Renal disease in (NZB × NZW) F1 hybrid mice treated with anti-lymphocytic antibody

Anti-lymphocytic IgG (ALG) in moderate doses postponed the onset of spontaneous renal disease in (NZB × NZW)F₁ hybrid (BW) mice decreasing their mortality. This result only occurred if antibody formation to the ALG was prevented by first rendering the mice tolerant to normal rabbit IgG (NRG), otherwise acceleration of renal disease occurred. This acceleration was particularly marked if γ-globulin, rather than a purified IgG preparation of ALG were used and was further enhanced by adult thymectomy. The development of antinuclear antibody was not delayed by ALG nor was the rise in the serum IgM levels.

The effects of ALG are seriously modified by alterations in factors such as dosage used, preceding tolerance to the NRG, thymectomy and strain of mouse used.

     

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.     

Normal induction and maintenance of tolerance in (NZB × NZW)F1 mice

(NZB × NZW) F₁ hybrid mice could be readily made tolerant to NRG by neonatal injection or, in adult life, by use of cyclophosphamide even with ALG as the inducing stimulus. Many could also be made tolerant by an injection of NRG alone. No difference was found between this group and mice of other strains in this regard, and tolerance when induced in the neonatal animals could be maintained for over nine months without any tendency to waning.     

Suppression of autoimmune disease in New Zealand mice associated with infection with malaria. I. (NZB × NZW) F1 hybrid mice

Female (NZB × NZW) F₁ hybrid (B/W) mice were infected with Plasmodium berghei at the age of 1 month. In contrast to the high mortality from renal disease observed in control female mice of this strain, all of the malaria-infected female mice were alive at 12 months of age and none had any clinical features of severe renal disease. This finding gives some support to the hypothesis that a background of repeated parasitic infection may modify the development of autoimmune disease in man.     

Age- and sex-dependent thymic abnormalities in NZB × SJL F1 hybrid mice

The cellular organization of the thymus was investigated in 3- and 12-month-old NZB × SJL F₁ hybrid (NS) mice. Age-dependent alterations were demonstrated which differed strikingly according to the sex of the animals. In female mice, marked abnormalities of the thymus developed during ageing. They consisted of a more or less pronounced hypertrophy accompanied by histological changes and modifications in the nature of the lymphocyte populations. Three types of qualitative changes were found at 12 months of age: (1) depletion of cortical thymocytes as evidenced by histology, by the evaluation of peanut-agglutinin (PNA) binding and by cell electrophoresis; (2) hyperplasia of the medullary lymphoid tissue, probably reflecting the expansion of a population of mature T lymphocytes. This was further suggested by a rise (up to 60%) in the frequency of lymphocytes lacking both PNA receptor and B cell markers, by an increased proportion (57%) of high electrophoretic mobility (EPM) lymphocytes and by an augmentation of in vitro reactivities to phytohaemagglutinin (PHA) and, although to a lesser extent, to concanavalin A (Con A). (3) The appearance of significant numbers of B lymphocytes (up to 20%) as assessed by surface immunoglobulin (sIg) and complement receptor (CR) detection which was accompanied by a vigorous responsiveness of thymus cells to lipopolysaccharide (LPS). None of these abnormalities was seen in the male mice. Instead, the thymus of NS males displayed a nearly normal age-related involution without major change in the proportions of its lymphocyte subpopulations. NS mice thus provide an interesting model of thymic disease influenced by sex-linked factors.     

Beneficial effect of Salmonella typhimurium infection and of immunoglobulins from S. typhimurium-infected mice on the autoimmune disease of (NZB × NZW)F1 mice

Various infections can precede or aggravate autoimmune diseases. Yet a beneficial effect of infection has also been described and various mechanisms have been postulated to explain this effect. The aim of this study was to examine the hypothesis that infection can have an immunoregulatory effect on the autoimmune process via the increased production of natural polyreactive antibodies. The effect of Salmonella typhimurium infection on the lupus-like disease of (NZB × NZW)F₁ (B/W) mice was therefore studied. The effect of IgM and IgG preparations isolated from the serum of S. typhimurium-infected C57Bl/6 and CBA mice on the autoimmune disease of B/W mice was also tested. C57Bl/6 and CBA mice were chosen because they are respectively genetically susceptible and resistant to S. typhimurium infection and they differ in their antibody response during the early phase of infection. CBA mice can mount a specific anti-bacterium antibody response, whereas C57Bl/6 mice present increased production of polyreactive antibodies. The infection effect was evaluated on several disease parameters, i.e. survival, incidence of high grade proteinuria and serum IgM and IgG antibody activity directed against a panel of autoantigens. Our main findings were: (i) infection of B/W mice with an attenuated strain of S. typhimurium delayed the course of the autoimmune disease when performed before the appearance of autoimmune symptoms; and (ii) IgM and IgG preparations from S. typhimurium-infected C57Bl/6 mice had a similar effect, whereas the IgM and IgG preparations from infected CBA mice, as well as from normal C57Bl/6 and CBA mice, were ineffective. These results suggest that S. typhimurium infection can beneficially influence the development of the autoimmune disease of B/W mice. The immunoregulatory effect of the infection seems to be related, at least partially, to the increase of a particular population of antibodies, the polyreactive antibodies.     

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.     

In vitro and in vivo functional analysis of CD5+ and CD5− B cells of autoimmune NZB × NZW F1 mice

T cell-mediated immune responses are likely to be important in the pathogenesis of systemic vasculitis. However, identifying the T cells involved has proved difficult, and there are conflicting reports regarding T cell proliferation in response to different autoantigens. Perinuclear (P) and cytoplasmic (C) anti-neutrophil cytoplasmic antibodies (ANCA) are closely associated with systemic vasculitis, and are generally specific for MPO or PR3, respectively. We studied the proliferative responses to MPO and PR3 of peripheral blood mononuclear cells from patients with P-ANCA or C-ANCA specific for these antigens by ELISA. These responses were compared with those of normal controls, and of disease controls with P- or C-ANCA not specific for MPO or PR3. The patient group as a whole showed significant T cell proliferation in response to the autoantigens compared with controls (P =0·005). Cells from nine of 13 P-ANCA-positive, anti-MPO-positive patients proliferated in response to MPO, compared with five of 16 controls (P =0·04). Cells from five of eight C-ANCA-positive, anti-PR3-positive patients proliferated in response to PR3, compared with two of 11 controls (P =0·05). These experiments demonstrate that patients with P-ANCA or C-ANCA possess T cells which respond to MPO or PR3, respectively. As in other autoimmune diseases, responses to both antigens were also seen in a proportion of healthy controls. Further analysis of these responses will be important in understanding the pathogenesis of systemic vasculitis and in designing specific immunotherapy.     

Studies on thymus products. IV. Absence of serum `thymic activity'' in adult NZB and (NZB × NZW) F1 mice

New Zealand Black (NZB) mice and (B/W) F1 hybrids have a normal level of serum `thymic activity' (TA) at birth but this level decreases prematurely between the third and sixth weeks of life. At 2 months, NZB and NZ (B/W) F1 mice have no significant TA, whereas TA is still at birth's level in six control mouse strains and remains at this level until the fourth to the sixth month. Six weeks after the decline of serum TA, NZB mice show disappearance of theta-positive lymph node rosette-forming cells (RFC) and 2 weeks later, progressive decrease in spleen RFC sensitivity to anti-theta serum (AΘS) and azathioprine (AZ) as in neonatally thymectomized CBA mice. Normal AZ and AΘS sensitivity of spleen and lymph node RFC is reconstituted by in vitro or in vivo treatment by thymic extracts. The early thymic abnormalities found in NZB mice are in keeping with the thymic medullar epithelium atrophy reported in newborn NZB 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.     

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.     

A study of the effect of low-dosage irradiation on NZB and NZB × NZW mice

NZB, NZB × NZW and random-bred mice were exposed to continuous low-dosage gamma irradiation, 20 R/yr, about 200 times normal background irradiation, throughout their adult lives. The aim was to test for the possibility that their autoimmune disorders are based on a defective DNA repair mechanism which permits development of forbidden clones of immunocytes through abnormal somatic mutation.

The irradiation had no effect on NZB × NZW mice, which died predominantly of renal failure, as usual, with a mean life span of 374 days. Both the NZB and the random-bred mice died significantly sooner than control, unirradiated animals. The reason for this is not clear. There was no increase in tumour formation. The NZB mice died in two clusters, several months apart.

It is concluded that any effect of the irradiation was insufficient to support the idea that defective DNA repair is the genetically determined defect causing autoimmunity in these mice. The possibility that the irradiation increased the rate of formation of harmful forbidden clones by somatic mutation in the NZB mice cannot be excluded. Alternatively, the shorter life-span of the irradiated animals may have been due to slight impairment of immune defence against micro-organisms or increased rate of ageing processes in the irradiated mice.

     

Activated B cells express increased levels of costimulatory molecules in young autoimmune NZB and (NZB x NZW)F(1) mice

Polyclonal B cell activation is a hallmark of autoimmune disease in NZB and (NZB x NZW)F(1) (NZB/W) mice. However, the mechanism by which this activated cell subset facilitates disease development is unknown. We recently showed that resting B cells from these mice demonstrate enhanced expression of costimulatory molecules in response to CD40 crosslinking (Jongstra-Bilen et al., J. Immunol. 159,5810-5820, 1997). This led us to question whether activated B cells expressed costimulatory molecules in vivo. Using flow cytometry we found that NZB and NZB/W mice have an increased proportion of splenic B cells expressing B7.1 and elevated levels of B7.2 and ICAM-1. These B cells isolate within the low-density activated population and possess the phenotypic characteristics of marginal zone B cells. The levels of B7.1 on the activated B cell population are similar to those induced by CD40 stimulation raising the possibility that activated B cells in NZB and NZB/W mice provide costimulatory signals to self-reactive T cells leading to loss of tolerance.     

Early hyperbaric oxygen therapy attenuates disease severity in lupus-prone autoimmune (NZB x NZW) F1 mice

The effects of hyperbaric oxygen (HBO(2)) therapy on the immune system are reported including potential changes to the CD4/CD8 ratio and a decreased proliferation of lymphocytes during exposure. The immunosuppressive effect of HBO(2) had been suggested to be applicable for the treatment of certain autoimmune diseases. (NZB x NZW) F1 hybrid mice, the unique lupus-prone mice, have been used for elucidating the pathogenesis of SLE. To investigate the effect of HBO(2) on NZB/W F1 lupus-prone mice, 32 female mice were divided into four groups. Three groups of mice were treated with HBO(2) (2.5 atm abs (ATA) for 90 min daily over 2 weeks) starting at (A) 3 months, (B) 6 months, or (C) 8 months of age, while the remaining group (D) served as control. Animals were followed until 11 months of age. Experimental parameters included life span, proteinuria, peripheral lymphocytes, anti-dsDNA antibody titers, and renal histopathology. HBO(2) treatment resulted in increased survival, decreased proteinuria, alterations in lymphocyte-subset redistribution, reduced anti-dsDNA antibody titers, and amelioration of immune-complex deposition in groups A and B. Our data demonstrated that HBO(2) therapy attenuated disease severity in NZB/W F1 mice. HBO(2) treatment may be of use in the clinical treatment of lupus patients and would benefit from further study.     

Abnormal development of B cells and B cell progenitors in autoimmune (NZB x NZW)F1 mice

The (NZB x NZW)F1 (BWF1) autoimmune strain displays reduced numbers of both c mu+ pre-B cells and Ly5(220)+ B cell progenitors in the bone marrow. The loss of these B cell precursor populations in the bone marrow increases with age. In contrast, the bone marrow of BWF1 mice possesses sIg+ B cells comparable in both number and surface phenotype to that observed in conventional strains. Analysis of the surface densities of both sIg and Ly5(220) antigens indicates that BWF1 bone marrow B cells comprise a heterogeneous population of both immature and mature B cells. In addition, BWF1 bone marrow still possessed progenitor cells capable of yielding newly generated B cell precursors and B cells in vitro. The diminished levels of intermediate B cell progenitors observed in BWF1 bone marrow may reflect abnormal regulation of B lineage differentiation during the life span of this autoimmune strain.     

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.     

A defect of B-lymphocyte transport of aggregated HGG into germinal centres in NZB and NZB×NZW F1 hybrid mice

Previous experiments have shown (i) that the localization of intravenously injected heat aggregated human γ-globulin (HGG) in the germinal centres of normal mice provides a model for studying the natural uptake of circulating immune complexes in these areas, and (ii) that the aggregated HGG is carried into germinal centres by lymphocytes which have receptors for altered γ-globlin.

Evidence from thymus-cell depletion experiments is now presented which suggests that the lymphocytes concerned are bone-marrow-derived B cells. Defective localization was found in NZB and NZB × NZW F₁ hybrids at different ages and the onset of the autoimmunity and the appearance of histological abnormalities in the spleen. As disease develops it progresses to a complete inability to localize complexes in germinal centres. It is concluded that a functional defect of the bone marrow-derived lymphocyte population exists in these mice.

     

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.