beginning at about 6 months of age. This was associated with the hyporesponsiveness to IL2 of 6B2-dull splenic B cell population in the old B/W F1 mice. This phenomenon seems to relate to further activation and differentiation, which occur in Ly1 B cells with aging.     

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.     

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.     

Clinical neurology and brain histopathology in NZB/NZW F1 lupus mice

Active generalized systemic lupus erythematosus (SLE), clinical neurological deficits and histological lesions in the brains were present in New Zealand Black/New Zealand White (NZB/W) F1 mice at 10 to 18 months of age. Clinical neurological abnormalities of the central nervous system (CNS) were detected with a standardized neurological examination and scoring procedure. Active generalized SLE was present in all mice of this group, as determined by elevated serum anti-DNA antibodies and by the presence of glomerulonephritis. High titres of serum anti-cardiolipin antibodies were present in almost all mice. On histopathological examination, most of the brains had prominent mononuclear cell infiltration around cerebral and hippocampal blood vessels and in the choroid plexus. A subgroup of these mice, having higher clinical neurological scores, had correspondingly higher brain histopathological scores. The neurological and histological abnormalities were compatible with a diagnosis of CNS SLE. In contrast, 2-month-old NZB/W, 5-month-old C57Bl/6 and 14-month-old C57Bl/6 mice had low neurological scores, low serum anti-DNA antibody titres, low or absent anti-cardiolipin antibodies and no evidence of brain or kidney pathological lesions.     

Monoclonal autoantibodies to histones from autoimmune NZB/NZW F1 mice

Fusion of spleen cells from autoimmune NZB/NZW female mice with drug-resistant myeloma cells (clones NSI/1, X63-Ag8.653 and NSO/1) produced hybrid clones which secreted antibodies to various nuclear components. Roughly 50% of the anti-nuclear hybridomas produced antibodies reacting with DNA, 20% with RNA and 30% reacted with other nuclear antigens. Two hybridomas of the latter group were cloned and studied in detail. They secreted antibodies which produced bright fluorescence staining of nuclei and metaphase chromosomes. The specificity of the antibodies was determined by testing them in an enzyme-linked immunosorbent assay and a radioimmunoassay against individual acid- and salt-extracted histones, against histones mixed two and three at a time and against histone complexes isolated as such from chromatin. One of the monoclonal antibodies was specific for histone H2B and reacted with the histone free in solution or when present as a H2A-H2B complex. The second monoclonal antibody recognized a specific conformation in the H3-H4 complex that was present only when the complex was obtained from chromatin by salt extraction. The same conformation, however, could be induced by adding histone H2B to a mixture of acid-extracted H3 and H4. Our findings show that the autoimmune syndrome in NZB/NZW mice resembles human systemic lupus erythematosus not only in the incidence of antibodies to DNA and RNA, but also in the production of autoantibodies to histones.     

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.     

Prolongation of life in female NZB/NZW (F1) hybrid mice by cyclosporin A.

Oral administration of cyclosporin A (Cy A), an immunosuppressive agent, prolonged the life span of female NZB/NZW (F1) hybrid mice (NZB/W). The most prominent feature of this study was the reduction of glomerular proliferation, proteinurea, and delay of renal failure. Protection from renal damage, in treated mice, occurred despite similar degrees of perivascular cellular infiltration in the kidney and other organs, and glomerular deposition of immunoglobulin and complement. Treatment did not influence the hypergammaglobulinaemia, high levels of circulating immune complexes and later development of autoimmune haemolytic anaemia typically associated with this murine disease. The levels of antibodies to double stranded DNA (dsDNA), an important disease marker, and the response to sheep red blood cells were reduced in the Cy A treated mice, in contrast with the untreated mice. Levels of rheumatoid factors were increased in Cy A treated mice, when compared with untreated mice, and this could play a role in prolongation of life associated with protection from glomerular damage, although it was considered that inhibitory effects on T cell function were a more likely mechanism.     

Suppressor cells and immunodeficiency in (NZB x NZW)F1 hybrid mice.

Old (15-20 month) male (NZB x NZW)F1 (B/W) mice have severely impaired spleen cell reactivity to phytohemagglutinin (PHA), a mitogen which stimulates mainly T lymphocytes. Spleen cells from old mice markedly suppressed the PHA response of splenocytes from young (3-4 month) B/W males. Similar suppressor activity was not present in the spleens of old mice of four nonautoimmune strains. The suppressor activity of old B/W spleen cells was mediated by a nonphagocytic, radioresistant, mononuclear leukocyte. Although this cell was eluted in the "T lymphocyte" fraction of nylon wool colums, it was not sensitive to treatment with anti-Thy-1 antiserum and complement. Suppressor activity was lost after 18 h incubation at 37 degrees C in tissue culture medium. Supernatants of these overnight cultures had no suppressive effect on fresh young B/W spleen cells. Old B/W spleen cells suppressed PHA reactivity more than concanavalin A or lipopolysaccharide reactivity. Kinetic studies demonstrated an increasing suppression with time over 72 h of culture. This study demonstrate that the severely impaired PHA reactivity of old B/W mice is mediated, at least in part, by active suppression.     

Estrogen receptor-alpha deficiency attenuates autoimmune disease in (NZB x NZW)F1 mice

Estrogens promote lupus in humans and some mouse models of this disease. Nonetheless, little is known about the role of estrogen receptors in lupus pathogenesis. Here, we report that in females on the lupus-prone (NZB x NZW)F(1) background, disruption of estrogen receptor-alpha (ER alpha or Esr1) attenuated glomerulonephritis and increased survival. ER alpha deficiency also retarded development of anti-histone/DNA antibodies, suggesting that ER alpha promotes loss of immunologic tolerance. Furthermore, ER alpha deficiency in (NZB x NZW)F(1) females attenuated the subsequent development of anti-double-stranded DNA (dsDNA) IgG antibodies, which are associated with glomerulonephritis in this model. We provide evidence that ER alpha may promote lupus, at least in part, by inducing interferon-gamma, an estrogen-regulated cytokine that impacts this disease. ER alpha deficiency in (NZB x NZW)F(1) males increased survival and reduced anti-dsDNA antibodies, suggesting that ER alpha also modulates lupus in males. These studies demonstrate that ER alpha, rather than ER beta, plays a major role in regulating autoimmunity in (NZB x NZW)F(1) mice. Furthermore, our results suggest for the first time that ER alpha promotes lupus, at least in part, by impacting the initial loss of tolerance. These data suggest that targeted therapy disrupting ER alpha, most likely within the immune system, may be effective in the prevention and/or treatment of lupus.     

Hyperglycemia in the NZB/NZW F1 hybrid mouse

Both the NZW and NZB mice exhibit an elevated fasting blood sugar level when compared to Swiss white mice. The NZB/NZW F₁ hybrid mouse shows still a higher fasting serum glucose level than either of its parental strains. This elevated glucose level is noted very early in the animals' life, long before definitive signs or symptoms of pathology are evident, and remains elevated at least until the fortieth week of life, the last testing period in our series before sacrificing the animals. There are two major peaking periods for the glucose levels, namely at 4–10 weeks of age and then again at 31–40 weeks of age. These age periods correspond to the very young animal and to the animal that is beyond its prime and most likely sick and near the end of its lifespan due to the animals' inherent disease processes. The NZB and NZB/NZW F₁ hybrid succumb to their disease processes at approximately 8–10 months of age. The NZW usually lives until 18 months. The questions are raised: does the altered immunologic state in the NZB and/or the NZB/NZW F₁ hybrid produce the elevated blood sugar levels and the pancreatic histopatholgy or is it the early hyperglycemic condition coupled with pancreatic pathology which are in some measure instrumental in producing the lesions discussed and observed, in the beginning of the three to four month period of age, in the various organs? Is there a relationship between the hyperglycemic condition and the immune state or are these independent phenomena in a genetically “mixed up” mouse?     

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.     

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.     

Pattern of age variability of the autoantibody levels in (NZB×NZW) F1 mice

The age variability of the levels of antibodies against structurally different antigens is studied in female (NZB×NZW) F1 mice. It is established that the pubertal period is critical for the number of features studied and the degree of variability of autoantibody levels dependes on the nature of the autoantigen. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 117, № 3, pp. 287–289, March, 1994     

Acceleration of autoimmunity in NZB/NZW F1 mice by graft-versus-host disease.

Chronic graft-versus-host (GVH) disease was induced in NZB/NZW F1 (B/W) hybrid female mice by the weekly injection of parental NZB spleen cells. Control mice received injections of syngeneic spleen cells only. The mice were assayed for antibodies to [3H]DNA and [3H]polyadenylic-polyuridylic acid by a cellulose ester filter radioimmunoassay, and for antibody to thymocytes by a cytotoxicity method. GVH disease accelerated the development of all three antibodies in B/W mice. In addition, sucrose density gradient ultracentrifugation of pooled sera suggested that an accelerated switch from 19S to 7S anti-DNA production may be an early effect of GVH. The mechanism of acceleration is discussed in terms of immunological and viral factors generated by the GVH reaction.     

Examination of the inhibitory effects of interferon-gamma on interleukin-4-induced stimulation of resting B cells from NZB/NZW F1 mice.

Percoll fractionation of NZB/NZW F1 mouse B cells revealed an increased ratio of low-density fraction ('activated' B cell fraction) and a decreased ratio of high-density fraction ('resting' B cell fraction), in comparison to normal mouse B cells. Resting B cells of B/W F1 mice were hyperresponsive to interleukin-4 (IL-4), with increased proliferation and IgG anti-DNA antibody production, in comparison to normal mice. The hyper-responses of resting B cells from NZB/NZW F1 mice to IL-4 were affected with dose dependency by interferon-gamma (INF-gamma); specifically, cell proliferation and anti-nuclear antibody production were suppressed in the presence of IFN-gamma. This suppressor action of IFN-gamma was most noticeable when it was added to the culture system simultaneously with IL-4. These findings suggested that the abnormally activated state of NZB/NZW F1 mice B cells can be attenuated by the action of IFN-gamma on resting B cells.     

Hyperglycemia in the NZB/NZW F1 hybrid mouse.

Both the NZW and NZB mice exhibit an elevated fasting blood sugar level when compared to Swiss white mice. The NZB/NZW F1 hybrid mouse shows still a higher fasting serum glucose level than either of its parental strains. The elevated glucose level is noted very early in the animals' life, long before definitive signs or symptoms of pathology are evident, and remains elevated at least until the fortieth week of life, the last testing period in our series before sacrificing the aminals. There are two major peaking periods for the glucose levels, namely at 4-10 weeks of age and then again at 31-40 weeks of age. These age periods correspond to the very young animal and to the animal that is beyond its prime and most likely sick and near the end of its lifespan due to the animals' inherent disease processes. The NZB and NZB/NZW F1 hybrid succumb to their disease processes at approximately 8-10 months of age. The NZW usually lives until 18 months. The questions are raised: does the altered immunologic state in the NZB and/or the NZB/NZW F1 hybrid produce the elevated blood sugar levels and the pancreatic histopathology or is it the early hyperglycemic condition coupled with pancreatic pathology which are in some measure instrumental in producing the lesions discussed and observed, in the beginning of the three to four month period of age, in the various organs? Is there a relationship between the hyperglycemic condition and the immune state or are these independent phenomena in a genetically "mixed up" mouse?