Leukocyte-endothelial cell interactions are enhanced in dermal postcapillary venules of MRL/fas(lpr) (lupus-prone) mice: roles of P- and E-selectin

MRL/fas(lpr) mice are affected by a systemic autoimmune disease that results in widespread leukocytic infiltration of the vasculature, including in the skin. The molecular pathways responsible for this leukocyte recruitment are poorly understood. Therefore, the aim of these experiments was to examine the mechanisms of leukocyte trafficking in the dermal microvasculature of MRL/fas(lpr) mice. Intravital microscopy was used to examine leukocyte rolling and adhesion in dermal postcapillary venules of MRL/fas(lpr) mice at 8, 12, and 16 wk of age. When compared with age-matched BALB/c and MRL(+/+) (nondiseased) mice, leukocyte rolling and adhesion in MRL/fas(lpr) mice were significantly enhanced at 12 wk of age, and remained elevated at 16 wk of age. At 8 and 12 wk, leukocyte rolling in all three strains was almost entirely inhibited by an anti-P-selectin mAb. In contrast, at 16 wk some (approximately 10%) leukocyte rolling persisted following P-selectin blockade. This residual rolling was predominantly inhibitable with an anti-E-selectin mAb; however, treatment with anti-E-selectin mAb alone had a minimal effect. P-selectin-deficient MRL/fas(lpr) mice also displayed leukocyte rolling that was significantly lower than in wild-type MRL/fas(lpr) mice. However, in these mice, leukocyte adhesion remained at the elevated levels observed in wild-type MRL/fas(lpr) mice. This adhesion was eliminated by chronic treatment with anti-E-selectin mAb. These findings indicate that leukocyte-endothelial cell interactions are enhanced in the dermal microvasculature of MRL/fas(lpr) mice above the age of 12 wk. Furthermore, the data suggest that the endothelial selectins share overlapping roles in mediating this enhanced leukocyte recruitment.     

Critical role of the alpha 4 integrin/VCAM-1 pathway in cerebral leukocyte trafficking in lupus-prone MRL/fas(lpr) mice

MRL/fas(lpr) mice are affected by a systemic autoimmune disease that results in leukocyte recruitment to a wide range of vascular beds, including the cerebral microvasculature. The mechanisms responsible for the leukocyte trafficking to the brain in these animals are not known. Therefore, the aim of this study was to directly examine the cerebral microvasculature in MRL/fas(lpr) mice and determine the molecular mechanisms responsible for this leukocyte recruitment. Intravital microscopy was used to assess leukocyte-endothelial cell interactions (rolling, adhesion) in the pial microcirculation of MRL(+/+) (control) and MRL/fas(lpr) mice at 8, 12, and 16 wk of age. Leukocyte rolling and adhesion were rarely observed in MRL(+/+) mice of any age. MRL/fas(lpr) mice displayed similar results at 8 and 12 wk. However, at 16 wk, significant increases in leukocyte rolling and adhesion were observed in these mice. Histological analysis revealed that the interacting cells were exclusively mononuclear. Leukocyte rolling was reduced, but not eliminated in P-selectin(-/-)-MRL/fas(lpr) mice. However, leukocyte adhesion was not reduced in these mice, indicating that P-selectin-dependent rolling was not required for leukocyte recruitment to the cerebral vasculature in this model of systemic inflammation. E-selectin blockade also had no effect on leukocyte rolling. In contrast, blockade of either the alpha4 integrin or VCAM-1 eliminated P-selectin-independent leukocyte rolling. alpha4 Integrin blockade also significantly inhibited leukocyte adhesion. These studies demonstrate that the systemic inflammatory response that affects MRL/fas(lpr) mice results in leukocyte rolling and adhesion in the cerebral microcirculation, and that the alpha4 integrin/VCAM-1 pathway plays a central role in mediating these interactions.     

Type I IFN protects against murine lupus

Both the type I (IFN-alpha beta) and type II (IFN-gamma) IFNs have been heavily implicated in the pathogenesis of systemic lupus erythematosus. To test the relative roles of these systems, congenic lupus-prone MRL/CD95(lpr/lpr) (MRL/lpr) mice lacking the type I IFN receptor (IFN-RI), type II IFN receptor (IFN-RII), or both, were derived. As expected, deficiency for IFN-RII protected MRL/lpr mice from the development of significant autoimmune-associated lymphadenopathy, autoantibodies, and renal disease. However, deficiency for the IFN-RI surprisingly worsened lymphoproliferation, autoantibody production, and end organ disease; animals doubly deficient for IFN-RI and IFN-RII developed an autoimmune phenotype intermediate between wild-type and IFN-RII-deficient animals, all correlating with an ability of type I IFN to suppress MRL B cell activation. Thus, type I IFNs protect against both the humoral and end organ autoimmune syndrome of MRL/lpr mice, independent of IFN-gamma. These findings warrant caution in the use of type I IFN antagonists in the treatment of autoimmune diseases and suggest further investigation into the interplay between the types I and II IFNs during the ontogeny of pathogenic autoantibodies.     

Undernutrition without malnutrition restricts the numbers and proportions of Ly-1 B lymphocytes in autoimmune (MRL/I and BXSB) mice

MRL/Mp-lpr/lpr (MRL/l) and BXSB mice represent inbred mouse strains in which lymphoproliferative disease and autoimmune disease that includes lethal renal disease routinely occurs by 6 months of age. Chronic energy intake restriction increases longevity and health span of MRL/l and BXSB mice as it does in mice of other short-lived as well as long-lived strains. Chronic energy intake restriction forestalls development of the lymphoproliferative process, prevents development of renal lesions, decreases levels of circulating immune complexes, and permits maintenance of vigorous immunologic function with age. We have reported that in autoimmune-prone mice, a population of Ly-1 B lymphocytes that is associated with autoimmune disease and is greatly expanded among cells of the spleen, peritoneal exudate, and peripheral blood can be reduced in proportion as a consequence of undernutrition without malnutrition. Herein, we demonstrate that in MRL/l and BXSB mice, chronic energy intake restriction imposed at weaning inhibited accumulation of Ly-1 B lymphocytes throughout the lymphoid system, i.e., among cells of the spleen, thymus, mesenteric lymph nodes, bone marrow, peritoneal exudate, and peripheral blood when these tissues or fluids were studied at age 3 or 5 months. These results extend our previous finding that autoimmune-prone mice possess unusually large numbers of Ly-1 B cells in their lymphoid tissues which can be reduced in frequency as a function of diet toward the levels present in long-lived autoimmune-resistant mice.     

The relationship between plasma fibronectin levels and autoimmune disease activity in MRL/l mice

Plasma fibronectin levels increased significantly over time in MRL/l mice with progressive autoimmune disease. At 100 and 120 days of age both male and female MRL/l mice exhibited significantly higher fibronectin (Fn) levels than the more resistant MRL/l controls. Male mice at early time points had Fn levels no greater than controls due perhaps to the later onset of disease in MRL/l males. In contrast, female MRL/l mice, when compared with MRL/n controls, had higher Fn levels from 40 days of age. The proteinuria in these animals was also above MRL/n controls from the first time point taken (Day 40). In a temporal study with female MRL/l mice, Fn levels peaked at age 120 days and reflected the pattern of the survival curve, indicating that plasma Fn levels have an association with disease activity.     

Treatment of autoimmune MRL/Ipr mice with monoclonal antibody to Thy-1.2: a single injection has sustained effects on lymphoproliferation and renal disease

MRL/Mp-lpr/lpr (MRL/lpr) mice spontaneously develop an autoimmune disease characterized by anti-DNA antibodies, immune-complex glomerulonephritis, and massive proliferation of a distinct population of T cells. The proliferating T cells have the phenotype Thy-1.2+, T200+, Lyt-1+,2-,3-, but Thy-1.2 and Lyt-1 are expressed in abnormally low density. These cells appear to function as helper cells, and neonatal thymectomy prevents both lymphoproliferation and autoimmunity, which suggests that autoimmunity in MRL/lpr mice is secondary to T cell proliferation. We therefore attempted to reduce lymphoproliferation by treating MRL/lpr mice with a single injection of rat monoclonal antibody (MAb) to Thy-1.2 (30-H12, IgG2b). Mice were treated at 8 wk, before the onset of overt disease. We found that MRL/lpr mice were resistant to depletion of circulating T cells (CTC) by anti-Thy-1.2; 0.6 mg of antibody totally depleted CTC from normal mice, but had little or no effect on CTC in MRL/lpr mice. However, treatment with 6 mg of MAb against Thy-1.2 reduced CTC in MRL/lpr mice by over 70%. Moreover, this single treatment markedly reduced the proliferation of CTC over the ensuing 3 mo, despite clearance of the anti-Thy-1.2 from the circulation within 3 wk. Treated mice maintained better renal function than untreated controls, as assessed by levels of blood urea nitrogen (BUN), although anti-DNA antibodies were not significantly reduced. The effect of anti-Thy-1.2 was specific; treatment with rat MAb to the common leukocyte antigen T200 produced only a transient effect on circulating lymphocytes and did not reduce renal disease. The prolonged effects of a single injection of anti-Thy-1.2 suggest that the MAb produces a sustained alteration in immune regulation. The improvement in renal disease is in accord with evidence that autoimmune disease in MRL/lpr mice is T cell dependent. Monoclonal anti-lymphocyte antibodies may be useful in the treatment of autoimmunity.     

Thymic dysfunction in the mutant diabetic (db/db) mouse

Thymic function has been explored in genetically diabetic homozygous C57BL/KsJ (db/db) mice by evaluating their serum thymic factor (FTS) levels with a rosette assay. As previously reported for other autoimmune mice (NZB or MRL/I mice), the age-dependent decline of FTS levels was significantly accelerated in diabetic mice when compared to heterozygous littermates. Furthermore, FTS inhibitory molecules were detected in db/db mouse sera (as early as 10 wk of age) as evaluated by their ability to absorb in vitro the activity of synthetic FTS in the rosette assay, and in vivo for their capacity to induce the disappearance of endogenous FTS when injected into normal mice. These inhibitors were shown to be immunoglobulins. Histologically, the thymus presented an accelerated involution starting with a cortical lymphocytic depletion and an increased number of Hassall's corpuscles. Ultrastructural studies showed alterations in thymic epithelial cells, mainly represented by an increasing number of cytoplasmic vacuoles. By means of indirect immunofluorescence with anti-FTS monoclonal antibodies, it was shown that the number of FTS+ cells was reduced in db/db mouse thymuses: at the age of 22 wk, diabetic mice had 10 times fewer FTS+ cells than heterozygotes of the same age. Taken together, these results indicate important abnormalities in the thymus of diabetic mice. It is possible that the associated lymphocyte dysfunction plays a role in the pathogenesis of the autoimmune disease presented by db/db mice.     

The lpr gene is associated with resistance to engraftment by lymphoid but not erythroid stem cells from normal mice

This study demonstrates cell lineage-specific resistance to engraftment involving lymphocytes but not erythrocytes by the spontaneously autoimmune MRL/lpr mouse strain. In these experiments, MRL/lpr mice were lethally irradiated (1000 R) and reconstituted with normal A-Thy bone marrow stem cells. Periodic analysis from 6 wk to 6 mo posttransplantation demonstrated that the T and B cells of these chimeras were derived from the MRL/lpr host. However, in the same A-Thy----MRL/lpr chimeras, erythrocyte repopulation was completely of A-Thy donor origin. In contrast, control MRL/+ (congenic mice that differ from MRL/lpr at the lpr locus and do not develop accelerated autoimmune disease) recipients were successfully repopulated in both the lymphoid and erythroid compartments by the A-Thy donor cells.     

Anti-CD4 monoclonal antibody therapy suppresses autoimmune disease in MRL/Mp-lpr/lpr mice.

MRL/Mp-lpr/lpr (MRL/lpr) mice spontaneously develop systemic autoimmune disease, characterized by vasculitis, lymphadenopathy, glomerulonephritis, and autoantibody formation. The target organ inflammatory lesions are composed largely of CD4+ "helper" T cells, while the massively enlarged lymph nodes are composed primarily of CD3+ CD4- CD8- TCR alpha/beta + "double-negative" T cells. In this study we investigated the effect of treatment of MRL/lpr mice with anti-CD4 monoclonal antibody (mAb); control groups consisted of animals treated with normal saline or rat immunoglobulin (Ig). Anti-CD4 mAb treatment, which was started at 4 weeks and continued through 20 weeks of age, resulted in a dramatic reduction of both the frequency and severity of the autoimmune disease, as demonstrated histologically and serologically. Anti-CD4 mAb therapy markedly reduced the frequency of glomerulonephritis and eliminated vasculitis of the major renal arterial branches. Glomerulonephritis was detected in 9 of 9 saline-treated, 9 of 9 rat Ig-treated, but in only 1 of 9 anti-CD4 mAb-treated mice; vasculitis was detected in 6 of 9 saline-treated, 7 of 9 rat Ig-treated, but in none of 9 anti-CD4 mAb-treated mice. The frequency of antinuclear antibodies, titer of anti-dsDNA antibodies, and total Ig levels were all significantly reduced by anti-CD4 mAb therapy. These data support the hypothesis that CD4+ T cells play a central role in the disease process in this autoimmune strain.     

T-cell mediated suppression in the MRL mouse

MRL/lpr mice possess an autosomal recessive gene, lpr, which is associated with lymphoproliferation and acceleration of autoimmune disease. Lymphoproliferation has been ascribed to a single gene defect predominantly affecting the T-lymphocyte component of the immune system. MRL/++ mice do not possess the lpr autosomal recessive mutation and do not develop early lymphadenopathy. T-lymphocyte functional activity was studied in these mice using the polyclonal T-cell mitogens PHA and Con A. Our results indicated a significant suppression of the spleen and lymph node response of MRL/lpr mice to these polyclonal mitogens as compared to the MRL/++ response noted as early as 6 weeks of age. In addition, there was a progressive decline in the MRL/lpr spleen and lymph node cell mitogenic responses with increasing age. Spleen and lymph node cells from 20-week MRL/lpr mice were also relatively unresponsive in the mixed lymphocyte culture reaction as compared to cells from MRL/ ++ or BALB/c mice. The in vitro proliferative response of the MRL mice was further examined with respect to possible accessory cell modulation by both macrophages and T cells. It was found that in 20-week MRL/lpr lymph nodes a significant degree of suppression of lymphocyte proliferation could be mediated by the MRL/lpr T cell. Increased lymphocyte proliferation to a mitogenic signal could only be demonstrated in those MRL/lpr mice 3 weeks of age.     

Effects of calorie restriction on immunologic functions and development of autoimmune disease in NZB mice.

Chronic energy (calorie) intake restriction (CEIR) prolonged life, inhibited autoimmune disease, and influenced immunologic and hematologic parameters in NZB mice. Abnormalities in numbers and proportions of T and B cells populations were corrected. Deficient responses to phytomitogens, mixed lymphocyte reactions, formation of plaque-forming cells to sheep red blood cells in vitro, production of cytotoxic T lymphocytes after in vitro stimulation, and interleukin 2 production were also corrected. CEIR prevented the extreme splenomegaly that normally occurs with age in NZB mice. This influence was associated with reduction of a greatly expanded non-T, non-B lymphoid cell population. Calorie restriction also prevented in NZB mice the rapid decrease in total numbers of colony-forming B cells in bone marrow that is also characteristic of mice of this strain. The influences of CEIR on immune parameters and hematopoiesis were generally less marked in non-autoimmune-prone DBA/2 mice than in autoimmune-prone NZB mice. CEIR has been shown to produce profound influences on several strains of autoimmune-prone mice (NZB x NZW)F1, MRL/lpr, BXSB, and NZB herein). In each of these strains, the pathogenesis and manifestations of autoimmune disease are dissimilar. Therefore, it seems likely that calorie restriction acts on an as yet elusive mechanism that operates to foster development of the diseases associated with aging common to each of these autoimmune strains as well as autoimmune-resistant mice and rats. Further investigation of the molecular and cellular bases of the benefits of CEIR seems urgent.     

Decreased expression of the Ets family transcription factor Fli-1 markedly prolongs survival and significantly reduces renal disease in MRL/lpr mice

Increased Fli-1 mRNA is present in PBLs from systemic lupus erythematosus patients, and transgenic overexpression of Fli-1 in normal mice leads to a lupus-like disease. We report in this study that MRL/lpr mice, an animal model of systemic lupus erythematosus, have increased splenic expression of Fli-1 protein compared with BALB/c mice. Using mice with targeted gene disruption, we examined the effect of reduced Fli-1 expression on disease development in MRL/lpr mice. Complete knockout of Fli-1 is lethal in utero. Fli-1 protein expression in heterozygous MRL/lpr (Fli-1(+/-)) mice was reduced by 50% compared with wild-type MRL/lpr (Fli-1(+/+)) mice. Fli-1(+/-) MRL/lpr mice had significantly decreased serum levels of total IgG and anti-dsDNA Abs as disease progressed. Fli-1(+/-) MRL/lpr mice had significantly increased splenic CD8(+) and naive T cells compared with Fli-1(+/+) MRL/lpr mice. Both in vivo and in vitro production of MCP-1 were significantly decreased in Fli-1(+/-) MRL/lpr mice. The Fli-1(+/-) mice had markedly decreased proteinuria and significantly lower pathologic renal scores. At 48 wk of age, survival was significantly increased in the Fli-1(+/-) MRL/lpr mice, as 100% of Fli-1(+/-) MRL/lpr mice were alive, in contrast to only 27% of Fli-1(+/+) mice. These findings indicate that Fli-1 expression is important in lupus-like disease development, and that modulation of Fli-1 expression profoundly decreases renal disease and improves survival in MRL/lpr mice.     

Immunoregulation in MRL/Mp-lpr/lpr mice: evidence for decreased helper-T-cell and increased suppressor-T-cell function with age

In vivo and in vitro plaque-forming cell (PFC) responsiveness to sheep erythrocytes (SRBC) was used to assess immunoregulatory function in the autoimmune MRL mouse strain. MRL/Mp-lpr/lpr (MRL/l) mice had good primary and secondary IgM and IgG responses in vivo compared to MRL/Mp-+/+ (MRL/n) mice when young, but with age the MRL/l responses declined markedly. In vitro primary SRBC-specific PFC responses in MRL/l mice declined at the same time as in vivo responses, indicating that the in vivo autoimmune environment could not account for cellular dysfunction. When varied mixtures of T and B cells from MRL/l and MRL/n mice were cultured, abnormalities in MRL/l T-cell function became apparent. T-helper-cell (T_H) function declined rapidly with age, beginning by 2 to $\text{2}\text{1}\text{2}$ months of age. T cells from MRL/l mice 2 months of age and older also had increased suppressor activity when cultured with B cells and MRL/n T cells. The degree of suppressor activity increased with age. The correlation of these findings with results of previous studies by others and with autoimmune disease is discussed.     

C1q deficiency and autoimmunity: the effects of genetic background on disease expression

Gene-targeted C1q-deficient mice have been shown to develop a syndrome reminiscent of human systemic lupus erythematosus with antinuclear Abs and proliferative glomerulonephritis. Initial phenotypic analysis conducted in (129 x C57BL/6) hybrid mice showed that background genes were a significant factor for the full expression of the autoimmune disease. To assess the contribution of background genes in the expression of the autoimmune phenotype, the disrupted C1qa gene was backcrossed for seven generations onto C57BL/6 and MRL/Mp(+/+) strains. These were intercrossed with C57BL/6.lpr/lpr and MRL/Mp-lpr/lpr strains to generate C1q-deficient substrains. In C1q-deficient C57BL/6 mice, no evidence of an autoimmune phenotype was found, and C1q deficiency in both the C57BL/6.lpr/lpr and MRL/Mp-lpr/lpr strains did not modify the autoimmune phenotype observed in wild-type controls. However, in C1q-deficient MRL/Mp(+/+) animals an acceleration of both the onset and the severity of antinuclear Abs and glomerulonephritis was seen. Disease was particularly pronounced in females, which developed severe crescentic glomerulonephritis accompanied by heavy proteinuria. In addition, the C1q-deficient MRL/Mp(+/+) mice had an impairment in the phagocytic clearance of apoptotic cells in vivo. These data demonstrate that the expression of autoimmunity in C1q-deficient mice is strongly influenced by other background genes. The work also highlights the potential value of the C1q-deficient MRL/Mp(+/+) strain as a tool with which to dissect further the underlying mechanisms of the autoimmune syndrome associated with C1q deficiency.     

Effects in chicks of arsenic,arginine, and zinc and their interaction on body weight,plasma uric acid,plasma urea,and kidney arginase activity

The interaction among arsenic, zinc, and arginine was studied in chicks using two fully crossed, three-way, two-by-two-by-two experiments. Arsenic at levels of 0 and 2 μg/g zinc at levels of 2.5 (zinc-deficient) and 25 (zinc-adequate) μg/g, and arginine at levels of 0 and 16 mg/g were supplemented to the diet. After 28 d in both experiments, growth was depressed in chicks fed diets either supplemented with arginine or deficient in zinc. Arsenic deprivation depressed growth of chicks fed diets containing the basal level of arginine and 25 μg supplemental Zn/g. Arsenic deprivation had little or no effect on growth of zinc-deprived chicks fed diets containing the basal level of arginine, or in zinc-deprived or zinc-adequate chicks fed supplemental arginine. Zinc-deficiency elevated urea in plasma and arginase activity in kidney. Those elevations, however, were more marked in arsenic-supplemented than in arsenic-deprived chicks. Also, plasma urea and kidney arginase activity were markedly elevated in chicks fed supplemental arginine; the elevations were more marked in zinc-deficient chicks. These findings support the concept that arsenic has a physiological role, associated with zinc, that can influence arginine metabolism in the chick.     

Effect of acute zinc deficiency on insulin receptor binding in rat adipocytes

Considerable data have been reported on the relationship between insulin resistance and zinc deficiency. In this study, insulin receptor binding was measured in isolated rat adipocytes. Two assays were carried out at 37°C (binding and internalization) and 16°C (binding) using¹²⁵I insulin 0.05–20 nM. A decreased insulin receptor binding was observed in zinc-deficient rat adipocytes, but we could not make any distinction between the specific zinc depletion effects and the effects of the caloric restriction induced by zinc deficiency.     

Pathogenesis of lupus dermatoses in autoimmune mice. X. Evaluation of histamine-N-methyltransferase activity in the skin of autoimmune

We measured histamine concentration and its metabolizing enzymes in the skin of MRL/Mp-lpr/lpr (MRL/l) and BXSB mice to clarify the contribution of histamine metabolism to the mechanisms of the development of lupus dermatoses. The concentration of histamine seemed to differ with the mouse strain. The activity of histamine-N-methyltransferase (HMT), one of two major metabolizing enzymes, was significantly lower in the tail and back skin of MRL/l mice at the age of 5 months than in the control MRL/Mp-+/+(MRL/n) mice, although there were no characteristic differences among several mouse strains of 1 mo of age. In the back skin of MRL/l mice, an age-dependent decrease of HMT activity was observed along with a corresponding decrease in histamine concentration, whereas an age-dependent increase of both HMT activity and histamine concentration was demonstrated in BXSB mice and other control mouse strains. Autoimmune-prone male BXSB mice and non-autoimmune female BXSB mice at 5 mo of age showed similar HMT activity. Corticosteroid treatment restored HMT activity in the skin of MRL/l mice but not in MRL/n mice. In addition, the change in HMT activity in MRL/l mice treated with corticosteroid appeared earlier than changes in clinicopathological examinations including skin eruptions, dermatopathology and proteinuria. Diamine oxidase (DAO) activity, another major metabolizing enzyme, was not detected in the skin of any autoimmune or control mouse strains. These findings suggest that the low activity of HMT in the skin of MRL/l mice plays a significant pathological role in the development of spontaneous lupus-like eruption. In other mouse strains, it is assumed that HMT activity is regulated by genetic factors.     

The Igh-V locus of MRL mice: restriction fragment length polymorphism in eleven strains of mice as determined with VH and D gene probes

The MRL strain of mice is a model system that closely parallels the human autoimmune disease systemic lupus erythematosus. Our analysis of the variable region genes of MRL mice showed that the MRL/lpr D genes were similar to those of the C3H mouse (Igh-C allotype j). This result was unexpected, because previous studies of the MRL/lpr and MRL/+ substrains suggested that they are allotype a at the Igh-1 (gamma 2a) locus of the constant region. The Igh-V (heavy chain variable region) locus of the MRL/lpr and MRL/+ strains of mice and their parents were therefore examined by restriction fragment length polymorphism with probes for the DSP2 and DFL16 gene families and with two cloned VH probes. Five other strains of mice were also included because the heavy chain locus of the LG mouse, which is the major progenitor of the MRL strains, has not been studied. The MRL substrains and the LG and C3H parents were indistinguishable at all the Igh-V loci studied. These results suggest that the MRL substrains and their LG parent are haplotype j at the Igh-V locus. The results obtained with D gene probes show that the DSP2 gene family is more polymorphic than the DFL16 gene family, which is relatively conserved. We have assigned Igh-V haplotypes for the four VH loci to the 11 strains of mice studied.     

Studies of lymphoproliferation in MRL-lpr/lpr mice

MRL-lpr/lpr mice develop massive lymphoproliferation and an associated autoimmune process that includes anti-DNA formation, vasculitis, and glomerulonephritis. We have investigated the lymphoproliferation of MRL-lpr/lpr mice and have found that multiple factors are operative. Although neonatal thymectomy markedly retards the syndrome, chronic injection of poly rI.rC could substitute for the thymus. The resulting cells had the phenotype characteristic of the abnormal MRL-lpr/lpr T cells, Thy-1+, dull Ly-1+, Lyt-2-, 6B2+, Ig-. Splenectomy at 2 wk of age markedly retarded the development of this syndrome; however, splenectomy at birth did not. Some protection was afforded by splenectomy at 5 wk. Thus, there appears to be a critical period in the life of an MRL-lpr/lpr mouse when the spleen contributes importantly to the lymphoproliferation. A most remarkable observation was that an MRL-lpr/lpr spleen graft under the kidney capsule could induce lymphadenopathy characteristic of lpr/lpr mice in MRL +/+ recipients. Cells within the graft had to be able to proliferate for the adenopathy to occur because irradiation of the spleen with 800 R just before grafting abrogated the lymphadenopathy-inducing potential. No adenopathy was induced by +/+ spleen grafts placed into +/+ mice. Although MRL-lpr/lpr males develop disease slightly more slowly than female littermates, the differences are small. Manipulations that retard disease, such as splenectomy at 2 wk or neonatal thymectomy, magnified the sex differences. Male MRL-lpr/lpr mice that were thymectomized and splenectomized and given polyclonal immune activators failed to develop either anti-DNA or lymphadenopathy, whereas their female littermates expressed both abnormalities. We conclude from these studies that multiple factors serve to modulate the magnitude of the lymphoproliferation and autoimmune syndrome of MRL-lpr/lpr mice.     

Analysis of C4 and the C4 binding protein in the MRL/lpr mouse

Systemic lupus erythematosus is a complement-mediated autoimmune disease. While genetic deficiencies of classical pathway components lead to an increased risk of developing systemic lupus erythematosus, end organ damage is associated with complement activation and immune complex deposition. The role of classical pathway regulators in systemic lupus erythematosus is unknown. C4 binding protein (C4bp) is a major negative regulator of the classical pathway. In order to study the role of C4bp deficiency in an established murine model of lupus nephritis, mice with a targeted deletion in the gene encoding C4bp were backcrossed into the MRL/lpr genetic background. Compared with control MRL/lpr mice, C4bp knockout MLR/lpr mice had similar mortality and similar degrees of lymphoproliferation. There were no differences in the extent of proteinuria or renal inflammation. Staining for complement proteins and immunoglobulins in the kidneys of diseased mice revealed no significant strain differences. Moreover, there was no difference in autoantibody production or in levels of circulating immune complexes. In comparison with C57BL/6 mice, MRL/lpr mice had depressed C4 levels as early as 3 weeks of age. The absence of C4bp did not impact serum C4 levels or alter classical pathway hemolytic activity. Given that immune complex renal injury in the MRL/lpr mouse is independent of Fc receptors as well as the major negative regulator of the classical pathway, new mechanisms for immune-complex-mediated renal injury need to be considered.