IL-33 mediates antigen-induced cutaneous and articular hypernociception in mice

IL-33, a new member of the IL-1 family, signals through its receptor ST2 and induces T helper 2 (Th2) cytokine synthesis and mediates inflammatory response. We have investigated the role of IL-33 in antigen-induced hypernociception. Recombinant IL-33 induced cutaneous and articular mechanical hype rn ociception in a time- and dose-dependent manner. The hypernociception was inhibited by soluble (s) ST2 (a decoy receptor of IL-33), IL-1 receptor antagonist (IL-1ra), bosentan [a dual endothelin (ET)(A)/ETB receptor antagonist], clazosentan (an ETA receptor antagonist), or indomethacin (a cyclooxygenase inhibitor). IL-33 induced hypernociception in IL-18(-/-) mice but not in TNFR1(-/-) or IFN gamma(-/-) mice. The IL-33-induced hypernociception was not affected by blocking IL-15 or sympathetic amines (guanethidine). Furthermore, methylated BSA (mBSA)-induced cutaneous and articular mechanical hypernociception depended on TNFR1 and IFN gamma and was blocked by sST2, IL-1ra, bosentan, clazosentan, and indomethacin. mBSA also induced significant IL-33 and ST2 mRNA expression. Importantly, we showed that mBSA induced hypernociception via the IL-33 -> TNF alpha -> IL-1 beta -> IFN gamma -> ET-1 -> PGE(2) signaling cascade. These results therefore demonstrate that IL-33 is a key mediator of immune inflammatory hype rn ociception normally associated with a Th1 type of response, revealing a hitherto unrecognized function of IL-33 in a key immune pharmacological pathway that may be amenable to therapeutic intervention.

Involvement of LTB4 in zymosan-induced joint nociception in mice: participation of neutrophils and PGE(2)

Leukotriene B-4 (LTB4) mediates different inflammatory events such as neutrophil migration and pain. The present study addressed the mechanisms of LTB4-mediated joint inflammation-induced hypernociception. It was observed that zymosan-induced articular hypernociception and neutrophil migration were reduced dose-dependently by the pretreatment with MK886 (1-9 mg/kg; LT synthesis inhibitor) as well as in 5-lypoxygenase-deficient mice (5LO(-/-)) or by the selective antagonist of the LTB4 receptor (CP105696; 3 mg/kg). Histological analysis showed reduced zymosan-induced articular inflammatory damage in 5LO(-/-) mice. The hypernociceptive role of LTB4 was confirmed further by the demonstration that joint injection of LTB4 induces a dose (8.3, 25, and 75 ng)-dependent articular hypernociception. Furthermore, zymosan induced an increase in joint LTB4 production. Investigating the mechanism underlying LTB4 mediation of zymosan-induced hypernociception, LTB4-induced hypernociception was reduced by indomethacin (5 mg/kg), MK886 (3 mg/kg), celecoxib (10 mg/kg), antineutrophil antibody (100 mu g, two doses), and fucoidan (20 mg/kg) treatments as well as in 5LO(-/-) mice. The production of LTB4 induced by zymosan in the joint was reduced by the pretreatment with fucoidan or antineutrophil antibody as well as the production of PGE(2) induced by LTB4. Therefore, besides reinforcing the role of endogenous LTB4 as an important mediator of inflamed joint hypernociception, these results also suggested that the mechanism of LTB4-induced articular hypernociception depends on prostanoid and neutrophil recruitment. Furthermore, the results also demonstrated clearly that LTB4-induced hypernociception depends on the additional release of endogenous LTs. Concluding, targeting LTB4 synthesis/action might constitute useful therapeutic approaches to inhibit articular inflammatory hypernociception.

15d-prostaglandin J(2) inhibits inflammatory hypernociception: Involvement of peripheral opioid receptor

The 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) is an endogenous ligand of peroxisome proliferator-activated receptors gamma (PPAR-gamma) and is now recognized as a potent anti-inflammatory mediator. However, information regarding the influence of 15d-PGJ(2) on inflammatory pain is still unknown. In this study, we evaluated the effect of 15d-PGJ(2) upon inflammatory hypernociception and the mechanisms involved in this effect. We observed that intraplantar administration of 15d-PGJ(2) (30-300 ng/paw) inhibits the mechanical hypernociception induced by both carrageenan (100 mu g/paw) and the directly acting hypernociceptive mediator, prostaglandin E-2 (PGE(2)). Moreover, 15d-PGJ(2) [100 ng/temporomandibular joint (TMJ)] inhibits formalininduced TMJ hypernociception. On the other hand, the direct administration of 15d-PGJ(2) into the dorsal root ganglion was ineffective in blocking PGE(2)- induced hypernociception. In addition, the 15d-PGJ(2) antinociceptive effect was enhanced by the increase of macrophage population in paw tissue due to local injection of thioglycollate, suggesting the involvement of these cells on the 15d-PGJ(2)-antinociceptive effect. Moreover, the antinociceptive effect of 15d-PGJ(2) was also blocked by naloxone and by the PPAR-gamma antagonist 2-chloro-5-nitro-N-phenylbenzamide (GW9662), suggesting the involvement of peripheral opioids and PPAR-gamma receptor in the process. Similar to opioids, the 15d-PGJ(2) antinociceptive action depends on the nitric oxide/cGMP/protein kinase G (PKG)/K-ATP(+) channel pathway because it was prevented by the pretreatment with the inhibitors of nitric-oxide synthase (N-G-monomethyl-L-arginine acetate), guanylate cyclase] 1H-(1,2,4)-oxadiazolo(4,2-alpha) quinoxalin-1- one[, PKG [indolo[2,3-a]pyrrolo[3,4-c]carbazole aglycone (KT5823)], or with the ATP-sensitive potassium channel blocker glibenclamide. Taken together, these results demonstrate for the first time that 15d-PGJ(2) inhibits inflammatory hypernociception via PPAR-gamma activation. This effect seems to be dependent on endogenous opioids and local macrophages.

Inhibition of Neutrophil Migration by Hemopexin Leads to Increased Mortality Due to Sepsis in Mice

Rationale: The reduction of neutrophil migration to the bacterial focus is associated with poor outcome in sepsis. Objectives: The objective of this study was to identify soluble substances in the blood of septic mice that inhibit neutrophil migration. Methods: A pool of serum obtained from mice 2 hours after the induction of severe sepsis by cecal ligation and puncture inhibited the neutrophil migration. The proteins with inhibitory activity on neutrophil migration were isolated by Blue-Sepharose chromatography, high-performance liquid chromatography, and electrophoresis, and identified by mass spectrometry. Measurements and Main Results: Hemopexin was identified as the serum component responsible for the inhibition of neutrophil migration. In sepsis, the pretreatment of wild-type mice with hemopexin inhibited neutrophil migration to the focus of infection and decreased the survival rate from 87.5 to 50.0%. Hemopexin-null mice subjected to severe sepsis presented normal neutrophil migration, low bacteremia, and an improvement of 40% in survival rate. Moreover, hemopexin inhibited the neutrophil chemotaxis response evoked by C5a or macrophage inflammatory protein-2 and induced a reduction of CXCR2 and L-selectin as well as the up-regulation of CD11b expression in neutrophil membranes. The inhibitory effect of hemopexin on neutrophil chemotaxis was prevented by serine protease inhibitors or ATP. In addition, serum levels of ATP were decreased 2 hours after severe sepsis. Conclusions: These data demonstrate for the first time the inhibitory role of hemopexin in neutrophil migration during sepsis and suggest that the therapeutic inhibition of hemopexin or its protease activity could improve neutrophil migration to the focus of infection and survival in sepsis.

Peroxisome proliferator-activated receptor-gamma ligand, 15-deoxy-Delta A(12,14)-prostaglandin J(2), reduces neutrophil migration via a nitric oxide pathway

Ligands for peroxisome proliferator-activated receptor gamma (PPAR-gamma), such as 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) have been implicated as a new class of anti-inflammatory compounds with possible clinical applications. Based on this concept, this investigation was designed to determine the effect of 15d-PGJ(2)-mediated activation of PPAR-gamma ligand on neutrophil migration after an inflammatory stimulus and clarify the underlying molecular mechanisms using a mouse model of peritonitis. Our results demonstrated that 15d-PGJ(2) administration decreases leukocyte rolling and adhesion to the inflammated mesenteric tissues by a mechanism dependent on NO. Specifically, pharmacological inhibitors of NO synthase remarkably abrogated the 15d-PGJ(2)-mediated suppression of neutrophil migration to the inflammatory site. Moreover, inducible NOS(-/-) mice were not susceptible to 15d-PGJ(2)-mediated suppression of neutrophil migration to the inflammatory sites when compared with their wild type. In addition, 15d-PGJ(2)-mediated suppression of neutrophil migration appeared to be independent of the production of cytokines and chemokines, since their production were not significantly affected in the carrageenan-injected peritoneal cavities. Finally, up-regulation of carrageenan-triggered ICAM-I expression in the mesenteric microcirculation vessels was abrogated by pretreatment of wild-type mice with 15d-PGJ(2), whereas 15d-PGJ(2) inhibited F-actin rearrangement process in neutrophils. Taken together these findings demonstrated that 15d-PGJ(2) suppresses inflammation-initiated neutrophil migration in a mechanism dependent on NO production in mesenteric tissues.

Quercetin Reduces Neutrophil Recruitment Induced by CXCL8, LTB(4), and fMLP: Inhibition of Actin Polymerization

Recent in vitro data have suggested that the flavonoid quercetin (1) does not affect the functioning of neutrophils. Therefore, we evaluated in vivo and in vitro whether or not 1 affects neutrophil function, focusing on recruitment. The in vivo treatment with 1 inhibited in a dose-dependent manner the recruitment of neutrophils to the peritoneal cavity of mice induced by known chemotatic factors such as CXCL1, CXCL5, LTB(4), and fMLP. Further-more, 1 also inhibited in a concentration-dependent manner the chemoattraction of human neutrophils induced by CXCL8, LTB(4), and fMLP in a Boyden chamber. In vitro treatment with 1 did not affect human neutrophil surface expression of CXCR1, CXCR2, BLT1, or FLPR1, but rather reduced actin polymerization. These results suggest that 1 inhibits actin polymerization, hence, explaining the inhibition of neutrophil recruitment in vivo and in vitro and highlighting its possible usefulness to diminish excessive neutrophil migration during inflammation.

IL-33 induces neutrophil migration in rheumatoid arthritis and is a target of anti-TNF therapy

Objectives Interleukin 33 (IL-33) is a new member of the IL-1 family of cytokines which signals via its receptor, ST2 (IL-33R), and has an important role in Th2 and mast cell responses. This study shows that IL-33 orchestrates neutrophil migration in arthritis. Methods and results Methylated bovine serum albumin (mBSA) challenge in the knee joint of mBSA-immunised mice induced local neutrophil migration accompanied by increased IL-33R and IL-33 mRNA expression. Cell migration was inhibited by systemic and local treatments with soluble (s) IL-33R, an IL-33 decoy receptor, and was not evident in IL-33R-deficient mice. IL-33 injection also induced IL-33R-dependent neutrophil migration. Antigen- and IL-33-induced neutrophil migration in the joint was dependent on CXCL1, CCL3, tumour necrosis factor a (TNF alpha) and IL-1 beta synthesis. Synovial tissue, macrophages and activated neutrophils expressed IL-33R. IL-33 induces neutrophil migration by activating macrophages to produce chemokines and cytokines and by directly acting on neutrophils. Importantly, neutrophils from patients with rheumatoid arthritis successfully treated with anti-TNF alpha antibody (infliximab) expressed significantly lower levels of IL-33R than patients treated with methotrexate alone. Only neutrophils from patients treated with methotrexate alone or from normal donors stimulated with TNF alpha responded to IL-33 in chemotaxis. Conclusions These results suggest that suppression of IL-33R expression in neutrophils, preventing IL-33-induced neutrophil migration, may be an important mechanism of anti-TNF alpha therapy of inflammation.

Hydrogen Sulfide Improves Neutrophil Migration and Survival in Sepsis via K(ATP)(+) Channel Activation

Rationale Recovering the neutrophil migration to the infectious focus improves survival in severe sepsis. Recently, we demonstrated that the cystathionine gamma-lyase (CSE)/hydrogen sulfide (H(2)S) pathway increased neutrophil recruitment to inflammatory focus during sterile inflammation. Objectives: To evaluate if H(2)S administration increases neutrophil migration to infectious focus and survival of mice. Methods. Sepsis was induced by cecal ligation and puncture (CLP) Measurements and Main Results. The pretreatments of mice with H2S donors (NaHS or Lawesson`s reagent) improved leukocyte rolling/adhesion in the mesenteric microcirculation as well as neutrophil migration. Consequently, bacteremia levels were reduced, hypotension and lung lesions were prevented, and the survival rate increased from approximately 13% to approximately 80% Even when treatment was delayed (6 h after CLP), a highly significant reduction in mortality compared with untreated mice was observed Moreover, H(2)S pretreatment prevented the down-regulation of CXCR2 and L-selectin and the up-regulation of CD11b and G protein-coupled receptor kinase 2 in neutrophils during sepsis. H(2)S also prevented the reduction of intercellular adhesion molecule-1 expression in the endothelium of the mesenteric microcirculation in severe sepsis Confirming the critical role of H(2)S on sepsis outcome, pretreatment with dl-propargylglycine (a CSE inhibitor) inhibited neutrophil migration to the infectious focus, enhanced lung lesions, and induced high mortality in mice subjected to nonsevere sepsis (from 0 to similar to 80%). The beneficial effects of H(2)S were blocked by glibenclamide (a ATP-dependent K(+) channel blocker). Conclusions: These results showed that H(2)S restores neutrophil migration to the infectious focus and improves survival outcome in severe sepsis by an ATP-dependent K(+) channel-dependent mechanism.

IL-17 mediates articular hypernociception in antigen-induced arthritis in mice

IL-17 is an important cytokine in the physiopathology of rheumatoid arthritis (RA). However, its participation in the genesis of nociception during RA remains undetermined. In this study, we evaluated the role of IL-17 in the genesis of articular nociception in a model of antigen (mBSA)-induced arthritis. We found that mBSA challenge in the femur-tibial joint of immunized mice induced a dose-and time-dependent mechanical hypernociception. The local IL-17 concentration within the mBSA-injected joints increased significantly over time. Moreover, co-treatment of mBSA challenged mice with an antibody against IL-17 inhibited hypernociception and neutrophil recruitment. In agreement, intraarticular injection of IL-17 induced hypernociception and neutrophil migration, which were reduced by the pre-treatment with fucoidin, a leukocyte adhesion inhibitor. The hypernociceptive effect of IL-17 was also reduced in TNFR1(-/-) mice and by pre-treatment with infliximab (anti-TNF antibody), a CXCR1/2 antagonist or by an IL-1 receptor antagonist. Consistent with these findings, we found that IL-17 injection into joints increased the production of TNF-alpha, IL-1 beta and CXCL1/KC. Treatment with doxycycline (non-specific MMPs inhibitor), bosentan (ET(A)/ET(B) antagonist), indomethacin (COX inhibitor) or guanethidine (sympathetic blocker) inhibited IL-17-induced hypernociception. IL-17 injection also increased PGE(2) production, MMP-9 activity and COX-2, MMP-9 and PPET-1 mRNA expression in synovial membrane. These results suggest that IL-17 is a novel pro-nociceptive cytokine in mBSA-induced arthritis, whose effect depends on both neutrophil migration and various pro-inflammatory mediators, as TNF-alpha, IL-1 beta, CXCR1/2 chemokines ligands, MMPs, endothelins, prostaglandins and sympathetic amines. Therefore, it is reasonable to propose IL-17 targeting therapies to control this important RA symptom. (C) 2009 International Association for the Study of Pain. Published by Elsevier B. V. All rights reserved.

Acute and persistent nociceptive paw sensitisation in mice: The involvement of distinct signalling pathways

Aims: Many fundamental pharmacological studies in pain and inflammation have been performed on rats. However, the pharmacological findings were generally not extended to other species in order to increase their predictive therapeutic value. We studied acute and chronic inflammatory nociceptive sensitisation of mouse hind paws by prostaglandin E(2) (PGE(2)) or dopamine (DA), as previously described in rats. We also investigated the participation of the signalling pathways in acute and persistent sensitisation. Main methods: Mechanical sensitisation (hypernociception) induced by intraplantar administrations of PGE(2) or DA was evaluated with an electronic pressure meter. The signalling pathways were pharmacologically investigated with the pre-administration of adenylyl cyclase (AC), cAMP-dependent protein kinase (PKA), protein kinase C epsilon (PKC epsilon), and the extracellular signal-related kinase (ERK) inhibitors. Key findings: Single or 14 days of successive intraplantar injections of PGE(2) or DA-induced acute and persistent hypernociception (lasting for more than 30 days), respectively. The involvement of AC, PKA or PKC epsilon was observed in the acute hypernociception induced by PGE(2), while PKA or PKC epsilon were continuously activated during the period of persistent hypernociception. The acute hypernociception induced by DA involves activation of ERK, PKC epsilon, AC or PKA, while persistent hypernociception implicated ERK activation, but not PKA, PKC epsilon or AC. Significance: In mice, acute and persistent paw sensitisation involves the different activation of kinases, as previously described for rats. This study opens the possibility of comparing pharmacological approaches in both species to further understand acute and chronic inflammatory sensitisation, and possibly associated genetic manipulations. (C) 2009 Elsevier Inc. All rights reserved.

Quercetin Reduces Inflammatory Pain: Inhibition of Oxidative Stress and Cytokine Production

Quercetin (1) is known to have both antioxidant and antinociceptive effects. However, the mechanism involved in its antinociceptive effect is not fully elucidated. Cytokines and reactive oxygen species have been implicated in the cascade of events resulting in inflammatory pain. Therefore, we evaluated the antinociceptive mechanism of 1 focusing on the role of cytokines and Oxidative stress. Intraperitoneal and oral treatments with 1 dose-dependently inhibited inflammatory nociception induced by acetic acid and phenyl-p-benzoquinone and also the second phase of formalin- and carrageenin-induced mechanical hypernociception. Compound I also inhibited the hypernociception induced by cytokines (e.g., TNF alpha and CXCL1), but not by inflammatory mediators that directly sensitize the nociceptor such as PGE(2) and dopamine. On the other hand, 1 reduced carrageenin-induced IL-1 beta production as well as carrageenin-induced decrease of reduced glutathione (GSH) levels. These results suggest that I exerts its analgesic effect by inhibiting pro-nociceptive cytokine production and the oxidative imbalance mediation of inflammatory pain.

Functional characterization of the hypothalamic-pituitary-adrenal axis of the Wistar Audiogenic Rat (WAR) strain

The Wistar Audiogenic Rat (WAR) strain is a genetic model of sound-induced reflex epilepsy which was selected starting from audiogenic seizures susceptible Wistar rats. Wistar resistant rats were used as WAR`s control in this study. In the acute situation, audiogenic seizures (AS) in WARs mimic tonic-clonic seizures and, in the chronic protocol, mimic temporal lobe epilepsy. AS have been shown to evoke neuroendocrine responses; however, the hypothalamic-pituitary-adrenal activity in the WAR has not been established. The aim of this study was to evaluate the hypothalamic-pituitary-adrenal axis (HPA) responses to exogenous ACTH stimulation (8 ng/rat), fifteen minute restraint stress and circadian variation (8 am and 8 pm) under rest conditions in these animals through plasma measurements of ACTH and corticosterone concentrations. We also measured the body weight from birth to the 9th week of life and determined adrenal gland weight. We found that WARs are smaller than Wistar and presented a higher adrenal gland weight with a higher level of corticosterone release after intravenous ACTH injection. They also showed altered HPA axis circadian rhythms and responses to restraint stress. Our data indicate that, despite the lower body weight, WARs have increased adrenal gland weight associated with enhanced pituitary and adrenal responsiveness after HPA axis stimulation. Thus, we propose WARs as a model to study stress-epilepsy interactions and epilepsy-neuropsychiatry comorbidities. (C) 2011 Elsevier B.V. All rights reserved.

Brood cell size of Apis mellifera modifies the reproductive behavior of Varroa destructor

We undertook a field study to determine whether comb cell size affects the reproductive behavior of Varroa destructor under natural conditions. We examined the effect of brood cell width on the reproductive behavior of V. destructor in honey bee colonies, under natural conditions. Drone and worker brood combs were sampled from 11 colonies of Apis mellifera. A Pearson correlation test and a Tukey test were used to determine whether mite reproduction rate varied with brood cell width. Generalized additive model analysis showed that infestation rate increased positively and linearly with the width of worker and drone cells. The reproduction rate for viable mother mites was 0.96 viable female descendants per original invading female. No significant correlation was observed between brood cell width and number of offspring of V. destructor. Infertile mother mites were more frequent in narrower brood cells.

Antimicrobial Activities of Indole Alkaloids from Tabernaemontana catharinensis

Tabernaemontana catharinensis root bark ethanol extract, EB2 fraction and the MMV alkaloid (12-methoxy-4-methylvoachalotine) were evaluated for their antimicrobial activities. T. catharinensis ethanol extract was effective against both strains of the dermatophyte Trichophyton rubrum at concentrations of 2.5 mg/mL (wild strain) and 1.25 mg/mL (mutant strain), while the EB2 fraction and MMV alkaloid showed a strong antifungal activity against wild and mutant strains with MIC values of <0.02 and 0.16 mg/mL, respectively. The EB2 fraction showed a strong antibacterial activity against ATCC strains of S. aureus, S. epidermidis, E. coli and P. aeruginosa with MICs from <0.02 to 0.04 mg/mL, as well as against resistant clinical isolates species of Enterococcus sp, Klebsiella oxytoca, Citrobacter, K. pneumoniae, P. mirabilis, S. aureus, S. epidermidis, E. coli and P. aeruginosa with MIC values ranging from 0.04 to 0.08 mg/mL. The MMV alkaloid presented a MIC of 0.16 mg/mL against the strains of S. aureus and E. coli ATCC. For the resistant clinical isolates Enterococcus sp, Citrobacter, S. aureus, S. epidermidis, E. coil and P. aeruginosa the MIC of MMV ranged from 0.08 to 0.31 mg/mL. The chromatography analysis of the EB2 fraction revealed the presence of indole alkaloids, including MMV, possibly responsible for the observed antimicrobial activity.

Increased cell proliferation in experimentally induced endometriosis in rabbits

Objective: To characterize the pattern of cell proliferation and apoptosis of eutopic and ectopic endometrium in rabbits after endometrium implantation for the experimental induction of endometriosis. Design: Animal experimental study. Setting: Sector of experimental surgery. Animal(s): Twenty-female New Zealand rabbits. Intervention(s): All animals underwent laparotomy for endometriosis induction by resection of one uterine horn, isolation of the endometrium, and fixation of tissue segment to the pelvic peritoneum. Two groups of 10 animals were sacrificed 4 and 8 weeks after endometriosis induction. The lesion was excised together with the opposite uterine horn for endometrial gland and stroma determination. Main Outcome Measure(s): Cell proliferation and apoptosis were determined in the eutopic and ectopic endometrium, and the cell proliferation index (CPI) and apoptotic index (AI) were calculated as the number of labeled cells per 1,000 cells. The tissue homeostasis index was the CPI/AI ratio. Glands and stroma were analyzed separately. Result(s): The CPI for ectopic tissue was increased compared with eutopic tissue, but there was no difference in the ectopic lesions between 4 and 8 weeks of induction. Considering only the AI, eutopic and ectopic endometrium did not differ after 4 weeks, but differed significantly in glandular tissue after 8 weeks. The tissue homeostasis index revealed cell proliferation in these tissues, with a CPI/AI more than 1. Conclusion(s): Ectopic lesions seem to have a higher CPI than eutopic endometrium, with uncontrolled tissue growth occurring in induced endometriotic lesions. (Fertil Steril (R) 2010;93:1637-42. (C)2010 by American Society for Reproductive Medicine.)