Reduced pain hypersensitivity and inflammation in mice lacking microsomal prostaglandin e synthase-1

We examined the in vivo role of membrane-bound prostaglandin E synthase (mPGES)-1, a terminal enzyme in the PGE2-biosynthetic pathway, using mPGES-1 knockout (KO) mice. Comparison of PGES activity in the membrane fraction of tissues from mPGES-1 KO and wild-type (WT) mice indicated that mPGES-1 accounted for the majority of lipopolysaccharide (LPS)-inducible PGES in WT mice. LPS-stimulated production of PGE2, but not other PGs, was impaired markedly in mPGES-1-null macrophages, although a low level of cyclooxygenase-2-dependent PGE2 production still remained. Pain nociception, as assessed by the acetic acid writhing response, was reduced significantly in KO mice relative to WT mice. This phenotype was particularly evident when these mice were primed with LPS, where the stretching behavior and the peritoneal PGE2 level of KO mice were far less than those of WT mice. Formation of inflammatory granulation tissue and attendant angiogenesis in the dorsum induced by subcutaneous implantation of a cotton thread were reduced significantly in KO mice compared with WT mice. Moreover, collagen antibody-induced arthritis, a model for human rheumatoid arthritis, was milder in KO mice than in WT mice. Collectively, our present results provide unequivocal evidence that mPGES-1 contributes to the formation of PGE2 involved in pain hypersensitivity and inflammation.     

Reduced incidence and severity of collagen-induced arthritis in mice lacking IL-18

We have recently reported the presence and a potential proinflammatory role of IL-18 in the synovium of patients with rheumatoid arthritis. To obtain direct evidence that IL-18 plays an influential role in articular inflammation, we investigated the development of collagen-induced arthritis in a strain of mice lacking IL-18 (IL-18(-/-)) of DBA/1 background. IL-18(-/-) mice developed markedly reduced incidence of arthritis compared with heterozygous or wild-type mice. Of the IL-18(-/-) mice that developed arthritis, the severity of the disease was significantly reduced compared with the intact mice. This was accompanied by reduced articular inflammation and destruction evident on histology. IL-18(-/-) mice also had significantly reduced Ag-specific proliferation and proinflammatory cytokine (IFN-gamma, TNF-alpha, IL-6, and IL-12) production by spleen and lymph node cells in response to bovine type II collagen (CII) in vitro compared with wild-type mice, paralleled in vivo by a significant reduction in serum anti-CII IgG2a Ab level. Treatment with rIL-18 completely reversed the disease of the IL-18(-/-) mice to that of the wild-type mice. These data directly demonstrate a pivotal role of IL-18 in the development of inflammatory arthritis and suggest that antagonists to IL-18 may have therapeutic potential in rheumatic diseases.     

Inhibition of matrix metalloproteinase-3 and -13 synthesis induced by IL-1beta in chondrocytes from mice lacking microsomal prostaglandin E synthase-1

Joint destruction in arthritis is in part due to the induction of matrix metalloproteinase (MMP) expression and their inhibitors, especially MMP-13 and -3, which directly degrade the cartilage matrix. Although IL-1β is considered as the main catabolic factor involved in MMP-13 and -3 expression, the role of PGE(2) remains controversial. The goal of this study was to determine the role of PGE(2) on MMP synthesis in articular chondrocytes using mice lacking microsomal PGE synthase-1 (mPGES-1), which catalyses the rate-limiting step of PGE(2) synthesis. MMP-3 and MMP-13 mRNA and protein expressions were assessed by real-time RT-PCR, immunoblotting, and ELISA in primary cultures of articular chondrocytes from mice with genetic deletion of mPGES-1. IL-1β-induced PGE(2) synthesis was dramatically reduced in mPGES-1(-/-) and mPGES-1(+/-) compared with mPGES-1(+/+) chondrocytes. A total of 10 ng/ml IL-1β increased MMP-3 and MMP-13 mRNA, protein expression, and release in mPGES-1(+/+) chondrocytes in a time-dependent manner. IL-1β-induced MMP-3 and MMP-13 mRNA expression, protein expression, and release decreased in mPGES-1(-/-) and mPGES-1(+/-) chondrocytes compared with mPGES-1(+/+) chondrocytes from 8 up to 24 h. Otherwise, MMP inhibition was partially reversed by addition of 10 ng/ml PGE(2) in mPGES-1(-/-) chondrocytes. Finally, in mPGES-1(-/-) chondrocytes treated by forskolin, MMP-3 protein expression was significantly decreased compared with wild-type, suggesting that PGE(2) regulates MMP-3 expression via a signaling pathway dependent on cAMP. These results demonstrate that PGE(2) plays a key role in the induction of MMP-3 and MMP-13 in an inflammatory context. Therefore, mPGES-1 could be considered as a critical target to counteract cartilage degradation in arthritis.     

Deletion of microsomal prostaglandin E2 (PGE2) synthase-1 reduces inducible and basal PGE2 production and alters the gastric prostanoid profile

Microsomal prostaglandin E synthase-1 (mPGES-1) is an inducible protein recently shown to be an important source of inflammatory PGE2. Here we have used mPGES-1 wild type, heterozygote, and null mice to assess the impact of reduction or absence mPGES-1 protein on the production of PGE2 and other prostaglandins in lipopolysaccharide (LPS)-treated macrophages and mice. Thioglycollate-elicited peritoneal macrophages with mPGES-1 deficiency were found to lose their ability to produce PGE2 upon LPS stimulation. Resident mPGES-1(-/-) peritoneal macrophages exhibited severely impaired PGE2-releasing activity but retained some LPS-inducible PGE2 production capacity. Both macrophage types showed a 50% decrease in PGE2 production with removal of one copy of the mPGES-1 gene. In vivo, mPGES-1 deletion abolished the LPS-stimulated production of PGE2 in spleen, kidney, and brain. Surprisingly, lack of mPGES-1 activity resulted in an 80-90% decrease in basal, cyclooxygenase-1 (COX-1)-dependent PGE2 production in stomach and spleen, and a 50% reduction in brain and kidney. Other prostaglandins (thromboxane B2, PGD2, PGF(2alpha), and 6-keto-PGF(1alpha)) were significantly elevated in stomachs of mPGES-1-null mice but not in other tissues. Examination of mRNA for several terminal prostaglandin synthases did not reveal changes in expression levels associated with mPGES-1 deficiency, indicating that gastric prostaglandin changes may be due to shunting of cyclooxygenase products to other terminal synthases. These data demonstrate for the first time a dual role for mPGES-1 in both inflammatory and COX-1-mediated PGE2 production and suggest an interdependence of prostanoid production with tissue-specific alterations of prostaglandin levels in the absence of mPGES-1.     

Collagen-induced arthritis is exacerbated in IL-10-deficient mice

IL-10 is a potent immunoregulatory cytokine attenuating a wide range of immune effector and inflammatory responses. In the present study, we assess whether endogenous levels of IL-10 function to regulate the incidence and severity of collagen-induced arthritis. DBA/1 wildtype (WT), heterozygous (IL-10+/-) and homozygous (IL-10-/-) IL-10-deficient mice were immunized with type II collagen. Development of arthritis was monitored over time, and collagen-specific cytokine production and anticollagen antibodies were assessed. Arthritis developed progressively in mice immunized with collagen, and 100% of the WT, IL-10+/-, and IL-10-/- mice were arthritic at 35 days. However, the severity of arthritis in the IL-10-/- mice was significantly greater than that in WT or IL-1+/- animals. Disease severity was associated with reduced IFN-gamma levels and a dramatic increase in CD11b-positive macrophages. Paradoxically, both the IgG1 and IgG2a anticollagen antibody responses were also significantly reduced. These data demonstrate that IL-10 is capable of controlling disease severity through a mechanism that involves IFN-gamma. Since IL-10 levels are elevated in rheumatoid arthritis synovial fluid, these findings may have relevance to rheumatoid arthritis.     

Effects of selective iNOS inhibition on type II collagen-induced arthritis in mice

Nitric oxide as well as prostaglandins has been reported to play an important role in inflammatory diseases including arthritis. In the present study, the effects of iNOS inhibition on development of disease were examined in type II collagen-induced arthritis (CIA) in male DBA/1J mice. From 4 weeks after the first immunization with bovine type II collagen, 1400W (10 mg/kg/day, p.o.), a selective iNOS inhibitor, indomethacin (1 mg/kg/day, p.o.), a cyclooxygenase (COX) inhibitor, or 1400W + indomethacin was administered for 8 weeks. Immunization with type II collagen evoked arthritic inflammation of paws and bone destruction accompanied by increases in urinary nitrite/nitrate (NOx) excretion, plasma NOx and PGE2 levels. Administration of 1400W reduced urinary NOx excretion and increased plasma PGE2 levels, while it had no effect on arthritic inflammation or bone destruction. Indomethacin slightly reduced the inflammatory signs and bone destruction with marked reduction of plasma PGE2. Combination of 1400W and indomethacin reduced urinary NOx and PGE2 levels, and showed greater amelioration of inflammatory signs and bone destruction than either alone. In conclusion, 1400W, a selective iNOS inhibitor, failed to prevent CIA probably due to its increasing effect on PGE2 production, but showed a synergistic ameliorative effect in combination with indomethacin.     

Genetic deletion of mPGES-1 abolishes PGE2 production in murine dendritic cells and alters the cytokine profile, but does not affect maturation or migration

We undertook this study to determine the role of Microsomal PGE Synthase-1 (mPGES-1), and mPGES-1-generated Prostaglandin (PG) E2 on Dendritic Cell (DC) phenotype and function. Using mPGES-1 KnockOut (KO) mice, we generated bone marrow derived DCs and determined their eicosanoid production profile, cell surface marker expression, and cytokine production. We also assessed DC migratory and functional capacity in vivo. Compared to wild-type, mPGES-1 deficient DCs exhibited a markedly attenuated increase in PGE2 production upon LPS stimulation, and displayed preferential shunting towards PGD2 production. mPGES-1 KO DCs did not display deficiencies in maturation, migration or ability to sensitize T cells. However, mPGES-1 deficient DCs generated reduced amounts of the Th1 cytokine IL-12, which may in part be due to increased PGD2 rather than decreased PGE2. These findings provide useful information on the effects of inducible PGE2 on the innate immune system, and have important implications regarding potential consequences of pharmacologic mPGES-1 inhibition.     

Lack of microsomal prostaglandin E synthase-1 reduces cardiac function following angiotensin II infusion

Our laboratory previously reported that inducible PGE(2) synthase, mPGES-1, contributes to micromolar production of PGE(2) in neonatal ventricular myocytes in vitro, which stimulates their growth. We therefore hypothesized that mPGES-1 contributes to cardiac hypertrophy following angiotensin II (ANG II) infusion. To test this hypothesis, we used 10- to 12-wk-old mPGES-1 knockout mice (mPGES-1 KO) and C57Bl/6 control mice infused for 8 wk with either 1.4 mg · kg(-1) · day(-1) ANG II or vehicle subcutaneously. Blood pressure [systolic blood pressure (SBP)] was measured throughout the study, and cardiac function was assessed by M-mode echocardiography at baseline and at 8 wk of infusion. At the conclusion of the study, immunohistochemistry was used to evaluate collagen fraction, myocyte cross-sectional area (MCSA), and apoptosis. At baseline, there was no difference in SBP between mPGES-1 KO mice and C57BL/6 controls. ANG II infusion increased SBP to similar levels in both strains. In control mice, infusion of ANG II increased MCSA and posterior wall thickness at diastole (PWTd) but had little effect on cardiac function, consistent with compensatory hypertrophy. In contrast, cardiac function was worse in mPGES-1 KO mice after ANG II treatment. Ejection fraction declined from 76.2 ± 2.7 to 63.3 ± 3.4% after ANG II, and left ventricular dimension at systole and diastole increased from 1.29 ± 0.02 to 1.78 ± 0.15 mm and from 2.57 ± 0.03 to 2.90 ± 0.13 mm, respectively. Infusion of ANG II increased both the LV-to-body weight and the mass-to-body weight ratios to a similar extent in both strains. However, PWTd increased by a lesser extent in KO mice, suggesting an impaired hypertrophic response. ANG II infusion increased collagen staining similarly in both strains, but TdT-dUTP nick end labeling staining was greater in mPGES-1 KO mice. Overall, these results are consistent with a beneficial effect for mPGES-1 in the maintenance of cardiac function in ANG II-dependent hypertension.     

mPGES-1 deletion potentiates urine concentrating capability after water deprivation

PGE(2) plays an important role in the regulation of fluid metabolism chiefly via antagonizing vasopressin-induced osmotic permeability in the distal nephron, but its enzymatic sources remain uncertain. The present study was undertaken to investigate the potential role of microsomal PGE synthase (mPGES)-1 in the regulation of urine concentrating ability after water deprivation (WD). Following 24-h WD, wild-type (WT) mice exhibited a significant reduction in urine volume, accompanied by a significant elevation in urine osmolality compared with control groups. In contrast, in response to WD, mPGES-1 knockout (KO) mice had much less urine volume and higher urine osmolality. Analysis of plasma volume by measurement of hematocrit and by using a nanoparticle-based method consistently demonstrated that dehydrated WT mice were volume depleted, which was significantly improved in the KO mice. WD induced a twofold increase in urinary PGE(2) output in WT mice, which was completely blocked by mPGES-1 deletion. At baseline, the KO mice had a 20% increase in V(2) receptor mRNA expression in the renal medulla but not the cortex compared with WT controls; the expression was unaffected by WD irrespective of the genotype. In response to WD, renal medullary aquaporin-2 (AQP2) mRNA exhibited a 60% increase in WT mice, and this increase was greater in the KO mice. Immunoblotting demonstrated increased renal medullary AQP2 protein abundance in both genotypes following WD, with a greater increase in the KO mice. Similar results were obtained by using immunohistochemistry. Paradoxically, plasma AVP response to WD seen in WT mice was absent in the KO mice. Taken together, these results suggest that mPGES-1-derived PGE(2) reduces urine concentrating ability through suppression of renal medullary expression of V(2) receptors and AQP2 but may enhance it by mediating the central AVP response.     

HLA-DRB1*0402 (DW10) transgene protects collagen-induced arthritis-susceptible H2Aq and DRB1*0401 (DW4) transgenic mice from arthritis

To investigate the role of HLA-DR4 in predisposition to arthritis, we generated transgenic mice carrying DRB1*0401 and DRB1*0402 genes. We have previously shown that DRB1*0401 molecule renders B10.RQB3 (H2A(q)) mice susceptible to porcine and human type II collagen-induced arthritis. We report that the introduction of DRB1*0402 transgene does not lead to development of arthritis in mice when they are immunized with porcine and human type II collagen. In addition, DRB1*0402 protects B10.RQB3 mice against developing arthritis with bovine type II collagen. These data show that DRB1 can modulate the disease mediated by A(q). In vivo depletion of DRB1*0402 did not lead to induction of collagen-induced arthritis in transgenic mice. In vitro cytokine analysis shows that mice protected from collagen-induced arthritis produce lower amounts of Th1 and higher levels of Th2 type cytokines upon immunization with type II collagen. Protection of mice was also related to higher apoptosis in DW10 mice as indicated by higher amounts of BclII in response to type II collagen. On the basis of our observations in HLA transgenic mice, we hypothesize that DRB1 polymorphism can modulate disease by shaping the T cell repertoire in thymus and select autoreactive T cells.     

Cytosolic phospholipase A2alpha regulates induction of brain cyclooxygenase-2 in a mouse model of inflammation

The products of arachidonic acid metabolism are key mediators of inflammatory responses in the central nervous system, and yet we do not know the mechanisms of their regulation. The phospholipase A(2) enzymes are sources of cellular arachidonic acid, and the enzymes cyclooxygenase-2 (COX-2) and microsomal PGE synthase-1 (mPGES-1) are essential for the synthesis of inflammatory PGE(2) in the brain. These studies seek to determine the function of cytosolic phospholipase A(2)alpha (cPLA(2)alpha) in inflammatory PGE(2) production in the brain. We wondered whether cPLA(2)alpha functions in inflammation to produce arachidonic acid or to modulate levels of COX-2 or mPGES-1. We investigated these questions in the brains of wild-type mice and mice deficient in cPLA(2)alpha (cPLA(2)alpha(-/-)) after systemic administration of LPS. cPLA(2)alpha(-/-) mice had significantly less brain COX-2 mRNA and protein expression in response to LPS than wild-type mice. The reduction in COX-2 was most apparent in the cells of the cerebral blood vessels and the leptomeninges. The brain PGE(2) concentration of untreated cPLA(2)alpha(-/-) mice was equal to their wild-type littermates. After LPS treatment, however, the brain concentration of PGE(2) was significantly less in cPLA(2)alpha(-/-) than in cPLA(2)alpha(+/+) mice (24.4 +/- 3.8 vs. 49.3 +/- 11.6 ng/g). In contrast to COX-2, mPGES-1 RNA levels increased equally in both mouse genotypes, and mPGES-1 protein was unaltered 6 h after LPS. We conclude that cPLA(2)alpha regulates COX-2 levels and modulates inflammatory PGE(2) levels. These results indicate that cPLA(2)alpha inhibition is a novel anti-inflammatory strategy that modulates, but does not completely prevent, eicosanoid responses.     

Membrane-bound prostaglandin E synthase-1-mediated prostaglandin E2 production by osteoblast plays a critical role in lipopolysaccharide-induced bone loss associated with inflammation

PGE(2) acts as a potent stimulator of bone resorption in several disorders including osteoarthritis and periodontitis. Three PGE synthases (PGES) were isolated for PGE(2) production, but which PGES has the major role in inflammatory bone resorption is still unclear. In this study, we examined the role of PGE(2) in LPS-induced bone resorption using membrane-bound PGES (mPGES)-1-deficient mice (mPges1(-/-)). In osteoblasts from wild-type mice, PGE(2) production was greatly stimulated by LPS following the expression of cyclooxygenase 2 and mPGES-1 mRNA, whereas no PGE(2) production was found in osteoblasts from mPges1(-/-). LPS administration reduced the bone volume in wild-type femur that was associated with an increased number of osteoclasts. In mPges1(-/-), however, LPS-induced bone loss was reduced. We next examined whether mPGES-1 deficiency could alter the alveolar bone loss in LPS-induced experimental periodontitis. LPS was injected into the lower gingiva and bone mineral density of alveolar bone was measured. LPS induced the loss of alveolar bone in wild-type, but not in mPges1(-/-) mice, suggesting an mPGES-1 deficiency resistant to LPS-induced periodontal bone resorption. To understand the pathway of LPS-induced PGE(2) production in osteoblast, we used C3H/HeJ mice with mutated tlr4. Osteoblasts from C3H/HeJ mice did not respond to LPS, and PGE(2) production was not altered at all. LPS-induced bone loss in the femur was also impaired in C3H/HeJ mice. Thus, LPS binds to TLR4 on osteoblasts that directly induce mPGES-1 expression for PGE(2) synthesis, leading to subsequent bone resorption. Therefore, mPGES-1 may provide a new target for the treatment of inflammatory bone disease.     

Combined effects of IL-12 and IL-18 on the induction of collagen-induced arthritis

IL-18 expression has recently been detected in rheumatoid arthritis (RA) synovial membrane. We investigated the mechanisms by which IL-18-induced collagen-induced arthritis in DBA/1 mice primed intradermally with type II bovine collagen in IFA and boosted i.p. 21 days later with CII in saline. Mice were injected i.p. with rIL-12, rIL-18, or both (100 ng) during days -1 to 4 and again on days 20-24. Control mice received PBS. Mice treated with IL-12 or IL-18 alone developed significantly higher incidence and more severe disease compared with controls. These were elevated further by combination treatment with IL-12 and IL-18. The cytokine treatments led to markedly enhanced synovial hyperplasia, cellular infiltration, and cartilage erosion compared with controls. Cytokine-treated mice produced significantly more IFN-gamma, TNF-alpha, and IL-6 than the controls. Interestingly, IL-18-treated mice produced more TNF-alpha and IL-6, but less IFN-gamma, compared with mice treated with IL-12. Furthermore, splenic macrophages from DBA/1 mice cultured in vitro with IL-18, but not IL-12, produced substantial amounts of TNF-alpha. Mice treated with IL-18 or IL-18 plus IL-12 produced markedly more IgG1 and IgG2a anti-collagen Ab compared with controls, whereas IL-12 treatment only led to an enhanced IgG2a response. Together these results demonstrate that IL-18 can promote collagen-induced inflammatory arthritis through mechanisms that may be distinct from those induced by IL-12.     

Anti-inflammatory activity of a novel selective cyclooxygenase-2 inhibitor, FR140423, on type II collagen-induced arthritis in Lewis rats

The mechanism of action of FR140423 (3-(difluoromethyl)-1-(4-methoxyphenyl)-5-[4-(methylsulfinyl)-phenyl]pyrazole), a novel and selective cyclooxygenase (COX)-2 inhibitor, in rat type II collagen-induced arthritis was investigated and compared with that of indomethacin. We tested the inhibitory effects of FR140423 on paw edema and the formation of arachidonic acid metabolites in inflamed paws immunized with type II collagen. Oral administration of FR 140423 showed a dose-dependent anti-inflammatory effect and was two-fold more potent than indomethacin. The increase of prostaglandin (PG) E2 and thromboxane (TX) B2 but not leukotriene B4 in inflamed paws was associated with the development of paw edema. FR140423 and indomethacin dose-dependently suppressed the levels of PGE2 and TXB2 in arthritic rat paws. Unlike indomethacin, FR140423 did not induce gastric lesions in arthritic rats. These results suggest that FR140423 shows a potent anti-inflammatory effect mediated by inhibition of prostanoids produced by COX-2 in inflamed tissues immunized with type II collagen, with a greatly improved safety profile compared to indomethacin.     

Prevention of collagen-induced arthritis in mice transgenic for the complement inhibitor complement receptor 1-related gene/protein y

The objective of these studies was to examine collagen-induced arthritis (CIA) in C57BL/6 mice transgenic for the rodent complement regulatory protein complement receptor 1-related gene/protein y (Crry) (Crry-Tg), a C3 convertase inhibitor. The scores for clinical disease activity and for histological damage in the joints were both significantly decreased in Crry-Tg mice in comparison to wild-type (WT) littermates. The production of both IgG1 and IgG2a anti-collagen Abs was reduced in the Crry-Tg mice, although spleen cell proliferation in response to collagen type II was not altered. The production of IFN-gamma, TNF-alpha, and IL-1beta by LPS-stimulated spleen cells was decreased, and IL-10 was increased, in cells from Crry-Tg mice in comparison to WT. The steady-state mRNA levels for IFN-gamma, TNF-alpha, and IL-1beta were all decreased in the joints of Crry-Tg mice in comparison to WT. The synovium from Crry-Tg mice without CIA contained the mRNA for the Crry transgene, by RT-PCR, and the synovium from transgenic mice with CIA exhibited little deposition of C3 protein by immunohistological analysis. These results suggest that suppression of CIA in Crry-Tg mice may be due to enhanced synthesis of Crry locally in the joint with decreased production of proinflammatory cytokines.     

Acceleration and increased severity of collagen-induced arthritis in P-selectin mutant mice.

P-selectin plays an important role in leukocyte adherence to microvascular endothelium and is expressed in synovial tissue from patients with rheumatoid arthritis (RA). However, the contribution of P-selectin to the initiation and chronicity of joint inflammation is not well understood. In these studies, collagen-induced arthritis (CIA) was induced in P-selectin mutant (-/-) mice to explore the role of P-selectin in the development of joint inflammation. Surprisingly, CIA onset was accelerated and severity was increased in P-selectin mutant mice, compared with wild-type mice (+/+). Increased levels of anti-type II collagen IgG were detected in both nonarthritic and arthritic P-selectin mutant mice from days 14-91. In addition, splenocytes isolated from immunized and nonimmunized P-selectin mutant mice produced significantly less IL-2 and IL-4, but significantly higher levels of IL-10 and IL-5 than splenocytes from wild-type mice. These observations show that P-selectin-mediated leukocyte rolling is not required for the development of murine CIA and that P-selectin expression exerts a controlling effect on the development of Ag-driven inflammatory joint disease, possibly by mediating the recruitment and/or trafficking of specific leukocyte subtypes into lymphoid tissue or inflammatory foci.     

The beta2-adrenergic agonist salbutamol is a potent suppressor of established collagen-induced arthritis: mechanisms of action

The therapeutic potential of salbutamol, a beta2-adrenergic agonist, was explored in collagen-induced arthritis. This study was based on a report that salbutamol, by elevating intracellular cAMP, inhibits IL-12 production by macrophages and dendritic cells, thus preventing Th1 development. Ten-week-old male DBA/1 mice were immunized by intradermal injection of type II collagen in CFA. Arthritis developed 15-30 days later and the mice were treated after onset of disease with salbutamol, 200 microgram i.p. After 10 days, the mice were sacrificed, and the hind paws were evaluated histologically. Salbutamol, 200 microgram daily or every other day, had a profound therapeutic effect on the clinical progression of arthritis, as assessed by clinical score and paw thickness. The therapeutic effect was dose dependent. Daily administration of 200 microgram of salbutamol offered the best protection against joint damage, as assessed by histology. In vitro, salbutamol reduced IL-12 and TNF-alpha release by peritoneal macrophages in a dose-dependent manner, as well as TNF release by synovial cells from arthritic mice. Ex vivo, draining lymph node cells of the salbutamol-treated arthritic mice showed a diminished CII-specific IFN-gamma production and proliferation. In vivo, salbutamol specifically blocked mast cell degranulation in joint tissues. In conclusion, salbutamol has important effects on the immunoinflammatory response and a significant therapeutic action in collagen-induced arthritis.     

THR0921, a novel peroxisome proliferator-activated receptor gamma agonist, reduces the severity of collagen-induced arthritis

THR0921 is a novel peroxisome proliferator-activated receptor gamma (PPARgamma) agonist with potent anti-diabetic properties. Because of the proposed role of PPARgamma in inflammation, we investigated the potential of orally active THR0921 to inhibit the pathogenesis of collagen-induced arthritis (CIA). CIA was induced in DBA/1J mice by the injection of bovine type II collagen in complete Freund's adjuvant on days 0 and 21. Mice were treated with THR0921 (50 mg/kg/day) starting on the day of the booster injection and throughout the remaining study period. Both clinical disease activity scores as well as histological scores of joint destruction were significantly reduced in mice treated with THR0921 compared to untreated mice. Proliferation of isolated spleen cells, as well as circulating levels of IgG antibody to type II collagen, was decreased by THR0921. Moreover, spleen cell production of IFN-gamma, tumor necrosis factor (TNF)-alpha and IL-1beta in response to exposure to lipopolysaccharide or type II collagen was reduced by in vivo treatment with THR0921. Steady state mRNA levels of TNF-alpha, IL-1beta, monocyte chemotactic protein-1 and receptor activator of nuclear factor kappaB ligand (RANKL) in isolated joints were all decreased in mice treated with THR0921. Finally, THR0921 inhibited osteoclast differentiation of bone marrow-derived cells stimulated with macrophage colony-stimulating factor and RANKL. In conclusion, THR0921 attenuates collagen-induced arthritis in part by reducing the immune response. As such, PPARgamma may be an important therapeutic target for rheumatoid arthritis.     

Microsomal prostaglandin E synthase-1 enhances bone cancer growth and bone cancer-related pain behaviors in mice

AimsNonsteroidal anti-inflammatory drugs are a therapeutic modality for chronic cancer pain arising from bone metastases. Chronic administration of a cyclooxygenase (COX)-2 inhibitor is effective to bone cancer-related pain. However, adverse cardiovascular effects have limited COX-2 inhibitor therapy, and elucidation of better targets for blocking prostaglandin (PG) biosynthesis is necessary. Microsomal PGE synthase-1 (mPGES-1) is an inducible enzyme that catalyzes isomerization of the endoperoxide PGH₂ to PGE₂. To investigate the validity of mPGES-1 as a therapeutic target, we evaluated bone cancer pain-related behaviors in mPGES-1 knockout (PGES-1?/?) mice.Main methodsLewis lung carcinoma cells (LLCCs) were injected into the intramedullary space of the femur of wild-type (WT) and PGES-1?/? mice. Pain-related behaviors were evaluated.Key findingsPGES-1?/? mice exhibited reduced tumor growth in bone marrow compared to WT. The expression of pro-calcitonin gene-related peptide (CGPR) in the dorsal root ganglia of L_1–5 was significantly higher in WT mice at day 14, whereas it was unchanged in mPGES-1 mice. In the observation of pain-related behaviors, mPGES-1?/? mice exhibited significantly fewer spontaneous flinches and their onset was several days later than WT. The appearance of other pain-related behaviors in mPGES-1?/? mice was also delayed as compared to WT. LLCC-injected WT mice treated with a COX-2 inhibitor, celecoxib, exhibited similar temporal changes to mPGES1?/?.SignificanceThe present results suggest that mPGES-1 plays a crucial role in the enhancement of bone cancer growth and bone cancer pain, and that inhibition of mPGES-1 may have clinical utility in the management of bone cancer pain.