Fernando Q. Cunha

The pivotal role of tumour necrosis factor α in the development of inflammatory hyperalgesia

1 The hyperalgesic activities in rats of interleukin‐1β (IL‐1β), IL‐6, IL‐8, tumour necrosis factor α (TNFα) and carrageenin were investigated. 2 IL‐6 activated the previously delineated IL‐1/prostaglandin hyperalgesic pathway but not the IL‐8/sympathetic mediated hyperalgesic pathway. 3 TNFα and carrageenin activated both pathways. 4 Antiserum neutralizing endogenous TNFα abolished the response to carrageenin whereas antisera neutralizing endogenous IL‐1β, IL‐6 and IL‐8 each partially inhibited the response. 5 The combination of antisera neutralizing endogenous IL‐1β + IL‐8 or IL‐6 + IL‐8 abolished the response to carrageenin. 6 These results show that TNFα has an early and crucial role in the development of inflammatory hyperaglesia. 7 The delineation of the roles of TNFα, IL‐1β, IL‐6 and IL‐8 in the development of inflammatory hyperalgesia taken together with the finding that the production of these cytokines is inhibited by steroidal anti‐inflammatory drugs provides a mechanism of action for these drugs in the treatment of inflammatory hyperalgesia.

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.

Feedback inhibition of nitric oxide synthase activity by nitric oxide

1 A murine macrophage cell line, J774, expressed nitric oxide (NO) synthase activity in response to interferon‐gamma (IFN‐γ, 10 u ml−1) plus lipopolysaccharide (LPS, 10 ng ml−1). The enzyme activity was first detectable 6 h after incubation, peaked at 12 h and became undetectable after 48 h. 2 The decline in the NO synthase activity was not due to inhibition by stable substances secreted by the cells into the culture supernatant. 3 The decline in the NO synthase activity was significantly slowed down in cells cultured in a low l‐arginine medium or with added haemoglobin, suggesting that NO may be involved in a feedback inhibitory mechanism. 4 The addition of NO generators, S‐nitroso‐acetyl‐penicillamine (SNAP) or S‐nitroso‐glutathione (GSNO) markedly inhibited the NO synthase activity in a dose‐dependent manner. The effect of NO on the enzyme was not due to the inhibition of de novo protein synthesis. 5 SNAP directly inhibited the inducible NO synthase extracted from activated J774 cells, as well as the constitutive NO synthase extracted from the rat brain. 6 The enzyme activity of J774 cells was not restored after the removal of SNAP by gel filtration, suggesting that NO inhibits NO synthase irreversibly.

Interleukin-8 as a mediator of sympathetic pain.

1 The hyperalgesic effects of interleukin‐8 (IL‐8), interleukin‐1β (IL‐1β) and carrageenin were measured in a rat paw pressure test. 2 IL‐8 evoked a dose‐dependent hyperalgesia which was attenuated by a specific antiserum, the β‐adrenoceptor antagonists atenolol and propranolol, the dopamine1 receptor antagonist SCH 23390 and the adrenergic neurone‐blocking agent guanethidine. The hyperalgesia was not attenuated by the cyclo‐oxygenase inhibitor indomethacin or the IL‐1β analogue Lys‐d‐Pro‐Thr. 3 IL‐1β‐evoked hyperalgesia was attenuated by indomethacin and Lys‐d‐Pro‐Thr but not by atenolol or SCH 23390. 4 Carrageenin‐evoked hyperalgesia was attenuated by atenolol, indomethacin and anti‐IL‐8 serum. The effects of atenolol and anti‐IL‐8 serum were not additive. The effects of indomethacin and anti‐IL‐8 serum were additive: this combination abolished carrageenin‐evoked hyperalgesia. 5 A new biological activity of IL‐8 is described, namely the capacity to evoke hyperalgesia by a prostaglandin‐independent mechanism. IL‐8 is the first endogenous mediator to be identified as evoking hyperalgesia involving the sympathetic nervous system. Since IL‐8 is released by activated macrophages and endothelial cells it may be a humoral link between tissue injury and sympathetic hyperalgesia.

Interleukin-33 attenuates sepsis by enhancing neutrophil influx to the site of infection

Sepsis is a systemic inflammatory condition following bacterial infection with a high mortality rate and limited therapeutic options. Here we show that interleukin-33 (IL-33) reduces mortality in mice with experimental sepsis from cecal ligation and puncture (CLP). IL-33–treated mice developed increased neutrophil influx into the peritoneal cavity and more efficient bacterial clearance than untreated mice. IL-33 reduced the systemic but not the local proinflammatory response, and it did not induce a T helper type 1 (TH1) to TH2 shift. The chemokine receptor CXCR2 is crucial for recruitment of neutrophils from the circulation to the site of infection. Activation of Toll-like receptors (TLRs) in neutrophils downregulates CXCR2 expression and impairs neutrophil migration. We show here that IL-33 prevents the downregulation of CXCR2 and inhibition of chemotaxis induced by the activation of TLR4 in mouse and human neutrophils. Furthermore, we show that IL-33 reverses the TLR4-induced reduction of CXCR2 expression in neutrophils via the inhibition of expression of G protein–coupled receptor kinase-2 (GRK2), a serine-threonine protein kinase that induces internalization of chemokine receptors. Finally, we find that individuals who did not recover from sepsis had significantly more soluble ST2 (sST2, the decoy receptor of IL-33) than those who did recover. Together, our results indicate a previously undescribed mechanism of action of IL-33 and suggest a therapeutic potential of IL-33 in sepsis.

A Role for IL-18 in Neutrophil Activation

IL-18 expression and functional activity has been identified in several autoimmune and infectious diseases. To clarify the potential role of IL-18 during early innate immune responses, we have explored the capacity of IL-18 to activate neutrophils. Human peripheral blood-derived neutrophils constitutively expressed IL-18R (α and β) commensurate with the capacity to rapidly respond to IL-18. IL-18 induced cytokine and chemokine release from neutrophils that was protein synthesis dependent, up-regulated CD11b expression, induced granule release, and enhanced the respiratory burst following exposure to fMLP, but had no effect upon the rate of neutrophil apoptosis. The capacity to release cytokine and chemokine was significantly enhanced in neutrophils derived from rheumatoid arthritis synovial fluid, indicating differential responsiveness to IL-18 dependent upon prior neutrophil activation in vivo. Finally, IL-18 administration promoted neutrophil accumulation in vivo, whereas IL-18 neutralization suppressed the severity of footpad inflammation following carrageenan injection. The latter was accompanied by reduction in tissue myeloperoxidase expression and suppressed local TNF-α production. Together, these data define a novel role for IL-18 in activating neutrophils and thereby promoting early innate immune responses.

Chemokines in Oral Inflammatory Diseases: Apical Periodontitis and Periodontal Disease

The inflammatory oral diseases are characterized by the persistent migration of polymorphonuclear leukocytes, monocytes, lymphocytes, plasma and mast cells, and osteoblasts and osteoclasts. In the last decade, there has been a great interest in the mediators responsible for the selective recruitment and activation of these cell types at inflammatory sites. Of these mediators, the chemokines have received particular attention in recent years. Chemokine messages are decoded by specific receptors that initiate signal transduction events, leading to a multitude of cellular responses, including chemotaxis and activation of inflammatory and bone cells. However, little is known about their role in the pathogenesis of inflammatory oral diseases. The purpose of this review is to summarize the findings regarding the role of chemokines in periapical and periodontal tissue inflammation, and the integration, into experimental models, of the information about the role of chemokines in human diseases.

THE MAJOR ROLE OF PERIPHERAL RELEASE OF HISTAMINE AND 5-HYDROXYTRYPTAMINE IN FORMALIN-INDUCED NOCICEPTION

Formalin injected subcutaneously into the paw is a widely used model of pain. This procedure evokes a short-lasting period of flinching (phase 1) and a long-lasting period of intense flinching (phase 2) following a very short period of quiescence. Phase 2 has been extensively used to support the involvement of central (spinal cord) sensitization in inflammatory hyperalgesia. The present study evaluated the contribution of stimulation of peripheral nociceptors by the release of endogenous mediators at the site of lesion. The participation of histamine and 5-hydroxytryptamine was demonstrated by the treatment of the rat hindpaws with selective histamine H1 (pyrilamine and meclizine) and histamine H2 (cimetidine) receptor antagonists or selective 5-hydroxytryptamine(1A) (WAY100,135) and 5-hydroxytryptamine(4/3) (tropisetron) receptor antagonists. The co-administration of pyrilamine or meclizine with formalin (1%) significantly reduced phases 1 and 2, while cimetidine had no effect. Pyrilamine administration during the period of quiescence (10min after formalin administration) caused strong dose-related inhibition of phase 2. The co-administration of tropisetron with formalin caused a blockade of both phases, while with WAY100,135 caused only inhibition of the phase 2. In contrast, tropisetron administrated during the period of quiescence did not cause antinociception. Histamine and 5-hydroxytryptamine receptors could be strongly activated in naïve animals by administration of a mixture of both agonists or compound 48/80 (2microg/paw) which is known to release both mediators from mast cells. Pretreatment of the paws with a mast cell stabilizer, sodium cromoglycate, significantly reduced the second phase of the formalin injection model. From these results we suggest that phases 1 and 2 of the formalin test are dependent upon the ongoing afferent input. Furthermore, while histamine H1 participates in both phases, 5-hydroxytryptamine(4/3) participates in phase 1 and 5-hydroxytryptamine(1A) in phase 2.

Cytokine‐mediated inflammatory hyperalgesia limited by interleukin‐4

The effect of IL‐4 on responses to intraplantar (i.pl.) carrageenin, bradykinin, TNFα, IL‐1β, IL‐8 and PGE2 was investigated in a model of mechanical hyperalgesia in rats. Also, the cellular source of the IL‐4 was investigated. IL‐4, 30 min before the stimulus, inhibited responses to carrageenin, bradykinin, and TNFα, but not responses to IL‐1β, IL‐8 and PGE2. IL‐4, 2 h before the injection of IL‐1β, did not affect the response to IL‐1β, whereas IL‐4, 12 or 12+2 h before the IL‐1β, inhibited the hyperalgesia (−30%, −74%, respectively). In murine peritoneal macrophages, murine IL‐4 for 2 h before stimulation with LPS, inhibited (−40%) the production of IL‐1β but not PGE2. Murine IL‐4 (for 16 h before stimulation with LPS) inhibited LPS‐stimulated PGE2 but not IL‐1β. Anti‐murine IL‐4 antibodies potentiated responses to carrageenin, bradykinin and TNFα, but not IL‐1β and IL‐8, as well as responses to bradykinin in athymic rats but not in rats depleted of mast cells with compound 40/80. These data suggest that IL‐4 released by mast cells limits inflammatory hyperalgesia. During the early phase of the inflammatory response the mode of action of the IL‐4 appears to be inhibition of the production TNFα, IL‐1β and IL‐8. In the later phase of the response, in addition to inhibiting the production of pro‐inflammatory cytokines, IL‐4 also may inhibit the release of PGs.

Crucial role of neutrophils in the development of mechanical inflammatory hypernociception

Neutrophil migration is responsible for tissue damage observed in inflammatory diseases. Neutrophils are also implicated in inflammatory nociception, but mechanisms of their participation have not been elucidated. In the present study, we addressed these mechanisms in the carrageenan‐induced mechanical hypernociception, which was determined using a modification of the Randall‐Sellito test in rats. Neutrophil accumulation into the plantar tissue was determined by the contents of myeloperoxidase activity, whereas cytokines and PGE2 levels were measured by ELISA and radioimmunoassay, respectively. The pretreatment of rats with fucoidin (a leukocyte adhesion inhibitor) inhibited carrageenan‐induced hypernociception in a dose‐ and time‐dependent manner. Inhibition of hypernociception by fucoidin was associated with prevention of neutrophil recruitment, as it did not inhibit the hypernociception induced by the direct‐acting hypernociceptive mediators, PGE2 and dopamine, which cause hypernociception, independent of neutrophils. Fucoidin had no effect on carrageenan‐induced TNF‐α, IL‐1β, and cytokine‐induced neutrophil chemoattractant 1 (CINC‐1)/CXCL1 production, suggesting that neutrophils were not the source of hypernociceptive cytokines. Conversely, hypernociception and neutrophil migration induced by TNF‐α, IL‐1β, and CINC‐1/CXCL1 was inhibited by fucoidin, suggesting that neutrophils are involved in the production of direct‐acting hypernociceptive mediators. Indeed, neutrophils stimulated in vitro with IL‐1β produced PGE2, and IL‐1β‐induced PGE2 production in the rat paw was inhibited by the pretreatment with fucoidin. In conclusion, during the inflammatory process, the migrating neutrophils participate in the cascade of events leading to mechanical hypernociception, at least by mediating the release of direct‐acting hypernociceptive mediators, such as PGE2. Therefore, the blockade of neutrophil migration could be a target to development of new analgesic drugs.