Conventional B cells, not B-1 cells, are responsible for producing autoantibodies in lpr mice.

Mice homozygous for the lpr gene develop autoantibodies and polyclonal B cell activation similar to what is seen in human systemic lupus erythematosus patients. We have previously shown that an lpr-specific intrinsic B cell defect was necessary for autoantibody production in this model. In the current study, we have further defined these autoantibody-producing B cells. Two major subsets of B cells have been described. B-1 cells (CD5+ B cells) can be distinguished from conventional B cells on the basis of phenotype, cytokine secretion, gene expression, anatomical location, and function. In addition, B-1 cells have been implicated in autoimmunity in several murine and human studies. To address the question of which B cell subset produces autoantibodies in lpr mice, we used immunoglobulin heavy chain (Igh) allotype-marked peritoneal (B-1 cell source) and bone marrow (conventional B cell source) cells from lpr mice to establish B cell chimeras. We used two general approaches. In one, we reconstituted sublethally irradiated mice with B-1 cells of one allotype and bone marrow cells of another allotype. In the second method, we suppressed endogenous B cells in neonatal mice with allotype-specific anti-IgM antibody, and injected peritoneal cells of another allotype. After antibody treatment was stopped, the mouse's conventional B cells recovered, but the B-1 subset was only reconstituted by the donor. In both types of chimeras, antichromatin, rheumatoid factor, and anti-single stranded DNA (ssDNA) autoantibodies were produced by the conventional B cell bone marrow source. In addition, an age-related decrease in peritoneal B-1 cells was seen, even in unmanipulated lpr mice. These data show that lpr B-1 cells are not important producers of autoantibodies. Conventional B cells are the source of autoantibodies directed at chromatin, ssDNA, and IgG.

A nonsteroid anti-inflammatory drug exacerbates Coxsackie B3 murine myocarditis.

Nonsteroid anti-inflammatory drugs are often used to treat myalgias and arthralgias in enteroviral infections, but their effects on acute viral myocarditis are unknown. The effect of the nonsteroidal anti-inflammatory drug, ibuprofen, on acute viral myocarditis was studied in 75 four week old male BALB/c mice infected with 1.75 X 10(7) plaque-forming units of Coxsackie virus B3 on day 0. Ibuprofen was given intraperitoneally at a dose of 15 mg/kg body weight daily. The mice were assigned to four groups--Group I, 18 uninfected mice given ibuprofen on days 1 to 14; Group II, 18 infected, untreated mice; Group III, 20 infected mice given ibuprofen on days 1 to 14; and Group IV, 17 infected mice given ibuprofen on days 7 to 14. Nine animals in Group I, eight in Group II and seven in Group III were killed on day 7; the remaining mice were killed on day 14. Heart viral cultures and histologic analysis were done. Cultures at days 7 and 14 were all negative. Inflammation and necrosis analyzed in each animal were graded 0 to 4, with grade 4 representing widespread inflammation and necrosis. The heart was histologically normal in all 18 uninfected mice (Group I) given ibuprofen only. Inflammation and necrosis were not significantly different in Group II (infected, untreated) and Group III (infected, treated beginning day 1) mice killed at day 7. Inflammation scores of mice killed on day 14 were 2.1 +/- 0.6 (Group II), 3.1 +/- 0.7 (Group III) and 2.9 +/- 1.0 (Group IV infected, treated days 7 to 14).(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of the immune response by dimethylglycine, a nontoxic metabolite.

The immunomodulating capacities of dimethylglycine (DMG) were examined in a rabbit model. Female New Zealand white rabbits were immunized on day 0 and were given booster inoculations on day 9 with either killed influenza virus or Salmonella typhi vaccine. Experimental animals were force fed 20 mg/kg body weight of DMG daily beginning 14 days prior to the first inoculation and continuing throughout the experiment. Control animals were force fed daily only distilled water. Blood was obtained on day 0, day 9, and day 30. Hemagglutination inhibition assays showed a more than fourfold increase in mean antibody titer to influenza antigen in the DMG-treated animals (p = 0.0006) after the first inoculation, and a fourfold increase in mean titer after the booster inoculation (p = 0.1000). A standard agglutination test for Salmonella typhi O (somatic) and H (flagella) antigens was performed on all sera from animals receiving the typhoid vaccine. Mean antibody titers to the O antigen were significantly higher (more than threefold) after the first inoculation (p = 0.0302) and more than fivefold higher after the booster inoculation (p = 0.0047) in DMG-treated animals. Mean antibody titers to the H antigen were also higher in DMG-treated animals compared with controls after both the first and second inoculation. Lymphocyte transformation assays on cells taken from DMG-treated animals immunized with the influenza vaccine showed a tenfold increase in mean proliferative response (p = 0.0024). Lymphocytes from DMG-treated animals immunized with the typhoid vaccine showed a fourfold increase (mean values) in thymidine uptake (p = 0.0180). No toxicity or adverse effects were observed at any time during the experiment.(ABSTRACT TRUNCATED AT 250 WORDS)

Fas/Fas ligand interactions are involved in ultraviolet-B-induced human lymphocyte apoptosis.

We wondered whether the apoptosis known to occur after UV-B irradiation might involve the Fas/Fas ligand (FasL) signaling pathway. We exposed PBLs from normal individuals, and also the Jurkat (E6-1) and U937 cell lines, to graded doses of UV-B irradiation and observed a prompt and marked increase in Fas expression at doses as low as 0.5 mJ/cm2. Increased Fas expression did not require new protein synthesis, since cycloheximide-treated cells also showed an increase in Fas after UV-B. UV-B-irradiated cells cultured in the presence of zinc showed inhibition of apoptosis coincident with a marked increase in Fas+ cells, apparently indicating the accumulation of Fas-bearing cells unable to undergo apoptosis. After UV-B irradiation, PBLs showed increased expression of Fas ligand; the E6-1 lymphocytic cell line also released soluble FasL. UV-B induced apoptosis could be partially blocked by neutralizing FasL Abs, and a FasL-resistant variant of E6-1 cell line showed reduced apoptosis after UV-B irradiation, implying that the increase in Fas expression signified a role for Fas in UV-induced apoptosis. UV-induced Fas expression may serve to target stress-injured cells for removal by FasL-bearing cells or by FasL produced by the cells themselves in response to the stimuli, and may represent a general function of the Fas/FasL pathway in facilitating the apoptosis and elimination of undesirable or harmful cells.

Expression and function of Fas on cells damaged by gamma-irradiation in B6 and B6/lpr mice.

Fas (CD95) is a cell surface protein that mediates apoptosis. lpr is a mutation of the Fas gene caused by a retroviral insertion resulting in premature termination of transcription and aberrant splicing of Fas mRNA. Mice homozygous for the lpr gene develop lymphoproliferation and produce autoantibodies closely resembling those of human systemic lupus erythematosus. While lpr mice have been reported to express low levels of normally spliced Fas mRNA, it is unknown whether they express functional Fas protein. Here we show that splenocytes from lpr mice that have been damaged by gamma-irradiation expressed Fas protein. Fas was up-regulated on irradiated B6 cells and could be detected on B6/lpr cells undergoing apoptosis following in vitro culture. Detection of Fas on live lpr cells was demonstrable when apoptosis was blocked by zinc. In a short term chimera system, Fas was shown to play a role, in vivo, in the disposition of radiation-injured cells from both normal and lpr mice. The addition of anti-Fas Ab to in vitro cultures resulted in an increase in apoptosis in both B6 and B6/lpr cells. Detection of intact Fas message and low levels of Fas protein in lpr mice has led to the consideration of lpr as a leaky mutation. This study demonstrates that lpr mice can produce functional Fas protein. This system is also appropriate for identifying the in vivo role of Fas/FasL in apoptosis following other cell manipulations.

The effects of cyclosporine on acute murine Coxsackie B3 myocarditis.

The effects of the immunosuppressant drug cyclosporine were studied in the murine model of Coxsackie B3 myocarditis. Ten BALB/c mice, given daily cyclosporine (15 mg/kg) intraperitoneally but not infected, were normal in all respects after 2 weeks. All 32 BALB/c mice infected, but given no cyclosporine, survived and had moderate myocardial mononuclear infiltrates and minimal necrosis at 7 and 14 days. In contrast, 24 mice concurrently infected and given cyclosporine had a high mortality rate (75%) and a significantly attenuated mononuclear infiltrate in the presence of enhanced necrosis when compared with control infected mice. Sixteen mice started on the drug 1 week after infection had a lower mortality rate (55%), but very similar histologic abnormalities. In contrast to negligible or no virus in the hearts of infected mice that were not given cyclosporine, drug treated, infected groups had easily detectable virus in their hearts 14 days after infection. An identical study in Swiss ICR mice yielded similar results. Cyclosporine, when given early during acute murine Coxsackie B3 myocarditis, causes a significant increase in myocardial necrosis and mortality, possibly secondary to enhanced viral survival.

Radiation and stress-induced apoptosis: a role for Fas/Fas ligand interactions.

The lpr gene encodes a defective form of Fas, a cell surface protein that mediates apoptosis. This defect blocks apoptotic deletion of autoreactive T and B cells, leading to lymphoproliferation and lupus-like autoantibody production. The effects of the lpr Fas mutation on other kinds of physiologically relevant apoptosis are largely undocumented. To assess whether some of the apoptosis known to occur after ionizing radiation might be mediated by Fas/Fas ligand (FasL) interactions, we quantitated in vitro apoptosis by flow cytometry measurement of DNA content in splenic T and B cells from irradiated 5- to 8-month-old B6/lpr mice. Total apoptosis of both lpr and control cells was substantial after treatment; however there was a significant difference between B6 (73%) and lpr (25%) lymphocyte apoptosis. Thy1, CD4, CD8, and IgM cells from lpr showed much lower levels of apoptosis than control cells after irradiation. Apoptosis induced by heat shock was also impaired in lpr. The finding that gamma-irradiation increased Fas expression on B6 cells and that irradiation-induced apoptosis could be blocked with a Fas-Fc fusion protein further supported the possible involvement of Fas in this form of apoptosis. Fas/FasL interactions may thus play an important role in identifying and eliminating damaged cells after gamma-irradiation and other forms of injury.

Apoptosis abnormalities of splenic lymphocytes in autoimmune lpr and gld mice.

The murine gene lpr encodes an aberrant form of the apoptosis-inducing receptor Fas. The gene gld, which causes an autoimmune syndrome phenotypically identical to that caused by lpr, encodes a mutant Fas ligand. Because the lpr gene must be expressed in both T and B cells to produce autoimmune disease, it might be anticipated that apoptosis abnormalities would be present in both. Therefore, we quantitated apoptosis in T and B cells from lpr, gld, and normal mice in a short-term in vitro culture system. Freshly isolated spleen cells from normal, lpr, or gld mice showed little or no apoptosis as assessed by quantitative DNA flow cytometry. However, after overnight culture, both T and B cells showed substantial spontaneous apoptosis. Such apoptosis increased strikingly with age in normal but not in autoimmune B cells. CD23low B cells, which are prominent in lpr and gld mice, were particularly notable for high levels of programmed cell death in normal mice. The apoptosis caused by the gld defect could not be corrected by coculture with normal spleen cells. The persistence with age of low levels of B cell apoptosis in lpr and gld mice presumably reflects deficient Fas/Fas ligand interactions. The further localization of the B cell apoptosis defect to the unusual CD23low B cells, which accumulate in lpr and gld mice, adds to the evidence that these cells may be of critical importance to autoimmunity.

Conventional B cells, not B1 cells, are the source of autoantibodies in chronic graft-versus-host disease.

B1 (CD5+) B cells have been implicated as a source of certain autoantibodies in several murine and human studies. We have previously shown in the lpr model of autoimmunity, however, that conventional B cells, not B1 cells, were the source of autoantibodies directed at chromatin, ssDNA, and IgG. In the current study, we have investigated the origin of autoantibodies in chronic graft-versus-host (GVH) disease, induced in nonautoimmune mice by transferring la-incompatible spleen cells. GVH mice develop multiple autoantibodies and significant kidney damage. Therefore, this model allowed us to examine the B cell subset involved in both autoantibody production and tissue injury. We used two protocols to establish B cell chimeras that possessed immunoglobulin heavy chain (lgh) allotype-marked peritoneal (B1-cell source) cells and bone marrow-derived (conventional B cell source) cells from nonautoimmune C57BL/6kh (B6) congenic mice. In both types of chimera, chronic GVH was induced by giving mice alloreactive T cells i.p. All of the subsequent anti-chromatin, RF, and anti-ssDNA autoantibodies were produced by the conventional B cells and not by B1 cells. In addition, glomerular immune complex deposits of both IgM and IgG originated from the conventional B cells and not from B1 cells. These findings thus parallel those from our previous work on autoantibodies in lpr, and extend those findings by demonstrating that antibodies within pathogenic immune complexes in the kidneys are also exclusively of conventional B cell origin.

The role of B cell abnormalities in the systemic autoimmune syndromes of lpr and gld mice.

B cell abnormalities play a central role in the systemic autoimmune syndromes of lpr and gld mice. In the lpr model, autoantibody production requires the intrinsic expression of the lpr gene in B cells, while in the gld mouse the genetic defect is extrinsic, yet probably results in a similar failure of B cell tolerance. Despite their abnormality, lpr B cells require lpr (abnormal) T cells in order to produce autoantibodies.

bcl-2 transgenic Lpr mice show profound enhancement of lymphadenopathy.

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.

Novel immunoregulatory B cell pathways revealed by lpr-+ mixed chimeras.

lpr, a murine mutation of the Fas apoptosis receptor, causes lymphadenopathy and autoantibody production, with lymphadenopathy primarily due to a population of CD4-CD8-B220+ T cells. Previous in vivo experiments, in which lpr and normal bone marrow cells were coinfused into lpr hosts, have demonstrated that only T cells of lpr origin accumulated abnormally and only B cells of lpr origin produced autoantibodies. Moreover, in these chimeras, B cells of normal origin were unable to respond to conventional, T cell-dependent exogenous Ag. To address the role of lpr B cells in regulation of lpr autoimmunity, we have prepared lpr-+ mixed chimeras and selectively eliminated lpr B cells using allele-specific, mAb treatment, thus allowing normal B cells to develop in an environment with lpr T cells. From these data, we arrived at four major conclusions: 1) Compared with control-treated chimeric mice, lpr B cell-depleted mice had greatly reduced total lymph node cell counts; 2) the T cells were derived equally from normal and lpr donors, and the percentage of lpr-derived CD4-CD8- T cells was greatly reduced; 3) despite the presence of the remaining lpr T cells, no autoantibodies were produced by the normal derived B cells; and 4) lpr T cells without lpr B cells were unable to prevent a normal B cell response to conventional Ag. These data demonstrate that B cells can play a critical and expansive regulatory role, not only for T cells, but for other B cells as well.

Phagocytosis and clearance of apoptotic cells is mediated by MER.

Apoptosis is fundamental to the development and maintenance of animal tissues and the immune system. Rapid clearance of apoptotic cells by macrophages is important to inhibit inflammation and autoimmune responses against intracellular antigens. Here we report a new function for Mer, a member of the Axl/Mer/Tyro3 receptor tyrosine kinase family. mer(kd) mice with a cytoplasmic truncation of Mer had macrophages deficient in the clearance of apoptotic thymocytes. This was corrected in chimaeric mice reconstituted with bone marrow from wild-type animals. Primary macrophages isolated from mer(kd) mice showed that the phagocytic deficiency was restricted to apoptotic cells and was independent of Fc receptor-mediated phagocytosis or ingestion of other particles. The inability to clear apoptotic cells adequately may be linked to an increased number of nuclear autoantibodies in mer(kd) mice. Thus, the Mer receptor tyrosine kinase seems to be critical for the engulfment and efficient clearance of apoptotic cells. This has implications for inflammation and autoimmune diseases such as systemic lupus erythematosus.

Phenotypic abnormalities of splenic and bone marrow B cells in lpr and gld mice.

Mice homozygous for the mutant Fas gene lpr develop generalized lymphoproliferation and produce autoantibodies resembling those found in human SLE. We have previously shown that these autoantibodies are produced by B2 cells rather than B1 cells and that the autoantibody- producing B cells are intrinsically abnormal. We investigated further the lpr B cell with a large panel of antibodies to B-cell surface markers to identify phenotypic abnormalities. B cells from spleen and bone marrow of age-matched congenic mice differing only at the lpr locus were examined by flow cytometry. Two consistent phenotypic differences were identified. First, spleen cells from older lpr mice had an increase in the number and percentage of IgM+ B cells expressing low levels of CD23. Second, lpr bone marrow had decreased numbers of B220hiIgM+-syndecan-1+CD23+ B cells. All other markers tested, except the previously identified modest increase of Ia on lpr spleen cells, showed no consistent differences. B cells from gld mice showed the same phenotypic abnormalities as those from lpr. Compared to T cells, the relative paucity of cell surface marker differences between lpr and +/+ B cells suggests that B cells may have fewer regulatory mechanisms to silence autoreactive specificities. The phenotypic differences identified may provide clues to the mechanism of autoantibody production in lpr mice, while the overwhelming phenotypic similarity between lpr and +/+ B cells suggests that the major abnormality of lpr B cells may lie in their specificity, that is, in their inability to delete autoreactive subsets.

MHC genes modify systemic autoimmune disease. The role of the I-E locus.

The MHC exerts an important influence on systemic autoimmune disease. In C57BL/6-lpr/lpr (B6/lpr) mice, substitution of the H-2d instead of the H-2b MHC haplotype results in a global reduction in autoantibody levels. Since H-2d expresses both I-A and I-E, while H-2b expresses only I-A, general down-regulation of autoimmunity in the d haplotype might be due to I-E expression. This was tested with I-E alpha d transgenic B6/lpr mice, which expressed a functional surface I-E molecule. Five-month-old transgene-positive B6/lpr mice had much lower total IgG, IgG anti-chromatin, anti-DNA, and IgM rheumatoid factor directed against IgG1 and against IgG2b than transgene-negative littermates (p < or = 0.002), as well as significantly lower spleen and lymph node weights (p < or = 0.002). Decreases in autoantibody levels in the transgenic lpr mice were not due to a nonspecific effect of the I-E alpha d transgene, since transgene-positive B6/lpr.H-2d mice had levels of autoantibodies comparable with transgene-negative B6/lpr.H-2d mice. To determine whether autoantibody was preferentially made by I-E-negative B cells, irradiated (B6/lpr.Igha x B6/lpr.I-E alpha d)F1 mice were reconstituted with equal amounts of B6/lpr.Igha and B6/lpr.I-E alpha d bone marrow. Allotype-specific ELISA showed that most autoantibody was produced by the I-E negative B cells (range 97% to 84%). The results show that a functional I-E molecule in lpr mice leads to generalized reduction in autoantibody levels through a direct effect on the B cell. The molecular mechanism of this effect remains to be determined.

The role of environmental antigens in the spontaneous development of autoimmunity in MRL-lpr mice.

It has been proposed that the "normal" stimulation of the immune system that occurs from interactions with environmental stimuli, whether infectious or dietary, is necessary for the initiation and/or continuation of autoimmunity. We tested this hypothesis by deriving a group of MRL-lpr mice into a germfree (GF) environment. At 5 mo of age, no differences between GF and conventional MRL-lpr mice were noted in lymphoproliferation, flow cytometric analysis of lymph node cells (LN), or histologic analysis of the kidneys. Autoantibody levels were comparably elevated in both groups. A second experiment tested the role of residual environmental stimuli by contrasting GF mice fed either a low m.w., ultrafiltered Ag-free (GF-AF) diet or an autoclaved natural ingredient diet (GF-NI). At 4 mo of age, both groups showed extensive lymphoproliferation and aberrant T cell formation, although the GF-AF mice had approximately 50% smaller LNs compared with sex-matched GF-NI controls. Autoantibody formation was present in both groups. Histologic analysis of the kidneys revealed that GF-AF mice had much lower levels of nephritis, while immunofluorescence analysis demonstrated no difference in Ig deposits but did reveal a paucity of C3 deposition in the kidneys of GF-AF mice. These data do not support a role for infectious agents in the induction of lymphoproliferation and B cell autoimmunity in MRL-lpr mice. Furthermore, they suggest that autoantibodies do not originate from B cells that were initially committed to exogenous Ags. They do suggest a possible contributory role for dietary exposure in the extent of lymphoproliferation and development of nephritis in this strain.