Treatment with Apocynin Limits the Development of Acute Graft-versus-Host Disease in Mice.

Graft-versus-host disease (GVHD) is the most serious complication limiting the clinical utility of allogeneic hematopoietic stem cell transplantation (HSCT), in which lymphocytes of donors (graft) are activated in response to the host antigen. This disease is associated with increased inflammatory response through the release of inflammatory mediators such as cytokines, chemokines, and reactive oxygen species (ROS). In this study, we have evaluated the role of ROS in GVHD pathogenesis by treatment of recipient mice with apocynin (apo), an inhibitor of intracellular translocation of cytosolic components of NADPH oxidase complex. The pharmacological blockade of NADPH oxidase resulted in prolonged survival and reduced GVHD clinical score. This reduction in GVHD was associated with reduced levels of ROS and TBARS in target organs of GVHD in apocynin-treated mice at the onset of the mortality phase. These results correlated with reduced intestinal and liver injuries and decreased levels of proinflammatory cytokines and chemokines. Mechanistically, pharmacological blockade of the NADPH oxidase was associated with inhibition of recruitment and accumulation of leukocytes in the target organs. Additionally, the chimerism remained unaffected after treatment with apocynin. Our study demonstrates that ROS plays an important role in mediating GVHD, suggesting that strategies aimed at blocking ROS production may be useful as an adjuvant therapy in patients subjected to bone marrow transplantation.

The Involvement of the Endocannabinoid System in the Peripheral Antinociceptive Action of Ketamine.

Ketamine has been widely used as an analgesic and produces dissociative anesthetic effects. The antinociceptive effects of ketamine have been studied, but the involvement of endocannabinoids in these effects has not yet been investigated. In this study, we evaluated the involvement of the endocannabinoid system in the peripheral antinociceptive effects induced by ketamine. All drugs were administered via the intraplantar route. To induce hyperalgesia, rat paws were injected with prostaglandin E2 (2 µg per paw). The nociceptive threshold for mechanical stimulation was measured in the right hind paw of Wistar rats using the Randall-Selitto test. The tissue levels of anandamide (AEA), 2-arachidonoylglycerol, palmitoylethanolamide, and oleoylethanolamide were measured using liquid chromatography coupled to single quadrupole mass spectrometry. The administration of the cannabinoid receptor type 1 (CB1) antagonist, N(piperidine-1yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl 1 pyrazolcarboxamide (20, 40, and 80 µg per paw), but not the cannabinoid receptor type 2 antagonist, 6-iodo-2-methyl-1-(2-morpholinoethyl)-1H-indol-3-yl) (4-methoxyphenyl) methanone (100 µg per paw), antagonized the ketamine-induced peripheral antinociception in a dose-dependent manner. Additionally, the administration of the endocannabinoid metabolizing enzyme inhibitor (.5 µg per paw) or an AEA reuptake inhibitor, (5Z,8Z,11Z,14Z)N(4Hydroxy2methylphenyl)5,8,11,14 eicosatetraenamide (2.5 µg per paw) significantly enhanced low-dose ketamine-induced peripheral antinociception. AEA paw levels were increased only after ketamine administration to prostaglandin E2-injected paws. These data suggest that ketamine, in the presence of a nociceptive stimulus, induces a selective release of AEA levels and subsequent CB1 cannabinoid activation at the peripheral level. PERSPECTIVE: This study suggests that ketamine antinociception depends at least in part on AEA release and CB1 cannabinoid receptor activation in inflammatory conditions. This study could potentially help clinicians in the use of ketamine as a peripheral analgesic for inflammatory pain.

Serotonin induces peripheral mechanical antihyperalgesic effects in mice.

The role of serotonin (5-HT) in nociception will vary according to the subtypes of receptors activated. When administered peripherally, it induces pain in humans and in rats by activation of 5-HT1, 5-HT2 and 5-HT3 receptors. In addition, endogenous 5-HT produced in situ, is involved in the nociceptive response induced by formalin in rat's paw inflammation, possibly via 5-HT3 receptors. Moreover, it has been shown that 5-HT released in the dorsal horn of the spinal cord by stimulation of the periaqueductal gray causes activation of inhibitory interneurons, resulting in inhibition of spinal neurons. In the present study we evaluated the effect of serotonin and its receptors at peripheral antinociception. The mice paw pressure test was used in animals that had increased sensitivity by an intraplantar injection of PGE2 (2 µg). We used selective antagonists of serotonin receptors (isamoltan 5-HT1B, BRL 15572 5-HT1D, ketanserin 5-HT2A, ondansetron 5-HT3 and SB-269970 5-HT7). Administration of serotonin into the right hind paw (62.5, 125, 250 and 500 ng and 1 µg) produced a dose-dependent peripheral mechanical antihyperalgesic effect of serotonin in mice. Selective antagonists for 5-HT1B, 5-HT2A, 5-HT3 receptors at doses of 0.1, 1 and 10 µg, reversed the antihyperalgesic effect induced by 250 ng serotonin. In contrast, selective antagonists for 5-HT1D and 5-HT7 receptors were unable to reverse the antihyperalgesic effect induced by serotonin. These results demonstrated for the first time, the peripheral mechanical antihyperalgesic effect of serotonin, and participation of 5-HT1B, 5-HT2A and 5-HT3 receptors in this event.

Angiotensin-(1-7) through Mas receptor activation induces peripheral antinociception by interaction with adrenoreceptors.

Angiotensin-(1-7) [Ang-(1-7)] develops its functions interacting with Mas receptor. Mas receptor was recently identified in the DRG and its activation by Ang-(1-7) resulted in peripheral antinociception against PGE2 hyperalgesia in an opioid-independent pathway. Nevertheless, the mechanism by which Ang-(1-7) induce peripheral antinociception was not yet elucidated. Considering that endogenous noradrenaline could induce antinociceptive effects by activation of the adrenoceptors the aim of this study was verify if the Ang-(1-7) is able to induce peripheral antinociception by interacting with the endogenous noradrenergic system. Hyperalgesia was induced by intraplantar injection of prostaglandin E2 (2µg). Ang-(1-7) was administered locally into the right hindpaw alone and after either agents, α2-adrenoceptor antagonist, yohimbine (5, 10 and 20 µg/paw), α2C-adrenoceptor antagonist rauwolscine (10, 15 and 20 µg/paw), α1-adrenoceptor antagonist prazosin (0.5, 1 and 2 µg/paw), ß-adrenoceptor antagonist propranolol (150, 300 and 600 ng/paw). Noradrenaline (NA) reuptake inhibitor reboxetine (30 µg/paw) was administered prior to Ang-(1-7) low dose (20 ng) and guanetidine 3 days prior to experiment (30 mg/kg/animal, once a day), depleting NA storage. Intraplantar Ang-(1-7) induced peripheral antinociception against hyperalgesia induced by PGE2. This effect was reversed, in dose dependent manner, by intraplantar injection of yohimbine, rauwolscine, prazosin and propranolol. Reboxetine intensified the antinociceptive effects of low-dose of Ang-(1-7) and guanethidine, which depletes peripheral sympathomimetic amines, reversed almost 70% the Ang-(1-7)-induced peripheral antinociception. Then, this study provides evidence that Ang-(1-7) induce peripheral antinociception stimulating an endogenous noradrenaline release that activates peripheral adrenoceptors inducing antinociception.

Nanocomposite treatment reduces disease and lethality in a murine model of acute graft-versus-host disease and preserves anti-tumor effects.

Graft versus host disease (GVHD) is an immunological disorder triggered by bone marrow transplantation that affects several organs, including the gastrointestinal tract and liver. Fullerenes and their soluble forms, fullerols, are nanocomposites with a closed symmetrical structure with anti-inflammatory and anti-oxidant properties. The present study evaluated the effects of treatment with the fullerol (C60(OH)18-20) in the development and pathogenesis of GVHD in a murine model. Mice with experimental GVHD that were treated with the fullerol showed reduced clinical signs of disease and mortality compared with untreated mice. Treatment with the fullerol decreased the hepatic damage associated with reduced hepatic levels of reactive oxygen species, pro-inflammatory cytokines and chemokines (IFN-γ TNF-α, CCL2, CCL3 and CCL5) and reduced leukocyte accumulation. The amelioration of GVHD after treatment with the fullerol was also associated with reduced intestinal lesions and consequent bacterial translocation to the blood, liver and peritoneal cavity. Moreover, the fullerol treatment alleviated the GVHD while preserving effects of the graft against a leukemia cell line (GFP+P815). In summary, the fullerol was effective in reducing the GVHD inflammatory response in mice and may suggest novel ways to treat this disease.

Lithothamnion muelleri controls inflammatory responses, target organ injury and lethality associated with graft-versus-host disease in mice.

Lithothamnion muelleri (Hapalidiaceae) is a marine red alga, which is a member of a group of algae with anti-inflammatory, antitumor, and immunomodulatory properties. The present study evaluated the effects of treatment with Lithothamnion muelleri extract (LM) in a model of acute graft-versus-host disease (GVHD), using a model of adoptive splenocyte transfer from C57BL/6 donors into B6D2F1 recipient mice. Mice treated with LM showed reduced clinical signs of disease and mortality when compared with untreated mice. LM-treated mice had reduced tissue injury, less bacterial translocation, and decreased levels of proinflammatory cytokines and chemokines (interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), chemokine (C-C motif) ligand 2 (CCL2), chemokine (C-C motif) ligand 3 (CCL3) and chemokine (C-C motif) ligand 5 (CCL5)). The polysaccharide-rich fraction derived from LM could inhibit leukocyte rolling and adhesion in intestinal venules, as assessed by intravital microscopy. LM treatment did not impair the beneficial effects of graft-versus-leukaemia (GVL). Altogether, our studies suggest that treatment with Lithothamnion muelleri has a potential therapeutic application in GVHD treatment.

Control of murine Ly6C(high) monocyte traffic and immunosuppressive activities by atypical chemokine receptor D6.

The atypical chemokine receptor D6 is a decoy and scavenger receptor for most inflammatory CC chemokines and prevents the development of exacerbated inflammatory reactions. Here we report that mice lacking D6 expression in the nonhematopoietic compartment have a selective increase in the number of Ly6C(high) monocytes in the circulation and in secondary lymphoid tissues. Under inflammatory conditions, Ly6C(high) monocytes accumulate in increased number in secondary lymphoid organs of D6(-/-) mice in a CCR2-dependent manner. Ly6C(high) monocytes derived from D6(-/-) mice have enhanced immunosuppressive activity, inhibit the development of adaptive immune responses, and partially protect mice from the development of GVHD. Thus, control of CCR2 ligands by D6 regulates the traffic of Ly6C(high) monocytes and controls their immunosuppressive potential.

The role of chemokines in mediating graft versus host disease: opportunities for novel therapeutics.

Bone marrow transplantation (BMT) is the current therapy of choice for several malignancies and severe autoimmune diseases. Graft versus host disease (GVHD) is the major complication associated with BMT. T lymphocytes and other leukocytes migrate into target organs during GVHD, become activated and mediate tissue damage. Chemokines are well known inducers of leukocyte trafficking and activation and contribute to the pathogenesis of GVHD. Here, we review the major animal models used to study GVHD and the role of chemokines in mediating tissue damage in these models. The role of these molecules in promoting potential beneficial effects of the graft, especially graft versus leukemia, is also discussed. Finally, the various pharmacological strategies to block the chemokine system or downstream signaling events in the context of GVHD are discussed.

Platelet-activating factor receptor plays a role in the pathogenesis of graft-versus-host disease by regulating leukocyte recruitment, tissue injury, and lethality.

PAF is a potent lipid mediator involved in several manifestations of acute inflammation, including leukocyte influx, leukocyte interaction with endothelium, and production of inflammatory cytokines. The present study evaluated the relevance of PAFR for the pathogenesis of acute GVHD using a model of adoptive transfer of splenocytes from WT or PAFR(-/-) C57BL/6J to B6D2F1 mice. Mice, which received PAFR(-/-) splenocytes or treatment with the PAFR antagonist, showed reduced clinical signs of disease and no mortality. In GVHD mice receiving PAFR(-/-) splenocytes, there was deceased bacterial translocation and tissue injury. Furthermore, production of proinflammatory cytokines and chemokines (TNF-α, IFN-γ, CCL2, CCL3, and CCL5) and accumulation of CD8(+) cells in intestine and liver were reduced in mice transplanted with the PAFR(-/-) splenocyte. Mechanistically, an absence or pharmacological blockade of PAFR was associated with decreased rolling and adhesion of leukocytes to the mesenteric microcirculation, as assessed by intravital microscopy. Despite decreased GVHD, there was maintained GVL activity when PAFR(-/-) leukocytes were transferred into WT mice. In conclusion, PAFR on donor leukocytes plays a critical role in GVHD by mediating leukocyte influx and cytokine production in target tissues. PAFR antagonist may potentially be useful in the treatment of GVHD in bone marrow-transplanted patients.

PI3Kγ controls leukocyte recruitment, tissue injury, and lethality in a model of graft-versus-host disease in mice.

PI(3)Kγ is thought to mediate leukocyte migration to injured tissues and may be important in the pathogenesis of various T-lymphocyte-dependent pathologies, including autoimmune and inflammatory diseases. The present study evaluated the relevance of PI(3)Kγ in donor cells for the pathogenesis of acute GVHD using a model of adoptive transfer of splenocytes from WT or PI(3)Kγ(-/-) C57BL/6J mice to B6D2F1 mice, and mice that received PI(3)Kγ(-/-) cells showed reduced clinical signs of disease, bacterial translocation, tissue injury, and lethality rates. This was associated with reduced production of proinflammatory cytokines and chemokines (TNF-α, IFN-γ, CCL2, CCL3, and CCL5) and reduced infiltration of CD8(+), CD4(+), and CD11c(+) cells in the small intestine. Mechanistically, in addition to decreasing production of proinflammatory mediators, absence or pharmacological blockade of PI(3)Kγ was associated with decreased rolling and adhesion of leukocytes to the mesenteric microcirculation, as assessed by intravital microscopy. Despite decreased GVHD, there was maintained GVL activity when PI(3)Kγ(-/-) leukocytes were transferred into WT mice. In conclusion, PI(3)Kγ plays a critical role in GVHD by mediating leukocyte influx and activation in tissues. PI(3)Kγ inhibitors may be useful in the treatment of GVHD in patients undergoing BMT.

The CCL3/macrophage inflammatory protein-1alpha-binding protein evasin-1 protects from graft-versus-host disease but does not modify graft-versus-leukemia in mice.

CCL3 is a protein of the CC chemokine family known to be important for T cell recruitment in inflammatory diseases. The aim of the current study was to evaluate the effects and putative mechanism of action of evasin-1, a novel CCL3-binding protein, in the pathogenesis of acute graft-versus-host disease (GVHD). GVHD was induced by the transplantation of splenocytes from C57BL/6J to B6D2F1 mice. Treatment of recipient mice with evasin-1 prevented mortality associated with GVHD. This was correlated with reduced weight loss and clinical disease severity. Analysis of the small intestine showed that evasin-1 treatment reduced the histopathological score and decreased levels of IFN-gamma and CCL5. Mechanistically, evasin-1 treatment reduced the number of CD4(+) and CD8(+) T cells infiltrating the small intestine, as assessed by immunohistochemistry, and the adhesion of leukocytes to intestinal venules of recipient mice, as assessed by intravital microscopy. Evasin-1 was also able to decrease liver damage, as seen by reduction of inflammatory infiltrate and IFN-gamma levels. Treatment with evasin-1 did not interfere with graft-versus-leukemia. Altogether, our studies demonstrate that CCL3 plays a major role in mediating GVHD, but not graft-versus-leukemia in mice and suggest that blockade of CCL3 with evasin-1 has potential therapeutic application in patients undergoing bone marrow transplantation.

Treatment with a novel chemokine-binding protein or eosinophil lineage-ablation protects mice from experimental colitis.

Eosinophils are multifunctional leukocytes implicated in numerous inflammatory diseases. The present study was conducted to clarify the precise role of eosinophils in the development of colitis by using eosinophil-depleted mice and a novel chemokine-binding protein that neutralizes CCL11 action. Colitis was induced by administration of dextran sodium sulfate (DSS) to wild-type and eosinophil-deficient DeltadblGATA-1 mice. Accumulation of eosinophils in the gut of mice given DSS paralleled worsening of clinical score and weight loss. In response to DSS, DeltadblGATA-1 mice showed virtual absence of eosinophil recruitment, amelioration of clinical score, weight loss, and tissue destruction, and no lethality. There was a decrease in CXCL1 and CCL3 production and decreased neutrophil influx in the intestine of DeltadblGATA-1 mice. Transfer of bone marrow cells from wild-type mice reconstituted disease manifestation in DSS-treated DeltadblGATA-1 mice, and levels of CCL11 were increased after DSS treatment and localized to inflammatory cells. Treatment with the chemokine-binding protein evasin-4 at a dose that prevented the function of CCL11 greatly ameliorated clinical score, weight loss, overall tissue destruction, and death rates. In conclusion, the influx of eosinophils is critical for the induction of colitis by DSS. Treatment with a novel chemokine-binding protein decreased eosinophil influx and greatly ameliorated colitis, suggesting that strategies that interfere with the recruitment of eosinophils may be useful as therapy for colitis.