The time course of inflammatory cytokine secretion in a rat model of postoperative pain does not coincide with the onset of mechanical hyperalgesia

We characterized the time course of inflammatory cytokine release at the site of injury and in plasma after surgery on the rat tail. Anesthetized Sprague-Dawley rats had a 20 mm long incision made through the skin and fascia of their tails. Control rats were anesthetized, but no incision was made. Blood and tissue samples were taken 2 h and 1, 2, 4, and 8 days after surgery and analysed by ELISA for interleukin-1beta (IL-1beta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and cytokine-induced neutrophil chemoattractant-1 (CINC-1). In another group of rats, daily behavioral measurements were made of the rats' responses to a blunt noxious mechanical stimulus (4 Newtons) applied to their tails. Primary hyperalgesia developed within 2 h of surgery and lasted for 6 days. The tissue concentrations of IL-1beta, IL-6, and CINC-1 increased within 24 h of surgery, and TNF-alpha concentration increased within 48 h of surgery. Thereafter, cytokine concentrations remained elevated for 4 (IL-1beta and IL-6) to 8 days (CINC-1, TNF-alpha) after surgery. Control animals did not develop hyperalgesia and no changes in cytokines concentrations were detected. Thus, in our model of postoperative pain, secretion of inflammatory cytokines IL-1beta, IL-6, TNF-alpha, and CINC-1 was not essential for the initiation of postoperative hyperalgesia.     

Cytokine-mediated inflammatory hyperalgesia limited by interleukin-13

The effect of interleukin-13 (IL-13) on hyperalgesic responses to intraplantar (i.pl.) injection of carrageenin, E. coli endotoxin (LPS), bradykinin, tumour necrosis factor a (TNF-alpha), interleukin-1 beta (IL-1 beta), interleukin-8 (IL-8) and prostaglandin E(2) (PGE(2)) was investigated in a model of mechanical hyperalgesia in rats. Also, the cellular source of the IL-13 was investigated. IL-13, administered 30 min before the stimulus, inhibited responses to carrageenin, LPS, bradykinin, and TNF-alpha, but not responses to IL-1 beta, IL-8 and PGE2. IL-13, administered 2 hours before the injection of IL-1b, did not affect the response to IL-1b, whereas IL-13, administered 12 hours or 12 + 2 hours before the IL-1 beta, inhibited the hyperalgesia (- 35%, - 77%, respectively). In murine peritoneal macrophages, IL-13 administered 2 hours before stimulation with LPS, inhibited the production of IL-1 beta (- 67%) and PGE(2) (- 56%). IL-13 administered 12 hours before stimulation with LPS inhibited LPS-stimulated PGE(2) but not IL-1 beta. An anti-IL-13 serum potentiated responses to carrageenin, LPS, bradykinin and TNF-alpha (but not IL-1 beta and IL-8), as well as responses to bradykinin in rats depleted of mast cells with compound 40/80, but not in athymic rats. These data suggest that IL-13, released by lymphocytes, limits inflammatory hyperalgesia by the inhibition of the production TNF-alpha, IL-1 beta, IL-8 and PGs.     

Tumor necrosis factor, interleukin-1 and interleukin-8 mediate the nociceptive activity of the supernatant of LPS-stimulated macrophages

It has been suggested that the supernatant of LPSstimulated macrophages (macrophage nociceptive factor, MNF) promotes nociception in mice. Intraperitoneal administration of MNF induced dose-related writhing, which reached a plateau between 18 and 26 min after injection and decreased within 60 min. The release of MNF was inhibited by the pretreatment of the macrophages with cycloheximide, a protein synthesis inhibitor, or with the glucocorticoid dexamethasone. Cyclooxygenase inhibitors, such as indomethacin or paracetamol, had no effect. The MNF-induced nociception was inhibited in a dose-related manner by pretreatment of the animals with indomethacin, paracetamol or dexamethasone. Pretreatment of the animals with the sympatholytics guanethidine and atenolol partially reduced the MNF nociception, which was abolished by the combination of guanethidine or atenolol with indomethacin. The preincubation of MNF with antisera against TNF-alpha, IL-1 or IL-8 partially inhibited its nociceptive effect. Intraperitoneal injection of a mixture of the recombinants cytokines TNF-alpha, IL-1 and IL-8 mimicked MNF nociception. The individual injection of these cytokines was unable to induce the nociceptive effect. In conclusion, our data suggest that the nociceptive activity of the supernatant of LPSstimulated macrophages is explained by the presence of TNF-alpha, IL-1 and IL-8, the nociceptive activity of which (in mice) seems to be due to the release of cyclooxygenase and sympathetic metabolites.     

Monocyte chemotactic protein-1 regulates leukocyte recruitment during gastric ulcer recurrence induced by tumor necrosis factor-alpha

TNF-alpha has numerous biological activities, including the induction of chemokine expression, and is involved in many gastric injuries. C-C chemokines [monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein (MIP)-1alpha] and C-X-C chemokines [MIP-2 and cytokine-induced neutrophil chemoattractant (CINC)-2alpha] mediate chemotaxis of monocytes and neutrophils, respectively. We examined the roles of TNF-alpha and dynamics of chemokine expression in gastric ulceration including ulcer recurrence and indomethacin-induced injury. Rats with healed chronic gastric ulcers received intraperitoneal TNF-alpha to induce ulcer recurrence. Some rats were given neutralizing antibodies against neutrophils or MCP-1 together with TNF-alpha. In a separate experiment, rats were orally administered 20 mg/kg indomethacin with or without pretreatment with pentoxifylline (an inhibitor of TNF-alpha synthesis) or anti-MCP-1 antibody. TNF-alpha (1 microg/kg) induced gastric ulcer recurrence after 48 h, which was completely prevented by anti-neutrophil antibody. TNF-alpha increased the number of macrophages and MCP-1 mRNA expression in scarred mucosa from 4 h, whereas it increased MPO activities (marker of neutrophil infiltration) and mRNA expression of MIP-2 and CINC-2alpha from 24 h. Anti-MCP-1 antibody inhibited leukocyte infiltration with reduction of the levels of C-X-C chemokines and prevented ulcer recurrence. Indomethacin treatment increased TNF-alpha/chemokine mRNA expression from 30 min and induced macroscopic erosions after 4 h. Pentoxifylline inhibited the indomethacin-induced gastric injury with reduction of neutrophil infiltration and expression of chemokine (MCP-1, MIP-2, and CINC-2alpha). Anti-MCP-1 antibody also inhibited the injury and these inflammatory responses but did not affect TNF-alpha mRNA expression. In conclusion, increased MCP-1 triggered by TNF-alpha may play a key role in gastric ulceration by regulating leukocyte recruitment and chemokine expression.     

Neutrophil migration induced by IL-8-activated mast cells is mediated by CINC-1

The aim of this study was to characterize the mediators released by mast cells responsible for IL-8-induced neutrophil migration. It was observed that IL-8 induces a dose-dependent neutrophil migration into peritoneal cavity of rats, but not into air-pouch cavity in which resident mast cells are not present. The transference of peritoneal mast cells to the air-pouch renders this cavity responsive to IL-8. The neutrophil migration induced by IL-8 into the peritoneal cavity was not observed when the peritoneal-resident mast cells were depleted by compound 48/80 or distilled water treatment. Confirming the importance of mast cells, IL-8-stimulated mast cells supernatant induced significant neutrophil migration when injected into peritoneal and air-pouch cavities. The IL-8-induced neutrophil migration was observed not to be dependent on LTB(4), prostaglandins or TNF-alpha, since MK886, indomethacin or thalidomide were unable to block the IL-8-induced neutrophil accumulation 'in vivo' or the release of neutrophil chemotactic factor "in vitro" by IL-8-stimulated mast cells. However, dexamethasone, an inhibitor of the synthesis of pro-inflammatory cytokines, blocked the neutrophil migration induced by IL-8 "in vivo" and also inhibited the release of the neutrophil chemotactic factor by IL-8-stimulated mast cells. Moreover, the incubation of IL-8-stimulated mast cells supernatant with antibody against cytokine-induced neutrophil chemoattractant 1 (CINC-1), but not against TNF-alpha or IL-1beta, inhibited its neutrophil chemotactic activity. Furthermore, we found a significant amount of CINC-1 in this supernatant. In conclusion, we demonstrated that the neutrophil migration induced by IL-8 is dependent on CINC-1 release from mast cells.     

Antihyperalgesic effects induced by the IL-1 receptor antagonist anakinra and increased IL-1beta levels in inflamed and osteosarcoma-bearing mice

Based on the well established involvement of IL-1beta in inflammatory hyperalgesia, we have assessed the possible role played by IL-1beta in a murine model of bone cancer-induced pain. With this aim, we measured IL-1beta levels at the region of the tibia and the spinal cord in mice bearing a tibial osteosarcoma induced by the inoculation of NCTC 2472 cells, and we tested whether the IL-1 receptor antagonist, anakinra, inhibits some hypernociceptive reactions evoked by the neoplastic injury. Parallel experiments were performed in mice with a chronic inflammatory process (intraplantar injection of complete Freund's adjuvant, CFA). IL-1beta levels were increased in the tibial region of osteosarcoma-bearing mice and in the paws of inflamed mice. To a lesser extent, the content of IL-1beta in the spinal cord was also augmented in both situations. Osteosarcoma-induced thermal hyperalgesia was inhibited by 30 and 100 mg/kg of systemic anakinra, but only 300 mg/kg prevented inflammatory thermal hyperalgesia. Mechanical hyperalgesia induced by the osteosarcoma was blocked by 100 and 300 mg/kg of anakinra, whereas a partial reversion of inflammatory mechanical hyperalgesia was induced by 300 mg/kg. Anakinra, intrathecally administered (1 and 10 microg) did not modify hyperalgesia of either origin. Besides, both tumoral and inflammatory mechanical allodynia remained unaltered after the administration of anakinra. In conclusion, some hyperalgesic symptoms observed in this model of bone cancer are mediated by the peripheral release of IL-1beta and may be inhibited by antagonists of type I IL-1 receptors with a similar or greater potency than symptoms produced by inflammation.     

Contribution of TNFalpha, IL-1beta and CINC-1 for articular incapacitation, edema and cell migration in a model of LPS-induced reactive arthritis

The protective effect of anti-CINC-1, -TNFalpha and -IL-1beta antisera on articular inflammatory incapacitation, articular diameter and synovial fluid cell content, which are correlated to nociception, edema and cell migration, respectively, were evaluated in a rat model of LPS-induced reactive arthritis. In this model, Escherichia coli lipopolysaccharide (LPS; 30 ng) was injected in a knee-joint previously primed with carrageenan (300 microg). Articular incapacitation was evaluated hourly by the automated registering of the knee-joint function during animal walking, and the knee-joint edema was evaluated by measuring the articular diameter increase. After 6 h, the animals were euthanized for collecting synovial fluid for the evaluation of cell migration. LPS produced dose-dependent incapacitation and edema. Anti-TNFalpha, -IL-1beta, and -CINC-1 antisera (20 and 40 microl) were used as pretreatment into knee-joint before LPS injection. At higher dose, Anti-TNFalpha and anti-CINC-1 were able to inhibit incapacitation, articular edema and mononuclear (MON) migration. Anti-IL1beta did not affect incapacitation at any dose, although inhibited edema and cell migration. Surprisingly, the higher dose of anti-IL1beta antisera did not inhibit cell migration, although inhibited articular edema. These findings corroborate the role TNFalpha has in different forms of arthritis, but points out the idea that CINC-1 (the homologue for human IL-8) may constitute a promising target for reactive arthritis management. Indeed, the potent antiedematogenic effect, and principally the anti-migration effect of anti-CINC-1, raises the possibility of a better control of disease progression than with anti-IL-1beta therapies.     

Stretch and CO2 modulate the inflammatory response of alveolar macrophages through independent changes in metabolic activity

Ventilatory-induced strain can exacerbate acute lung injury (ALI). Current ventilation strategies favour low tidal volumes and high end-expiratory volumes to 'rest' the lung, but can lead to an increase in CO2. Alveolar macrophages (AM) play a pivotal role in ALI through the release of inflammatory mediators. The effect of physical strain and CO2 on the release of pro-inflammatory mediators was examined in isolated rat AM. AM were cultured on IgG-coated silastic membranes with or without lipopolysaccharide (LPS) and 5% or 20% CO2 and subjected to a repetitive sinusoidal mechanical strain (30%, 60 cycles/min) for 4 h. Cell viability and metabolic activity were assessed. In both the presence and absence of LPS, physical strain increased metabolic activity by approximately 5%, while 20% CO2 decreased metabolic activity by approximately 40%. Twenty per cent CO2 decreased TNF-alpha secretion by approximately 45%, without affecting cell viability. Physical strain enhanced LPS-induced secretion of TNF-alpha by 1.5%, but not IL-6 or CINC-1. Hence, the effects of both CO2 and physical strain are mediated independently through changes in AM metabolic activity. Physical strain is not a major determinant of TNF-alpha, IL-6 or CINC-1 in AM. Our results confirm that high CO2 can lessen the TNF-alpha inflammatory response of AM.     

Differential effects of ebselen on neutrophil recruitment,chemokine, and inflammatory mediator expression in a rat model of lipopolysaccharide-induced pulmonary inflammation

We postulated that the seleno-organic compound ebselen would attenuate neutrophil recruitment and activation after aerosolized challenge with endotoxin (LPS) through its effect as an antioxidant and inhibitor of gene activation. Rats were given ebselen (1-100 mg/kg i.p.) followed by aerosolized LPS exposure (0.3 mg/ml for 30 min). Airway inflammatory indices were measured 4 h postchallenge. Bronchoalveolar lavage (BAL) fluid cellularity and myeloperoxidase activity were used as a measure of neutrophil recruitment and activation. RT-PCR analysis was performed in lung tissue to assess gene expression of TNF-alpha, cytokine-induced neutrophil chemoattractant-1 (CINC-1), macrophage-inflammatory protein-2 (MIP-2), ICAM-1, IL-10, and inducible NO synthase. Protein levels in lung and BAL were also determined by ELISA. Ebselen pretreatment inhibited neutrophil influx and activation as assessed by BAL fluid cellularity and myeloperoxidase activity in cell-free BAL and BAL cell homogenates. This protective effect was accompanied by a significant reduction in lung and BAL fluid TNF-alpha and IL-1 beta protein and/or mRNA levels. Ebselen pretreatment also prevented lung ICAM-1 mRNA up-regulation in response to airway challenge with LPS. This was not a global effect of ebselen on LPS-induced gene expression, because the rise in lung and BAL CINC-1 and MIP-2 protein levels were unaffected as were lung mRNA expressions for CINC-1, MIP-2, IL-10, and inducible NO synthase. These data suggest that the anti-inflammatory properties of ebselen are achieved through an inhibition of lung ICAM-1 expression possibly through an inhibition of TNF-alpha and IL-1 beta, which are potent neutrophil recruiting mediators and effective inducers of ICAM-1 expression.     

Estrogen prevents intestinal inflammation after trauma-hemorrhage via downregulation of angiotensin II and angiotensin II subtype I receptor

Although angiotensin II (Ang II) plays a key role in development of organ ischemia-reperfusion injury, it remains unclear whether it is involved in development of intestinal injury following trauma-hemorrhage (T-H). Studies have shown that 17beta-estradiol (E2) administration following T-H improves small intestinal blood flow; however, it is unclear whether Ang II plays a role in this E2-mediated salutary effect. Male Sprague-Dawley rats underwent laparotomy and hemorrhagic shock (removal of 60% total blood volume, fluid resuscitation after 90 min). At onset of resuscitation, rats were treated with vehicle, E2, or E2 and estrogen receptor antagonist ICI 182,780 (ICI). A separate group of rats was treated with Ang II subtype I receptor (AT1R) antagonist losartan. At 24 h after T-H, plasma Ang II, IL-6, TNF-alpha, intercellular adhesion molecule (ICAM)-1, cytokine-induced neutrophil chemoattractant (CINC)-1 and CINC-3 levels, myeloperoxidase (MPO) activity, and AT1R expression were determined. T-H significantly increased plasma and intestinal Ang II, IL-6, TNF-alpha levels, intestinal ICAM-1, CINC-1, CINC-3 levels, MPO activity, and AT1R protein compared with shams. E2 treatment following T-H attenuated increased intestinal MPO activity, Ang II level, and AT1R protein expression. ICI administration abolished the salutary effects of E2. In contrast, losartan administration attenuated increased MPO activity without affecting Ang II and AT1R levels. Thus Ang II plays a role in producing small intestine inflammation following T-H, and the salutary effects of E2 on intestinal inflammation are mediated in part by Ang II and AT1R downregulation.     

The role of estrogen receptor subtypes on hepatic neutrophil accumulation following trauma-hemorrhage: direct modulation of CINC-1 production by Kupffer cells

Although 17beta-estradiol (E2) administration following trauma-hemorrhage (T-H) reduces liver injury by decreasing neutrophil accumulation via estrogen receptor (ER)-alpha, it remains unclear whether cytokine-induced neutrophil chemoattractant (CINC)-1 production by Kupffer cells (KC) is directly modulated by ER-alpha under such condition. Male rats underwent laparotomy and hemorrhagic shock (40 mmHg for 90 min), followed by resuscitation with four times the shed blood volume in the form of Ringer's lactate. ER-alpha agonist propyl pyrazole triol (PPT; 5 microg/kg), ER-beta agonist diarylpropionitrile (DPN; 5 microg/kg), E2 (50 microg/kg), or vehicle (10% DMSO) was administered subcutaneously during resuscitation; rats were sacrificed 24h thereafter. KC were isolated and cultured with ER agonists to examine if they directly affect CINC-1 production. T-H increased plasma alanine aminotransferase (ALT; hepatic injury) and hepatic myeloperoxidase (MPO) activity. E2, PPT and DPN administration reduced increased ALT; however, PPT was more effective than DPN. PPT and E2, but not DPN significantly attenuated increased hepatic MPO activity and CINC-1 levels. PPT addition in vitro (10(-7) and 10(-6)M) significantly reduced KC CINC-1 production. In summary, the salutary effects of E2 against hepatic injury are mediated predominantly via ER-alpha which directly modulates KC CINC-1 production and hepatic neutrophil accumulation following T-H.     

Sequential appearance of IL-1 and IL-6 activities in rat carrageenin-induced pleurisy.

IL-1 and IL-6 activities were measured in the pleural exudate of rats during carrageenin-induced pleurisy to examine the relationship of the local production of cytokines to the inflammatory reaction. Time courses of appearance of the cytokines and inflammatory parameters in the exudate were compared. IL-1 activity and exudate volume started to increase at 1 h after the carrageenin injection, and then slightly later IL-6 activity and leukocyte number began to increase. IL-1 showed peak activity of approximately 700 U at 3 h and IL-6, of 6000 U at 5 h in the exudate, whereas exudate volume and number of polymorphonuclear leukocytes continued to increase thereafter. Furthermore, IL-6 level in the plasma of the carrageenin-injected rats showed a peak at 4 h (30 U/ml), and when rhIL-1 alpha (100,000 U) was intrapleurally injected, the more rapid increase in plasma IL-6 level was demonstrated at 1 h (30 U/ml). This latter rise was neutralized with simultaneous injection of anti-rhIL-1 alpha antibody. These facts indicate the possibility that IL-1 produced in the exudate or injected could rapidly propagate a signal to induce IL-6 production in the circulation. It took several hours to transmit an inflammatory signal that stimulated the liver to synthesize the acute-phase protein, T-kininogen. The time lag from the peak induction of IL-1 to the T-kininogen-increase in the pleurisy corresponded well to the interval for T-kininogen-increase by exogenous rhIL-1 alpha injection. These results strongly suggest that the initial inflammatory stimulus induces sequentially IL-1, IL-6, and T-kininogen production in this carrageenin inflammation.     

LPS inhibits fasted plasma ghrelin levels in rats: role of IL-1 and PGs and functional implications

LPS injected intraperitoneally decreases fasted plasma levels of ghrelin at 3 h postinjection in rats. We characterized the inhibitory action of LPS on plasma ghrelin and whether exogenous ghrelin restores LPS-induced suppression of food intake and gastric emptying in fasted rats. Plasma ghrelin and insulin and blood glucose were measured after intraperitoneal injection of LPS, intravenous injection of IL-1beta and urocortin 1, and in response to LPS under conditions of blockade of IL-1 or CRF receptors by subcutaneous injection of IL-1 receptor antagonist (IL-1Ra) or astressin B, respectively, and prostaglandin (PG) synthesis by intraperitoneal indomethacin. Food intake and gastric emptying were measured after intravenous injection of ghrelin at 5 h postintraperitoneal LPS injection. LPS inhibited the elevated fasted plasma ghrelin levels by 47.6 +/- 4.9%, 58.9 +/- 3.3%, 74.4 +/- 2.7%, and 48.9 +/- 8.7% at 2, 3, 5, and 7 h postinjection, respectively, and values returned to preinjection levels at 24 h. Insulin levels were negatively correlated to those of ghrelin, whereas there was no significant correlation between glucose and ghrelin. IL-1Ra and indomethacin prevented the first 3-h decline in ghrelin levels induced by LPS, whereas astressin B did not. IL-1beta inhibited plasma ghrelin levels, whereas urocortin 1 had no influence. Ghrelin injected intravenously prevented an LPS-induced 87% reduction of gastric emptying and 61% reduction of food intake. These data showed that IL-1 and PG pathways are part of the early mechanisms by which LPS suppresses fasted plasma ghrelin and that exogenous ghrelin can normalize LPS-induced-altered digestive functions.     

Inhibitory effect of amygdalin on lipopolysaccharide-inducible TNF-alpha and IL-1beta mRNA expression and carrageenan-induced rat arthritis

Amygdalin is a cyanogenic glycoside plant compound found in the seeds of rosaceous stone fruits. We evaluated the antiinflammatory and analgesic activities of amygdalin, using an in vitro lipopolysaccharide (LPS)-induced cell line and a rat model with carrageenan-induced ankle arthritis. One mM amygdalin significantly inhibited the expression of TNF-alpha and IL-1beta mRNAs in LPS-treated RAW 264.7 cells. Amygdalin (0.005, 0.05, and 0.1 mg/kg) was intramuscularly injected immediately after the induction of carrageenan-induced arthritic pain in rats, and the anti-arthritic effect of amygdalin was assessed by measuring the weight distribution ratio of the bearing forces of both feet and the ankle circumference, and by analyzing the expression levels of three molecular markers of pain and inflammation (c-Fos, TNF-alpha, and IL-1beta) in the spinal cord. The hyperalgesia of the arthritic ankle was alleviated most significantly by the injection of 0.005 mg/kg amygdalin. At this dosage, the expressions of c-Fos, TNF-alpha, and IL-1beta in the spinal cord were significantly inhibited. However, at dosage greater than 0.005 mg/kg, the painrelieving effect of amygdalin was not observed. Thus, amygdalin treatment effectively alleviated responses to LPStreatment in RAW 264.7 cells and carrageenan-induced arthritis in rats, and may serve as an analgesic for relieving inflammatory pain.     

The citrus flavonone naringenin reduces lipopolysaccharide-induced inflammatory pain and leukocyte recruitment by inhibiting NF-κB activation

Lipopolysaccharide (LPS) is the major structural component of Gram-negative bacteria cell wall and a highly pro-inflammatory toxin. Naringenin is found in Citrus fruits and exhibits antioxidant and anti-inflammatory properties through inhibition of NF-κB activation but its effects in LPS-induced inflammatory pain and leukocyte recruitment were not investigated yet. We investigated the effects of naringenin in mechanical hyperalgesia, thermal hyperalgesia and leukocyte recruitment induced by intraplantar injection of LPS in mice. We found that naringenin reduced hyperalgesia to mechanical and thermal stimuli, myeloperoxidase (MPO, a neutrophil and macrophage marker) and N-acetyl-β-D-glucosaminidase (NAG, a macrophage marker) activities, oxidative stress and cytokine (TNF-α, IL-1β, IL-6, and IL-12) production in the paw skin. In the peritoneal cavity, naringenin reduced neutrophil and mononuclear cell recruitment, and abrogated MPO and NAG activity, cytokine and superoxide anion production, and lipid peroxidation. In vitro, pre-treatment with naringenin inhibited superoxide anion and cytokine (TNF-α, IL-1β, IL-6, and IL-12) production by LPS-stimulated RAW 264.7 macrophages. Finally, we demonstrated that naringenin inhibited NF-κB activation in vitro and in vivo. Therefore, naringenin is a promising compound to treat LPS-induced inflammatory pain and leukocyte recruitment.     

Acute alcohol intoxication increases interleukin-18-mediated neutrophil infiltration and lung inflammation following burn injury in rats

In this study, we examined whether IL-18 plays a role in lung inflammation following alcohol (EtOH) and burn injury. Male rats ( approximately 250 g) were gavaged with EtOH to achieve a blood EtOH level of approximately 100 mg/dl before burn or sham injury ( approximately 12.5% total body surface area). Immediately after injury, rats were treated with vehicle, caspase-1 inhibitor AC-YVAD-CHO to block IL-18 production or with IL-18 neutralizing anti-IL-18 antibodies. In another group, rats were treated with anti-neutrophil antiserum approximately 16 h before injury to deplete neutrophils. On day 1 after injury, lung tissue IL-18, neutrophil chemokines (CINC-1/CINC-3), ICAM-1, neutrophil infiltration, MPO activity, and water content (i.e., edema) were significantly increased in rats receiving a combined insult of EtOH and burn injury compared with rats receiving either EtOH intoxication or burn injury alone. Treatment of rats with caspase-1 inhibitor prevented the increase in lung tissue IL-18, CINC-1, CINC-3, ICAM-1, MPO activity, and edema following EtOH and burn injury. The increase in lung IL-18, MPO, and edema was also prevented in rats treated with anti-IL-18 antibodies. Furthermore, administration of anti-neutrophil antiserum also attenuated the increase in lung MPO activity and edema, but did not prevent the increase in IL-18 levels following EtOH and burn injury. These findings suggest that acute EtOH intoxication before burn injury upregulates IL-18, which in turn contributes to increased neutrophil infiltration. Furthermore, the presence of neutrophils appears to be critical for IL-18-meditaed increased lung tissue edema following a combined insult of EtOH and burn injury.     

Diclofenac-induced peripheral antinociception is associated with ATP-sensitive K+ channels activation

In order to investigate to the contribution of K+ channels on the peripheral antinociception induced by diclofenac, we evaluated the effect of several K+ channel blockers, using the rat paw pressure test, in which sensitivity is increased by intraplantar injection (2 microg) of prostaglandin E2. Diclofenac administered locally into the right hindpaw (25, 50, 100 and 200 microg) elicited a dose-dependent antinociceptive effect which was demonstrated to be local, since only higher doses produced an effect when injected in the contralateral paw. This blockade of PGE2 mechanical hyperalgesia induced by diclofenac (100 microg/paw) was antagonized in a dose-dependent manner by intraplantar administration of the sulphonylureas glibenclamide (40, 80 and 160 microg) and tolbutamide (80, 160 and 320 microg), specific blockers of ATP-sensitive K+ channels, and it was observed even when the hyperalgesic agent used was carrageenin, while the antinociceptive action of indomethacin (200 microg/paw), a typical cyclo-oxygenase inhibitor, over carrageenin-induced hyperalgesia was not affected by this treatment. Charybdotoxin (2 microg/paw), a blocker of large conductance Ca2+-activated K+ channels and dequalinium (50 microg/paw), a selective blocker of small conductance Ca2+-activated K+ channels, did not modify the effect of diclofenac. This effect was also unaffected by intraplantar administration of non-specific voltage-dependent K+ channel blockers tetraethylammonium (1700 microg) and 4-aminopyridine (100 microg) or cesium (500 microg), a non-specific K+ channel blocker. The peripheral antinociceptive effect induced by diclofenac was antagonized by NG-Nitro L-arginine (NOarg, 50 microg/paw), a NO synthase inhibitor and methylene blue (MB, 500 microg/paw), a guanylate cyclase inhibitor, and this antagonism was reversed by diazoxide (300 microg/paw), an ATP-sensitive K+ channel opener. We also suggest that an endogenous opioid system may not be involved since naloxone (50 microg/paw) did not affect diclofenac-induced antinociception in the PGE2-induced hyperalgesia model. This study provides evidence that the peripheral antinociceptive effect of diclofenac may result from activation of ATP-sensitive K+ channels, possible involving stimulation of L-arginine/NO/cGMP pathway, while Ca2+-activated K+ channels, voltage-dependent K+ channels as well as endogenous opioids appear not to be involved in the process.     

Balance of tumor necrosis factor alpha and interleukin-10 in a buccal infection in a streptozotocin-induced diabetic rat model

This study evaluates the local levels of proinflammatory cytokine, tumor necrosis factor alpha (TNF-alpha), and anti-inflammatory cytokine, interleukin-10 (IL-10), in an experimental buccal abscess of a diabetic rat model. We prepared a buccal cavity induced by injection of carrageenin in a diabetic rat (blood glucose, 460.6 +/- 54.7 mg/dl, mean +/- SE) induced by streptozotocin (STZ). The buccal abscess was formed by the direct inoculation of Streptococcus pyogenes S-8 (2 x 10(7) cfu) into the buccal cavity at day 5 after carrageenin injection. Cytokine levels in the exudate of the buccal abscess were measured by enzyme-linked immunosorbent assay for 48 h after infection. Bacterial counts, weighing of exudate, and histological analysis were also performed. The mean TNF-alpha levels in the buccal abscess exudate of the diabetic group, which were generally higher than those of the control group, tended to increase over time until 48 h after infection, while the TNF-alpha levels in the control group peaked at 24 h after infection and then decreased. The IL-10 levels in the diabetic group remained almost unchanged until 48 h after infection, while the IL-10 levels in the control group were significantly higher than in the diabetic group at 12-24 h after infection. The mean ratio of TNF-alpha to IL-10 levels was 1.17-1.67 in the diabetic group, which was higher than the 0.26-0.69 of the control group. The bacterial counts in the buccal abscess and the weight of exudate were significantly higher in the diabetic group compared to the control group at 36-48 h. Histological findings showed that inflammatory cell infiltration was remarkable in the diabetic group compared to that of the control group. These results suggest that the elevated proinflammatory TNF-alpha levels and decreased anti-inflammatory IL-10 levels, which are produced at local infection sites, may at least in part be related to the severity of inflammation in this rat model with diabetes induced by STZ.     

Effect of forskolin on alterations of vascular permeability induced with bradykinin, prostaglandin E1, adenosine, histamine and carrageenin in rats

The effect of the diterpene forskolin on vascular permeability alone and in combination with bradykinin, prostaglandin E1, adenosine or histamine has been investigated in rats. Vascular permeability in rat skin was measured using [125I]-labelled bovine serum albumin ([125I]BSA) as a tracer. In addition, the effect of forskolin on footpad edema induced by the injection of a mixture of 2% carrageenin was determined. Forskolin caused a marked potentiation of the increase in vascular permeability in rat skin elicited by the intradermal injection of histamine or bradykinin. However, forskolin caused a significant suppression of the prostaglandin E1-induced vascular permeability response and at a low concentration suppressed the response to adenosine. Forskolin greatly potentiated the footpad edema induced with carrageenin in rats. Intravenous administration of the enzyme bromelain, which reduces plasma kininogen levels, inhibited the footpad edema induced with carrageenin or with a mixture of carrageenin and forskolin. Parenteral administration of a prostaglandin synthetase inhibitor, indomethacin, suppressed the footpad edema induced with carrageenin, but did not inhibit the footpad edema induced with a mixture of carrageenin and forskolin. An antihistamine, cyproheptadine, had no effect on carrageenin-induced footpad edema either in the presence or absence of forskolin. These results suggest that both bradykinin and prostaglandins are essential for the development of carrageenin-induced footpad edema and that bradykinin plays an important role in the potentiative effect of forskolin on footpad edema induced with carrageenin in rats.     

Moxifloxacin but not ciprofloxacin or azithromycin selectively inhibits IL-8, IL-6, ERK1/2, JNK, and NF-kappaB activation in a cystic fibrosis epithelial cell line

Cystic fibrosis (CF) is associated with severe neutrophilic airway inflammation. We showed that moxifloxacin (MXF) inhibits IL-8 and MAPK activation in monocytic and respiratory epithelial cells. Azithromycin (AZM) and ciprofloxacin (CIP) are used clinically in CF. Thus we now examined effects of MXF, CIP, and AZM directly on CF cells. IB3, a CF bronchial cell line, and corrected C38 cells were treated with TNF-alpha, IL-1beta, or LPS with or without 5-50 microg/ml MXF, CIP, or AZM. IL-6 and IL-8 secretion (ELISA), MAPKs ERK1/2, JNK, p38, and p65 NF-kappaB (Western blot) activation were measured. Baseline IL-6 was sixfold higher in IB3 than C38 cells but IL-8 was similar. TNF-alpha and IL-1beta increased IL-6 and IL-8 12- to 67-fold with higher levels in IB3 than C38 cells post-TNF-alpha (P < 0.05). Levels were unchanged following LPS. Baseline phosphorylated form of ERK1/2 (p-ERK1/2), JNK, and NF-kappaB p65 were higher in IB3 than C38 cells (5-, 1.4-, and 1.4-fold), and following TNF-alpha increased, as did the p-p38, by 1.6- to 2-fold. MXF (5-50 microg/ml) and CIP (50 microg/ml), but not AZM, suppressed IL-6 and IL-8 secretion by up to 69%. MXF inhibited TNF-alpha-stimulated MAPKs ERK1/2, 46-kDa JNK, and NF-kappaB up to 60%, 40%, and 40%, respectively. In contrast, MXF did not inhibit p38 activation, implying a highly selective pretranslational effect. In conclusion, TNF-alpha and IL-1beta induce an exaggerated inflammatory response in CF airway cells, inhibited by MXF more than by CIP or AZM. Clinical trials are recommended to assess efficacy in CF and other chronic lung diseases.