Evidence for a B lymphocyte defect underlying the anti-X anti-erythrocyte autoantibody response of NZB mice.

The autoimmune hemolytic anemia of NZB mice is pathogenetically mediated by a genetically prescribed anti-erythrocyte autoantibody response directed to the X erythrocyte autoantigen. The cellular locus of the immunoregulatory defect underlying the anti-X response was explored by adoptively transferring bone marrow cells (BMC) from NZB mice to lethally irradiated histocompatible recipients. Before adoptive transfer, BMC from donor mice were assayed for antigen-binding lymphocytes with receptors for the X autoantigen (X-ABL) by immunocytoadherence assays and for anti-X autoantibody-secreting cells (X-PFC) by plaque-forming cell assays. Twelve weeks after adoptive transfer, splenic lymphocytes from recipient mice were assayed for X-PFC and humoral anti-X autoantibody by Coombs' tests. Transfer of 15 to 30 x 10(6) BMC containing 6 to 12 x 10(3) X-ABL but no X-PFC from 6- to 8-week-old NZB mice to lethally irradiated BALB/c, B10.D2, C57BL/Ks, and DBA/2 mice produced X-PFC in 70% of the recipients. Development of X-PFC was not simply dependent upon available X-ABL since transfer of 15-30 x 10(6) BMC, containing comparable numbers of X-ABL, from BALB/c, B10.D2, C57BL/Ks, or DBA/2 mice to NZB or syngeneic recipients did not produce X-PFC. Transfer of BMC from NZB mice to BALB/c, B10.D2, and DBA/2 mice with weekly administrations of AKR anti-theta antiserum had no effect on the development of X-PFC; Tlymphocyte ablation was evidenced by the absence of theta+ spleen cells. These results suggest that the pathogenetic anti-X response is not genetically prescribed at the level of macrophages, humoral factors, or T cells, but rather appears to be a phenotypic expression of a primary B lymphocyte defect permitting or promoting differentiation of NZB X-ABL.     

Unidirectional IgG allotype- and isotype-specific suppressor cells in congeneic mice

By the use of allotypic markers on immunoglobulin molecules of isotypes IgG1 and IgG2a, in transfers of spleen cells between Igh haplotype congeneic partner strains BALB/c (Igha) and CB20 (=BALB/c-Ighb), the expression of donor and recipient lymphocytes could be followed differentially. BALB/c donor's allotype a was produced in nonirradiated CB20 recipients for months. By contrast, CB20 donor's allotype b disappeared in nonirradiated BALB/c recipients shortly after transfer. These BALB/c recipients of CB20 spleen cells ("CB20-primed") developed lymphocytes which were able to suppress the autochthoneous allotype b production of CB20 irradiated or CB20 nu/nu or neonatal F1 (BALB/c female X CB20 male) recipients immediately after transfer. Titers decreased with a half life of about 4 days, resembling that of immunoglobulin molecules. The suppression was restricted to the IgG2a isotype of allotype b. Neither the other isotype IgG1 of allotype b, nor, in the reciprocal transfer experiment, IgG1 or IgG2a of allotype a was affected. Analogous transfers between Igh congeneic partners on a C57B1/6 genomic background revealed the same susceptibility of allotype b-producing cells from C57B1/6 donors toward suppression by C57B1/6-Igha mice as recipients. Allotype suppression, induced by cell transfer, is thus unidirectional in that Igha haplotype mice react against allotype b but not vice versa, and it is isotype-specific, only directed against IgG2a, and not IgG1.     

Suppressor T cells and self-tolerance. Active suppression required for normal regulation of anti-erythrocyte autoantibody responses in spleen cells from nonautoimmune mice

Spleen cells from young, nonautoimmune strains of mice cultured with syngeneic E do not develop a significant anti-mouse E response in vitro, consistent with a state of self-tolerance to this Ag. In order to study the role of active suppression in regulating mouse RBC-(MRBC) specific cells in nonautoimmune cell populations, the effect of depleting T cell subsets on the generation of anti-MRBC autoantibodies by nonautoimmune spleen cells was determined. Spleen cells from young BALB/c and C57BL/6 mice were found to generate significant numbers of IgM and IgG anti-MRBC autoantibody-forming cells in culture with MRBC after depletion of Ly-2+ cells by anti-Ly-2 and C treatment. The response which develops is Ag dependent, Ag specific, and dependent upon L3T4+ Th. The magnitude and isotype of this response is similar to the anti-MRBC response generated by spleen cells from 12-mo-old, autoimmune NZB mice and young NZB mice also treated to remove Ly-2+ cells. Addition of isolated Ly-2+ T cells, but not L3T4+ or Ly-2- T cells, to spleen cells depleted of Ly-2+ cells restores apparently normal regulation of the anti-MRBC response in vitro. These data demonstrate that control of a specific autoantibody response to MRBC by nonautoimmune spleen cell populations requires active regulation by an Ly-2+ T cell subset.     

Distribution of anti-histone-antibody-secreting cells in NZB/NZW mice

Using a histone-specific plaque assay, we examined anti-histone-antibody (AHA) production at the organ level in the autoimmune NZB/NZW strain. The spleen had the highest absolute numbers of AHA-secreting cells. High percentages of immunoglobulin-secreting cells producing AHA were characteristic of spleen and bone marrow but not lymph node. AHA-secreting cells were detected in NZB/NZW mice with elevated serum activity but not in mice with normal serum levels. Serum AHA activity correlated with the number of AHA-secreting cells in the spleen but not with the total number of immunoglobulin-secreting cells in the spleen nor with the total serum immunoglobulin level. These findings concerning the organ distribution of AHA-secreting cells contrast with results of other investigators studying autoantibodies of other specificities. Furthermore, our results suggest that AHA production does not solely result from a generalized increase in total immunoglobulin synthesis present in NZB/NZW mice.     

Expression and regulation of a recurrent anti-erythrocyte autoantibody idiotype in spleen cells from neonatal and adult BALB/c mice.

BALB/c spleen cells depleted of CD8+ T cells generate an autoantibody response to mouse RBC (MRBC) when cultured 5 days in the presence of syngeneic RBC. More than 80% of the cells secreting anti-MRBC antibody are blocked by an antiidiotypic mAb that recognizes the G8 Id. This G8 Id was originally identified in an autoimmune NZB mouse derived anti-MRBC mAb and later characterized as a dominant Id in NZB anti-MRBC autoantibodies. Furthermore, the CD8+ regulatory T cells that control this autoimmune response in BALB/c mice are specifically eliminated by cytotoxic treatment with the G8 mAb + C, suggesting that the regulatory cells recognize the G8 Id. Spleen cells from neonatal BALB/c mice, which lack those regulatory cells can generate an in vitro antibody response to MRBC without depletion of CD8+ cells. More than 80% of these AFC were also found to express the G8 Id. We propose that Id determinants on autoantibodies that are produced neonatally induce Id-specific regulatory cells that maintain peripheral tolerance to self-RBC throughout the life of normal animals.     

Defective smooth muscle regulation in cGMP kinase I-deficient mice.

Regulation of smooth muscle contractility is essential for many important biological processes such as tissue perfusion, cardiovascular haemostasis and gastrointestinal motility. While an increase in calcium initiates smooth muscle contraction, relaxation can be induced by cGMP or cAMP. cGMP-dependent protein kinase I (cGKI) has been suggested as a major mediator of the relaxant effects of both nucleotides. To study the biological role of cGKI and its postulated cross-activation by cAMP, we inactivated the gene coding for cGKI in mice. Loss of cGKI abolishes nitric oxide (NO)/cGMP-dependent relaxation of smooth muscle, resulting in severe vascular and intestinal dysfunctions. However, cGKI-deficient smooth muscle responded normally to cAMP, indicating that cAMP and cGMP signal via independent pathways, with cGKI being the specific mediator of the NO/cGMP effects in murine smooth muscle.     

Demonstration of active suppressor cells in spleens of young NZB mice

NZB mice, a strain prone to the development of autoimmune disease, have during the first 2 weeks of life suppressor cells in their spleens which can in coculture with adult spleen cells suppress the antibody response to sheep red blood cells (SRBC) generated in culture by the adult cells. The suppressive activity of spleen cells from NZB mice in the first week after birth is similar to that of spleen cells from 4-day-old C57BL/6 mice, a strain which does not spontaneously develop autoimmune disease. As in “normal” strains of mice, suppressor cell activity in NZB mice is diminished at 2 weeks and undetectable at 3 weeks of age. The data indicate that there is no defect inherent in the suppressor cells detected in the spleens of newborn and young NZB mice and suggest that the development of autoimmune responses does not result from a lack of suppressor cells in the young animals.     

Modulation of autoimmunity in NZB mice by cyclophosphamide-induced, nonspecific suppressor cells

A group of NZB mice received six biweekly injections of cyclophosphamide-induced nonspecific suppressor cells, with treatment commencing at 2 mo of age. Mice were evaluated for Coombs and natural thymocytotoxic antibody at 6-wk intervals thereafter, and for anti-DNA autoantibodies, total IgM and IgG levels, and renal histology at selected time points. The administration of suppressor cells resulted in marked and prolonged suppression of both Coombs and natural thymocytotoxic antibody reactivity in the majority of animals while not measurably affecting the levels of anti-DNA autoantibodies, the total IgM and IgG levels, or the life span of the mice.     

Properties of fractionated spleen cells from NZB/W mice.

The unit gravity sedimentation technique was used to separate spleen cells from sevveral strains of mice. Settling patterns (plot of cell number against settling rate) were similar for BALB/c, DBA/2, C3H/He, and NZB/W mice of different ages. In particular, no subpopulation was found by this technique to be missing from the spleens of old NZB/W mice.A number of functional studies performed with the separated cells proved more informative than the settling patterns themselves. Fractions of cells which sedimented at a rate of between about 6 mm/hr and 10 mm/hr were enriched in responsiveness to PHA, Con A, and allogeneic cells. These fractions obtained from old NZB/W mice lacked such activities. However, the active fractions from young NZB/W spleens, which were enriched in θ-bearing cells, could restore the responsiveness of old NZB/W mice to primary immunization with sheep erythrocytes. These studies indicate that functional separation of spleen cells from NZB/W mice is possible and that activities lacking in whole spleens from old NZB/W mice are also lacking in the separate fractions. The ability to restore helper T cell function in old NZB/W mice with active fractions from young NZB/W mice has implications for further study and treatment of their autoimmune disease.     

Defective B cell clonal regulation and autoantibody production in New Zealand black mice

By using the splenic fragment assay in a KLH-primed host, we have evaluated the clonal anergy model of tolerance in DBA/2 and spontaneously autoimmune NZB mice. Unlike immature B cells from DBA/2 mice (which are tolerized by encounter with TNP-OVA), SIg- B cells from NZB mice respond to TNP-KLH with equal precursor frequency in TNP-OVA-tolerized or control fragments. In additional experiments, SIg- bone marrow or mature spleen cells of DBA/2 or NZB origin were adoptively transferred into irradiated (DBA/2 X NZB) F1 X xid hosts, and host-derived splenic fragments were stimulated in vitro with LPS and growth factors. These experiments revealed a substantial anti-ssDNA precursor frequency in NZB marrow and spleen (2.5 and 5.1, respectively, per 10(7) transferred cells). In DBA/2 SIg- marrow cells, there was an anti-ssDNA precursor frequency of 1.3 to 3.5/10(7) transferred cells; however, anti-ssDNA-producing clones were reduced in fragments derived from recipients of DBA/2 as compared with NZB spleen cells (0.2 to 1.9/10(7) transferred cells). By using a replica plate technique, we evaluated fragments from recipients of DBA/2 SIg- marrow cells or mature spleen cells for anti-TNP reactivity. In fragments derived from recipients of DBA/2 SIg- marrow cells, 92% of anti-TNP-producing fragments also bound ssDNA. In fragments derived from recipients of DBA/2 spleen cells, only 43% of anti-TNP-producing fragments also bound ssDNA. Our findings document that NZB marrow-derived immature B cells abnormally resist tolerance induction, and that clonal anergy/selection operates in directing the B cell repertoire away from autoantibody formation.     

DM loss in k haplotype mice reveals isotype-specific chaperone requirements

DM actions as a class II chaperone promote capture of diverse peptides inside the endocytic compartment(s). DM mutant cells studied to date express class II bound by class II-associated invariant chain-derived peptide (CLIP), a short proteolytic fragment of the invariant chain, and exhibit defective peptide-loading abilities. To evaluate DM functional contributions in k haplotype mice, we engineered a novel mutation at the DMa locus via embryonic stem cell technology. The present experiments demonstrate short-lived A(k)/CLIP complexes, decreased A(k) surface expression, and enhanced A(k) peptide binding activities. Thus, we conclude that DM loss in k haplotype mice creates a substantial pool of empty or loosely occupied A(k) conformers. On the other hand, the mutation hardly affects E(k) activities. The appearance of mature compact E(k) dimers, near normal surface expression, and efficient Ag presentation capabilities strengthen the evidence for isotype-specific DM requirements. In contrast to DM mutants described previously, partial occupancy by wild-type ligands is sufficient to eliminate antiself reactivity. Mass spectrometry profiles reveal A(k)/CLIP and a heterogeneous collection of relatively short peptides bound to E(k) molecules. These experiments demonstrate that DM has distinct roles depending on its specific class II partners.     

Genetic studies in NZB mice. II. Hyperdiploidy in the spleen of NZB mice and their hybrids.

The presence of hyperdiploidy was studied in New Zealand black (NZB) mice and the progeny of NZB X DBA/2 crosses and backcrosses. Hyperdiploidy was observed in the spleens of a majority of NZB mice but not in DBA/2 mice at 1 year of age. In crosses of NZB with the DBA/2 strain, hyperploidy was observed only in backcrosses to NZB. Hyperdiploidy appeared to be determined by a recessivley inherited trait and was not related to the presence of other immunological abnormalities, including splenomegaly, hypergammaglobulinemia, and spontaneous antibodies cytotoxic for T cells and reactive with single-stranded DNA. Abnormal cells were not present in Concanavalin A-stimulated 48-h spleen cultures. There was no difference in the in vitro sister chromatid exchange rate between the autoimmune NZB strain and the non-autoimmune DBA/2 strain. Identification of NZB chromosomes by banding analysis showed that chromosomes 15 and 17 were frequently present in more than two copies in hyperdiploid spleen cells. NZB chromsomes also had reduced C-banding in an autosomal pair. These studies indicate that chromosomal abnormalities which occur in NZB mice may be useful as genetic and cytogenetic markers.     

B-B cell interactions in the spontaneous activation of B cells in autoimmune NZB mice.

We analyzed the mechanism of spontaneous B cell activation in lupus mice by using anticlass-II antibody in vitro. The in vitro culture of B cells from old NZB mice markedly produced Ig without any stimulation, while B cells from NZW mice did not. The addition of anticlass-II antibody (anti-Iad antibody) to the culture inhibited Ig production of NZB B cells in a concentration-dependent manner. On the other hand, the addition of anticlass-I antibody (anti-H-2Dd antibody) and anticlass-II antibody with different specificity (anti-Iak) gave no effect on the Ig production of NZB B cells. When mitomycin C-treated B cells were added to in vitro culture of responder B cells as a stimulator, Ig production of responder B cells was enhanced in a concentration-dependent manner. However, the enhancing effect of the stimulator B cells was abrogated by the pretreatment with anticlass-II antibody. The stimulator B-cell activity to NZB B cells was marked in NZB B cells, moderate in NZB/W F1 B cells, and weak in NZW B cells. Furthermore, the stimulator B-cell activity with regard to NZB B cells was marked in old female NZB B cells, moderate in old male NZB B cells, and weak in young NZB B cells. The expression of class II antigens on the surface of old female NZB B cells was significantly higher than that of old male NZB and young NZB B cells. These results suggest that in lupus mice the spontaneous B-cell activation is induced by an abnormal B-B cell interaction mediated by class II antigens.     

Transferred B cells from autoimmune NZB/N mice fail to activate T suppressor cells

T-cell-mediated suppression of the antibody response of autoimmune NZB/N mice to Type III pneumococcal polysaccharide (SSS-III) can readily be induced in situ by priming with a subimmunogenic dose of SSS-III; however, the transfer of either "young" (8 weeks old) or "old" (42 weeks old) SSS-III-primed B cells, which activates suppressor T cells in normal BALB/cByJ mice, fails to induce suppression of the antibody response in recipient NZB/N mice, regardless of the number of cells transferred or the time interval between transfer and immunization. Transfer of 51Cr-labeled B cells demonstrated that syngeneic primed B cells home to the spleens of NZB/N mice in somewhat lower numbers than in BALB/cByJ mice, although the differences observed may not be sufficient to explain the complete absence of activation of suppressor T cells. These findings suggest that B cells from autoimmune NZB/N mice are unable to activate T suppressor cells upon transfer; this disorder in a normal regulatory mechanism may be important in the pathogenesis of disease.