The mouse mast cell-restricted tetramer-forming tryptases mouse mast cell protease 6 and mouse mast cell protease 7 are critical mediators in inflammatory arthritis

OBJECTIVE: Increased numbers of mast cells (MCs) that express beta tryptases bound to heparin have been detected in the synovium of patients with rheumatoid arthritis (RA). The corresponding tryptases in mice are mouse MC protease 6 (mMCP-6) and mMCP-7. Although MCs have been implicated in RA and some animal models of arthritis, no direct evidence for a MC-restricted tryptase in the pathogenesis of inflammatory arthritis has been shown. We created transgenic mice that lack heparin and different combinations of mMCP-6 and mMCP-7, to evaluate the roles of MC-restricted tryptase-heparin complexes in an experimental model of arthritis. METHODS: The methylated bovine serum albumin/interleukin-1beta (mBSA/IL-1beta) experimental protocol was used to induce inflammatory monarthritis in different mouse strains. Mice were killed at the time of peak disease on day 7, and histochemical methods were used to assess joint pathology. RESULTS: Arthritis was induced in the knee joints of mBSA/IL-1beta-treated mMCP-6(+)/mMCP-7(-) and mMCP-6(-)/mMCP-7(+) C57BL/6 mice, and numerous activated MCs that had exocytosed the contents of their secretory granules were observed in the diseased mice. In contrast, arthritis was markedly reduced in heparin-deficient mice and in mMCP-6(-)/mMCP-7(-) C57BL/6 mice. CONCLUSION: MC-derived tryptase-heparin complexes play important roles in mBSA/IL-1beta-induced arthritis. Because mMCP-6 and mMCP-7 can compensate for each other in this disease model, the elimination of both tryptases is necessary to reveal the prominent roles of these serine proteases in joint inflammation and destruction. Our data suggest that the inhibition of MC-restricted tryptases could have therapeutic potential in the treatment of RA.     

Molecular and cellular mediators of interleukin-1-dependent acute inflammatory arthritis

OBJECTIVE: To examine the molecular and cellular mechanisms in a model of acute inflammatory monarticular arthritis induced by methylated bovine serum albumin (mBSA) and interleukin-1 (IL-1). METHODS: Mice were injected intraarticularly with mBSA on day 0 and subcutaneously with recombinant human IL-1beta on days 0-2. At day 7, knee joints were removed and assessed histologically. Flow cytometry and RNase protection were used to analyze IL-1-dependent events. RESULTS: C57BL/6 (B6), 129/Sv, and (B6 x 129/ Sv)F1 hybrid mice, all H-2b strains, were susceptible to mBSA/IL-1-induced arthritis, whereas C3H/HeJ (H-2k) mice were not. B6 mice lacking T and B cells (RAG1-/-) or major histocompatibility complex (MHC) class II antigens (MHCII-/-), and B6 mice treated with a CD4+ T cell-depleting monoclonal antibody, were resistant to disease. In contrast, B cell-deficient (muMT/ muMT) mice developed arthritis at an incidence and severity similar to that of controls. RelB-deficient (RelB-/-) bone marrow chimeric mice had arthritis that was significantly reduced in incidence and severity. In B6 mice, flow cytometry demonstrated an IL-1-dependent leukocyte infiltration into the synovial compartment and RNase protection assays revealed induction of messenger RNA (mRNA) for the chemokines monocyte chemoattractant protein 1, macrophage inhibitory protein 2 (MIP-2), RANTES, MIP-1alpha, and MIP-1beta, in vivo and in vitro. CONCLUSION: Arthritis induced by mBSA/IL-1 is strain specific and dependent on CD4+ T lymphocytes and at least partially on RelB, but not on B lymphocytes or antibody. IL-1 contributes to leukocyte recruitment to the synovium and directly induces chemokine mRNA production by synovial cells. This model of acute monarticular arthritis is particularly suitable for further investigations into cell-mediated immunity in arthritis and the role of IL-1.     

Antigen‐specific suppression of established arthritis in mice by dendritic cells deficient in NF‐κB

Objective

NF‐κB inhibitors applied to animal models of rheumatoid arthritis (RA) demonstrate the important role of NF‐κB in the production of mediators of inflammation in the joint and their antiinflammatory effects. Because NF‐κB is involved in the differentiation, activation, and survival of almost all cells, its prolonged inhibition might have unwanted adverse effects. Therefore, we sought to apply NF‐κB inhibitors more specifically, targeting dendritic cell (DC) differentiation, in order to influence the outcome of the autoimmune response, rather than to produce a broad antiinflammatory effect. We tested whether DCs treated with the NF‐κB inhibitor BAY 11‐7082 and exposed to arthritogenic antigen would suppress established arthritis in C57BL/6 mice.

Methods

Antigen‐induced arthritis was generated in C57BL/6 mice by injection of methylated bovine serum albumin (mBSA). After mBSA challenge, mouse knee joints were injected with antigen‐exposed BAY 11‐7082–treated DCs or with soluble tumor necrosis factor receptor (sTNFR). Intraarticular injection of interleukin‐1 (IL‐1) was used to induce disease flare.

Results

Inflammation and erosion were suppressed in mice that received mBSA‐exposed BAY 11‐7082–treated DCs, but not in those that received keyhole limpet hemocyanin–exposed BAY 11‐7082–treated DCs. Clinical improvement was dependent on IL‐10 and was associated with antigen‐specific suppression of the delayed‐type hypersensitivity (DTH) reaction and switching of anti‐mBSA antibody isotype from IgG2b to IgG1 and IgA. Suppression of the DTH reaction or arthritic disease was not impaired by concomitant administration of sTNFR. Suppression could be reversed with intraarticular administration of IL‐1β and could be restored by a second injection of mBSA‐exposed BAY 11‐7082–treated DCs.

Conclusion

BAY 11‐7082–treated DCs induce antigen‐specific immune suppression in this model of inflammatory arthritis, even after full clinical expression of the disease. Such DCs have potential as antigen‐specific therapy for autoimmune inflammatory arthritis, including RA.

     

Molecular and cellular mediators of interleukin‐1–dependent acute inflammatory arthritis

Objective

To examine the molecular and cellular mechanisms in a model of acute inflammatory monarticular arthritis induced by methylated bovine serum albumin (mBSA) and interleukin‐1 (IL‐1).

Methods

Mice were injected intraarticularly with mBSA on day 0 and subcutaneously with recombinant human IL‐1β on days 0–2. At day 7, knee joints were removed and assessed histologically. Flow cytometry and RNase protection were used to analyze IL‐1–dependent events.

Results

C57BL/6 (B6), 129/Sv, and (B6 × 129/Sv)F₁ hybrid mice, all H‐2^b strains, were susceptible to mBSA/IL‐1–induced arthritis, whereas C3H/HeJ (H‐2^k) mice were not. B6 mice lacking T and B cells (RAG‐1^−/−) or major histocompatibility complex (MHC) class II antigens (MHCII^−/−), and B6 mice treated with a CD4+ T cell–depleting monoclonal antibody, were resistant to disease. In contrast, B cell–deficient (μMT/μMT) mice developed arthritis at an incidence and severity similar to that of controls. RelB‐deficient (RelB^−/−) bone marrow chimeric mice had arthritis that was significantly reduced in incidence and severity. In B6 mice, flow cytometry demonstrated an IL‐1–dependent leukocyte infiltration into the synovial compartment and RNase protection assays revealed induction of messenger RNA (mRNA) for the chemokines monocyte chemoattractant protein 1, macrophage inhibitory protein 2 (MIP‐2), RANTES, MIP‐1α, and MIP‐1β, in vivo and in vitro.

Conclusion

Arthritis induced by mBSA/IL‐1 is strain specific and dependent on CD4+ T lymphocytes and at least partially on RelB, but not on B lymphocytes or antibody. IL‐1 contributes to leukocyte recruitment to the synovium and directly induces chemokine mRNA production by synovial cells. This model of acute monarticular arthritis is particularly suitable for further investigations into cell‐mediated immunity in arthritis and the role of IL‐1.

     

Antigen-specific suppression of established arthritis in mice by dendritic cells deficient in NF-kappaB

OBJECTIVE: NF-kappaB inhibitors applied to animal models of rheumatoid arthritis (RA) demonstrate the important role of NF-kappaB in the production of mediators of inflammation in the joint and their antiinflammatory effects. Because NF-kappaB is involved in the differentiation, activation, and survival of almost all cells, its prolonged inhibition might have unwanted adverse effects. Therefore, we sought to apply NF-kappaB inhibitors more specifically, targeting dendritic cell (DC) differentiation, in order to influence the outcome of the autoimmune response, rather than to produce a broad antiinflammatory effect. We tested whether DCs treated with the NF-kappaB inhibitor BAY 11-7082 and exposed to arthritogenic antigen would suppress established arthritis in C57BL/6 mice. METHODS: Antigen-induced arthritis was generated in C57BL/6 mice by injection of methylated bovine serum albumin (mBSA). After mBSA challenge, mouse knee joints were injected with antigen-exposed BAY 11-7082-treated DCs or with soluble tumor necrosis factor receptor (sTNFR). Intraarticular injection of interleukin-1 (IL-1) was used to induce disease flare. RESULTS: Inflammation and erosion were suppressed in mice that received mBSA-exposed BAY 11-7082-treated DCs, but not in those that received keyhole limpet hemocyanin-exposed BAY 11-7082-treated DCs. Clinical improvement was dependent on IL-10 and was associated with antigen-specific suppression of the delayed-type hypersensitivity (DTH) reaction and switching of anti-mBSA antibody isotype from IgG2b to IgG1 and IgA. Suppression of the DTH reaction or arthritic disease was not impaired by concomitant administration of sTNFR. Suppression could be reversed with intraarticular administration of IL-1beta and could be restored by a second injection of mBSA-exposed BAY 11-7082-treated DCs. CONCLUSION: BAY 11-7082-treated DCs induce antigen-specific immune suppression in this model of inflammatory arthritis, even after full clinical expression of the disease. Such DCs have potential as antigen-specific therapy for autoimmune inflammatory arthritis, including RA.     

Proinflammatory role of the Th17 cytokine interleukin‐22 in collagen‐induced arthritis in C57BL/6 mice

Objective

To investigate the role of interleukin‐22 (IL‐22) in collagen‐induced arthritis (CIA), an animal model of rheumatoid arthritis.

Methods

C57BL/6 mice were immunized with type II collagen (CII) in Freund's incomplete adjuvant with added Mycobacterium tuberculosis, and levels of IL‐22 and its specific receptor, IL‐22 receptor type I (IL‐22RI), were measured in sera and tissue by enzyme‐linked immunosorbent assay and real‐time quantitative polymerase chain reaction analysis. Clinical and histologic signs of arthritis were recorded and compared with those in C57BL/6 mice deficient in the IL‐22 gene (IL‐22^–/–). Humoral and cellular immune responses against CII were analyzed. In vitro osteoclastogenesis assays were performed on splenocytes.

Results

Upon immunization with CII in Freund's incomplete adjuvant plus heat‐killed Mycobacterium tuberculosis, sera from C57BL/6 mice were found to contain high levels of IL‐22, and the specific IL‐22RI was expressed in lymphoid tissue, including splenocytes. IL‐22^–/– mice were less susceptible to CIA than were wild‐type mice, as evidenced by their decreased incidence of arthritis and decreased pannus formation. Remarkably, the less severe form of arthritis in IL‐22^–/– mice was associated with increased production of CII‐specific and total IgG antibodies, whereas cellular CII responses were unchanged. In vitro, IL‐22 was found to promote osteoclastogenesis, a process that might contribute to its proinflammatory activity in CIA.

Conclusion

Endogenous IL‐22 plays a proinflammatory role in CIA in C57BL/6 mice. Our data also indicate that IL‐22 promotes osteoclastogenesis and regulates antibody production.

     

The effect of an interleukin-1 receptor antagonist protein on type ii collagen–induced arthritis and antigen-induced arthritis in mice

Objective. To investigate the anti-arthritic effect of recombinant human interleukin-1 receptor antagonist protein (IRAP) in two experimental models of arthritis. Methods. Recombinant IRAP was administered daily to mice with type II collagen–induced arthritis (CIA) or with antigen-induced arthritis (AIA) provoked by methylated bovine serum albumin (mBSA). Disease incidence and severity were assessed by a clinical index and histologic features. Serum antibody to type II collagen, spleen cell proliferation to mBSA, and anti-IRAP antibodies were measured as indices of immune function. Results. IRAP reduced the incidence and delayed the onset of CIA and suppressed the antibody response to type II collagen. In contrast, IRAP did not affect the pathogenesis of AIA and had no effect on either humoral or cellular immune responses to mBSA in arthritic mice. Conclusion. These observations suggest that interleukin-1 may play a prominent role in the development of some, but not all, forms of arthritis.     

Dependence of interleukin‐1–induced arthritis on granulocyte–macrophage colony‐stimulating factor

Objective

To determine whether granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and macrophage CSF (M‐CSF or CSF‐1) are involved in the methylated bovine serum albumin/interleukin‐1 (mBSA/IL‐1)–induced arthritis model.

Methods

Following systemic injection, IL‐1 has been shown to augment a weak inflammatory response to mBSA in murine joints and to induce an acute erosive arthritis. GM‐CSF and M‐CSF have been implicated in inflammatory reactions, including those in joints, and have recently been shown to exacerbate murine arthritis. Since in vitro studies have found that IL‐1 can enhance GM‐CSF and M‐CSF production, we reasoned that they might be playing a part in IL‐1–mediated arthritis. GM‐CSF–deficient (GM‐CSF−/−) and M‐CSF–deficient (op/op) mice were injected intraarticularly with mBSA and subcutaneously with IL‐1. Arthritis was monitored histologically on day 7. Normal mice were also treated intraperitoneally with blocking monoclonal antibodies to GM‐CSF and M‐CSF, and to the M‐CSF receptor. Numbers of macrophages (Mac‐2 and F4/80 staining) were monitored, as was the number of cycling (bromodeoxyuridine‐positive) cells.

Results

GM‐CSF−/− mice and normal mice treated with anti–GM‐CSF antibody did not show IL‐1–induced arthritis progression. There was a dramatic reduction in synovial cellularity, including reduced numbers of macrophages and cycling cells. The op/op mice did not develop mBSA/IL‐1–induced disease, but blocking antibody to M‐CSF or to the M‐CSF receptor failed to diminish disease in normal mice.

Conclusion

GM‐CSF is involved in the IL‐1–induced arthritis that follows mBSA injection; M‐CSF involvement in the model is also suggested, since op/op mice did not develop arthritis. These studies provide the first in vivo evidence for a role of GM‐CSF, and possibly M‐CSF, in the proinflammatory actions of IL‐1.

     

Multiple low-dose streptozotocin-induced hyperglycemia and insulitis in C57BL mice: Influence of inbred background, sex, and thymus

Insulin-dependent diabetes induced in susceptible strains of mice by multiple, low-dose streptozotocin treatment has been proposed to entail a thymus-dependent, autoimmune destruction of beta cells. In this study, thymectomized and genetically athymic mice have been tested for susceptibility to streptozotocin. Thymectomy was performed on newborn (day 1) to 3-day-old C57BL/KsJ mice. At 8 wk of age, thymectomized and sham-operated mice of both sexes were tested for susceptibility to diabetes induction by multiple, low-dose streptozotocin treatment (35 mg/kg of body weight per day for 6 consecutive days). Thymectomy failed to block susceptibility of males to induction of severe hyperglycemia. Beta cell necrosis and inflammatory cell infiltrates (insulitis) were consistent histopathological features. In general, females—both thymus-intact and thymectomized—were less susceptible than males to streptozotocin-induced hyperglycemia, and females exhibited an equally severe insulitis by experimental day 14; thus, the detection of an underlying insulitis did not predict the development of a more severe hyperglycemia because most streptozotocin-treated females at experimental day 35 continued to show only a modest hyperglycemia (about 200 mg/dl) compared to males (>400 mg/dl). That streptozotocin-induced hyperglycemia could occur in the absence of an intact thymus was further demonstrated in genetically athymic C57BL/6J NIcrOu nu/nu males and thymus-intact +/? littermate controls. C57BL/6J mice were resistant to streptozotocin-induced insulitis. This study shows that the presence of insulitis does not necessarily presage onset of severe hyperglycemia (e.g., C57BL/KsJ females), and conversely, the presence of severe hyperglycemia after low-dose streptozotocin treatment is not necessarily diagnostic of an underlying insulitis (e.g., C57BL/6J +/? and nu/nu males). These data stress the need for caution in the interpretation of studies of streptozotocin-insulitis sensitivities of nude mice.     

Transfer of experimental autoimmune insulitis by spleen cells in mice

Summary We have tested whether experimental insulitis induced by multiple subdiabetogenic injections of streptozotocin can be transferred by lymphocytes to normal recipients. C57BL/6J mice were treated on 5 consecutive days with 40 mg streptozotocin /kg body weight. 5×10⁷ nucleated spleen cells from 20 animals which had developed hyperglycaemia with concomitant insulitis three weeks after the first streptozotocin-injection, were transferred into congenic thymusless C57BL/6J-nu/nu mice. The cell transfer led to lymphocytic infiltrations of pancreatic islets in 75% of the recipients. Hyperglycaemia was not observed. It is concluded that lowdose streptozotocin treatment induces cellular immune reactions against pancreatic islets.     

Dependence of interleukin-1-induced arthritis on granulocyte-macrophage colony-stimulating factor

OBJECTIVE: To determine whether granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage CSF (M-CSF or CSF-1) are involved in the methylated bovine serum albumin/interleukin-1 (mBSA/IL-1)-induced arthritis model. METHODS: Following systemic injection, IL-1 has been shown to augment a weak inflammatory response to mBSA in murine joints and to induce an acute erosive arthritis. GM-CSF and M-CSF have been implicated in inflammatory reactions, including those in joints, and have recently been shown to exacerbate murine arthritis. Since in vitro studies have found that IL-1 can enhance GM-CSF and M-CSF production, we reasoned that they might be playing a part in IL-1-mediated arthritis. GM-CSF-deficient (GM-CSF-/-) and M-CSF-deficient (op/op) mice were injected intraarticularly with mBSA and subcutaneously with IL-1. Arthritis was monitored histologically on day 7. Normal mice were also treated intraperitoneally with blocking monoclonal antibodies to GM-CSF and M-CSF, and to the M-CSF receptor. Numbers of macrophages (Mac-2 and F4/80 staining) were monitored, as was the number of cycling (bromodeoxyuridine-positive) cells. RESULTS: GM-CSF-/- mice and normal mice treated with anti-GM-CSF antibody did not show IL-1-induced arthritis progression. There was a dramatic reduction in synovial cellularity, including reduced numbers of macrophages and cycling cells. The op/op mice did not develop mBSA/IL-1-induced disease, but blocking antibody to M-CSF or to the M-CSF receptor failed to diminish disease in normal mice. CONCLUSION: GM-CSF is involved in the IL-1-induced arthritis that follows mBSA injection; M-CSF involvement in the model is also suggested, since op/op mice did not develop arthritis. These studies provide the first in vivo evidence for a role of GM-CSF, and possibly M-CSF, in the proinflammatory actions of IL-1.     

Absence of posttraumatic arthritis following intraarticular fracture in the MRL/MpJ mouse

OBJECTIVE: Posttraumatic arthritis is a frequent long-term complication of intraarticular fractures. A model of a closed intraarticular fracture in C57BL/6 mice that progresses to posttraumatic arthritis has been developed. The MRL/MpJ mouse has shown unique regenerative abilities in response to injury. The objective of this study was to determine if the MRL/MpJ mouse is protected from posttraumatic arthritis after intraarticular fractures. METHODS: Intraarticular fractures were created in MRL/MpJ mice and C57BL/6 control mice (n = 16 each). Limbs were analyzed for posttraumatic arthritis 4 and 8 weeks after fracture using microfocal computed tomography bone morphology, subchondral bone thickness evaluation, and histologic evaluation of cartilage degeneration. Serum cytokines and biomarkers were measured after the mice were killed. RESULTS: Intraarticular fractures were successfully created in all 32 mice. In the experimental fractured limbs, C57BL/6 mice had a decrease in bone density, increased subchondral bone thickness, and increased cartilage degeneration compared with normal contralateral control limbs. In the MRL/MpJ mice, no differences in bone density, subchondral bone thickness, or histologic grading of cartilage degeneration were seen between fractured and contralateral control limbs. Cytokine analysis showed lower systemic levels of the proinflammatory cytokine interleukin-1alpha (IL-1alpha) and higher levels of the antiinflammatory cytokines IL-4 and IL-10 in the MRL/MpJ mice. CONCLUSION: This study shows that the MRL/MpJ mouse is relatively protected from posttraumatic arthritis after intraarticular fracture. Further investigation into the mechanism involved in this response will hopefully provide new insight into the pathogenesis, prevention, and treatment of posttraumatic arthritis after intraarticular fracture.     

Clonal analysis of the anti-DNA repertoire of murine B lymphocytes.

The present studies characterize at the clonal level the repertoire of lipopolysaccharide-responsive murine B lymphocytes committed to the production of antibodies reactive with denatured DNA. This repertoire is vast in normal mice as 1-5% of total mitogen-induced antibody-forming cell clones secreted denatured DNA-reactive antibodies when the splenocyte donors were CBA (Ighj), BALB/c (Igha), C57BL/6 (Ighb), CBA nu/nu, and C57BL/6 nu/nu athymic mice. The autoimmune NZB (Ighe) strain did not display elevated proportions of anti-denatured DNA antibody-forming cell precursors. Cross-reactions shown by CBA anti-denatured DNA antibodies suggest that many antibodies might derive significant binding energy from interaction with the bases or similar hydrophobic moieties. Cross-reactions with other tested polynucleotides were frequent, but cross-reactions with phospholipids and phosphocholine were undetectable. Most anti-DNA antibodies bound preferentially or exclusively to single-stranded denatured DNA as compared to double-stranded native DNA. The frequency of anti-denatured DNA antibody-forming cell precursors among CBA peritoneal cells was not elevated. Fluorescence-activated cell sorter-selected Ly-1-positive NZB splenic B cells were not enriched, and Ly-1 negative B cells were not depleted of anti-DNA antibody-forming cell precursors. These results show that antibody-forming cell precursors specific for denatured DNA are not restricted to the Ly-1 positive B-cell subset.     

Mucosal‐associated invariant T cells promote inflammation and exacerbate disease in murine models of arthritis

Objective

The function of mucosal‐associated invariant T (MAIT) cells remains largely unknown. We previously reported an immunoregulatory role of MAIT cells in an animal model of multiple sclerosis. The aim of this study was to use animal models to determine whether MAIT cells are involved in the pathogenesis of arthritis.

Methods

MR1^−/− and MR1^+/+ DBA/1J mice were immunized with bovine type II collagen (CII) in complete Freund's adjuvant to trigger collagen‐induced arthritis (CIA). To assess CII‐specific T cell recall responses, lymph node cells from mice with CIA were challenged with CII ex vivo, and cytokine production and proliferation were evaluated. Serum levels of CII‐specific antibodies were measured by enzyme‐linked immunosorbent assay. Collagen antibody–induced arthritis (CAIA) was induced in MR1^−/− and MR1^+/+ C57BL/6 mice by injection of anti‐CII antibodies followed by injection of lipopolysaccharide. To demonstrate the involvement of MAIT cells in arthritis, we induced CAIA in MR1^−/− C57BL/6 mice that had been reconstituted with adoptively transferred MAIT cells. MAIT cell activation in response to cytokine stimulation was investigated.

Results

The severity of CIA was reduced in MR1^−/− DBA/1J mice. However, T and B cell responses to CII were comparable in MR1^−/− and MR1^+/+ DBA/1J mice. MR1^−/− C57BL/6 mice were less susceptible to CAIA, and reconstitution with MAIT cells induced severe arthritis in MR1^−/− C57BL/6 mice, demonstrating an effector role of MAIT cells in arthritis. MAIT cells became activated upon stimulation with interleukin‐23 (IL‐23) or IL‐1β in the absence of T cell receptor stimuli.

Conclusion

These results indicate that MAIT cells exacerbate arthritis by enhancing the inflammation.

     

Evaluation of protease‐activated receptor 2 in murine models of arthritis

Objective

Protease‐activated receptor 2 (PAR‐2) activation has been linked to pro‐ and antiinflammatory cellular responses. We undertook this study to explore the importance of PAR‐2 activation in 4 murine models of arthritis and to analyze the expression of PAR‐2 in human arthritic synovium.

Methods

Zymosan‐induced arthritis (ZIA), K/BxN serum–induced arthritis, and Freund's complete adjuvant (CFA)–induced arthritis were generated in naive PAR‐2^−/− mice and PAR‐2^+/+ littermates. Antigen‐induced arthritis (AIA) was generated in immunized mice using methylated bovine serum albumin (mBSA). The severity of arthritis was assessed by clinical scoring, technetium uptake measurement, and histologic analysis. Immune responses to mBSA were also evaluated from AIA. The expression of PAR‐2 in synovial tissues from rheumatoid arthritis (RA) and osteoarthritis (OA) patients was compared.

Results

In AIA, arthritis was significantly decreased in PAR‐2–deficient mice and was associated with decreased levels of anti‐mBSA IgG antibodies and lymph node cell proliferation. No difference in arthritis severity was seen in mice with ZIA, K/BxN serum–induced arthritis, and CFA‐induced arthritis. Synovial biopsy specimens from RA patients demonstrated significantly increased expression of PAR‐2 compared with those from OA patients.

Conclusion

PAR‐2 deficiency was found to modulate articular inflammation in murine models of arthritis that require prior immunization and was associated with reduced levels of anti‐mBSA IgG and lymph node cell proliferation in AIA. Expression of PAR‐2 in RA synovium was significantly higher than that in OA synovium, and this suggests that PAR‐2 is implicated in the pathogenesis of immune‐mediated forms of arthritis.

     

Oral Vaccination Against <Emphasis Type="Italic">Helicobacter pylori</Emphasis>Infection Is Not Effective in Mice With Fas Ligand Deficiency

The aim of this study was to delineate the role of the Fas pathway in vaccination against Helicobacter pylori. C57BL/6 and Fas ligand–deficient (gld) mice were divided into 3 groups: control, H. pyloriinfected, and orally vaccinated (H. pyloriwhole cell sonicate and cholera toxin adjuvant). Oral vaccination prevented H. pyloricolonization in 78% of C57BL/6 mice compared to only 18% of gldmice. Vaccination did not alter the degree of apoptosis in either strain of mice. Vaccination led to significant increase in interleukin (IL)-5 and IL-10 in C57BL/6 but not gldmice. H. pyloriinfection increased interferon (IFN)-γ levels in C57BL/6 but not in gldmice while vaccination had no effect on IFN-γ levels in either strain. Oral vaccination is not effective in Fas ligand–deficient mice likely owing to lack of effective cytokine responses. This indicates that the Fas pathway plays a critical role in promoting an appropriate effector response following H. pylorivaccination.     

Genetically based resistance to the antiinflammatory effects of methotrexate in the air‐pouch model of acute inflammation

Objective

Low‐dose methotrexate (MTX), a mainstay in the treatment of rheumatoid arthritis, is effective in only 60–70% of patients, a finding mirrored by poor antiinflammatory efficacy in some animal models, most notably collagen‐induced arthritis. To determine whether genetic factors or the model itself is responsible for the poor response to MTX, we directly compared the responses of 4 inbred mouse strains to MTX in the air‐pouch model of acute inflammation.

Methods

The exudate leukocyte count and adenosine concentration were determined in inbred mice treated with MTX (0.75 mg/kg intraperitoneally every week for 4 weeks) or vehicle 4 hours after injection of carrageenan into the air pouch using previously described methods. Quantitative trait locus mapping was performed using an in silico, or computer‐based, method to identify loci potentially associated with each phenotype.

Results

MTX significantly reduced the exudate leukocyte count in C57BL/6J and BALB/cJ mice, but not DBA/1J (the strain used in the collagen‐induced arthritis model) or DBA/2J mice. In a parallel manner, MTX increased adenosine concentration in inflammatory exudates of C57BL/6J and BALB/cJ mice, but not DBA/1J or DBA/2J mice. Antiinflammatory and adenosine responses to MTX in DBA/1J × C57BL/6J F₁ and F₂ offspring were most consistent with single genetic loci being responsible for each phenotype. In silico mapping identified partially overlapping loci containing candidate genes involved in both responses.

Conclusion

Genetic factors contribute to the antiinflammatory efficacy of MTX, and a single locus involved in MTX‐induced adenosine up‐regulation is likely responsible for the observed resistance to MTX in DBA/1J mice.

     

Acute CD4+ T lymphocyte-dependent interleukin-1-driven arthritis selectively requires interleukin-2 and interleukin-4, joint macrophages, granulocyte-macrophage colony-stimulating factor, interleukin-6, and leukemia inhibitory factor

OBJECTIVE: To further investigate the effects of interleukin-1 (IL-1) in immune-mediated joint inflammation, we examined the role of IL-2, Th1 interferon-gamma (IFNgamma), and Th2 (IL-4) cytokines, joint macrophages, and macrophage-derived cytokines (IL-12 p40, IL-6, leukemia inhibitory factor [LIF], oncostatin M [OSM], and granulocyte-macrophage colony-stimulating factor [GM-CSF]) in a CD4+ T lymphocyte-dependent model of acute arthritis. METHODS: Methylated bovine serum albumin (mBSA)/IL-1-induced arthritis was elicited in wild-type, gene-knockout, and monoclonal antibody-treated mice. Synovial lining macrophages were selectively depleted by intraarticular injection of clodronate liposomes prior to disease induction. The severity of arthritis was assessed histologically. RESULTS: Mice deficient in IL-2 were almost completely protected from arthritis, and neutralization of IL-4 reduced the severity of disease. In contrast, arthritis severity and resolution appeared to be independent of IFNgamma. Synovial lining macrophage depletion markedly reduced arthritis severity. IL-6 or LIF deficiency was only modestly protective, although as previously reported, GM-CSF deficiency conferred profound disease resistance. IL-12 p40-deficient mice (which lack IL-12 and IL-23) and OSM receptor-deficient mice were susceptible to mBSA/IL-1-induced arthritis. CONCLUSION: Acute mBSA/IL-1-induced arthritis is dependent on IL-2 and IL-4, but not IFNgamma. In vivo, the Th1/Th2 paradigm may be distorted by the presence of macrophage-derived cytokines such as IL-1. Synovial lining macrophages are essential in mBSA/IL-1-induced arthritis. However, the requirement for macrophage-derived cytokines is selective; that is, IL-6, LIF, and especially GM-CSF are necessary, but IL-12, IL-23, and OSM are dispensable. IL-1 may therefore influence both adaptive and innate immune mechanisms in acute inflammatory arthritis.     

Gut microbiota,tight junction protein expression,intestinal resistance,bacterial translocation and mortality following cholestasis depend on the genetic background of the host

Failure of the intestinal barrier is a characteristic feature of cholestasis. We have previously observed higher mortality in C57BL/6J compared with A/J mice following common bile duct ligation (CBDL). We hypothesized the alteration in gut barrier function following cholestasis would vary by genetic background. Following one week of CBDL, jejunal TEER was significantly reduced in each ligated mouse compared with their sham counterparts; moreover, jejunal TEER was significantly lower in both sham and ligated C57BL/6J compared with sham and ligated A/J mice, respectively. Bacterial translocation to mesenteric lymph nodes was significantly increased in C57BL/6J mice vs. A/J mice. Four of 15 C57BL/6J mice were bacteremic; whereas, none of the 17 A/J mice were. Jejunal IFN-γ mRNA expression was significantly elevated in C57BL/6J compared with A/J mice. Western blot analysis demonstrated a significant decrease in occludin protein expression in C57BL/6J compared with A/J mice following both sham operation and CBDL. Only C57BL/6J mice demonstrated a marked decrease in ZO-1 protein expression following CBDL compared with shams. Pyrosequencing of the 16S rRNA gene in fecal samples showed a dysbiosis only in C57BL/6J mice following CBDL when compared with shams. This study provides evidence of strain differences in gut microbiota, tight junction protein expression, intestinal resistance and bacterial translocation which supports the notion of a genetic predisposition to exaggerated injury following cholestasis.