Ronaldo A. Ribeiro

Involvement of resident macrophages and mast cells in the writhing nociceptive response induced by zymosan and acetic acid in mice.

Intraperitoneal administration of zymosan and acetic acid induced a dose-dependent nociceptive writhing response in mice. Lavage of the peritoneal cavities with saline reduced the number of total resident peritoneal cells and caused a proportional decrease in the nociceptive responses induced by these stimuli. Furthermore, the specific reduction of the peritoneal mast cell population by intraperitoneal administration of compound 48/80 also reduced the nociceptive responses induced by zymosan and acetic acid. In contrast, enhancement of the peritoneal macrophage population by pretreatment of the cavities with thioglycollate caused an increase in the number of writhes induced by both stimuli. These data suggest that the nociceptive responses induced by zymosan and acetic acid are dependent upon the peritoneal resident macrophages and mast cells. These cells modulate the nociceptive response induced by zymosan and acetic acid via release of tumour necrosis factor alpha (TNF-alpha), interleukin 1beta and interleukin 8. This suggestion is supported by the following observations: (a) pretreatment of the peritoneal cavities with antisera against these cytokines reduced the nociceptive responses induced by these stimuli; (b) peritoneal cells harvested from cavities injected with zymosan or acetic acid released both interleukin 1beta and TNF-alpha; (c) although individual injection of TNF-alpha, interleukin 1beta or interleukin 8 did not induce the nociceptive effect, intraperitoneal injection of a mixture of these three recombinant cytokines caused a significant nociceptive writhing response. In conclusion, our results suggest that the nociceptive activity of zymosan and acetic acid in the writhing model is due to the release of TNF-alpha, interleukin 1beta and interleukin 8 by resident peritoneal macrophages and mast cells.

Analgesic effect of thalidomide on inflammatory pain

Tumor necrosis factor alpha (TNF-alpha) may have a pivotal role in the genesis of mechanical inflammatory hyperalgesia in rats and in the nociceptive writhing response in mice. Thalidomide has been shown to selectively inhibit TNF-alpha production. We therefore investigated the effect of thalidomide on these responses as well as on the hot plate response in mice. Hyperalgesic responses to intraplantar (i.pl.) injections of carrageenin or bradykinin, which act by stimulating TNF-alpha release, but not responses to TNF-alpha or prostaglandin E(2), were inhibited in a dose-dependent manner by pretreatment of the animals with thalidomide. The nociceptive writhing responses induced by intraperitoneal (i.p.) injections of zymosan or acetic acid were also inhibited in a dose-dependent manner by pretreatment of mice with thalidomide. Moreover, the thalidomide pretreatment also reduced the TNF-alpha mRNA levels in the peritoneal cells induced by injection of zymosan in mice. The analgesic effect of thalidomide is not due to a central effect, since the drug had no effect in the hot plate test. The demonstration that thalidomide is able to inhibit inflammatory hyperalgesia in rats and the writhing nociceptive response in mice suggests that these analgesic effects seem to be a consequence of the inhibition of TNF-alpha production, and indicates the need for investigations on the possibility of the use of thalidomide for the treatment of pain refractory to classical non-narcotic analgesics.

Role of the NO/cGMP/K(ATP) pathway in the protective effects of sildenafil against ethanol-induced gastric damage in rats.

Sildenafil is a selective inhibitor of cGMP‐specific phosphodiesterase. Sildenafil, acting via NO‐dependent mechanisms, prevents indomethacin‐induced gastropathy. Activation of ATP‐sensitive potassium channels (KATP) is involved in gastric defence. Our objective was to evaluate the role of the NO/cGMP/KATP pathway in the protective effects of sildenafil against ethanol‐induced gastric damage.

Intestinal barrier function and secretion in methotrexate-induced rat intestinal mucositis

Chemotherapy-induced mucositis is an important dose-limiting and costly side effect for which there is no definitive prophylaxis or treatment. This is due in part to the lack of understanding of its pathophysiology and impact on intestinal function. The objectives of this study were to investigate the small intestine barrier function and electrolyte and water transport in an experimental model of methotrexate-induced mucositis, and to correlate these alterations with histological damage. Wistar rats were treated with methotrexate (1.5–3.5 mg/kg) for 3 days to induce mucositis. Intestinal permeability was measured by the urinary excretion rate of lactulose and mannitol following administration by gavage. Intestinal perfusion was performed in vivo for evaluation of water and electrolyte transports. Methotrexate-treated rats lost a significant amount of weight and presented a marked reduction in food intake. Methotrexate induced significant and dose-dependent villous atrophy and elongation of crypts in duodenum, jejunum, and ileum. Methotrexate also induced an increase in sodium and potassium secretion and an important reduction of the mucosa absorptive surface area, shown by the decrease in the mannitol excretion ratio. In conclusion, methotrexate caused major changes in small bowel function by disrupting intestinal permeability and inducing electrolyte secretion in parallel with substantial histological damage.

Tumor Necrosis Factor-α and Interleukin-1β Mediate the Production of Nitric Oxide Involved in the Pathogenesis of Ifosfamide Induced Hemorrhagic Cystitis in Mice

PURPOSE We investigated the participation of nitric oxide in ifosfamide induced hemorrhagic cystitis in mice, and the involvement of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta in the induction of nitric oxide production in this model. MATERIALS AND METHODS Hemorrhagic cystitis was induced in mice by 100 to 400 mg./kg. ifosfamide and evaluated 6, 12, 24 or 48 hours thereafter by certain parameters, including vesical edema measurements, microscopic analysis and immunohistochemical testing for inducible nitric oxide synthase. Ifosfamide injected mice were pretreated with 10 to 40 mg./kg. of the nitric oxide synthesis inhibitor L-NG-nitroarginine methyl ester, 80 mg./kg. of mesna, a chemical antagonist of acrolein and the urotoxic metabolite of ifosfamide, 50 microl. antiserum against TNF-alpha and IL-1beta per mouse, 45 mg./kg. of the selective TNF-alpha synthesis inhibitor thalidomide or 200 mg./kg. of the TNF-alpha and IL-1beta synthesis inhibitor pentoxifylline. RESULTS Ifosfamide induced vesical edema, which peaked 12 hours after ifosfamide injection. Microscopic analysis revealed vascular congestion, edema, hemorrhage, fibrin deposition, neutrophil infiltration and epithelial denudation. Inducible nitric oxide synthase immunolocalization demonstrated intense reactivity to inducible nitric oxide synthase in the cytoplasm of bladder epithelial cells, which showed diffuse necrosis. Pretreatment with mesna reduced the increases in vesical edema, while treatment with L-NG-nitroarginine methyl ester, antiserum to TNF-alpha or IL-1beta, thalidomide or pentoxifylline inhibited vesical edema and microscopic alterations. Antiserum treatments also inhibited the expression of inducible nitric oxide synthase in the urothelium. CONCLUSIONS Nitric oxide produced by inducible nitric oxide synthase is involved in urothelial damage and in the inflammatory events leading to hemorrhagic cystitis after ifosfamide administration in mice. The induction of inducible nitric oxide synthase in the urothelium appears to depend on the synergistic effect of IL-1beta and TNF-alpha.

Anti-inflammatory effect of glucose—mannose binding lectins isolated from Brazilian beans

Selectins are essential for leukocyte recruitment in inflammation. Because of a lectin domain present in the selectin structure, we investigated the anti-inflammtory activity of six mannose–glucose binding lectins from brazilian beans: Dioclea guianensis-DguiL; D. grandiflora-DgL; Cratylia floribunda-CfL; D. violacea-D.vL; D. virgata-DvirL and Canavalia brasiliensis-ConBr. The lectins were injected intravenously (i.v.) into rats (0.1 and 1.0 mg/kg; 30 min before irritants) and its activities compared to E. coli endotoxin (LPS,30 μg/kg i.v.). Three lectins (DvL, CfL and DguiL), although less intense than LPS, inhibited the neutrophil migration induced by carrageenan (Cg, 300 μg) in a dose-dependent manner (0.1 and 1.0 mg/kg). DvL activity was reversed by 0.1 M α-D-methyl-mannoside (α-CH3), but not by 0.1 M α-D-galactose. The fMLP (44 ng)-induced neutrophil migration was also reduced by these lectins. Endotoxin contamination of lectin samples could be excluded since α-CH3 treatment reversed the DvL effect, but did not modify LPS inhibitory activity. Carrageenan (300 μg)-induced paw oedema was also reduced by LPS or lectin treatments. Conversely, none of the tested lectins inhibited dextran (Dex, 300 μg)-induced paw oedema, a classical leukocyte independent model, or zymosan (Zy, 1.0 mg)-induced peritonitis and paw oedema. LPS showed no effect upon Dex-induced paw oedema and barely reduced (25%) the oedematogenic effects of zymosan. As proposed for LPS, the lectin inhibitory activity was better observed on neutrophil-mediated inflammatory reactions. We speculate that the plant lectin antiinflammatory activity is probably due to a competitive blockage of a common leukocyte and/or endothelial selectin carbohydrate ligand.

Hydrogen sulfide prevents ethanol-induced gastric damage in mice: role of ATP-sensitive potassium channels and capsaicin-sensitive primary afferent neurons.

The aim of this study was to evaluate the protective effect of hydrogen sulfide (H2S) on ethanol-induced gastric lesions in mice and the influence of ATP-sensitive potassium (KATP) channels, capsaicin-sensitive sensory afferent neurons, and transient receptor potential vanilloid (TRPV) 1 receptors on such an effect. Saline and l-cysteine alone or with propargylglycine, sodium hydrogen sulfide (NaHS), or Lawessons reagent were administrated for testing purposes. For other experiments, mice were pretreated with glibenclamide, neurotoxic doses of capsaicin, or capsazepine. Afterward, mice received l-cysteine, NaHS, or Lawessons reagent. After 30 min, 50% ethanol was administrated by gavage. After 1 h, mice were sacrificed, and gastric damage was evaluated by macroscopic and microscopic analyses. l-Cysteine, NaHS, and Lawessons reagent treatment prevented ethanol-induced macroscopic and microscopic gastric damage in a dose-dependent manner. Administration of propargylglycine, an inhibitor of endogenous H2S synthesis, reversed gastric protection induced by l-cysteine. Glibenclamide reversed l-cysteine, NaHS, or Lawessons reagent gastroprotective effects against ethanol-induced macroscopic damage in a dose-dependent manner. Chemical ablation of sensory afferent neurons by capsaicin reversed gastroprotective effects of l-cysteine or H2S donors (NaHS or Lawessons reagent) in ethanol-induced macroscopic gastric damage. Likewise, in the presence of the TRPV1 antagonist capsazepine, the gastroprotective effects of l-cysteine, NaHS, or Lawessons reagent were also abolished. Our results suggest that H2S prevents ethanol-induced gastric damage. Although there are many mechanisms through which this effect can occur, our data support the hypothesis that the activation of KATP channels and afferent neurons/TRPV1 receptors is of primary importance.

Role of resident mast cells and macrophages in the neutrophil migration induced by LTB4, fMLP and C5a des arg.

In the present study, we have investigated the participation of resident peritoneal cells (macrophages and mast cells) in the neutrophil migration induced in rats by the intraperitoneal administration of LTB4, fMLP or C5a des arg. The intraperitoneal injection of LTB4 (10 nmol), fMLP (10 nmol) and C5a des arg (zymosan-activated plasma, 1 ml) caused an intense neutrophil migration compared to the saline control (1,000, 1,500 and 2,000%, respectively). An 83% depletion in the number of resident cells following peritoneal lavage reduced the LTB4-stimulated neutrophil migration by 73.6% without affecting that caused by fMLP and C5a des arg. Increasing the peritoneal macrophage population (236%) by pretreating the cavities with thioglycollate enhanced the neutrophil migration induced by LTB4 (129%), but did not alter that induced by fMLP and C5a des arg. Similarly, reducing the population of peritoneal mast cells containing toluidine-blue-staining granules by subchronically pretreating the cavities with compound 48/80 diminished the LTB4-induced NM by 69% but had no effect on the responses to fMLP and C5a des arg. Pretreating the animals with dexamethasone strongly inhibited (70%) the neutrophil migration induced by the intraperitoneal injection of LTB4, fMLP and C5a des arg. Indomethacin, BW A4C and NDGA had no such effect. The incubating medium from peritoneal macrophages and mast cells stimulated with LTB4 induced neutrophil migration when injected into the peritoneal cavity of rats. This migration was strongly reduced (70%) by treating the cells with dexamethasone. In contrast, stimulating the cells with fMLP or C5a des arg did not result in the release of any promigratory activity into the incubating fluid. Our results suggest that LTB4 induces neutrophil migration via a mechanism dependent on resident mast cells and macrophages while that induced by C5a des arg and fMLP seems to be independent of such cellular involvement. The neutrophil migration induced by LTB4 is apparently mediated by factor(s) whose release is blocked by dexamethasone. fMLP and C5a appear to cause in vivo migration by the formation of a concentration gradient and by a glucocorticoid-sensitive mechanism different from that stimulated by LTB4.

Effect of Novel A2A Adenosine Receptor Agonist ATL 313 on Clostridium difficile Toxin A-Induced Murine Ileal Enteritis

ABSTRACT Clostridium difficile is a spore-forming, anaerobic, gram-positive bacillus that releases two main virulence factors: toxins A and B. Toxin A plays an important pathogenic role in antibiotic-induced diarrhea and pseudomembranous colitis, a condition characterized by intense mucosal inflammation and secretion. Agonist activity at A2A adenosine receptors attenuates inflammation and damage in many tissues. This study evaluated the effects of a new selective A2A adenosine receptor agonist (ATL 313) on toxin A-induced injury in murine ileal loops. ATL 313 (0.5 to 5 nM) and/or the A2A adenosine receptor antagonist (ZM241385; 5 nM) or phosphate-buffered saline (PBS) were injected into ileal loops immediately prior to challenge with toxin A (1 to 10 μg/loop) or PBS. Intestinal fluid volume/length and weight/length ratios were calculated 3 h later. Ileal tissues were collected for the measurement of myeloperoxidase, adenosine deaminase activity, tumor necrosis factor alpha (TNF-α) production, histopathology, and detection of cell death by the TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) method. Toxin A significantly increased volume/length and weight/length ratios in a dose-dependent fashion. ATL 313 treatment significantly (P < 0.05) reduced toxin A-induced secretion and edema, prevented mucosal disruption, and neutrophil infiltration as measured by myeloperoxidase activity. ATL 313 also reduced the toxin A-induced TNF-α production and adenosine deaminase activity and prevented toxin A-induced cell death. These protective effects of ATL 313 were reversed by ZM241385. In conclusion, the A2A adenosine receptor agonist, ATL 313, reduces tissue injury and inflammation in mice with toxin A-induced enteritis. The finding of increased ileal adenosine deaminase activity following the administration of toxin A is new and might contribute to the pathogenesis of the toxin A-induced enteritis by deaminating endogenous adenosine.

Antihyperalgesic effect of pentoxifylline on experimental inflammatory pain

The antihyperalgesic effect of pentoxifylline was investigated in three experimental pain models. Pentoxifylline (0.5–1.6 mg kg−1) given 30 min before the stimulus significantly inhibited the writhing response induced by the intraperitoneal (i.p.) administration of either acetic acid (−90%) or zymosan (−83%), but not that of iloprost, in mice, as well as the zymosan‐induced articular hyperalgesia in the zymosan arthritis in rats (−50%). Pentoxifylline also inhibited the mechanical hypernociception in rats induced by the intraplantar injection of either carrageenin (−81%), bradykinin (−56%) or tumor necrosis factor α (TNF‐α; −46%), but not that induced by interleukin‐1β (IL‐1β) or prostaglandin E2 (PGE2). Pentoxifylline did not inhibit the nociceptive response in the hot plate test in mice. Further, the antinociceptive effect of pentoxifylline in the writhing test in mice and the zymosan‐induced articular hyperalgesia were not reversed by the coadministration of the opioid receptor antagonist naloxone. Thus, pentoxifylline antinociceptive effect is probably not mediated at a central level. Pentoxifylline significantly reduced TNF‐α (−43%) and IL‐1β (−42%) concentrations in the joint exudates of rats stimulated by intra‐articular injection of zymosan and the production of both cytokines (−66 and −86%, respectively) by mouse peritoneal macrophages stimulated in vivo with zymosan as well as the expression of TNF‐α at the tissue level in carrageenin‐injected rat paws. In conclusion, the antinociceptive activity of pentoxifylline is associated with the inhibition of the release of both TNF‐α and IL‐1β.