Abstract. Antibodies which bind to different nuclear antigens in tissue sections or in permeabilized cell cultures are useful markers of subsets of connective tissue disease, especially of lupus erythematosus (LE), but whether these antibodies are able to react with these intracellular sequestered antigens in vivo and cause immunologic tissue damage has been a matter of much debate. We report experiments which show that ultraviolet light-irradiated, cultured human keratinocytes bind IgG antibodies from the sera of LE patients with either monospecific anti-SSA/Ro, anti-RNP, or anti-Sm activity, which implies that these antigens have been made accessible on the cell surface by ultraviolet irradiation. Normal human sera or LE patient's sera with antidouble-stranded DNA, anti-single-stranded DNA, or antihistone activity do not bind to the surface of irradiated human keratinocytes. In control experiments, all antisera produced the expected patterns of nuclear and cytoplasmic staining of fixed permeabilized, unirradiated keratinocytes. Careful study of the viability and permeability of irradiated keratinocytes by several techniques showed that this apparent cell membrane expression of extractable nuclear antigens (SSA/Ro, RNP, and Sm) following irradiation was seen on injured keratinocytes whose cell membranes were intact, but not on dead cells. It is particularly significant that all six monospecific

The induction of immunoglobulin (Ig) synthesis in the autologous MLR has an absolute requirement for helper/inducer (Leu-3) T cells, whereas an excess of suppressor/cytotoxic (leu-2) cells suppresses the response. The current study was an effort to assess the immunoregulatory potential to T cells activated in the autologous mixed-leukocyte response (MLR). T cells were cultured with autologous non-T cells for 8-9 d, after which the activated T cells were fractionated into subsets with monoclonal antibodies to T cell markers and HLA-DR antigen. Each population was co-cultured in fresh autologous MLR, and on the 8th day of culture, Ig-secreting cells were measured in a reverse hemolytic plaque assay. The results show that activated Leu-2, DR+ T cells, but neither Leu-2, DR- nor Leu-3 T cells, were at least 50 times more potent as suppressors of IgM and IgG synthesis than fresh Leu-2 cells alone. The activation of this Leu-2, DR+ subpopulation required Leu-3 cells in the primary culture. Furthermore, in the absence of Leu-2 cells in the second culture, little or no suppression was observed, suggesting that the Leu-2, DR+ cells act to amplify or induce suppressor effects of fresh Leu-2 cells. This indicates that at least two distinct subpopulations of Leu-2 cells are required for maximal suppression of an immune response, and that immunoregulatory circuits analogous to those described in the mouse exist in man.

No abstract

During Ag-driven development of memory B cells, Ab V genes are modified by somatic mutagenesis. Although V gene somatic mutations have important biologic consequences in both physiologic and autoimmune Ab responses, little is known about the mechanism of mutation, or whether it operates normally in autoreactive B cells. To approach these issues, we analyzed somatic mutations in Ab genes for evidence of sequence-specific target preferences. Our analysis was confined to noncoding segments of V genes so that the intrinsic characteristics of the somatic mutation process could be reliably dissociated from the indirect but substantial influences of cellular selection. We consistently observed that some dinucleotides, GC and TA in particular, mutated at frequencies that were higher than expected based on their frequency of occurrence. Most of the dinucleotide mutation preferences could not be extrapolated directly from mononucleotide mutation preferences. Specific trinucleotides, including AGC, TAC, and their inverse repeats (GCT, GTA), also mutated more frequently than expected. These and other mutation characteristics were virtually indistinguishable in V genes of normal and autoreactive B cells. An analysis of mutations in published flanking sequences confirmed the target preferences, as did an examination of reported "hot spots" within coding V sequences. The shared preferences in coding and noncoding regions of V genes suggests that somatic mutations are generated de novo. Collectively, our findings indicate that the somatic mutation process exhibits sequence-specific preferences, consistent with an untemplated mechanism, and appears to operate similarly in normal and autoreactive B cells.

The lpr gene encodes a defective form of the fas gene that mediates apoptosis, and its expression results in autoantibodies and massive lymphadenopathy. bcl-2, another gene locus that affects programmed cell death, acts to inhibit apoptosis. Since multiple mechanisms controlling programmed cell death may contribute to systemic autoimmunity, the effect of the bcl-2 transgene on the lpr model was examined by crossing bcl-2 transgenic and C57BL/6-lpr mice. Compared with bcl-2-/lpr mice, bcl-2+/lpr showed dramatic increases in lymphadenopathy and T cell accumulation, but not in autoantibodies or B cell numbers. Short term transfer studies demonstrated that double negative T cells normally have a limited lifespan, and their survival is enhanced by the bcl-2 transgene. Thus, defects in separate apoptosis mechanisms may combine to produce enhanced pathologic effects.

New Zealand Black (NZB) mice spontaneously develop autoimmune disease, usually characterized by an autoimmune hemolytic anemia, and NZB genes are essential for a severe systemic lupus-like disease in (NZB x NZW)F1 mice. We have found that resting B cells from NZB mice demonstrate a pronounced defect, compared with five normal strains, in apoptosis induction after cross-linking with anti-IgM Abs. In contrast, spontaneous apoptosis of NZB B cells in culture was similar to normal strains. B cells from young (NZB x SM/J)F1 and (NZB x NZW)F1 mice underwent apoptosis normally, indicating that the NZB defect in apoptosis is a recessive trait. However, older (8-32 wk) predisease (NZB x NZW)F1 mice manifested a similar defect in apoptosis induction. The analysis of NXSM recombinant inbred mice derived from NZB and SM/J, in addition to backcross mice, suggested that the NZB apoptosis defect is a multigenic trait. Interestingly, resting B cells form B6.lpr and B6gld mice underwent apoptosis following anti-IgM treatment at a level similar to that of the C57BL/6 parental strain. Thus, the induced apoptosis of resting B cells and the NZB defect are likely not related to either Fas or Fas ligand. We propose that this phenotypic defect in apoptosis induction, or the biochemical alteration that underlies the defect, may be casually related to autoimmune disease in NZB mice and its contribution to lupus-like disease in (NZB x NZW)F1 mice.

Sera from 20 patients with systemic lupus erythematosus (SLE), selected for elevated titers of antibody to native DNA (nDNA), were examined by indirect immunofluorescence (IF) on tissue culture Hep-2 and rabbit kidney cells. Twelve sera showed a particulate cytoplasmic staining, in addition to nuclear IF. Double IF staining by using a mouse monoclonal anti-nDNA and a human serum containing anti-mitochondrial antibody as probes showed that the cytoplasmic structures recognized by these 12 SLE sera were mitochondria. SLE sera showing mitochondrial staining had high anti-nDNA levels, as assessed by ELISA (3.5 +/- 1.9 O.D.), compared with those not showing this staining pattern (0.8 +/- 0.4 O.D.). Mitochondrial staining was abolished by DNase I pretreatment of the substrates. Liquid phase absorption of serum anti-nDNA with S1 nuclease-treated calf thymus DNA or purified mitochondrial DNA also removed staining. These findings demonstrate that anti-nDNA antibodies from patients with SLE bind to DNA in intact mitochondria. Therefore, mitochondrial IF staining on tissue culture cells in the presence of nuclear staining should be interpreted with caution, because the phenomenon could be entirely related to anti-native DNA. These observations might also provide new insights concerning the nature of immunogenic cellular components stimulating anti-DNA production.

Patients with intractable rheumatoid arthritis (RA) were treated with total lymphoid irradiation (TLI, 2000 rad). We previously reported long-lasting clinical improvement in this group associated with a persistent decrease in circulating Leu-3 (helper subset) T cells and marked impairment of in vitro lymphocyte function. In the present experiments, we studied the mechanisms underlying the decrease in pokeweed mitogen stimulated immunoglobulin (Ig) secretion observed after TLI. Peripheral blood mononuclear cells (PBL) from TLI-treated patients produced 10-fold less Ig (both IgM and IgG) in response to pokeweed mitogen than before radiotherapy. This decrease in Ig production was associated with the presence of suppressor cells in co-culture studies. By using responder cells obtained from normal individuals (allogeneic system), PBL from eight of 12 patients after TLI suppressed Ig synthesis by more than 50%. In contrast, PBL from the same patients before TLI failed to suppress Ig synthesis. Suppression by post-TLI PBL was also demonstrated in an autologous system by using responder cells cryopreserved before TLI. Again, only cells obtained after TLI were suppressive in four of seven patients. PBL with suppressive activity contained suppressor T cells, and the latter cells bore the Leu-2 surface antigen. In 50% of the patients studied, suppressor cells were also found in the non-T fraction and were adherent to plastic. Interestingly, the Leu-2+ cells from TLI-treated patients were no more potent on a cell per cell basis than purified Leu-2+ cells obtained before TLI. Additional experiments suggested that the suppression mediated by T cells after TLI is related to the increased ratio of Leu-2 to Leu-3 cells observed after radiotherapy.