Neurology, Brigham and Women's Hospital

The endogenous opioids mediate the analgesic effects of celecoxib in a model of mechanical hyperalgesia in rats. As responses to thermal stimuli may differ from those to mechanical stimuli, we have here assessed celecoxib in a rat model of thermal hyperalgesia and the possible involvement of endogenous opioids and their corresponding receptors in these effects. Injection of carrageenan (CG) into one hind paw induced a dose-related hyperalgesia (decreased time for paw withdrawal) to thermal stimuli (infra-red light beam), over 6h. Celecoxib (sc) 30 min before CG (250 microg per paw) induced a dose-dependent reversal of hyperalgesia, with withdrawal times well above basal levels, characterizing development of hypoalgesia. Indomethacin (sc) reversed CG-induced hyperalgesia only to basal levels (an anti-hyperalgesic effect). Naltrexone (sc) prevented hypoalgesia after celecoxib but did not change the response to indomethacin. Local (intraplantar) injection of either a selective antagonist of mu-(beta-funaltrexamine), kappa-(nor-binaltorphimine) or of delta-(naltrindole) opioid receptors also reversed the hypoalgesic effects of celecoxib, without modifying the hyperalgesia due to CG or affecting the nociceptive thresholds in the non-injected paw. Our data show that celecoxib, unlike indomethacin, was hypoalgesic in this model of thermal hyperalgesia, and that this effect was mediated by peripheral mu-, kappa- and delta-opioid receptors.

In this study we analyzed the mechanisms underlying celecoxib-induced analgesia in a model of inflammatory pain in rats, using the intracerebroventricular (i.c.v.) administration of selective opioid and cannabinoid antagonists. Analgesic effects of celecoxib were prevented by selective μ-(β-funaltrexamine) and δ-(naltrindole), but not κ-(nor-binaltorphimine) opioid antagonists, given i.c.v. 30 min before celecoxib. Similar pretreatment with AM 251, but not SR 144528, cannabinoid CB(1) and CB(2) receptor antagonists, respectively, prevented celecoxib-induced analgesia. The fatty acid amide hydrolase inhibitor, URB 597, also prevented celecoxib-induced analgesia. Our data provided further evidence for the involvement of endogenous opioids and revealed a new cannabinoid component of the mechanism(s) underlying celecoxib-induced analgesia.

Seizures are due to excessive, synchronous neuronal firing in the brain and are characteristic
of epilepsy, the fourth most prevalent neurologic disease. Herein, we report handling-induced
and spontaneous seizures in mice deficient of CD39, a cell-surface ATPase highly expressed
on microglial cells. CD39-/- mice with handling-induced seizures had normal input-output
curves and paired-pulse ratio measured from hippocampal slices and lacked microgliosis,
astrogliosis or overt cell loss in the hippocampus and cortex. As expected, however, the
cerebrospinal fluid of CD39-/- mice contained increased levels of ATP and decreased levels of
adenosine. To determine if immune activation was involved in seizure progression, we
challenged mice with lipopolysaccharide (LPS) and measured the effect on microglial
activation and seizure severity. Systemic LPS challenge resulted in increased cortical staining
of Iba1/CD68 and gene array data from purified microglia predicted increased expression of
IL-8, Trem1, p38, pattern recognition receptors, death receptor, NF-κB, complement, acute
phase, and IL-6 signaling pathways in CD39-/- versus CD39+/+ mice. However, LPS
treatment did not affect handling-induced seizures. In addition, microglia-specific CD39
deletion in adult mice was not sufficient to cause seizures, suggesting instead that altered
expression of CD39 during development or on non-microglial cells such as vascular
endothelial cells may promote the seizure phenotype. In summary, we show a correlation
between altered extracellular ATP/adenosine ratio and a previously unreported seizure
phenotype in CD39-/- mice. This work provides groundwork for further elucidation of the
underlying mechanisms of epilepsy.

Oral administration of Ag induces regulatory T cells that express latent membrane-bound TGF-b (latency-associated peptide
[LAP]) and have been shown to play an important role in the induction of oral tolerance. We developed an in vitro model to study
modulation of LAP+ on CD4+ T cells. The combination of anti-CD3 mAb, anti-CD28 mAb, and recombinant IL-2 induced
expression of LAP on naive CD4+ T cells, independent of Foxp3 or exogenous TGF-b. In vitro generated CD4+LAP+Foxp32
T cells were suppressive in vitro, inhibiting proliferation of naive CD4+ T cells and IL-17A secretion by Th17 cells. Assessing
the impact of different cytokines and neutralizing Abs against cytokines, we found that LAP induction was decreased in the
presence of IL-6 and IL-21, and to a lesser extent by IL-4 and TNF-a. IL-6 abrogated the in vitro induction of CD4+LAP+ T cells
by STAT3-dependent inhibition of Lrrc32 (glycoprotein A repetitions predominant [GARP]), the adapter protein that tethers
TGF-b to the membrane. Oral tolerance induction was enhanced in mice lacking expression of IL-6R by CD4+ T cells and by
treatment of wild-type mice with neutralizing anti-IL-6 mAb. These results suggest that proinflammatory cytokines interfere with
oral tolerance induction and that blocking the IL-6 pathway is a potential strategy for enhancing oral tolerance in the setting of
autoimmune and inflammatory diseases.

Heat shock proteins (Hsps) are highly expressed at all sites of inflammation. As they are ubiquitous and immunodominant antigens, these molecules represent good candidates for the therapeutic use of oral tolerance in autoimmune and chronic inflammatory diseases. Evidences from human and animal studies indicate that inflammatory bowel disease (IBD) results from uncontrolled inflammatory responses to intestinal microbi-ota. Hsps are immunodominant proteins expressed by several immune cells and by commensal bacteria. Using an IBD mouse model, we showed that oral pretreatment with genetically modified Lactococcus lactis that produces and releases Mycobacterium Hsp65, completely prevented DSS-induced colitis in C57BL/6 mice. Protection was associated with reduced pro-inflammatory cytokines, such as IFN-γ, IL-6, and TNF-α; increased IL-10 production in colonic tissue; and expansion of CD4 + Foxp3 + and CD4 + LAP + regulatory T cells in spleen and mesenteric lymph nodes. This effect was dependent on IL-10 and toll-like receptor 2. Thus, this approach may open alternative options for long-term management of IBD.