Sindynara Ferreira

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.

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.

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.

Cytokine-mediated inflammatory hyperalgesia limited by interleukin-1 receptor antagonist

The effect of IL‐1ra on response to intraplantar (i.pl.) injection of LPS, carrageenin, bradykinin, TNFα, IL‐1β, IL‐8, PGE2 and dopamine was investigated in a model of mechanical hyperalgesia in rats. IL‐1ra inhibited hyperalgesic response to LPS, carrageenin, bradykinin, TNFα, and IL‐1β, but not responses to IL‐8, PGE2 and dopamine. A sheep anti‐rat IL‐1ra serum potentiated response to LPS, carrageenin, bradykinin, TNFα and IL‐1β but not IL‐8. Carrageenin and LPS stimulated and production of immunoreactive TNFα, IL‐1β and IL‐1ra in the skin of injected paws. The inhibition by IL‐1ra of the hyperalgesic response to carrageenin was not affected by antibodies neutralizing IL‐4 and IL‐10. In mice, IL‐1ra inhibited the nociceptive response to i.p. injection of acetic acid. These data suggest that IL‐1ra, released at sites of inflammation, limits inflammatory hyperalgesia. This effect is independent of (IL‐1ra‐induced) IL‐4 and IL‐10 and appears to be the result of antagonism by IL‐1ra of IL‐1β‐stimulated eicosanoid production.

Blockade by antimacrophage serum of the migration of PMN neutrophils into the inflamed peritoneal cavity

The effect of rat antimacrophage serum (rAMS) was tested on the influence of normal or thioglycollate-stimulated macrophage populations of the rat peritoneal cavity on the migration of polymorphonuclear neutrophils (PMN) induced by carrageenin, heterologous serum (rabbit) and sheep red blood cells. The rAMS used did not cross-react with PMN or lymphocytes nor did it affect circulating white cells, complement levels or lysed PMN present in the inflammatory exudate. It did, however, give a positive immunofluorescence reaction with resident and stimulated macrophages. The rAMS inhibited macrophage function as tested by sheep red blood cell phagocytosisin vivo and release of a PMN chemotactic factor(s)in vitro. Thioglycollate-stimuated peritoneal cavities showed an increased macrophage population and responded with increased PMN migration when challenged with heterologous serum or carrageenin, as compared with control rats. The presence of rat antimacrophage antibodies inhibited PMN migration induced by heterologous serum, sheep red blood cells and carrageenin. It is concluded that resident macrophages participate in the control of PMN migration to the site of an acute inflammation by acting as ‘alarm cells’ and triggering several defence mechanisms which ultimately protect the host from injurious stimuli.

Pharmacological modulation of secondary mediator systems–cyclic AMP and cyclic GMP–on inflammatory hyperalgesia

The objective of the present paper was to evaluate the relevance of neuronal balance of cyclic AMP and cyclic GMP concentration for functional regulation of nociceptor sensitivity during inflammation. Injection of PGE2 (10–100 ng paw−1) evoked a dose‐dependent hyperalgesic effect which was mediated via a cyclic AMP‐activated protein kinase (PKA) inasmuch as hyperalgesia was blocked by the PKA inhibitor H89. The PDE4 inhibitor rolipram and RP73401, but not PDE3 and PDE5 inhibitors potentiated the hyperalgesic effects of PGE2. The hyperalgesic effect of dopamine was also enhanced by rolipram. Moreover, rolipram significantly potentiated hyperalgesia induced by carrageenan, bradykinin, TNFα, IL‐1β, IL‐6 and IL‐8. This suggests that neuronal cyclic AMP mediates the prostanoid and sympathetic components of mechanical hyperalgesia. Moreover, in the neuron cyclic AMP is mainly metabolized by PDE4. To examine the role of the NO/cyclic GMP pathway in modulating mechanical hyperalgesia, we tested the effects of the soluble guanylate cyclase inhibitor, ODQ. This substance counteracts the inhibitory effects of the NO donor, SNAP, on the hyperalgesia induced by PGE2. The ODQ potentiated hyperalgesia induced by carrageenan, bradykinin, TNFα, IL‐1β, IL‐6 and IL‐8. In contrast, ODQ had no significant effect on the hyperalgesia induced by PGE2 and dopamine. This indicates that the hyperalgesic cytokines may activate soluble guanylate cyclase, which down‐regulate the ability of these substances to cause hyperalgesia. This event appears not to be mediated by prostaglandin or dopamine. In conclusion, the results presented in this paper confirm an association between (i) hyperalgesia and elevated levels of cyclic AMP as well as (ii) antinociception and elevated levels of cyclic GMP. The intracellular levels of cyclic AMP that enhance hyperalgesia are controlled by the PDE4 isoform and appear to result in activation of protein kinase A whereas the intracellular levels of cyclic GMP results from activation of a soluble guanylate cyclase.

Recombinant interleukin-1 and tumor necrosis factor induce neutrophil migration “in vivo” by indirect mechanisms

The α and β forms of recombinant interleukin-1 (IL-1α and IL-1β) and of recombinant Tumor Necrosis Factor (TNFα and TNFβ) induced dose-dependent neutrophil migration into rat peritoneal cavities. Migration induced by both IL-1s showed a bell-shaped dose-response curve and IL-1β was 3-fold more potent than IL-1α. Pretreatment of the animals with dexamethasone or depletion of the peritoneal macrophage population, abolished the neutrophil migration induced by the four cytokines. “In vitro” stimulation of macrophage monolayers with IL-1β and the TNFs released a factor into the supernatant which, unlike these cytokines, induced neutrophil migration in dexamethasone pretreated animals. These results suggest that the neutrophil migration induced by IL-1α, IL-1β and TNFβ is not due to a direct effect on neutrophils, but occurs via the release of a chemotactic factors(s) from resident macrophages.

Heme oxygenase/carbon monoxide-biliverdin pathway down regulates neutrophil rolling, adhesion and migration in acute inflammation

Heme oxygenase (HO) activity is known to down‐regulate inflammatory events. Here, we address the role of HO and its metabolites, carbon monoxide (CO) and biliverdin (BVD), in leukocyte rolling, adhesion and neutrophil migration during inflammatory processes.

Tumour necrosis factor‐alpha and leukotriene B4 mediate the neutrophil migration in immune inflammation

We investigated the mediators responsible for neutrophil migration induced by ovalbumin (OVA) in immunized mice and the mechanisms involved in their release. OVA administration promoted dose‐ and time‐dependent neutrophil migration in immunized, but not in non‐immunized mice, which was mediated by leukotriene B4 (LTB4) and tumour necrosis factor (TNF)α, since it was inhibited by LTB4 synthesis inhibitor (MK 886) or by LTB4 receptor antagonist (CP 105,696), by dexamethasone and by antiserum to TNFα (82, 85, 63 and 87%, respectively). Confirming TNFα involvement, OVA challenge in immunized p55 TNF receptor deficient mice (p55−/−) did not promote neutrophil migration (control: 2.90±0.68; p55−/−: 0.92±0.23×106 neutrophils cavity−1). OVA‐stimulated peritoneal cells from immunized mice released a neutrophil chemotactic factor which mimicked, in naive mice, neutrophil migration induced by OVA. Supernatant chemotactic activity is due to TNFα and LTB4, since its release was inhibited by MK 886 (93%) and dexamethasone (90%), and significant amounts of these mediators were detected. TNFα and LTB4 released by OVA challenge seem to act through a sequential mechanism, since MK 886 inhibited (88%) neutrophil migration induced by TNFα. Moreover, peritoneal cells stimulated with TNFα released LTB4. CD4+ T cells are responsible for TNFα release, because the depletion of this subset prevented the release of TNFα (control: 400±25; immunized: 670±40; CD4+ depleted: 435±18 pg ml−1). In conclusion, neutrophil migration induced by OVA depends on TNFα released by CD4+ cells, which acts through an LTB4‐dependent mechanism.

Neutrophil migration induced by inflammatory stimuli is reduced by macrophage depletion

Previous experiments of our group have shown that neutrophil migration induced by inflammatory stimuli is reduced by agents which block the release from macrophages of a specific factor for neutrophil migration (MNCF, [1, 2]). The present paper evaluated the influence of macrophage depletion induced by lavage of the peritoneal cavity on neutrophil migration. In both normal and thioglycollatestimuled peritoneal cavities, lavage with saline reduced the resident macrophage population by about 80% and significantly blocked neutrophil migration induced by inflammatory stimuli such as carrageenin, zymosan andE. coli endotoxin. Peritoneal lavage, however, did not affect neutrophil migration induced by MNCF. Thus, these results support the suggestion that macrophages participate in the control of neutrophil migration induced by acute inflammatory stimuli.