Effects of dietary omega3 and omega6 lipids and vitamin E on chemokine levels in autoimmune-prone MRL/MpJ-lpr/lpr mice

Elevated levels of chemokines, such as Regulated upon Activation, Normal T cell Expressed and Secreted (RANTES), Monocyte Chemotactic Protein-1 (MCP-1), Macrophage Inflammatory Protein-1alpha (MIP-1alpha), and Macrophage Inflammatory Protein-1beta (MIP-1beta) have been found in rheumatoid arthritis (RA) and juvenile arthritis (JA), and they may be associated with the pathogenesis of these diseases. These chemokines are implicated in the migration of specific leukocytes into the joints. Omega-3 (omega3) fatty acid rich-fish oil (FO) and vitamin E may delay the progress of certain autoimmune diseases. The present study was designed to understand the effects of dietary lipids (omega-6 and omega-3 fatty acids) and vitamin E on the production of chemokines in autoimmune-prone MRL/lpr (a mouse model for RA) and congenic control MRL/++ mice. The MRL mice were fed for 4.5 months omega-6 and omega-3 diets that varied in lipid sources (corn oil; CO and fish oil; FO) and vitamin E levels (269 I.U./kg and 694 I.U./kg diet). Spleen cells were isolated and cultured aseptically in the presence of PHA for 48 h at 37 degrees C and the levels of chemokines (RANTES, JE/MCP-1 and MIP-1alpha) were determined in the cell-free supernatants. The levels of RANTES and JE/MCP-1 were significantly higher in MRL/lpr mice compared to MRL/++ mice. The FO had differential effect on RANTES and MCP-1 production by spleen cells. The production of RANTES and JE/MCP-1 by spleen cells in mice fed the FO diets was significantly lower than in mice fed the CO diets (p < 0.0001). The levels of vitamin E did not affect the production of RANTES and JE/MCP-1. The levels of vitamin E had a significant effect on MIP-1alpha as the spleen cells of mice fed diets containing 694 IU/kg diet of vitamin E produced significantly higher levels of MIP-1alpha compared to the group of mice fed the diets containing 269 IU of vitamin E (p < 0.0001). The data obtained from this study in MRL/lpr and MRL/++ mice suggest that FO diets containing omega-3 fatty acids are beneficial in decreasing the levels of certain pro-inflammatory chemokines (RANTES and MCP-1) thereby delaying the onset of and severity of autoimmune symptoms in MRL/lpr mouse model.     

Methotrexate chronotherapy is effective against rheumatoid arthritis

Methotrexate (MTX) is the most important drug for treating rheumatoid arthritis (RA). It has been stated that cytokines play an important role in the pathogenesis of RA, and that cytokine levels increase and show 24-h rhythms in RA patients. Previously, we found that arthritis was relieved after the administration of MTX at specific times in synchronization with the 24-h rhythm of tumor necrosis factor (TNF)-α in collagen-induced arthritis (CIA) animals. Based on our findings in an earlier study of the dosing time-dependent effects of MTX in MRL/lpr mice, which develop autoimmune disorders that share similarities with human RA, we examined here the utility of MTX chronotherapy in Japanese RA patients. In an initial animal modeling study, we collected blood from MRL/lpr mice at different times (2, 6, 10, 14, 18, or 22 hours after the light was turned on [HALO]), and we measured TNF-α mRNA expression in leukocytes. MTX was administered to the mice at two different dosing times (6 or 18 HALO), and various blood parameters were measured to estimate arthritis activity. TNF-α mRNA levels showed a clear 24-h rhythm with a peak at 22 HALO and a trough at 18 HALO after RA had developed. In these MRL/lpr mice, inflammation and TNF-α were markedly reduced when the MTX dosing time was matched to the time (18 HALO) when the TNF-α level began to increase. We then applied these findings to Japanese RA patients by switching them from the standard MTX three times/wk (day 1: after breakfast and supper; day 2: after breakfast schedule), to chronotherapy, in which the dose and number of doses/wk were not changed but MTX was administered once-a-day at bedtime. Disease Activity Score (DAS)28, modified health assessment questionnaire (MHAQ), and adverse effects were assessed. With MTX chronotherapy, DAS28, which is commonly used to quantitatively assess RA symptoms, was significantly improved at all follow-up clinical visit times compared with the baseline (vs. 1 mo: p?=?.0197, 2 mos: p?=?.0107, 3 mos: p?=?.0087). Significant symptom recovery was observed in 41.2% of patients, and 23.5% of patients achieved clinical remission during the 3 mos of follow-up. Functional capacity of RA patients, as indicated by the MHAQ, was markedly improved by chronotherapy. There were no severe adverse effects. Thus, we demonstrated (i) inflammation and plasma TNF-α concentrations were significantly reduced in MRL/lpr mice treated with MTX at 18 HALO, the time when TNF-α mRNA level began to increase; and (ii) MTX bedtime chronotherapy was safe, markedly reduced disease activity, and improved the functional capacity of RA patients. The findings on RA patients show that bedtime MTX chronotherapy can improve RA symptoms compared to the current standard dosing methods.     

Functional defects of culture-grown bone marrow-derived macrophages from autoimmune MRL/MpJ-lpr/lpr mice

To investigate the primary defects and development of macrophages in MRL/MpJ-/pr/lpr (MRL/l) mice, we used a pure population of macrophages derived from bone marrow precursor cells cultured in the presence of L-cell conditioned medium (LCM) as a source of colony stimulating factor. Bone marrow-derived macrophages (BMM phi) from MRL/l mice had lower antigen presenting activity as detected by the induction of antigen-specific T cell proliferation, than age- and sex-matched control mice (CBA/J). Cell surface antigens (Ia and Mac-1) were determined quantitatively by a cell sorter as markers of macrophage differentiation. The BMM phi from MRL/l contained a much smaller number of Ia antigen-positive macrophages than those from normal mice. Treatment of BMM phi with an Ia-inducing of factor (IFN-gamma) markedly increased the expression of Ia antigens. This increase was significantly greater in BMM phi from MRL/l mice than in BMM phi from control mice. Expression of Mac-1 antigen was not different in BMM phi from the two strains. The Fc-mediated phagocytosis of IgG-coated sheep red blood cells was decreased in BMM phi from MRL/l mice compared with those from control mice. The function of nonspecific phagocytosis as measured by latex-bead incorporation was also impaired in MRL/l mice. The functional defects of MRL/l BMM phi found in these experiments are not secondary defects acquired under the influence of environmental signals during development, but are derived from the primary abnormalities which already exist in myeloid stem cells.     

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.     

Increase in potential activities of protein phosphatases PP1 and PP2A in lymphoid tissues of autoimmune MRL/MpJ-lpr/lpr mice.

The differential assay conditions for protein phosphatases PP1, PP2A, and PP2C were extensively studied by using crude extracts from mouse lymphoid tissues as enzyme sources. Under these conditions, the protein phosphatase activities were measured in MRL/MpJ-lpr/lpr mice (MRL/lpr mice), autoimmune-prone mice, and MRL/MpJ(-)+/+ mice (MRL/+/+ mice) and C3H/HeJ mice as the controls. In MRL/lpr mice, significant alterations in protein phosphatase activities from those in the control mice were demonstrated. In spleen and liver from MRL/lpr mice, potential activities of PP1 and PP2A were distinctly elevated over those of the control mice. These elevations appeared to be due to accumulation of the abnormal lymphocytes that emerged in MRL/lpr mice. Although the PP1 activity in MRL/lpr lymph nodes was lower than those of normal spleen and thymus, it was greatly increased by Co(2+)-trypsin treatment so that the PP1 activity of MRL/lpr lymph nodes was the highest among those of all the tissues examined. In contrast, PP2C activity showed no remarkable alteration in the autoimmune disease model mice as compared with that in the control mice. These results demonstrated a specific elevation in potency of protein dephosphorylation in the tissues of MRL/lpr mice, suggesting a new explanation for the defect in signal transduction in this disease.     

Effects of dietary ω3 and ω6 lipids and vitamin E on chemokine levels in autoimmune-prone MRL/MpJ-lpr/lpr mice

Elevated levels of chemokines, such as Regulated upon Activation, Normal T cell Expressed and Secreted (RANTES), Monocyte Chemotactic Protein-1 (MCP-1), Macrophage Inflammatory Protein-1α (MIP-1α), and Macrophage Inflammatory Protein-1β (MIP-1β) have been found in rheumatoid arthritis (RA) and juvenile arthritis (JA), and they may be associated with the pathogenesis of these diseases. These chemokines are implicated in the migration of specific leukocytes into the joints. Omega-3 (ω3) fatty acid rich-fish oil (FO) and vitamin E may delay the progress of certain autoimmune diseases. The present study was designed to understand the effects of dietary lipids (ω-6 and ω-3 fatty acids) and vitamin E on the production of chemokines in autoimmune-prone MRL/lpr (a mouse model for RA) and congenic control MRL/++ mice. The MRL mice were fed for 4.5 months ω-6 and ω-3 diets that varied in lipid sources (corn oil; CO and fish oil; FO) and vitamin E levels (269 I.U./kg and 694 I.U./kg diet). Spleen cells were isolated and cultured aseptically in the presence of PHA for 48 h at 37°C and the levels of chemokines (RANTES, JE/MCP-1 and MIP-1α) were determined in the cell-free supernatants. The levels of RANTES and JE/MCP-1 were significantly higher in MRL/lpr mice compared to MRL/++ mice. The FO had differential effect on RANTES and MCP-1 production by spleen cells. The production of RANTES and JE/MCP-1 by spleen cells in mice fed the FO diets was significantly lower than in mice fed the CO diets (p < 0.0001). The levels of vitamin E did not affect the production of RANTES and JE/MCP-1. The levels of vitamin E had a significant effect on MIP-1α as the spleen cells of mice fed diets containing 694 IU/kg diet of vitamin E produced significantly higher levels of MIP-1α compared to the group of mice fed the diets containing 269 IU of vitamin E (p < 0.0001). The data obtained from this study in MRL/lpr and MRL/++ mice suggest that FO diets containing ω-3 fatty acids are beneficial in decreasing the levels of certain pro-inflammatory chemokines (RANTES and MCP-1) thereby delaying the onset of and severity of autoimmune symptoms in MRL/lpr mouse model.     

Methotrexate Chronotherapy is Effective Against Rheumatoid Arthritis

Methotrexate (MTX) is the most important drug for treating rheumatoid arthritis (RA). It has been stated that cytokines play an important role in the pathogenesis of RA, and that cytokine levels increase and show 24-h rhythms in RA patients. Previously, we found that arthritis was relieved after the administration of MTX at specific times in synchronization with the 24-h rhythm of tumor necrosis factor (TNF)-α in collagen-induced arthritis (CIA) animals. Based on our findings in an earlier study of the dosing time–dependent effects of MTX in MRL/lpr mice, which develop autoimmune disorders that share similarities with human RA, we examined here the utility of MTX chronotherapy in Japanese RA patients. In an initial animal modeling study, we collected blood from MRL/lpr mice at different times (2, 6, 10, 14, 18, or 22 hours after the light was turned on [HALO]), and we measured TNF-α mRNA expression in leukocytes. MTX was administered to the mice at two different dosing times (6 or 18 HALO), and various blood parameters were measured to estimate arthritis activity. TNF-α mRNA levels showed a clear 24-h rhythm with a peak at 22 HALO and a trough at 18 HALO after RA had developed. In these MRL/lpr mice, inflammation and TNF-α were markedly reduced when the MTX dosing time was matched to the time (18 HALO) when the TNF-α level began to increase. We then applied these findings to Japanese RA patients by switching them from the standard MTX three times/wk (day 1: after breakfast and supper; day 2: after breakfast schedule), to chronotherapy, in which the dose and number of doses/wk were not changed but MTX was administered once-a-day at bedtime. Disease Activity Score (DAS)28, modified health assessment questionnaire (MHAQ), and adverse effects were assessed. With MTX chronotherapy, DAS28, which is commonly used to quantitatively assess RA symptoms, was significantly improved at all follow-up clinical visit times compared with the baseline (vs. 1 mo: p?=?.0197, 2 mos: p?=?.0107, 3 mos: p?=?.0087). Significant symptom recovery was observed in 41.2% of patients, and 23.5% of patients achieved clinical remission during the 3 mos of follow-up. Functional capacity of RA patients, as indicated by the MHAQ, was markedly improved by chronotherapy. There were no severe adverse effects. Thus, we demonstrated (i) inflammation and plasma TNF-α concentrations were significantly reduced in MRL/lpr mice treated with MTX at 18 HALO, the time when TNF-α mRNA level began to increase; and (ii) MTX bedtime chronotherapy was safe, markedly reduced disease activity, and improved the functional capacity of RA patients. The findings on RA patients show that bedtime MTX chronotherapy can improve RA symptoms compared to the current standard dosing methods. (Author correspondence: hide-to@umin.net)     

Modulation of renal disease in MRL/lpr mice genetically deficient in the alternative complement pathway factor B

In systemic lupus erythematosus, the renal deposition of complement-containing immune complexes initiates an inflammatory cascade resulting in glomerulonephritis. Activation of the classical complement pathway with deposition of C3 is pathogenic in lupus nephritis. Although the alternative complement pathway is activated in lupus nephritis, its role in disease pathogenesis is unknown. To determine the role of the alternative pathway in lupus nephritis, complement factor B-deficient mice were backcrossed to MRL/lpr mice. MRL/lpr mice develop a spontaneous lupus-like disease characterized by immune complex glomerulonephritis. We derived complement factor B wild-type (B+/+), homozygous knockout (B-/-), and heterozygous (B+/-) MRL/lpr mice. Compared with B+/- or B+/+ mice, MRL/lpr B-/- mice developed significantly less proteinuria, less glomerular IgG deposition, and decreased renal scores as well as lower IgG3 cryoglobulin production and vasculitis. Serum C3 levels were normal in the B-/- mice compared with significantly decreased levels in the other two groups. These results suggest that: 1) factor B plays an important role in the pathogenesis of glomerulonephritis and vasculitis in MRL/lpr mice; and 2) activation of the alternative pathway, either by the amplification loop or by IgA immune complexes, has a prominent effect on serum C3 levels in this lupus model.     

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.     

High levels of anti-mitochondrial antibodies in MRL mice. Influence of anti-DNA antibodies on anti-mitochondrial antibodies measured by an enzyme-linked immunosorbent assay

We developed an easy enzyme-linked immunosorbent assay (ELISA) for anti-mitochondrial antibodies, and detected high levels of anti-mitochondrial antibodies in MRL/Mp-lpr/lpr (MRL1/l) mice. The influence of the presence of anti-DNA antibodies in the tested sera on this assay was also evaluated. The monoclonal anti-DNA antibodies, which were made from non-immunized MRL1/l mice, did not react with the mitochondrial antigens adhering to polystyrene plates. Absorption with DNA had little effect on the levels of mitochondrial antibodies in MRL1/l sera. Our assay for mitochondria-binding antibody was very easy and sensitive, although it could not differentiate among heterogeneous anti-mitochondrial antibodies.     

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.     

Age Associated Changes in the Distribution of Ipr Gene-Induced B220-Positive T Cells in Lymphoid Organs of MRL/Mp-Ipr/Ipr Mice Using Dual Exposure Microphotographs of Double Immunofluorescence Staining

Homozygous MRL/Mp-1pr/1pr (MRL/1pr) mice, which have an autosomal recessive mutant 1pr gene and exhibit defects in Fas antigen, spontaneously develop autoimmune disease with progressive expansion and accumulation of characteristic abnormal CD4-CD8-double negative T cells that express B220 surface antigen, a B cell-specific surface marker in normal mice. We analyzed the distribution and age related changes of lpr gene-induced abnormal T cells (B220-positive lpr T cells) In the lymphoid organs of MRL/1pr mice. We studied cryostat sections of the spleen, peripheral lymph nodes, mesenteric lymph nodes, and Peyer's patches at different stages using FITC (fluorescein isothiocyanate)-conjugated monoclonal antibodies directed against B220 (RA3-6B2) and PE (phycoerythrin)-conjugated anti-mouse CD3 (2C11) monoclonal antibody, examining dual-exposure microphotographs of double-immunofluorescence stained preparations. We observed that in aged MRL/lpr mice, B220-positive abnormal 1pr T cells were not present in the thymus-dependent area, and the majority of the follicular area cells were displaced by 1pr T cells. These findings suggest that the cellular trafficking of B220-positive lpr T cells differs from that of conventional T cells and that these 1pr-derived T cells play a role in the follicle.     

The liver of MRL/lpr mice contains defective accessory cells and a population of immunosuppressive lymphocytes

Immunoregulatory abnormalities in the MRL/lpr mouse strain include activation of macrophages and hepatic natural killer cells, spontaneous production of tumor necrosis factor, defective oral tolerance, and impaired production of interleukin-2. Because the liver is the major organ responsible for the clearance, degradation, and presentation of foreign antigens from the gastrointestinal tract, we have investigated antigen presentation activity of hepatic nonparenchymal cells (NPC) from MRL/lpr, MRL/++, and C3H/HeN female mice in the primary immune response as measured by stimulation of allogeneic one-way mixed lymphocyte response (MLR), and allogeneic cell-mediated lympholysis (CML). Whereas adherent NPC from C3H/HeN, MRL/++, and young MRL/lpr mice were effective stimulators, NPC from MRL/lpr mice older than 9 weeks were defective stimulators of both MLR and CML responses. This abnormality was not observed in splenic accessory cells from these mice. Moreover, a population of hepatic NPC from older MRL/lpr mice are immunosuppressive: mixing of MRL/lpr NPC with splenic stimulators from MRL/++ mice profoundly inhibited primary allogeneic CML responses. The inhibitory hepatic nonparenchymal cell population was nonadherent, radioresistant and was removed by pretreatment with antibodies to either asialoAGM-1 or Lyt-2 plus complement. This inhibition was not observed with the addition of MRL/++ NPC or supernates from cultured MRL/lpr NPC. These findings suggest a selective organ-specific and age-dependent impairment of antigen presentation and the presence of an immunosuppressive lymphocyte population in the liver of MRL/lpr mice which may contribute to the autoimmune process.     

Age Associated Changes in the Distribution of Ipr Gene-Induced B220-Positive T Cells in Lymphoid Organs of MRL/Mp-Ipr/Ipr Mice Using Dual Exposure Microphotographs of Double Immunofluorescence Staining

Homozygous MRL/Mp-1pr/1pr (MRL/1pr) mice, which have an autosomal recessive mutant 1pr gene and exhibit defects in Fas antigen, spontaneously develop autoimmune disease with progressive expansion and accumulation of characteristic abnormal CD4-CD8-double negative T cells that express B220 surface antigen, a B cell-specific surface marker in normal mice. We analyzed the distribution and age related changes of lpr gene-induced abnormal T cells (B220-positive lpr T cells) In the lymphoid organs of MRL/1pr mice. We studied cryostat sections of the spleen, peripheral lymph nodes, mesenteric lymph nodes, and Peyer's patches at different stages using FITC (fluorescein isothiocyanate)-conjugated monoclonal antibodies directed against B220 (RA3-6B2) and PE (phycoerythrin)-conjugated anti-mouse CD3 (2C11) monoclonal antibody, examining dual-exposure microphotographs of double-immunofluorescence stained preparations. We observed that in aged MRL/lpr mice, B220-positive abnormal 1pr T cells were not present in the thymus-dependent area, and the majority of the follicular area cells were displaced by 1pr T cells. These findings suggest that the cellular trafficking of B220-positive lpr T cells differs from that of conventional T cells and that these 1pr-derived T cells play a role in the follicle.     

Aberrant expression of the autoantigen heterogeneous nuclear ribonucleoprotein-A2 (RA33) and spontaneous formation of rheumatoid arthritis-associated anti-RA33 autoantibodies in TNF-alpha transgenic mice

Human TNF-alpha transgenic (hTNFtg) mice develop erosive arthritis closely resembling rheumatoid arthritis (RA). To investigate mechanisms leading to pathological autoimmune reactions in RA, we examined hTNFtg animals for the presence of RA-associated autoantibodies including Abs to citrullinated epitopes (anti-cyclic citrullinated peptide), heterogeneous nuclear ribonucleoprotein (hnRNP)-A2 (anti-RA33), and heat shock proteins (hsp) (anti-hsp). Although IgM anti-hsp Abs were detected in 40% of hTNFtg and control mice, IgG anti-hsp Abs were rarely seen, and anti-cyclic citrullinated peptide Abs were not seen at all. In contrast, >50% of hTNFtg mice showed IgG anti-RA33 autoantibodies, which became detectable shortly after the onset of arthritis. These Abs were predominantly directed to a short epitope, which was identical with an epitope previously described in MRL/lpr mice. Incidence of anti-RA33 was significantly decreased in mice treated with the osteoclast inhibitor osteoprotegerin and also in c-fos-deficient mice lacking osteoclasts. Pronounced expression of hnRNP-A2 and a smaller splice variant was seen in joints of hTNFtg mice, whereas expression was low in control animals. Although the closely related hnRNP-A1 was also overexpressed, autoantibodies to this protein were infrequently detected. Because expression of hnRNP-A2 in thymus, spleen, brain, and lung was similar in hTNFtg and control mice, aberrant expression appeared to be restricted to the inflamed joint. Finally, immunization of hTNFtg mice with recombinant hnRNP-A2 or a peptide harboring the major B cell epitope aggravated arthritis. These findings suggest that overproduction of TNF-alpha leads to aberrant expression of hnRNP-A2 in the rheumatoid joint and subsequently to autoimmune reactions, which may enhance the inflammatory and destructive process.     

Epimorphic regeneration in mice is p53-independent

The process of regeneration is most readily studied in species of sponge, hydra, planarian, and salamander (i.e., newt and axolotl). The closure of MRL mouse ear pinna through-and-through holes provides a model of unusual wound healing/regeneration in which a blastema-like structure closes the ear hole and cartilage and hair follicles are replaced. Recent studies, based on a broad level of DNA damage and a cell cycle pattern of G2/M "arrest," showed that p21^Cip1/Waf1 was missing from the MRL mouse ear and that a p21-null mouse could close its ear holes. Given the p53/p21 axis of control of DNA damage, cell cycle arrest, apoptosis, and senescence, we tested the role of p53 in the ear hole regenerative response. Using backcross mice, we found that loss of p53 in MRL mice did not show reduced healing. Furthermore, cross sections of MRL.p53^-/- mouse ears at 6 weeks post-injury showed an increased level of adipocytes and chondrocytes in the region of healing whereas MRL or p21^-/- mice show chondrogenesis alone in this same region, though at later time points. In addition, we also investigated other cell cycle-related mutant mice to determine how p21 was being regulated. We demonstrate that p16 and Gadd45 null mice show little healing capacity. Interestingly, a partial healing phenotype in mice with a dual Tgfb/Rag2 knockout mutation was seen. These data demonstrate an independence of p53 signaling for mouse appendage regeneration and suggest that the role of p21 in this process is possibly through the abrogation of the Tgfb/Smad pathway.     

Macrophage migration inhibitory factor deficiency attenuates macrophage recruitment, glomerulonephritis, and lethality in MRL/lpr mice

Systemic lupus erythematosus (SLE) is a serious systemic autoimmune disease of unknown etiology. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that is operative in innate and adaptive immunity and important in immune-mediated diseases such as rheumatoid arthritis and atherosclerosis. The functional relevance of MIF in systemic autoimmune diseases such as SLE is unknown. Using the lupus-prone MRL/lpr mice, we aim to examine the expression and function of MIF in this murine model of systemic autoimmune disease. These experiments revealed that renal MIF expression was significantly higher in MRL/lpr mice compared with nondiseased control mice (MRL/MpJ), and MIF was also markedly up-regulated in skin lesions of MRL/lpr mice. To examine the effect of MIF on development of systemic autoimmune disease, we generated MRL/lpr mice with a targeted disruption of the MIF gene (MIF(-/-)MRL/lpr), and compared their disease manifestations to MIF(+/+)MRL/lpr littermates. MIF(-/-)MRL/lpr mice exhibited significantly prolonged survival, and reduced renal and skin manifestations of SLE. These effects occurred in the absence of major changes in T and B cell markers or alterations in autoantibody production. In contrast, renal macrophage recruitment and glomerular injury were significantly reduced in MIF(-/-)MRL/lpr mice, and this was associated with reduction in the monocyte chemokine MCP-1. Taken together, these data suggest MIF as a critical effector of organ injury in SLE.