Unique surface phenotype of T cells in lymphoproliferative autoimmune MRL/Mp-lpr/lpr mice

MRL/l mice, which carry the mutant gene lpr, develop massive T cell lymphoproliferation and an autoimmune syndrome. The surface antigen profile of MRL/l lymphocytes was analyzed with rat monoclonal antibodies. They included YE1/9.9, anti-transferrin receptor; YE1/7.1, which reacts with a population of proliferating immature T cells; and YE1/19.1, a newly identified antibody reactive with a surface antigen on EL-4 cells, which consists of two disulfide-bonded polypeptide chains, each of 115,000 m.w. Flow cytometric analysis of lymphocytes from the enlarged lymph nodes of MRL/1 mice showed the majority of them to be Lyt-1+, YE1/7.1+, YE1/19.1+, YE1/9.9-. In contrast, YE1/7.1 and YE1/19.1 antibodies did not significantly react with lymphocytes from the congenic MRL/n mice, which lack the lpr gene and do not develop lymphoproliferation. Lymphocytes from younger MRL/l mice, which have almost normal size lymph nodes, were also YE1/7.1-, YE1/19.1-. A small proportion of mitogen-stimulated MRL/n lymph node cells appeared to express these antigens at low densities, but it is not known whether they represent a unique cell population. Among the several lymphoid cell lines tested, only the T lymphoma line EL-4 had the Lyt-1+, YE1/7.1+, YE1/19.1+ phenotype. These results suggest that the lpr mutation induces the expansion of a unique T cell subset.     

T cell lineages in the thymus of lpr/lpr mice. Evidence for parallel pathways of normal and abnormal T cell development

Autoimmune mice homozygous for the lpr/lpr (lpr) gene develop a profound lymphadenopathy resulting from the accumulation of immature Thy-1+ Lyt-2- L3T4- cells in peripheral lymphoid tissues. The source of these cells is not known although the presence of a thymus is necessary to manifest both the lymph node enlargement and the autoimmunity. For this reason and the fact that the abnormal lpr cell phenotypically resembles immature thymocytes, we studied the thymus in lpr mice. Adult lpr thymuses were found to contain an immature population phenotypically identical to the peripherally accumulating cells, including the expression of B220 and Pgp-1 antigens as well as the presence of surface T cell receptor molecules as defined by the antibody KJ16-133. Evidence is presented that some of these lpr precursor T cells are capable of intrathymic differentiation, whereas the vast majority are exported unchanged to the lymph nodes where a portion differentiate further into mature T cells. This lpr-specific lineage could be distinguished from a normal component of the lpr thymus by surface phenotype and immunohistology. The results suggest that the massive accumulation of cells in lpr lymph nodes is not so much the result of abnormal proliferation of T cells as abnormal intrathymic differentiation. In addition, a minor subpopulation of normal Lyt-2- L3T4- thymocytes was identified that resembles the phenotype of the lpr cell and similarly expresses surface T cell receptor molecules. The presence of two parallel lineages in the lpr thymus thus also provides insight into normal T cell development.     

Estrogen accelerates immune complex glomerulonephritis but ameliorates T cell-mediated vasculitis and sialadenitis in autoimmune MRL lpr/lpr mice.

Estrogen is known to influence immune responses in healthy subjects in a dichotomous fashion. Thus, in number of previous studies we and others have demonstrated that B cell activities are augmented after exposure to estrogen whereas T cell reactivity is suppressed. Furthermore, it has been shown that this hormone has significant impact on the course of certain human and experimental autoimmune diseases. In this study we report that treatment with physiological doses of estradiol exerts dichotomous effects on different manifestations of the lupus disease in MRL/l mice. On one hand immune complex-mediated glomerulonephritis was significantly accelerated. This outcome was due to polyclonal B cell activation with increased production of antibodies to double-stranded DNA and formation of circulating immune complexes. In contrast, T cell-mediated lesions such as focal sialadenitis, renal vasculitis, and periarticular inflammation were all significantly ameliorated in MRL/l mice exposed to estrogen. Thus, we were able to demonstrate that, within one subject and even within one organ, administration of estrogen leads to differential outcome of SLE morbidity. We propose that the differential effect of estrogen on the manifestations of the autoimmune disease of MRL/l mice is due to its dichotomous effects on B and T cell-mediated immune responses.     

Autoantibody in MRL/Mp-lpr/lpr mice. A monoclonal antibody specific for the abnormal T cells from mice bearing the lpr/lpr gene

We generated mAb from an unimmunized autoimmune MRL/Mp-lpr/lpr mouse. One of these mAb A108, reacted with cell surface Ag present on abnormal T cells from MRL/Mp-lpr/lpr, C3H-lpr/lpr, and C57BL/6-lpr/lpr mice. We failed to detect significant numbers of A108 bearing cells in the lymph nodes of MRL-Mp/+/+, normal C3H or normal C57BL/6 mice. Therefore, the expression of A108 correlates with the presence of the lpr/lpr gene. A108 binds to a variety of murine T cell tumor lines (e.g., EL4, BW5147, and YAC-1) and human T cell tumor lines (e.g., MOLT-3, Sup T1, and Jurkat). A108 does not bind to normal human PBL. Immunoprecipitation of surface iodinated EL-4 and BW5147 with A108 identified one major protein with a Mr of about 17.5-kDa. The significance of these findings with respect to the development of lymphoid proliferation and autoimmune disease in mice bearing the lpr/lpr gene will be discussed.     

Selection of anti-double-stranded DNA B cells in autoimmune MRL-lpr/lpr mice

Abs to DNA and nucleoproteins are expressed in systemic autoimmune diseases, whereas B cells producing such Abs are edited, deleted, or inactivated in healthy individuals. Why autoimmune individuals fail to regulate is not well understood. In this study, we investigate the sources of anti-dsDNA B cells in autoimmune transgenic MRL-lpr/lpr mice. These mice are particularly susceptible to lupus because they carry a site-directed transgene, H76R that codes for an anti-DNA H chain. Over 90% of the B cells are eliminated in the bone marrow of these mice, and the few surviving B cells are associated with one of two Vkappa editors, Vkappa38c and Vkappa21D. Thus, it appears that negative selection by deletion and editing are intact in MRL-lpr/lpr mice. However, a population of splenic B cells in the H76R MRL-lpr/lpr mice produces IgG anti-nuclear Abs, and these mice have severe autoimmune organ damage. These IgG Abs are not associated with editors but instead use a unique Vkappa gene, Vkappa23. The H76R/Vkappa23 combination has a relatively high affinity for dsDNA and an anti-nuclear Ab pattern characteristic of lupus. Therefore, this Vkappa gene may confer a selective advantage to anti-DNA Abs in diseased mice.     

Alpha 2,3-linked sialic acids are more abundant in CD45 antigens and leukosialins of abnormal T cells of lpr mice than in those of normal T cells

T cells from enlarged lymph nodes of MRL/MpJ-lpr/lpr (lpr) mice were found to express more binding sites for strongly hemagglutinating Phaseolus vulgaris agglutinin (PHA-E4) and fewer binding sites for Ricinus communis aglutinin (RCA) than those from normal MRL/MpJ-+/+ (+/+) mouse lymph node. We found that high-molecular-weight (180K-220K) glycoproteins on lpr T cells were strongly stained with these lectins on Western-blotting. These glycoproteins were found to belong to the CD45 family, by absorption with monoclonal anti-CD45 antibody. We also found that the other glycoproteins (105K and 120K glycoproteins on lpr T cells and a 105K glycoprotein on +/+ T cells) were strongly stained with the lectins which preferentially bind to mucin-type (O-linked) sugar chains on the cell surface. These glycoproteins were found to be leukosialins, by absorption with anti-leukosialin serum. From the results of the lectin-binding to these glycoproteins after sialidase treatment, CD45 antigens and leukosialin molecules on lpr T cells were found to have many more terminal alpha 2,3-linked sialic acids than those on +/+ T cells, and this fact explains why lpr T cells have more binding sites for PHA-E4 but fewer binding sites for RCA.     

Cloning of B cells from autoimmune MRL-lpr/lpr and MRL.xid mice

The relationship between colony formation (cloning) of B cells and their activation in murine autoimmunity was investigated in MRL-lpr/lpr and MRL.xid mice. Cells from MRL-lpr/lpr mice showed similar requirements for in vitro growth as normal CBA/J and BALB/c cells, with maximal colony formation in the presence of the supporting factors lipopolysaccharide and sheep red blood cells. The frequency of colony-forming cells from MRL-lpr/lpr spleens or hapten-specific B-cell preparations was slightly greater than the two normal control strains, with this difference significant only for a comparison of BALB/c and MRL-lpr/lpr spleens. In contrast, MRL-lpr/lpr mice bearing the xid gene for B-cell immunodeficiency (MRL.xid) had markedly reduced B-cell colony formation. These mice nevertheless expressed anti-DNA antibodies, although at levels reduced from that of MRL-lpr/lpr controls. These results indicate that enhanced in vitro colony formation need not accompany B-cell hyperactivity in murine autoimmune disease and that autoantibody production can occur in mice with impairment in this growth property.     

Transplanted islets from lpr mice are resistantto autoimmune destruction in a model of streptozotocin-induced type I diabetes

The mechanism by which β-cells die during autoimmune diabetes has remained a subject of intense investigation. The loss of β-cells in the disease is T cell mediated and thought to result from a number of different insults including apoptosis induction through the death receptor CD95. However, the role of CD95 in autoimmune diabetes, studied primarily in the non-obese diabetic (NOD) mouse model, has been controversial. We have used an alternative model of autoimmune diabetes triggered by repeated low doses of streptozotocin. In this model, islet grafts from C3H mice that carry the lpr mutation, and therefore lack the ability to undergo apoptosis through CD95-CD95L interaction, were completely protected when grafted in autoimmune diabetic mice despite periinsulitis (infiltration of T cells) which however did not progress to islet destruction. In contrast wild-type grafts were rapidly eliminated in autoimmune recipients. Our data provide strong support for a major role of CD95 in the destruction of islets in autoimmune mice.     

Lymphocytes from autoimmune MRL lpr/lpr mice are hyperresponsive to IL-18 and overexpress the IL-18 receptor accessory chain

MRL lpr/lpr mice spontaneously develop a severe autoimmune lupus syndrome characterized by strong autoantibody production and massive lymphoproliferation, in which IFN-gamma plays a major pathogenic effect. The role of the IFN-gamma-inducing cytokine IL-18 in the autoimmune syndrome of lpr/lpr mice has been investigated. In response to IL-18, lymph node cells of lpr/lpr mice produce significant amounts of IFN-gamma and proliferate more potently as compared with cells from +/+ mice. Cells likely responsible for such hyperresponsiveness to IL-18 include NK cells and the CD4(+)/CD8(+) self-reactive T lymphocytes characteristically present in lymph nodes of lpr/lpr mice. Analysis of the expression of IL-18R complex revealed that mRNA for the IL-18R alpha-chain is constitutively expressed at similar level both in +/+ and lpr/lpr lymphocytes. In contrast, the expression of the accessory receptor chain IL-18R beta is low in unstimulated +/+ cells but significantly high in lpr/lpr cells. Thus, the abnormally high expression of the IL-18R chain IL-18R beta could be one of the causes of the hyperresponsiveness of lpr/lpr cells to IL-18 at the basis of consequent enhancement of IFN-gamma production and development of IFN-gamma-dependent autoimmune pathology.     

Bone marrow-derived cells are responsible for the development of autoimmune arthritis in human T cell leukemia virus type I-transgenic mice and those of normal mice can suppress the disease.

Previously, we reported that human T cell leukemia virus type I env-pX region-introduced transgenic (pX-Tg) mice developed an inflammatory polyarthropathy associated with a development of autoimmunity. To elucidate roles of autoimmunity in the development of arthritis, the immune cells were reciprocally replaced between pX-Tg mice and non-transgenic (Tg) mice. When bone marrow (BM) cells and spleen cells from pX-Tg mice were transferred into irradiated non-Tg mice, arthritis developed in these mice. In contrast, arthritis in pX-Tg mice was completely suppressed by non-Tg BM and spleen cells. Similar results were obtained with BM cells only. After the transplantation, T cells, B cells, and macrophages were replaced completely, whereas cells in the joints were replaced partially. In those mice, serum Ig and rheumatoid factor levels correlated with the disease development, and inflammatory cytokine expression was elevated in the arthritic joints. Furthermore, involvement of T cells in the joint lesion was suggested, because the incidence was greatly reduced in athymic nu/nu mice although small proportion of the mice still developed arthritis. These observations suggest that BM stem cells are abnormal, causing autoimmunity in pX-Tg mice, and this autoimmunity plays an important, but not absolute, role in the development of arthritis in this Tg mouse.     

Unusual expression of IL 2 receptors and both the c-myb and c-raf oncogenes in T cell lines and clones derived from autoimmune MRL-lpr/lpr mice

Concomitant with their disease, autoimmune MRL-lpr/lpr mice develop a profound lymphadenopathy composed of an unusual dull Lyt-1+ population of T cells. To examine the unusual growth properties and origin of these T cells, as well as their potential role in disease, very rapidly growing T cell lines and clones have been developed from cultures of MRL-lpr/lpr spleen and LN cells. These were studied for growth receptors, oncogene expression, and surface markers. The results further demonstrate the unique nature of lpr-derived T cells and show that i) all lines and clones exhibit greatly elevated expression of both the c-myb and the c-raf oncogenes, ii) despite the reported defect in IL 2 receptor expression of mitogen-activated fresh MRL-lpr/lpr T cells, all long-term lines or clones bear large numbers of IL 2 receptors continuously and without stimulation, although iii) unlike slower growing IL 2-dependent lines from MRL-lpr/lpr mice, these rapidly growing lines and clones are poorly inhibited by anti-IL 2 receptor antibody. Such IL 2 receptor-bearing, nontransformed T cells that are easily maintained have been useful in growth factor studies.     

Functional alterations of macrophages in autoimmune MRL-lpr/lpr mice

To assess the role of macrophages (MAC) in the pathogenesis of systemic lupus erythematosus, we investigated functional aspects of peritoneal MAC obtained from autoimmune MRL/MpJ-lpr/lpr (MRL-lpr) mice. MRL-lpr and control C3H/HeN MAC were obtained from untreated mice or mice injected i.p. with 1 ml of 10% sterile peptone 3 days before cell harvest. MRL-lpr mice had significantly more peritoneal cells (MAC and lymphocytes) than did control mice. In endotoxin-free conditions, MRL-lpr MAC were similar to C3H/HeN MAC in their baseline, and IFN-gamma and/or LPS enhanced cytolysis of 3T12 fibrosarcoma tumor cells. Compared with C3H/HeN MAC, MRL-lpr MAC had a significant increase in antibody-dependent cellular cytotoxicity activity against sheep erythrocytes. This enhanced activity was not accompanied by a similar increase in adherence and/or phagocytosis of the same targets. Finally, in response to phorbol myristate acetate stimulation, both resident and peptone-induced MAC from MRL-lpr mice produced significantly more hydrogen peroxide than did those from control mice. These results indicate that MAC from MRL-lpr mice display features of selective "activation", and suggest that MAC or their products may play a role in the pathogenesis of inflammatory disorders seen in autoimmune diseases.     

Expansion of the population of double negative CD4-8- T alpha beta-cells in the liver is a common feature of autoimmune mice.

There have been several reports that double negative (DN) CD4-8- T alpha beta-cells might be responsible for the onset of autoimmune diseases in humans and mice. We previously revealed that such DN T alpha beta-cells are generated in the liver of autoimmune MRL-lpr/lpr mice. In the present study, we further characterize the histology of the liver in these mice by light and electron microscopic studies. An intensive accumulation of mononuclear cells in the liver was demonstrated and a significant proportion of these mononuclear lymphocytes was found to intimately interact with Kupffer cells or endothelial cells of the hepatic sinusoids. The majority of such lymphocytes were TcR+CD4-8-Pgp-1+ alpha beta-cells. Identification of DN T alpha beta-cells was then performed in various autoimmune model mice. Interestingly, all autoimmune mice tested (i.e., MRL-lpr/lpr, C3H/HeJ-gld/gld, BXSB, NOD, MRL(-)+/+ and NZB/W F1 mice), showed an increased proportion of DN T alpha beta-cells (greater than 11% among all MNC) in the liver when they became old and diseased. On the other hand, young and old normal mice and young autoimmune mice before the onset of disease did not have such a high proportion of DN T alpha beta-cells (less than 10%) in the liver. Among autoimmune mice, MRL-lpr/lpr and C3H/HeJ-gld/gld mice had lymphadenopathy, which consisted of DN T alpha beta-cells (greater than 25%), after the onset of disease. Autoimmune mice of the other strains had neither lymphadenopathy nor DN T alpha beta-cells in the periphery, even when they were diseased. These results suggest that the expansion of the DN T alpha beta-cell population in the liver is a common feature of autoimmune mice, irrespective of the information of lymphadenopathy.     

Development of the autoimmune B cell repertoire in MRL-lpr/lpr mice

The processes responsible for the production of autoantibodies have been shown to include both Ag-specific and generalized (polyclonal) forms of B cell activation. The relative contribution and temporal association of these processes to the genesis of systemic autoimmunity are incompletely understood. To study this relationship, the B cell repertoires of MRL-lpr/lpr mice were analyzed by ELISA spot assay over an 8-mo period. Between 6 and 12 wk of age, the number of splenic lymphocytes producing antibodies reactive with both autoantigens and conventional Ag increased proportionately. The repertoires of MRL-lpr/lpr mice under 12 wk were dominated by IgM-secreting B cells that showed no bias toward the production of specific autoantibodies. From 12 to 38 wk of age, an increasing proportion of animals developed repertoires dominated by IgG-secreting B cells that were skewed toward reactivity against one or very few (auto)antigens. Although there was no single Ag against which all mice developed skewed reactivity, 55% of MRL-lpr/lpr adults had increased numbers of B cells producing antibodies to the Sm Ag and 13 to 16% developed increased reactivity toward DNA, myosin, histone, thyroglobulin, or T cells. These data indicate that generalized (polyclonal) B cell activation dominates early repertoire development whereas (auto)-antigen-specific responses become increasingly important during the latter stages of disease in these autoimmune-prone mice.     

Heterologous cross-linking of Lyt-2 (CD8) to the alpha beta-T cell receptor is more effective in T cell activation than homologous alpha beta-T cell receptor cross-linking

Ag recognition of Lyt-2 (CD8)-positive T lymphocytes requires the presentation by APC of a suitably processed Ag in association with MHC class I molecules. In previous studies we have obtained evidence that, for optimal activation, both the alpha beta-TCR and Lyt-2 have to participate in this recognition process. In the current study we investigate the functional consequences of limited cross-linking of these cell surface molecules by using soluble, dimeric hetero- and homoconjugates of mAb to Lyt-2 and to the TCR beta-chain (F23.1). Heterologous cross-linking of Lyt-2 to the TCR induced a vigorous, selective Lyt-2+ T cell proliferative response. Functionally active cytotoxic cells were generated, and a high frequency of responding cells was observed in limiting dilution analyses. In contrast, homologous TCR cross-linking initiated a less pronounced proliferation with a relatively low frequency of response, whereas Lyt-2 cross-linking resulted in no cellular proliferation. Significant T cell activation occurred with exposure to anti-Lyt-2: F23.1 mAb dimers at concentrations an order of magnitude lower than those required for stimulation by F23.1:F23.1 mAb dimers. The induction of proliferation by mAb dimers occurred in the absence of Fc components and in rigorously APC depleted, purified T cell preparations. Effective stimulation of resting T cells could be induced also by heterodimers of monovalent Fab fragments. Heterologous cross-linking of Lyt-2 to the TCR was superior to homologous TCR cross-linking primarily with respect to proliferation in IL-2 containing media and to IL-2R expression, whereas proliferation in response to other lymphokines and the production of IL-2 itself were similar under both cross-linking regimens. Thus, when linked to the TCR, Lyt-2 contributed a strong, positive signal toward IL-2-dependent growth of resting T cells. We assume that in the case of Ag-driven T cell activation, the class I MHC molecule acts as the physiologic cross-linking ligand for Lyt-2 and the TCR.     

Granulocyte-colony stimulating factor treatment of lupus autoimmune disease in MRL-lpr/lpr mice.

G-CSF not only functions as an endogenous hemopoietic growth factor for neutrophils, but also displays pro-Th2 and antiinflammatory properties that could be of therapeutic benefit in autoimmune settings. We evaluated the effect of treatment with G-CSF in a murine model of spontaneous systemic lupus erythematosus, a disease in which G-CSF is already administered to patients to alleviate neutropenia, a common complication. Chronic treatment of lupus-prone MRL-lpr/lpr mice with low doses (10 microg/kg) of recombinant human G-CSF, despite the induction of a shift toward the Th2 phenotype of the autoimmune response, increased glomerular deposition of Igs and accelerated lupus disease. Conversely, high-dose (200 microg/kg) treatment with G-CSF induced substantial protection, prolonging survival by >2 mo. In the animals treated with these high doses of G-CSF, neither the Th1/Th2 profile nor the serum levels of TNF-alpha and IL-10 were modified. Despite the presence of immune complexes in their kidney glomeruli, no inflammation ensued, and serum IL-12 and soluble TNF receptors remained at pre-disease levels. This uncoupling of immune complex deposition and kidney damage resulted from a local down-modulation of FcgammaRIII (CD16) expression within the glomeruli by G-CSF. Our results demonstrate a beneficial effect of high doses of G-CSF in the prevention of lupus nephritis that may hold promise for future clinical applications, provided caution is taken in dose adjustment.     

Terminal deoxynucleotidyl transferase deficiency decreases autoimmune disease in MRL-Fas(lpr) mice.

The neonatal Ab and TCR repertoires are much less diverse, and also very different from, the adult repertoires due to the delayed onset of terminal deoxynucleotidyl transferase (TdT) expression in ontogeny. TdT adds nontemplated N nucleotides to the junctions of Igs and TCRs, and thus its absence removes one of the major components of junctional diversity in complementarity-determining region 3 (CDR3). We have generated TdT-deficient MRL/lpr, Fas-deficient (MRL-Fas(lpr)) mice, and show that they have an increased lifespan, decreased incidence of skin lesions, and much lower serum levels of anti-dsDNA, anti-chromatin, and IgM rheumatoid factors. The generalized hypergammaglobulinemia characteristic of MRL-Fas(lpr) mice is also greatly reduced, as is the percentage of CD4(-)CD8(-)B220(+) (double-negative) T cells. IgG deposits in the kidney are significantly reduced, although evidence of renal disease is present in many mice at 6 mo. CDR3 regions of both IgH and TCR from peripheral lymphocytes of MRL-Fas(lpr) mice are shorter in the absence of TdT, and there is a paucity of arginines in the IgH CDR3 regions of the MRL-Fas(lpr) TdT(-/-) mice. Because the amelioration of symptoms is so widespread, it is likely that the absence of N regions has more of an affect than merely decreasing the precursor frequency of anti-dsDNA B cells. Hence, either the T or B cell repertoires, or more likely both, require N region diversity to produce the full spectrum of autoimmune lupus disease.     

The differential ability of splenic adherent cells from B6.lpr mice to induce suppressor T cells

Hapten-coupled splenic adherent cells or resident peritoneal cells from autoimmune B6.lpr mice that are over 5 mo of age fail to induce first-order inducer suppressor T cells (Ts1). However, the same population of hapten-coupled cells can induce both delayed-type hypersensitivity responses and third-order effector suppressor T cells (Ts3). Thus, splenic and peritoneal antigen-presenting cells from B6.lpr mice display a defined defect in the ability to induce certain suppressor T cell responses. The cellular defect in Ts1 induction is controlled by the lpr gene, since age-matched congenic B6 mice do not display this defect. The splenic adherent cell defect is temporarily correlated with the autoimmunity that develops in B6.lpr animals. The antigen-presenting defect in the B6.lpr splenic adherent population for Ts1 induction is reversible by culturing the cells in interferon-gamma. The results are discussed as an illustration of the relationship between experimental models of autoimmunity and defects in a suppressor T cell cascade.