Autoimmune vasculitis in MRL/Mp-lpr/lpr mice: orthochromatic basophils participate in the development of delayed-type hypersensitivity angiitis.

The pathogenesis of autoimmune vasculitis is poorly understood. Understanding the immunologic mechanisms governing this disease requires precise identification of the cells which comprise the lesion. In this report, we have evaluated tissue sections from MRL/lpr mice from 16 to 45 weeks of age, representing all stages of clinical vasculitis. We demonstrate that basophil myelocytes participate in the evolution of the delayed-type hypersensitivity (DTH) response which initiates and perpetuates autoimmune vasculitis in these mice. These findings raise questions regarding the immunologic mechanisms by which basophils develop in this lesion and the interaction of basophils. VSMCs and lymphocytes in vasculitic angiodestruction.     

Beneficial effects of non-depleting anti-CD4 in MRL/Mp-lpr/lpr mice with active systemic lupus erythematosus and microscopic angiitis.

MRL/Mp-lpr/lpr (MRL/lpr) mice spontaneously develop lymphoproliferation and systemic autoimmune disease. The majority of mice in any cohort develop systemic lupus erythematosus (SLE) and up to a quarter of them develop a syndrome that resembles microscopic angiitis (MPA). Both conditions are characterized by vasculitis, glomerulonephritis and autoantibody formation. Depleting anti-CD4 monoclonal antibody (mAb) treatment is ineffective in MRL/lpr mice after disease onset. The present study investigates the effects of a non-depleting anti-CD4 mAb in MRL/lpr mice with active SLE or MPA. This antibody increased survival compared to control Ig but did not prolong survival compared to non-treated mice. However, anti-CD4 significantly reduced lymphoproliferation in the mice and furthermore reduced the vasculitis component of the autoimmune disease following several weeks of treatment.     

Systemic mononuclear-cell vasculitis in MRL/Mp-lpr/lpr mice. A histologic and immunocytochemical analysis.

The cellular mechanisms governing the expression of mononuclear cell vasculitis are poorly understood. For determination of the precise sequence of events in the development of vasculitis in autoimmune MRL/lpr mice, histologic sections from 4-20-week-old mice were evaluated with a panel of cytochemical and immunohistochemical stains. The results show that vascular disease in MRL/lpr mice develops as follows: Thy 1+, Ly 1+, L3T4- T cells assemble around predominantly small-to-medium muscular arteries at approximately 8 weeks of age. At 12 weeks of age, an adventitial inflammatory focus forms, composed of large "reactive" mononuclear inflammatory cells adjacent to hypertrophied vascular smooth muscle cells (VSMCs). Blastic Thy 1+, Ly 1+, L3T4- T cells subsequently infiltrate the tunica media, and selective VSMC karyolysis results. Occasional cytotoxic/suppressor T cells, macrophages, and possibly NK cells are noted primarily distal to the infiltration site. The outer zone of the inflammatory infiltrate is composed of mature B cells and occasional B-cell precursors. These findings suggest that cellular constituents of the immune response mediate mononuclear cell vasculitis in MRL/lpr mice.     

Resistance of MRL/Mp-lpr/lpr mice to tolerance induction

Mice of strain MRL/Mp-lpr/lpr develop resistance against tolerance induction with aggregate-freed rabbit gamma-globulin as early as 6 weeks of age and show very complete resistance when they are 10 weeks old; congenic mice MRL-Mp+/+ of 6 and 10 weeks can be rendered tolerant. By reconstitution experiments, it was shown that T cells from MRL/Mp-lpr/lpr are responsible for the resistance to tolerance induction. A cell which is not colchicine-sensitive contributes to tolerance of MRL/Mp-+/+, but not to MRL/Mp-lpr/lpr mice. Resistance of T cells against tolerance depends on expression of the lpr gene.     

Myeloperoxidase autoantibodies distinguish vasculitis mediated by anti-neutrophil cytoplasm antibodies from immune complex disease in MRL/Mp- lpr/lprmice: a spontaneous model for human microscopic angiitis

Anti-neutrophil cytoplasm antibodies (ANCA) with specificity for myeloperoxidase (MPO) occur in the sera of patients with microscopic angiitis, an autoimmune disease characterized by necrotizing vasculitis and crescentic glomerulonephritis. These autoantibodies have been shown to stimulate neutrophil degranulation and are believed to participate in pathogenesis. A neutrophilic vasculitis has been reported in MRL- lpr mice which has histological appearances similar to microscopic angiitis. In the present study we show that 22 % of female MRL- lpr mice develop MPO autoantibodies. These animals develop a clinical syndrome of vasculitis and glomerulonephritis that is distinct from immune complex disease. Anti-MPO monoclonal antibodies derived from these mice are polyreactive and react with double-stranded DNA. They bind a conformational epitope on human MPO which is also expressed by activated human neutrophils. The results suggest that a subset of MRL- lpr mice develop ANCA-related vasculitis rather than systemic lupus erythematosus and may be used as a model for human microscopic angiitis.     

Treatment of autoimmune MRL/Mp-lpr/lpr mice with cholera toxin.

The autoimmune manifestations of MRL/Mp-lpr/lpr(MRL/l), a murine model of systemic lupus erythematosus (SLE), were alleviated by administering 1 microgram cholera toxin (CT) every 14 days. The beneficial effects were: (i) significant prolongation of survival time, (ii) prevention of lymphadenopathy, (iii) improvement of T cell mitogenic responses and suppression of a B cell mitogenic response, (iv) decrease in serum anti-DNA and anti-Sm antibodies, (v) increase in IL-2 production by stimulation of spleen cells with concanavalin A (Con A). It is possible that CT may be effective for treatment of murine lupus nephritis by modulating polyclonal lymphocyte activation. This type of immunomodulation may pave the way toward treatment of lupus and other autoimmune diseases.     

Effects of immunomodulating treatment on autoimmune sialadenitis in MRL/Mp-lpr/lpr mice

The autoimmune MRL/Mp-lpr/lpr (MRL/l) mouse spontaneously develops sialadenitis with a morphological and phenotypical pattern similar to that seen in human Sjögren's syndrome (SS). This makes the MRL/1 mouse a suitable model for therapeutical studies of autoimmune sialadenitis. We have, by histological and immunohistochemical techniques, analyzed the therapeutical effect of treatment with LS2616, a recently synthesized oxokinolinamide derivative, on sialadenitis in submandibular glands of MRL/1 mice. The results were compared with effects obtained after treatment with cyclophosphamide (CY) and physiologic saline. Administration of both LS2616 and CY to MRL/1 mice has previously been found to result in prolongation of survival and amelioration of organ pathology. However, only CY treatment reduced sialadenitis, while LS2616 increased the semiquantitatively assessed focal inflammation of salivary glands in 6 months old mice. No differences in T-cell phenotypes of infocal the frequency of B-cells in the sialadenitis was decreased in the CY treated group. In contrast, CY but not LS2616 treatment normalized expression of T-helper and cytotoxic T-cell phenotypes as well as reduced the B-cell portion in lymph nodes. It is concluded that CY treatment can suppress sialadenitis although both LS2616 and CY are effective in prolongation lifespan of MRL/1 mice. This may implicate different immunopathogenic mechanisms for development of sialadenitis versus other organ lesions in the autoimmune disease of MRL/1 mice.     

Cellular interactions for the in vitro production of anti-chromatin autoantibodies in MRL/Mp-lpr/lpr mice

Anti-chromatin autoantibodies are spontaneously produced by autoimmune but not by normal mice. An in vitro system was developed to study the cellular mechanisms of anti-chromatin production in MRL/Mp-lpr/lpr (MRL/lpr) mice. In such cultures, spleen cells from MRL/lpr mice with active autoimmune disease generated substantial amounts of anti-chromatin, as measured by ELISA of culture supernatants and by ELISA spot assay of anti-chromatin-producing cells. In vitro production of anti-chromatin autoantibodies was independent of T cells, even when spleen cells from animals as young as 1 month were examined. In contrast, anti-Sm production under the same conditions was highly T cell dependent. Macrophages and/or macrophage-derived factors were necessary for the in vitro production of anti-chromatin autoantibodies. The lack of anti-chromatin production by cells from nonautoimmune mice could not be ascribed to the presence of suppressor cells. These studies indicate that individual autoantibodies may arise through distinct cellular mechanisms in systemic lupus erythematosus mice. MRL/lpr mice develop global T lymphocyte deficiency along with their autoimmunity. The progressive increase in relatively thymus independent antibodies such as anti-chromatin is consistent with the lack of functional T lymphocytes in aging MRL/lpr mice.     

Androgen control of autoimmune expression in lacrimal glands of MRL/Mp-lpr/lpr mice

The purpose of the present study was to determine, by utilizing an animal model of Sj?gren's syndrome, whether androgen therapy might ameliorate autoimmune sequelae in the lacrimal gland. Age-matched female MRL/Mp-lpr/lpr mice were administered subcutaneous implants of placebo- or testosterone-containing pellets after the onset of disease. Lacrimal glands and, for comparison, submandibular glands were collected from sacrificed mice immediately prior to androgen administration and following 17 and 34 days of maintained hormone exposure. Tissues were processed for light microscopy and examined with a computer-assisted image analysis system. Results demonstrated that testosterone exposure dramatically reduced lymphocyte infiltration in lacrimal tissue: following 34 days of treatment, the percentage infiltrate had undergone a 12-fold decrease. This hormone action, which was time dependent, involved significant abrogations in both infiltrate size and number. Testosterone administration also induced a significant 2- to 3-fold rise in lacrimal gland weight and acinar area and a 2-fold reduction in acinar density/field, compared to values in placebo-treated controls. In addition, androgen administration significantly decreased the magnitude of lymphocyte infiltration in submandibular glands. Overall, our findings demonstrate that androgen therapy may reverse autoimmune sequelae in lacrimal, as well as submandibular, glands in a mouse model of Sj?gren's syndrome.     

Autoimmune kidney disease in MRL/Mp-lpr/lpr mice inhibited by OK-432, a streptococcal preparation

Autoimmune MRL/Mp-lpr/lpr (MRL/l) mice were treated with the immunostimulating anti-cancer drug OK-432 (a streptococcal preparation), a potent inducer of tumour necrosis factor. Treatment was initiated at 8 weeks of age, before the onset of the autoimmune disease. OK-432 prevented the development of immune complex-mediated glomerulonephritis in a dose-dependent manner, and prolonged the life in this strain of mice. At 36 weeks of age, the incidence of proteinuria was 90% in the controls, 60% in the 0.5-KE(1 KE = 0.1 mg) treatment group, and 33% in the 2.0-KE group. The 50% survival time was 23 weeks for the controls; 32 weeks for the 0.5-KE group; and greater than 36 weeks for the 2.0-KE group. Immune complex deposition in glomeruli was significantly reduced in the treated groups. The IgM class of serum autoantibody levels was significantly increased by OK-432 treatment but the IgG class was almost unchanged. Furthermore, lymphadenopathy and splenomegaly were not suppressed. The results indicate that OK-432 may be useful in the treatment of autoimmune disease in humans.     

Unusual cell surface properties of the T lymphocyte population expanding in MRL/Mp-lpr/lpr mice.

MRL/Mp-lpr/lpr (lpr/lpr) mice but not the congenic MRL/Mp-+/+ (+/+) mice, develop a generalized lymph node (LN) hypertrophy reflecting the expansion of a T-cell population that acts as an enhancing factor for autoimmunity. In order to characterize better this T-cell population, we investigated some of its surface properties in comparison with those of +/+ T cells. Electrophoretic measurements revealed that lpr/lpr T cells possess a lower electronegative surface charge than %/% T cells which indicates that the two cell types differ in the molecular composition of their plasmic membrane periphery. This notion was substantiated by the quantification of T- and B-cell markers and of lectin-binding sites on these cells using single- and two-colour flow cytofluorimetry. In agreement with recent observations by Lewis, Giorgi & Warner (1981) lpr/lpr T cells exhibited lower levels of Thy-1 and Lyt-1 antigens than +/+ T cells and were mostly devoid of Lyt-2 antigen. Although lpr/lpr lymph node (LN) cells displayed similar amounts of surface receptors for peanut agglutinin as +/+ LN cells, the expression of surface receptors for other lectins were either lower (Limulus polyphemus agglutinin, Maclura pomifera agglutinin, Concanavalin A) or higher (Helix pomatia agglutinin, Soya bean agglutinin, Bandeiraea simplicifolia agglutinin I, Phytohaemagglutinin L) on lpr/lpr T cells than on +/+ T cells. These data indicate that the T cells accumulating in hypertrophied lpr/lpr LN are endowed with unique surface characteristics which may explain some of the functional abnormalities of these cells.     

Phenotypic and functional analyses on T-cell subsets in lymph nodes of MRL/Mp-lpr/lpr mice

By means of killing and/or FACS sorting the double-negative (DN) Lyt2-, L3T4- cells, Lyt2+ or L3T4+ cells and B220- cell populations were separated from T-cell-enriched lymph node (LN) cells of 4- to 5-month-old MRL/Mp-lpr/lpr mice. These highly purified cell populations were examined for their proliferative responses, interleukin 2 (IL2) production and expression of IL2 receptor (IL2R) in response to phorbol myristate acetate (PMA) and the calcium ionophore A23187 (A2) or PMA plus concanavalin A. The DNT-cell population was unable to respond to the stimuli and did not express IL2R. Thus the DN T cells, the major population responsible for the lymphadenopathy, possess fundamental defects in signal transduction as well as in the IL2-IL2R-mediated function. On the other hand, Lyt2+ or L3T4+ T cells obtained by sorting or B220- cells purified by the sorting after killing B220+ cells, exhibited proliferative responses indistinguishable from that of LN cells of the congenic MRL/Mp-+/+(+/+) mice. These cells also expressed IL2R after stimulation, however, the amount of IL2 produced was significantly lower than that produced by congenic +/+ cells. This suggested that phenotypically normal Lyt2+ or L3T4+ T cells of lpr LNs also possess a partial defect in the signal transduction system for IL2 production under the influence of the lpr gene.     

Pulmonary inflammation in autoimmune MRL/Mp-lpr/lpr mice. Histopathology and bronchoalveolar lavage evaluation.

Early detection of lupus pneumonitis is difficult because it requires lung biopsy. The authors describe here in detail the age-related histologic changes in pulmonary inflammation, the age-related changes in bronchoalveolar lavage (BAL), and the effect of cyclophosphamide (8 mg/kg) on pulmonary inflammation and bronchoalveolar lavage in MRL/Mp-lpr/lpr mouse, an animal model of systemic lupus erythematosus. To assess the evolution of pulmonary inflammation and response to cyclophosphamide therapy, they compared the age-related progression of pulmonary inflammation with sequential changes in BAL cell populations in this autoimmune mouse model. A striking similarity was noted between age-related changes in pulmonary inflammation and lymphocyte counts in BAL. A trend to reduction in histologic evidence of inflammation was reflected by lymphocytes in BAL in cyclophosphamide-treated (8 mg/kg/day) males but not in females. There was a striking sex-related difference in that the histologic evidence of pulmonary inflammation and bronchoalveolar lavage lymphocyte count in cyclophosphamide-treated males was significantly lower than cyclophosphamide-treated females of the same age.     

Variable region genes of anti-histone autoantibodies from a MRL/Mp-lpr/lpr mouse

The variable region nucleotide sequences of seven (five IgM and two IgG) anti-histone monoclonal antibodies from a single MRL/Mp-lpr/lpr mouse have been determined. These antibodies are not clonally related and used diverse V, D and J genes. However, six of the seven antibodies have VH segments encoded by genes from the J558 family, two of these (an IgM and an IgG) share an identical VH gene. The isoelectric points of MRA3 and MRA12, the two IgG antibodies of the panel, range from 6.3 to 7.0 and from 6.0 to 6.3, respectively. The second conplementarity-determining region (CDR) of the VH gene of MRA12 (the most acidic and the most strongly histone-reactive antibody) includes only two positively charged but five negatively charged amino acid residues. This feature is unusual since the equivalent CDR in most VHJ558 genes are not comprised predominantly of acidic residues and suggests that such negatively charged residues are important for antibody binding to histones.     

Naturally occurring antibodies to poly(ADP-ribose) in autoimmune MRL/Mp-lpr/lpr mice.

The presence of antibodies to poly(ADP-ribose) was demonstrated in the sera of MRL/Mp-lpr/lpr (MRL/l) mice by an enzyme linked immunosorbent assay (ELISA). Antibody to poly(ADP-ribose) was strongly inhibited by single stranded DNA (ssDNA) and poly(ADP-ribose), and less by double stranded DNA (dsDNA). Affinity purified anti-poly(ADP-ribose) antibodies bound more with immobilized ssDNA than with poly(ADP-ribose), and were significantly inhibited by soluble ssDNA, although poly(ADP-ribose) was the best soluble inhibitor. On the contrary, affinity purified anti-ssDNA antibodies bound best with ssDNA and significantly but less with poly(ADP-ribose); however, they were scarcely inhibited by poly(ADP-ribose). These results suggest that similar antigenic determinants exist in poly(ADP-ribose) and ssDNA. It is conceivable, however, at the present moment that 'naturally occurring antibodies to poly(ADP-ribose)' in MRL/l mice are subpopulations of anti-ssDNA antibodies that react equally well with poly(ADP-ribose) and ssDNA.     

Splenocytes from MRL/Mp-lpr/lpr mice spontaneously produce antibodies against a cell-surface protein cross-reacting with DNA

Anti-double-stranded (ds) DNA autoantibodies are considered as pathogenic in systemic lupus erythematosus (SLE). Anti-DNA antibodies have been shown to be released by B cells from lupus mice or patients in vitro. A monoclonal anti-DNA antibody (PME 77) specific for dsDNA has previously been shown to react with a cell-surface protein called LAMP (lupus-associated membrane protein), which is present on the cell surface of various cell types involved in SLE pathogenesis. Using an immunoreplica analysis technique, we show here that spleen cells from MRL/Mp-lpr/lpr lupus mice, cultured in vitro, spontaneously produce anti-LAMP IgG antibodies. Conversely, anti-LAMP antibodies were not detected in spleen-cell culture supernatants from nonautoimmune CBA/Ca mice. Taken together with our previous reports, this result adds a new argument for a pathogenic role of anti-LAMP autoantibodies in SLE.     

Functional analyses of lpr gene in MRL/Mp-lpr/lpr mice. Role of lymph node stromal cells in lpr-lymphadenopathy.

To clarify the mechanism by which the lpr gene causes lymphadenopathy, we established an experimental system to induce lymph node (LN) swelling in unaffected mice. In MRL-(+)/+ mice that had been 5 Gy-irradiated and grafted with bone marrow cells (BMCs) plus LN from MRL-lpr/lpr mice, a remarkable enlargement of the LN grafts was seen. The enlarged grafts lacked normal LN structure and were indistinguishable from LNs of MRL-lpr/lpr mice. The induction of LN swelling by this method was achieved not only in [MRL-lpr/lpr-->MRL-(+)/+] but also in [MRL-lpr/lpr-->BALB/c], [MRL-lpr/lpr-->C3H], [B6-lpr/lpr-->B10.Thy1.1], and [B6-lpr/lpr-->BALB/c] combinations. Furthermore, the lpr/lpr LN grafts developed lymph node swelling even without the transplantation of BMCs. Most cells in the grafted LNs disappeared within a few days, and large clear fibroblast-like cells then became dominant for 1 to 4 weeks. Thereafter, lymphoid cells increased and had filled the graft by the 8th week. The LN grafts obtained from MRL-lpr/lpr (but not MRL-(+)/+) mice showed the ability to transfer LN node swelling into the secondary MRL-(+)/+ hosts two weeks after the primary transplantation. These results strongly suggest that the fibroblast-like LN stromal cells play a crucial role in lpr-associated lymphadenopathy.     

Detection of anti-neutrophil cytoplasmic antibodies in MRL/Mp-lpr/lpr mice and analysis of their target antigens

Anti-neutrophil cytoplasmic antibodies (ANCA) have been widely studied and recognized to be clinically very important for some human diseases including systemic rheumatic diseases. We analyzed ANCA response and their target antigens in MRL/Mp-lpr/lpr (MRL-lpr) mice, an animal model of systemic rheumatic disease. P-ANCA was detected in 57% of the mice. Antibodies to the known P-ANCA target antigens at the same age were examined. Among these, antibodies to high mobility group (HMG) proteins HMG1 and HMG2 were detected in 57% of the mice, 75% of which were also positive for P-ANCA. These anti-HMG1/HMG2 activities were absorbed by preincubation with a mixture of HMG1 and HMG2. In contrast, antibodies to myeloperoxidase and cathepsin G were detected in 14% and 7%, respectively, but these activities were not inhibited by preincubation with corresponding antigens. In addition, the titers of P-ANCA and anti-HMG1/HMG2 antibodies in MRL-lpr mice were significantly correlated with each other. Thus, HMG1 and HMG2 were considered to be significant target antigens of P-ANCA in MRL-lpr mice.     

Fate of immune complexes, glomerulonephritis, and cell-mediated vasculitis in lupus-prone MRL/Mp lpr/lpr mice

Immune complex formation was induced by the injection of (125)I-BSA into female MRL/Mp lpr/lpr mice, which develop spontaneous systemic lupus erythematosus (SLE)-like disease, and MRL/Mp +/+ mice, which do not. At designated intervals following the injection of 10 mg of (125)I-bovine serum albumin (BSA), the nonlupus mice developed sparse, small electron-dense deposits in mesangial areas and subepithelial immune deposits that underwent partial resolution. By contrast, glomeruli of the SLE-prone mouse kidneys revealed proliferation of mesangial cells and some increase in mesangial matrix material. Numerous subepithelial and mesangial electron-dense deposits were present. Some subendothelial and intramembranous deposits were also demonstrated. Capillary lumens contained massive electron-dense deposits. The resolving subepithelial deposits observed were fewer than half the number found in kidneys of the non-SLE mice. Whole body counts were also recorded daily following the injection of (125)I-BSA. Whereas, both lupus-prone and non-SLE control mice eliminated (125)I-BSA at equivalent rates through day 12 postinoculation, those with SLE-like disease showed a decreased (125)I-BSA elimination rate between days 6 and 12. Results suggest an impairment in the ability of SLE-prone mice to resolve immune complexes, whether they are nuclear-antinuclear or from an exogenous source, i.e., BSA-anti-BSA, compared to controls in this experimental model of the superimposition of exogenous immune complex formation on systemic lupus erythematosus-like disease.