Air Pouch Model Of Inflammation Research Articles

Evaluation of the effect of the degree of acetylation on the inflammatory response to 3D porous chitosan scaffolds

The effect of the degree of acetylation (DA) of 3D chitosan (Ch) scaffolds on the inflammatory reaction was investigated. Chitosan porous scaffolds with DAs of 4 and 15% were implanted using a subcutaneous air-pouch model of inflammation. The initial acute inflammatory response was evaluated 24 and 48 h after implantation. To characterize the initial response, the recruitment and adhesion of inflammatory cells to the implant site was studied. The fibrous capsule formation and the infiltration of inflammatory cells within the scaffolds were evaluated for longer implantation times (2 and 4 weeks). Chitosan with DA 15% attracted the highest number of leukocytes to the implant site. High numbers of adherent inflammatory cells were also observed in this material. For longer implantation periods Ch scaffolds with a DA of 15% induced the formation of a thick fibrous capsule and a high infiltration of inflammatory cells within the scaffold. Our results indicate that the biological response to implanted Ch scaffolds was influenced by the DA. Chitosan with a DA of 15% induce a more intense inflammatory response when compared with DA 4% Ch. Because inflammation and healing are interrelated, this result may provide clues for the relative importance of acetyl and amine functional groups in tissue repair and regeneration.

Effect of canine hyperimmune plasma on TNFα and inflammatory cell levels in a lipopolysaccharide-mediated rat air pouch model of inflammation

The purpose of this study was to test the effect of canine hyperimmune frozen plasma (HFP), which is known to contain elevated levels of soluble TNFα receptor 1 (sTNFR1), on TNFα and inflammatory cell levels in a LPS-mediated rat air pouch model of inflammation. There appears to be a correlation between elevated levels of sTNFR1 and depression of TNFα and neutrophil levels in the pouch fluid of HFP dosed rats (r = -0.73, P < 0.0001). The data suggest that canine HFP, which has been demonstrated to contain elevated levels of sTNFR1 compared with FFP, has a direct effect on depressing TNFα levels and neutrophil sequestration in the rat air pouch model of inflammation. These data suggest that HFP may be worthy of further investigation to determine whether such preparations have a therapeutic potential for treatment of acute inflammatory diseases in which TNFα is implicated.

Anti-Inflammatory Effects of an Ethanol Extract of Angelica gigas in a Carrageenan-Air Pouch Inflammation Model

Anti-inflammatory effects of an ethanol extract of Angelica gigas (EAG; 50, 160, or 500 mg/kg) were investigated in a carrageenan-induced air pouch inflammation model. Injection of 1 ml of carrageenan (1%) into mouse air pouches markedly increased the exudate volume and exudate albumin concentration, which were significantly attenuated by oral pretreatment with EAG. EAG also markedly reduced carrageenan-induced infiltrations of neutrophils, monocytes, and lymphocytes, but did not influence eosinophils or basophils. Carrageenan dramatically increased levels of tumor necrosis factor-alpha and interleukin-6, which might be derived from the infiltrated cells. It also elevated nitric oxide, and slightly increased prostaglandin E(2). EAG pretreatment significantly lowered tumor necrosis factor-alpha and nitric oxide, but did not alter interleukin-6 or prostaglandin E(2) levels. These results indicate that EAG attenuates some inflammatory responses by blocking the tumor necrosis factor-alpha-nitric oxide pathway, and that EAG could be a promising anti-inflammatory drug candidate for inflammatory diseases.

A combination of aspirin and γ-tocopherol is superior to that of aspirin and α-tocopherol in anti-inflammatory action and attenuation of aspirin-induced adverse effects

Nonsteroidal anti-inflammatory drugs such as aspirin are used for pain relief and chemoprevention against cancer, but frequently cause gastric mucosal injury. We examined whether combinations of aspirin and α-tocopherol (αT) or aspirin and γ-tocopherol (γT), with αT and γT being the two major forms of vitamin E, are better anti-inflammatory agents than aspirin alone, and whether these combinations alleviate aspirin-associated side effects. In the carrageenan-induced air-pouch inflammation model in the rat, aspirin (150 mg/kg) or a combination of aspirin and γT (33 mg/kg) inhibited proinflammatory prostaglandin E 2 (PGE 2) by 70% ( P<.02) at the inflammation site 6 h after inflammation was initiated. However, at 18 h, only the combination decreased exudate volume (15%; P<.05) and showed modest inhibition of PGE 2 (40%; P<.07) and lactate dehydrogenase activity (30%; P=.07) in the fluid collected at the inflammation site. γT, but not αT, spared aspirin-induced reduction in food intake, partially reversed aspirin-depressed gastric PGE 2 and attenuated stomach lesions. Surprisingly, the combination of aspirin and αT (33 mg/kg) did not show more benefits than aspirin alone, but worsened gastric injury and food intake reduction. Our study demonstrated that a combination of aspirin and γT, but not a combination of aspirin and αT, has some advantage over aspirin alone in terms of anti-inflammatory effects and attenuation of aspirin-induced adverse effects. This combination may be useful in complementing aspirin in the treatment of chronic inflammatory conditions and cancer.

Unique Role of Junctional Adhesion Molecule-A in Maintaining Mucosal Homeostasis in Inflammatory Bowel Disease

Junctional adhesion molecule-A (JAM-A) is localized at the tight junctions and controls leukocyte migration into the tissues. However, its functional role in inflammatory bowel disease (IBD) is unexplored. Control, Crohn's disease (CD), and ulcerative colitis (UC) tissue specimens were studied for JAM-A expression, as well as the colon of mice given dextran sodium sulfate (DSS). Wild-type and JAM-A(-/-), Tie-2-Cre-JAM-A(-/-) (endothelial/hematopoietic-specific JAM inactivation) mice were studied for susceptibility to DSS. Disease activity and colonic inflammation were assessed using a disease activity index histology and endoscopy, and mucosal cytokines were measured by enzyme-linked immunosorbent assay. JAM-A function was investigated by RNA silencing in epithelial cells, and apoptosis was measured. In both CD and UC, as well as in experimental colitis, there is a loss of epithelial but not endothelial JAM-A expression. Deletion of JAM-A results in a dramatic increase in susceptibility to DSS colitis, as assessed by weight loss, disease activity index, histologic and endoscopic severity, and strikingly high mortality rates. This is not caused by the absence of JAM-A in the endothelial or hematopoietic compartments because Tie-2-Cre-JAM-A(-/-) mice are no more susceptible to DSS colitis than wild-type animals. JAM-A(-/-) mice displayed increased intestinal permeability and inflammatory cytokine production, and marked epithelial apoptosis. Silencing of JAM-A in intestinal epithelial cells resulted in increased permeability in vitro. Our results show a nonredundant and novel role of JAM-A in controlling mucosal homeostasis by regulating the integrity and permeability of epithelial barrier function.

Evaluation of COX-1/COX-2 selectivity and potency of a new class of COX-2 inhibitors

A new class of selective cyclooxygenase-2 (COX-2) inhibitors has been identified by high throughput screening. Structurally distinct from previously described selective COX-2 inhibitors, these benzopyrans contain a carboxylic acid function and CF 3 functionality. The compound SC-75416 is a representative of this class. A range if in vitro and in vivo tests were employed to characterize its potency and selectivity. Using human recombinant enzymes, this compound displays a concentration that provides 50% inhibition (IC 50) of 0.25 μM for COX-2 and 49.6 μM for COX-1. A mutation of the side pocket residues in COX-2 to COX-1 had little effect on potency suggesting that these inhibitors bind in a unique manner in COX-2 distinct from COX-2 inhibiting diaryl heterocycles. Using rheumatoid arthritic synovial cells stimulated with interleukin-1β (IL-1β) and washed platelets the compound displayed IC 50 of 3 nM and 400 nM respectively. Potency and selectivity was maintained but predictably right shifted in whole blood with IC 50 of 1.4 μM for lipopolysaccharide (LPS) stimulated induction of COX-2 and > 200 μM for inhibition of platelet thromboxane production. SC-75416 is 89% bioavailable and its in vivo half life is sufficient for once a day dosing. In the rat air pouch model of inflammation, the compound inhibited PGE 2 production with an effective dose that provides 50% inhibition (ED 50) of 0.4 mg/kg, while sparing gastric prostaglandin E2 (PGE 2) production with an ED 50 of 26.5 mg/kg. In a model of acute inflammation and pain caused by carrageenan injection into the rat paw, the compound reduced edema and hyperalgesia with ED 50s of 2.7 and 4 mg/kg respectively. In a chronic model of arthritis the compound demonstrated an ED 50 of 0.081 mg/kg and an ED 80 of 0.38 mg/kg. In a model of neuropathic pain, SC-75416 had good efficacy. This compound's unique chemical structure and effect on COX enzyme binding and activity as well as its potency and selectivity may prove useful in treating pain and inflammation.

Low-frequency electroacupuncture suppresses zymosan-induced peripheral inflammation via activation of sympathetic post-ganglionic neurons

Electroacupuncture (EA) is used to treat a variety of inflammatory diseases; however, the neurophysiological mechanisms underlying EA's anti-inflammatory effect remain unclear. Accumulating evidence suggests that the sympathetic nervous system regulates immunologic and inflammatory responses and thus we hypothesized that this system could be involved in EA's anti-inflammatory effect (EA-AI). The goal of the present study was to evaluate whether the sympathetic nervous system plays a critical role in EA-AI using a mouse air pouch inflammation model. We found that bilateral low-frequency (1 Hz) EA applied to the Zusanli acupoint significantly suppressed the number of zymosan-induced leukocytes migrating into the air pouch. Furthermore, double-labeling immunohistochemical experiments showed that EA stimulation increased Fos expression in choline acetyltransferase (ChAT)-positive sympathetic pre-ganglionic neurons in the intermediolateral region of thoracic spinal cord segments. Chemical sympathetic denervation by intraperitoneal injection of 6-hydroxydopamine (which spares sympathetic adrenal medullary innervation) significantly inhibited EA-AI. In contrast, adrenalectomy did not alter EA-AI. Finally, systemic propranolol administration significantly inhibited EA's anti-inflammatory effect, suggesting that β-adrenoceptors are involved. Collectively, these results suggest that EA produces an anti-inflammatory effect in this mouse air pouch model by activating the sympathetic nervous system leading to the release of catecholamines from post-ganglionic nerve terminals, which act on β-adrenoceptors on immune cells to inhibit their migration.

Reduction of urate crystal-induced inflammation by root extracts from traditional oriental medicinal plants: elevation of prostaglandin D2 levels.

Dried roots of the plants Acanthopanax senticosus, Angelica sinensis and Scutellaria baicalensis are used in traditional oriental medicine and reportedly possess anti-inflammatory properties. Using the murine air pouch model of inflammation, we investigated the efficacy and mode of action of an extract from these three plants in crystal-induced inflammation. Air pouches were raised on the backs of 8-week-old BALB/c mice. Mice were fed 100 mg/kg body weight of root extracts (A. senticosus:A. sinensis:S. baicalensis mixed in a ratio of 5:4:1 by weight) or vehicle only on days 3–6. Inflammation was elicited on day 6 by injecting 2 mg of monosodium urate (MSU) crystals into the pouch. Neutrophil density and IL-6 and TNF-α mRNA levels were determined in the pouch membrane, and the leukocyte count and IL-6, prostaglandin E2 (PGE2) and prostaglandin D2 (PGD2) levels were determined in the pouch exudate. Treatment with the root extracts led to a reduction in all inflammatory parameters: the leukocyte count in the pouch exudate decreased by 82%; the neutrophil density in the pouch membrane decreased by 68%; IL-6 and TNF-α mRNA levels in the pouch membrane decreased by 100%; the IL-6 concentration in the pouch fluid decreased by 50%; and the PGE2 concentration in the pouch fluid decreased by 69%. Remarkably, the concentration of the potentially anti-inflammatory PGD2 rose 5.2-fold in the pouch exudate (p < 0.005), which led to a normalization of the PGD2:PGE2 ratio. A 3.7-fold rise in hematopoietic PGD synthase (h-PGDS) mRNA paralleled this rise in PGD2 (p = 0.01).Thus, the root extracts diminished MSU crystal-induced inflammation by reducing neutrophil recruitment and expression of pro-inflammatory factors and increasing the level of the potentially anti-inflammatory PGD2. These results support a need for further studies of the efficacy of these extracts in the treatment of inflammatory arthropathies and suggest elevation of PGD2 levels as a novel mechanism for an anti-inflammatory agent.

Evaluation of the anti-inflammatory efficacy of Passiflora edulis

The aim of this study was to investigate the anti-inflammatory effect of the aqueous extract of Passiflora edulis forma flavicarpa Degenerer leaves in the air pouch inflammation model induced by carrageenan, histamine or substance P, in mice. An aqueous extract was prepared by infusion and lyophilized. Part of the extract was fractionated with n -butanol, resulting in the butanolic and aqueous residual fractions. The anti-inflammatory activities of the lyophilized extract and these two derived fractions were evaluated. In the inflammation induced by carrageenan, aqueous extract (100 mg/kg, i.p.), butanolic fraction (50 mg/kg, i.p.), aqueous residual fraction (100 mg/kg, i.p.) and dexamethasone (0.5 mg/kg, i.p.) inhibited the leukocyte, neutrophil, myeloperoxidase, nitric oxide, and interleukin-1 beta (IL-1β) levels ( p < 0.05). The aqueous extract and butanolic and aqueous residual fractions, but not dexamethasone, decreased macrophage inflammatory protein-2 (MIP-2) levels ( p < 0.05). Only dexamethasone inhibited mononuclear cells ( p < 0.01). In the inflammation induced by histamine, the aqueous extract, butanolic and aqueous residual fractions, and dexamethasone inhibited total and differential leukocytes ( p < 0.01). In the inflammation induced by substance P, the aqueous extract, butanolic and aqueous residual fractions, and dexamethasone also inhibited total leukocytes and mononuclears ( p < 0.01). Neutrophils were only inhibited by aqueous extract, butanolic fraction, and dexamethasone ( p < 0.05). In conclusion, the active principle(s) present in the P. edulis aqueous extract and its two fractions showed pronounced anti-inflammatory properties, inhibiting cell migration, proinflammatory cytokines, enzymes and mediators.

S100A8 Triggers Oxidation-sensitive Repulsion of Neutrophils

The inflammatory response to tissue injury is a multi-faceted process. During this process, neutrophils migrate in the extravascular spaces, directed to the site of injury by chemical gradients generated by chemotactic molecules. S100A8, a protein associated with a wide variety of inflammatory conditions, is heavily over-expressed in association with inflammation. We hypothesized that human S100A8 possesses neutrophil-repelling properties that result in an anti-inflammatory effect in vivo. The chemotactic activity of S100A8 on neutrophils was tested in Transwell chemotaxis assays. Analysis of the data indicates that S100A8 causes a repulsion of peripheral neutrophils, an activity that S100A8 loses upon its oxidation. Using a mutant of S100A8 resistant to oxidation and consistent with the in vitro findings, we demonstrated that S100A8 causes a strong anti-inflammatory effect in the rat air-pouch model of inflammation in vivo. These data highlight a naturally occurring novel anti-inflammatory pathway and provide potential molecular targets for the development of novel anti-inflammatory therapeutics. Abbrevations: ethylene diamine tetraacetic acid (EDTA); limulus amoebocyte lysate assay (LAL); pertussis toxin (PTX); forward scatter (FSC); Interleukin-8 (IL-8); formyl-Met-Leu-Phe (fMLP); monocyte chemotactic protein 1 (MCP1).

Induction of triggering receptor expressed on myeloid cells 1 in murine resident peritoneal macrophages by monosodium urate monohydrate crystals

Triggering receptor expressed on myeloid cells 1 (TREM-1) is a cell surface molecule that was recently identified on monocytes and neutrophils. TREM-1 has been implicated in the early inflammatory responses induced by microbes, but its pathophysiologic role in nonmicrobial inflammation remains unknown. In the present study, we investigated the role of TREM-1 in acute inflammation induced by monosodium urate monohydrate (MSU) crystals. Induction of TREM-1 expression by MSU crystal-stimulated murine resident peritoneal macrophages and infiltrating leukocytes in a murine air-pouch model of crystal-induced acute inflammation was determined. The biologic role of TREM-1 in crystal-induced cytokine production by resident peritoneal macrophages was also investigated. TREM-1 expression by resident peritoneal macrophages and infiltrating leukocytes in a murine air-pouch model was determined by quantitative real-time polymerase chain reaction, Western blot analysis, and flow cytometry. Cytokine production by resident peritoneal macrophages after incubation with MSU crystals in the presence or absence of an anti-TREM-1 agonist antibody was determined by enzyme-linked immunosorbent assay. TREM-1 expression by resident peritoneal macrophages was significantly induced after stimulation with the crystals. Maximum expression of TREM-1 transcripts and protein occurred at 1 and 4 hours after exposure to the crystals, respectively. Costimulation of resident peritoneal macrophages with MSU crystals and an anti-TREM-1 agonist antibody synergistically increased the production of both interleukin-1beta and monocyte chemotactic protein 1 compared with stimulation with the crystals alone. MSU crystals also induced TREM-1 expression in infiltrating leukocytes in a murine air-pouch model of crystal-induced acute inflammation. These findings suggest that rapid induction of TREM-1 expression on resident peritoneal macrophages and neutrophils by MSU crystals may contribute to the development of acute gout through enhancement of inflammatory responses.

Suppression of inflammation by low-dose methotrexate is mediated by adenosine A2A receptor but not A3 receptor activation in thioglycollate-induced peritonitis

Prior studies demonstrate that adenosine, acting at one or more of its receptors, mediates the anti-inflammatory effects of methotrexate in animal models of both acute and chronic inflammation. Both adenosine A2A and A3 receptors contribute to the anti-inflammatory effects of methotrexate treatment in the air pouch model of inflammation, and the regulation of inflammation by these two receptors differs at the cellular level. Because different factors may regulate inflammation at different sites we examined the effect of low-dose weekly methotrexate treatment (0.75 mg/kg/week) in a model of acute peritoneal inflammation in adenosine A2A receptor knockout mice and A3 receptor knockout mice and their wild-type littermates. Following intraperitoneal injection of thioglycollate there was no significant difference in the number or type of leukocytes, tumor necrosis factor alpha (TNF-α) and IL-10 levels that accumulated in the thioglycollate-induced peritoneal exudates in adenosine A2A knockout mice or wild-type control mice. In contrast, there were more leukocytes, TNF-α and IL-10 in the exudates of the adenosine A3 receptor-deficient mice. Low-dose, weekly methotrexate treatment increased the adenosine concentration in the peritoneal exudates of all mice studied, and reduced the leukocyte accumulation in the wild-type mice and A3 receptor knockout mice but not in the A2A receptor knockout mice. Methotrexate reduced exudate levels of TNF-α in the wild-type mice and A3 receptor knockout mice but not the A2A receptor knockout mice. More strikingly, IL-10, a critical regulator of peritoneal inflammation, was increased in the methotrexate-treated wild-type mice and A3 knockout mice but decreased in the A2A knockout mice. Dexamethasone, an agent that suppresses inflammation by a different mechanism, was similarly effective in wild-type mice, A2A mice and A3 knockout mice. These findings provide further evidence that adenosine is a potent regulator of inflammation that mediates the anti-inflammatory effects of methotrexate. Moreover, these data provide strong evidence that the anti-inflammatory effects of methotrexate and adenosine are mediated by different receptors in different inflammatory loci, an observation that may explain why inflammatory diseases of some organs but not of other organs respond to methotrexate therapy.

Inhibitory effect of the coffee diterpene kahweol on carrageenan-induced inflammation in rats

Previous studies reported that kahweol, a coffee-specific diterpene, inhibits cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression in cultured lipopolysaccharide-activated macrophages. The aim of this study was to confirm the anti-inflammatory effects of kahweol by examining its effect on the inflammatory response induced by carrageenan in a rat using an acute air pouch inflammation model. Kahweol significantly reduced the levels of the inflammatory process markers in the air pouch, such as the volume of exudates, the amount of protein and the number of leukocytes and neutrophils. The levels of nitrite, TNF-alpha and prostaglandin E2 (PGE2) were also markedly lower in the air pouch of the kahweol-treated animals than in the controls. Immunoblot analysis showed that kahweol reduced the COX-2 and iNOS expression level in the exudate cells. The histological examination showed that there was a lower inflammatory response in the pouch tissues from the kahweol-treated animals. In addition, kahweol significantly reduced the paw edema induced by carrageenan and also markedly reduced the level of PGE2 production in the inflamed paw. These results suggest that kahweol has significant anti-inflammatory effects in vivo, which might be due to the inhibition of iNOS and COX-2 expression in the inflammatory sites.

Inhibitory Effect of the Saponins Derived from Roots ofPlatycodon grandiflorumon Carrageenan-Induced Inflammation

Previous studies have reported that the saponins isolated from the roots of Platycodon grandiflorum A. DC (Campanulaceae), Changkil saponins (CKS), inhibited cyclooxygenase-2 (COX-2) expression in cultured lipopolysaccharide-activated macrophages. The aim of this presented study was to confirm the anti-inflammatory effects of CKS by examining their effect on the inflammatory response induced by carrageenan in a rat by using an acute air pouch inflammation model. CKS significantly reduced the levels of the inflammatory process markers in the air pouch, such as the volume of exudates, the amount of protein and the number of leukocytes and neutrophils. The levels of TNF-alpha and prostaglandin E2 (PGE2) were also markedly lower in the air pouch of the CKS-treated animals than in the controls. An immunoblot analysis showed that CKS reduced the COX-2 expression level in the exudate cells. In addition, CKS significantly reduced the paw edema induced by carrageenan and also markedly reduced the level of PGE2 production in the inflamed paw. These results suggest that CKS had significant anti-inflammatory effects in vivo.

The Anti-Inflammatory Effects of Low- and High-Frequency Electroacupuncture Are Mediated by Peripheral Opioids in a Mouse Air Pouch Inflammation Model

Although acupuncture has been widely used for complementary therapeutic approaches to treat inflammatory diseases and inflammation-induced pain, the potential anti-inflammatory effects of acupuncture treatment remain controversial in clinical trials, and the underlying mechanisms are still unclear. The objective was to determine whether electroacupuncture (EA) is able to suppress the peripheral inflammatory response (e.g., zymosan-induced leukocyte migration into air pouch). As part of a mechanistic approach, it was further evaluated whether endogenous opioid systems are involved in the "EA-induced anti-inflammatory effect" (EA-AI). EA (1 or 120 Hz) was performed bilaterally in the Zusanli acupoint (ST36) or in a nonacupoint (gluteal muscle) for 30 min in ICR mice under anesthetic condition. The number of leukocytes that migrated into the air pouch was counted 4 hours after zymosan injection. EA was performed at 0, 0.5, 1, or 2 hours prior to zymosan injection, respectively. To evaluate opioid involvement in EA-AI, intrathecal naloxone (36 microg/mouse) and intraperitoneal naloxone methiodide (30 mg/kg) were administered 10 min before EA stimulation. Both the 1 and 120 Hz frequencies of EA into Zusanli acupoint at the same time with zymosan injection significantly reduced leukocyte migration into the air pouch as compared with those of control groups (i.e., anesthetic control and needling control into Zusanli acupoint without electrical stimulation). The EA stimulation into nonacupoint did not produce any significant anti-inflammatory effect. EA treatment at 0.5 hours prior to zymosan injection also produced an anti-inflammatory effect but 1 and 2 hours prior to zymosan injection did not elicit any effect. Peripheral opioid blockage significantly reversed EA-AI, whereas spinal opioid blockage did not alter EA-AI. EA can suppress peripheral inflammation through a peripheral opioid mechanism. To achieve the full effectiveness of EA, repeated application is recommended for the treatment of a variety of inflammatory diseases.

Comparative study of anti-inflammatory and ulcerogenic activities of different cyclo-oxygenase inhibitors

The aim of the present work was to study the in vivo anti-inflammatory activity of six NSAIDs, ibuprofen, diclofenac, nimesulide, meloxicam, celecoxib and rofecoxib, using the rat air-pouch model of inflammation to characterize the ability of these drugs to induce gastric damage and PGE(2) inhibition. Selective compounds were observed to have no ulcerogenic properties at anti-inflammatory doses; however, these drugs were weaker inhibitors of several inflammatory aspects such as cell influx and exudate formation. In contrast, the non-selective and preferential compounds present anti-inflammatory properties at lower doses than presented by selective drugs. At anti-inflammatory doses, only meloxicam and ibuprofen produced gastric damage and inhibition of PGE(2) synthesis, suggesting that ulcerogenic properties of NSAIDs cannot be predicted by their selectivity index, since meloxicam demonstrates ulcerogenic properties despite its preferential profile.

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

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. 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. 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 x C57BL/6J F(1) and F(2) 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. 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.

The anti-inflammatory effect of peripheral bee venom stimulation is mediated by central muscarinic type 2 receptors and activation of sympathetic preganglionic neurons

The anti-inflammatory effect (AI) induced by peripheral injection of diluted bee venom (dBV) involves activation of spinal cord circuits and is mediated by catecholamine release from adrenal medulla, but the precise neuronal mechanisms involved are not fully understood. In a recent study, we demonstrated that an increase in spinal acetylcholine is involved in mediating the anti-inflammatory effect of dBV and that this mediation also involves adrenomedullary activation. The present study utilized the mouse air pouch inflammation model to evaluate the involvement of spinal acetylcholine receptors and sympathetic preganglionic neurons (SPNs) in dBV's anti-inflammatory effect (dBVAI). Intrathecal (IT) pretreatment with atropine (muscarinic cholinergic antagonist) but not hexamethonium (nicotinic cholinergic antagonist) significantly suppressed dBVAI on zymosan-evoked leukocyte migration. Subsequent experiments showed that IT pretreatment with methoctramine (a muscarinic receptor type 2; M 2 antagonist), but not pirenzepine (an M 1 antagonist) or 4-DAMP (an M 3 antagonist), suppressed the dBVAI. In addition, dBV stimulation specifically increased Fos expression in SPNs of the T7–T11, but not the T1–T6 or T12–L2 spinal cord segments, in animals with zymosan-induced inflammation. Moreover, IT methoctramine pretreatment suppressed this dBV-induced Fos expression specifically in SPNs of T7–T11 level. Peripheral sympathetic denervation using 6-hydroxydopamine (6-OHDA) treatment (which spares sympathetic adrenal medullary innervation) did not alter dBVAI. Collectively these results indicate that dBV stimulation leads to spinal cord acetylcholine release that in turn acts on spinal M 2 receptors, which via a hypothesized disinhibition mechanism activates SPNs that project to the adrenal medulla. This activation ultimately leads to the release of adrenal catecholamines that contribute to dBVAI.

3-[4-(Methylsulfonyl)phenyl]-5-(trifluoromethyl)(2-pyridyl) Phenyl Ketone as a Potent and Orally Active Cyclooxygenase-2 Selective Inhibitor: Synthesis and Biological Evaluation

Incorporation of a spacer group between the central scaffold and the aryl ring resulted in a new cyclooxygenase-2 (COX-2) selective inhibitor core structure, 3-[4-(methylsulfonyl)phenyl]-5-(trifluoromethyl)(2-pyridyl) phenyl ketone (20), with COX-2 IC50 = 0.25 microM and COX-1 IC50 = 14 microM (human whole blood assay). Compound 20 was orally active in the rat air pouch model of inflammation, inhibiting white blood cell infiltration and COX-2-derived PG production. Our data support the identification of a novel COX-2 selective inhibitor core structure exemplified by 20.

Potentiation of neutrophil cyclooxygenase-2 by adenosine: an early anti-inflammatory signal

Neutrophils, which are often the first to migrate at inflamed sites, can generate leukotriene B(4) from the 5-lipoxygenase pathway and prostaglandin E(2) through the inducible cyclooxygenase-2 pathway. Adenosine, an endogenous autacoid with several anti-inflammatory properties, blocks the synthesis of leukotriene B(4) while it potentiates the cyclooxygenase-2 pathway in fMLP-treated neutrophils, following activation of the A(2A) receptor. Using the murine air pouch model of inflammation, we observed that inflammatory leukocytes from mice lacking the A(2A) receptor have less cyclooxygenase-2 induction than wild-type animals. In human leukocytes, A(2A) receptor activation specifically elicited potentiation of cyclooxygenase-2 in neutrophils, but not in monocytes. Signal transduction studies indicated that the cAMP, ERK1/2, PI-3K and p38K intracellular pathways are implicated both in the direct upregulation of cyclooxygenase-2 and in its potentiation. Together, these results indicate that neutrophils are particularly important mediators of adenosine's effects. Given the uncontrolled inflammatory phenotype observed in knockout mice and in view of the potent inhibitory actions of prostaglandin E(2) on inflammatory cells, an increased cyclooxygenase-2 expression resulting from A(2A) receptor activation, observed particularly in neutrophils, may take part in an early modulatory mechanism promoting anti-inflammatory activities of adenosine.