Studies of defective tolerance induction in NZB mice. Evidence for a marrow pre-T cell defect.

NZB mice manifest a defect in tolerance induction by deaggregated heterologous gamma globulins. We have used an adoptive transfer system to study the defect. Thymectomized, intact, or thymectomized recipients given thymic epithelial grafts were studied after lethal irradiation and reconstitution with NZB, DBA/2, or (NZB x DBA(F1 marrow depleted of mature T cells. NZB thymocytes were responsible for the tolerance defect of NZB mice. The information for the defect was present in the NZB marrow prethymocyte. That defect could only be expressed when there was further maturation in association with a thymus. However, the normal DBA/2 thymic epithelium served as well as the abnormal NZB thymic epithelium. These studies resolve existing conflicts as to whether the NZB marrow or thymus is responsible for the loss of tolerance in association with autoimmunity.

T cell abnormalities in NZB mice occur independently of autoantibody production.

By means of a series of crosses and backcrosses, ZB.CBA/N mice were prepared bearing largely NZB autosomal genes, but having X chromosomes derived only from CBA/N mice. The CBA/N X chromosome carries a gene, xid, that is associated with the lack of a B cell subset necessary for most of the spontaneous autoantibody production by NZB mice. These ZB.CBA/N mice failed to develop autoantibodies to T cells, erythrocytes, or DNA. The availability of mice that were mostly NZB, but which failed to make autoantibodies, especially anti-T cell antibodies, allowed us to study possible T cell regulatory defects in NZB mice in the absence of either antibodies reactive with such T cells or other autoantibodies. We found that such mice had derangements of T cell regulation as did the NZB mice. These observations strongly suggest that the t cell abnormalities of NZB mice are not caused by the B cell hyperactivity of these mice, but rather represent independent defects. Thus, NZB mice appear to have primary defects in both the B cell population and the T cell population. Whether or not these are separate, or derive from a common precursor cell abnormality, remains to be determined.

Ability of the xid gene to prevent autoimmunity in (NZB X NZW)F1 mice during the course of their natural history, after polyclonal stimulation, or following immunization with DNA.

F1 hybrid offspring of New Zealand Black mothers and New Zealand White fathers [(NZB X NZW)F1] female mice develop antibodies to single-stranded (ss) and native DNA, immune complex glomerulonephritis, massive proteinuria, and premature death with renal failure. By a series of matings, congenic (NZB X NZW)F1 . xid/xid mice were prepared. These mice were different from (NZB X NZW)F1 mice in having the X chromosome-linked immune deficiency gene, xid, in homozygous form. Such congenic (NZB X NZW)F1 . xid/xid females failed to develop antibodies to single-stranded or native DNA. They also failed to develop fatal renal disease as measured by proteinuria, glomerular histology, glomerular immunofluorescence, and survival. To control for unknown genetic factors, studies were performed with littermates that were derived by mating NZB . xid/+ females with NZW . xid/Y males such that the resulting offspring were either (NZB X NZW)F1 . xid/xid (and therefore "defective") or (NZB X NZW)F1 . xid/+ [phenotypically like (NZB X NZW)F1]. In these and in additional studies, mice were housed in the same cages and identified by ear tagging so as to avoid possible environmental variations from cage to cage. In these studies, xid/xid mice failed to develop the characteristic signs of autoimmunity, whereas the controls did. Similar results were also obtained with (NZW X NZB)F1 xid/xid mice compared with (NZW X NZB)F1 xid/+ mice. The effect of xid/xid upon (NZB X NZW)F1 mice was further investigated by assessing responses to immunization and polyclonal B cell activation in vivo. The xid/xid mice failed to produce anti-ssDNA following immunization with ssDNA complexed to a protein carrier in fluid form or even emulsified in adjuvant. Finally, the xid/xid mice failed to produce antiDNA in response to multiple injections of the polyclonal activator, bacterial lipopolysaccharide (LPS), or the polyclonal activator, polyribose inosinic acid . polyribose cytidylic acid. However, the xid/xid mice were neither generally hyporesponsive nor unable to recognize LPS because they made normal antibody responses following immunization with LPS to which multiple trinitrophenyl groups were chemically attached. We conclude from these studies that xid/xid, which is known to cause the deletion of a B cell subset, has a profound affect upon (NZB X NZW)F1 mice, rendering them insusceptible to the naturally occurring autoimmune disease characteristic of (NZB X NZW)F1 mice, and preventing them from producing antibodies to DNA despite purposeful immunization and polyclonal B cell activation. These results force a reevaluation of previous concepts regarding the mechanisms by which xid/xid might interfere with the development of autoimmunity, and a consideration of therapeutic implications.

Virus-induced diabetes in autoimmune New Zealand mice.

Infection of autoimmune New Zealand mice with the D variant of encephalomyocarditis (EMC) virus results in beta-cell damage and clinical diabetes. The induction of diabetes in parental NZB and NZW strains was independent of sex. However, the susceptibility to virus-induced diabetes in their F1 offspring was sex dependent. This susceptibility was significantly higher in male (NZB X NZW) F1 mice as compared with female F1 mice. Castration of male F1 mice significantly reduced the susceptibility to diabetes. These results suggest that parental NZB and NZW strains have recessive genes at different loci which do not allow sex hormones to influence the susceptibility to diabetes. It is concluded that both the genetic background of the host and sex hormones influence the development of virus-induced diabetes in autoimmune New Zealand mice.

Evidence for abnormalities in separate lymphocyte populations in NZB mice.

Neonatally thymectomized, lethally irradiated NZB X DBA/2 and DBA/2 X NZB F1 mice were reconstituted with NZB or DBA/2 bone marrow cells and NZB or DBA2 thymocytes. Of the four resulting groups of recipient F1 mice, those given NZB bone marrow cells developed high serum IgM levels, irrespective of the thymocyte donor strain. In contrast, recipients of NZB thymocytes were resistant to induction of tolerance to BCG, irrespective of the bone marrow donor strain. Only recipients of NZB bone marrow cells made spontaneous antierythrocyte autoantibodies; of these, the responses of NZB thymocyte recipients were greater and more consistent than those of DBA/2 thymocyte recipients. Recipients of either NZB bone marrow cells or NZB thymocytes made antibody responses to ssDNA; the highest anti-ssDNA responses occurred in recipients of both NZB bone marrow cells and NZB thymocytes. We conclude that functional abnormalities in separate lymphocyte populations underlie different immune abnormalities in NZB mice.

Evidence for thymic regulation of autoimmunity in BXSB mice: acceleration of disease by neonatal thymectomy.

BXSB mice spontaneously develop an autoimmune syndrome characterized by autoantibody production, immune complex renal disease, and lymphadenopathy containing B cells. The male BSXB mouse has a rapid onset of disease that is determined by defective stem cells. Although the B cell hyperactivity has been traced to the stem cell, previous studies did not evaluate the T cells of BXSB mice with regard to a role in modifying their natural history. The present study was carried out in an attempt to determine whether or not thymic regulation played a role in modulating the stem-B cell abnormalities in BXSB mice. It was found that neonatal thymectomy led to a marked increase in autoantibody production (anti-RBC, anti-DNA), a dramatic increase in lymphadenopathy, and worsening of renal disease. Flow cytometric analysis of the lymph node cells from intact and thymectomized mice demonstrated a loss of Lyt-2+ cells from the thymectomized mice and an increased proportion of Ly-1+, Thy-1.2- cells. These results indicate that although the B cell hyperactivity of BXSB mice is determined by a marrow stem cell defect, thymic regulation normally serves to dampen that B cell hyperactivity; neonatal thymectomy reduces the thymic regulation of autoimmunity in the BXSB males and leads to excessive B cell numbers and function.

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.

NZB cells actively interfere with the establishment of tolerance to BGG in radiation chimeras.

We report experiments designed to determine if the tolerance defect in NZB mice results from i) failure of NZB cells to become tolerant, or ii) the ability of NZB cells to interfere actively with the development of tolerance. The results indicate that NZB cells are primed by the tolerogen itself and actively interfere with the expression of tolerance by DBA/2 cells, which normally can be rendered tolerant.

Studies of congenic MRL-Ipr/Ipr.xid mice.

Highly inbred MRL-Ipr/Ipr.xid congenic mice were bred and compared with their + littermates. The xid-bearing congenics developed lymphadenopathy consisting of dull Ly-1+ T cells and impairment of cellular proliferation and IL 2 production in response to the T cell mitogen Con A. Thus, the lpr gene was fully expressed. The xid gene, however, was also expressed as indicated by the failure to respond to immunization with TNP-Ficoll and flow cytometric analysis of splenic B cells. The xid gene was associated with a marked reduction in IgM anti-ssDNA and anti-nDNA of both classes, and serum Ig-bound gp 70. Kidney disease was markedly retarded as was death from the autoimmune process. These studies suggest that the T cell lymphoproliferation and dysfunction characteristic of MRL-Ipr/Ipr mice is not sufficient to induce accelerated autoimmunity; xid is able to markedly slow the process. The xid gene interferes with the development of a B cell subset necessary for maximum autoantibody production, anti-gp 70 production, and the resultant immune complex renal and cardiac disease. The present finding of protection against accelerated autoimmunity in MRL-Ipr/Ipr mice by xid, coupled with previous demonstrations of protection against autoimmunity in other autoimmune mouse strains, suggests that a common approach to the therapy of systemic lupus may be possible.

Induction of autoimmunity in normal mice by thymectomy and administration of polyclonal B cell activators: association with contrasuppressor function.

In order to gain insight into the role of polyclonal B cell activation in the development of autoimmunity, non-autoimmune mice were given chronic injections of polyclonal B cell activators (PBA). In addition, to assess the contribution of T cell regulation of such PBA-induced B cell hyperactivity, the additional effect of postnatal thymectomy was studied. Mice that were postnatally thymectomized and given PBA (LPS +/- poly rI X rC) thrice weekly were found to have elevated levels of IgG and significantly increased serum concentrations of anti-ssDNA. This anti-DNA production was greater than that observed with PBA alone or with thymectomy alone. The entire experiment was repeated with different non-autoimmune mice with the same result. Numbers of proliferating cells in the spleens of the mice in the various groups were analysed by flow cytometry. The number of cells in S+G2+M phases of the cell cycle was significantly increased by PBA or thymectomy alone as well as by the combination. As a result, B cell proliferation was not sufficient to result in maximal anti-ssDNA; an additional T cell defect was required. This was further studied in an in vitro assay for suppressor and contrasuppressor activity. In three separate experiments, mice which were clinically autoimmune were found to have defective suppressor function and the presence of abnormal contrasuppressor activity, whereas non-autoimmune controls had normal suppressor function and no contrasuppressor function. These results indicate that the combination of PBA and thymectomy can most easily induce autoimmunity. The autoimmune state so induced in non-autoimmune strains was associated with a failure of normal suppressor function and the abnormal presence of contrasuppressor function. These results have important implications for spontaneously occurring autoimmune diseases.

Studies of lymphoproliferation in MRL-lpr/lpr mice.

MRL-lpr/lpr mice develop massive lymphoproliferation and an associated autoimmune process that includes anti-DNA formation, vasculitis, and glomerulonephritis. We have investigated the lymphoproliferation of MRL-lpr/lpr mice and have found that multiple factors are operative. Although neonatal thymectomy markedly retards the syndrome, chronic injection of poly rI.rC could substitute for the thymus. The resulting cells had the phenotype characteristic of the abnormal MRL-lpr/lpr T cells, Thy-1+, dull Ly-1+, Lyt-2-, 6B2+, Ig-. Splenectomy at 2 wk of age markedly retarded the development of this syndrome; however, splenectomy at birth did not. Some protection was afforded by splenectomy at 5 wk. Thus, there appears to be a critical period in the life of an MRL-lpr/lpr mouse when the spleen contributes importantly to the lymphoproliferation. A most remarkable observation was that an MRL-lpr/lpr spleen graft under the kidney capsule could induce lymphadenopathy characteristic of lpr/lpr mice in MRL +/+ recipients. Cells within the graft had to be able to proliferate for the adenopathy to occur because irradiation of the spleen with 800 R just before grafting abrogated the lymphadenopathy-inducing potential. No adenopathy was induced by +/+ spleen grafts placed into +/+ mice. Although MRL-lpr/lpr males develop disease slightly more slowly than female littermates, the differences are small. Manipulations that retard disease, such as splenectomy at 2 wk or neonatal thymectomy, magnified the sex differences. Male MRL-lpr/lpr mice that were thymectomized and splenectomized and given polyclonal immune activators failed to develop either anti-DNA or lymphadenopathy, whereas their female littermates expressed both abnormalities. We conclude from these studies that multiple factors serve to modulate the magnitude of the lymphoproliferation and autoimmune syndrome of MRL-lpr/lpr mice.

Studies of the induction of anti-DNA in normal mice.

Injection of PBA has previously been demonstrated to induce anti-DNA. In the present study, we found that the combination of neonatal thymectomy and chronic administration of PBA (LPS + poly rI . rC) led to significantly higher anti-DNA levels than either PBA or thymectomy separately. These results suggested that a thymic regulatory process normally serves to suppress anti-DNA after chronic PBA exposure. Indeed, antigen-nonspecific suppressor function was found to be deficient in such thymectomy + PBA-treated mice. In addition, the cells of such mice in vitro interfered with the development of normal suppressor function by control cells.

Approach to the study of the role of sex hormones in autoimmunity.

Investigators from this laboratory have been studying sex hormones in normal and autoimmune mice for the past 10 years. We have found that immune responses to DNa are influenced by sex hormones. Androgens reduce and estrogens increase both spontaneous and immunization-induced antibodies to single-stranded DNA in NZB X NZW, NZB X C3H, NZB X CBA, NZB X DBA mice. Treatment of female NZB/W mice with testosterone or 5 alpha dihydrotestosterone retards the progress of autoimmunity. Castration is not necessary for this effect. In contrast, danazol has no favorable effect on the disease process. Estrogens cause a marked acceleration of autoimmunity and a reduction in thymus weight. During the course of these studies, we found that a number of problems or variables arise in studying sex hormone effects, including: 1) X-linked genes, 2) metabolism of testosterone to estrogens, 3) dose of hormone, 4) age at which administration is initiated, 5) differential effects of sex hormones on different autoantibodies and various immune responses.