Glória E.P. Souza

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.

Kinin B1 Receptor Up-Regulation after Lipopolysaccharide Administration: Role of Proinflammatory Cytokines and Neutrophil Influx

Several studies have now clearly established the ability of LPS to induce bradykinin B1 receptor up-regulation in vivo and the functional relevance of this up-regulation for the pathophysiological effects of LPS. Using an in vivo system in which LPS is injected locally into the rat paw, we have examined the potential contribution of proinflammatory cytokines, NF-κB activation, and neutrophil influx for the functional and molecular up-regulation of the bradykinin B1 receptor. Treatment with LPS resulted in a rapid and sustained functional up-regulation of B1 receptors in the rat paw that correlated with the increase in B1 receptor mRNA levels. B1 receptor up-regulation is preceded by the rapid activation of the transcription factor NF-κB and the production of proinflammatory cytokines, including TNF-α and IL-1β. More importantly, blockade of NF-κB translocation, TNF-α, or IL-1β prevented the functional and molecular up-regulation of B1 receptors. Injection of LPS also induced the influx of neutrophils that followed the peak of cytokine production and associated with the persistent activation of NF-κB and functional B1 receptor up-regulation. Blockade of neutrophil influx with platelet-activating factor receptor antagonists or cell adhesion molecule blockers prevented B1 receptor up-regulation. Thus, by acting in cooperation and in a coordinated, timely manner, TNF-α, IL-1β, neutrophils, and the transcription factor NF-κB are major and essential players in the ability of LPS to induce B1 receptor expression in vivo.

Endothelin-1-induced ETA receptor-mediated nociception, hyperalgesia and oedema in the mouse hind-paw: modulation by simultaneous ETB receptor activation

Endothelin‐1 causes ETA receptor‐mediated enhancement of capsaicin‐induced nociception in mice. We have assessed if this hyperalgesic effect of endothelin‐1 is also accompanied by other pro‐inflammatory effects, namely nociception and oedema, and characterized the endothelin ET receptors involved. Intraplantar (i.pl.) hind‐paw injection of endothelin‐1 (0.3–30 pmol) induced graded nociceptive responses (accumulated licking time: vehicle, 20.5±3.3 s; endothelin‐1 at 30 pmol, 78.1±9.8 s), largely confined to the first 15 min. Endothelin‐1 (1–10 pmol) potentiated ipsilateral capsaicin‐induced (0.1 μg, i.pl.; at 30 min) nociception (vehicle, 40.2±2.6 s; endothelin‐1 at 10 pmol, 98.4±5.8 s, but 30 pmol was inactive), and caused oedema (increase in paw weight 5 min after capsaicin: vehicle, 46.3±2.3 mg; endothelin‐1 at 30 pmol, 100.3±6.1 mg). Selective ETB receptor agonists sarafotoxin S6c (up to 30 pmol) and IRL 1620 (up to 100 pmol) were inactive, whereas endothelin‐3 (up to 30 pmol) induced only modest oedema. ETA receptor antagonists BQ‐123 (1 nmol, i.pl.) or A‐127722‐5 (6 μmol kg−1, i.v.) prevented all effects of endothelin‐1 (10 pmol), but the ETB receptor antagonist BQ‐788 (1 or 10 nmol, i.pl.) was ineffective. BQ‐788 (10 nmol, i.pl.) unveiled hyperalgesic effects of 30 pmol endothelin‐1 and endothelin‐3. Sarafotoxin S6c (30 pmol, i.pl.) did not modify endothelin‐1‐induced (10 pmol) nociception or oedema, but abolished hyperalgesia. Thus, endothelin‐1 triggers ETA receptor‐mediated nociception, hyperalgesia and oedema in the mouse hind‐paw. Simultaneous activation of ETB receptors by endothelin‐1 or selective agonists can limit the hyperalgesic, but not the nociceptive or oedematogenic, effects of the peptide.

In vivo B1 kinin-receptor upregulation. Evidence for involvement of protein kinases and nuclear factor κB pathways

Intradermal (i.d.) injection of cytokines, IL‐1β and TNFα (5 ng, 60 and 30 min prior) produces a rapid onset up‐regulation of des‐Arg9‐BK‐mediated rat paw oedema. Here we analyse the mechanisms involved in des‐Arg9‐BK‐induced oedema in animals pre‐treated with IL‐1β or TNFα. Co‐injection of anti‐IL‐1β, anti‐TNFα and anti‐IL‐8 (50 ng) significantly inhibited des‐Arg9‐BK‐induced oedema in animals pre‐treated with IL‐1β (65, 37 and 42%) or TNFα (39, 64, 25%). IL‐1 receptor antagonist (IRA, 100 μg) or IL‐10 (10 ng) inhibited the oedema caused by des‐Arg9‐BK, in rats that had received either IL‐1β (67 and 63%) or TNFα (46 and 35%). Co‐injection of the PKC inhibitors, staurosporine (10 nmol) or RO 318220 (30 nmol) inhibited des‐Arg9‐BK‐induced paw oedema (44 and 42% for IL‐1β and, 53 and 30% for TNFα, respectively). Genistein (tyrosine kinase inhibitor, 2.5 mg kg−1, s.c.) or PD 098059 (MAP‐kinase inhibitor, 30 nmol) produced marked inhibition of des‐Arg9‐BK‐induced oedema (58 and 39% for IL‐1β and 31 and 35% for TNFα respectively). The NF‐κB inhibitors TLCK (2 mg kg−1, i.p.) and PDCT (100 mg kg−1, i.p.) significantly inhibited the oedema of des‐Arg9‐BK in IL‐1β (27 and 83%) or TNFα (28 and 80%) pre‐treated animals. It is concluded that up‐regulation of B1 receptors modulated by IL‐1β or TNFα involves the release of other cytokines, activation of PKC and tyrosine kinase pathways, co‐ordinated with the activation of MAP‐kinase and nuclear factor κB, reinforcing the view that B1 receptors may exert a pivotal role in modulating chronic inflammatory processes.

Analgesic activity of the lignans from Lychnophora ericoides.

Lychnophora ericoides is a Brazilian medicinal plant that is commercially available as an analgesic and anti-inflammatory agent. The extract from roots, which yielded 10 lignans, showed analgesic activity in the mouse writhing test and the lignan, cubebin, was one of the most active. Anti-inflammatory and anti-pyretic activities from cubebin (10 mg/kg) revealed no significant effects. In addition two previously unknown methyl clusin derivatives are reported.

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.

Upregulation of B1 receptor mediating des-Arg9-BK-induced rat paw oedema by systemic treatment with bacterial endotoxin.

1 The effect of pretreatment with bacterial endotoxin (LPS, 10 μg, i.v., 24 h) on the bradykinin B1 and B2 receptor‐induced oedema in the rat paw, and the interaction of B1‐mediated responses with other inflammatory mediators, was investigated. 2 Intraplantar (i.pl.) injection of the selective B1 agonist, des‐Arg9‐BK (DABK, 100 nmol) in naive animals pretreated with the angiotensin converting enzyme inhibitor, captopril caused a small increase in paw volume (0.04±0.003 ml, mean±s.e.mean, n = 6), while the B2‐selective agonist, tyrosine8‐bradykinin (T‐BK, 3 nmol) induced marked oedema (0.36±0.02 ml). However, i.pl. injection of DABK (3–300 nmol) in rats pretreated with LPS (24 h beforehand) resulted in a marked dose‐ and time‐related increase in paw volume, with mean ED50 of 24.1 nmol. In contrast, oedema caused by T‐BK (3 nmol) was reduced by 79±4% in animals treated with LPS when compared with naive animals. 3 Oedema caused by prostaglandin E2 (PGE2, 10 nmol) was unaffected by LPS treatment, while oedema induced by histamine (100 nmol), 5‐hydroxytryptamine (5‐HT, 10 nmol) and substance P (SP, 3 nmol) was reduced (P<0.05). 4 The selective B1 antagonist, des‐Arg9[Leu8]‐BK (100–300 nmol), produced dose‐dependent inhibition of DABK (100 nmol)‐induced paw oedema in LPS‐treated animals with mean IC50 of 134 nmol, while the selective B2 antagonists, Hoe 140 and NPC 17731 (each 10 nmol), had no effect. 5 Treatment of animals with dexamethasone (0.5 mg kg−1, s.c.) 24 or 48 h prior to LPS injection resulted in a graded inhibition of DABK (100 nmol)‐induced oedema formation (58±3 and 82±2%, respectively), and almost reversed to control value oedema formation induced by T‐BK (3 nmol) in LPS‐pretreated rats. Cycloheximide (1 mg kg−1, s.c.) or indomethacin (2 mg kg−1, i.p.) pretreatment 24 and 1 h prior to LPS injection, respectively, markedly inhibited DABK (100 nmol)‐induced paw oedema (98±2 and 50±4%, respectively). 6 Intraplantar injection of submaximal dose of DABK (10 nmol) in LPS‐treated rats produced modest paw oedema (0.09±0.03 ml). However, i.pl. injections of PGE2, prostacyclin (PGI2), calcitonin‐gene‐related peptide (CGRP), SP, 5‐HT, or platelet activating factor (PAF) (each 1 nmol), which alone caused little or no paw oedema, resulted in a potentiation of the DABK‐induced oedema. The increases in paw volume (in ml) were: PGE2 + DABK (0.31±0.03), PGI2 + DABK (0.39±0.02), CGRP + DABK (0.35±0.04), DABK + SP (0.33±0.04), DABK + 5‐HT (0.40±0.02) and DABK + PAF (0.38±0.016) ml. In contrast, histamine (1 nmol) was ineffective in potentiating the response to DABK. 7 The selective B1 receptor antagonist, DALBK (100–300 nmol), produced dose‐dependent inhibition of paw oedema potentiation induced by co‐injection of DABK and other mediators with mean ID50s (nmol) of: 180, 160, 139 and 135 in the presence of PGE2, PGI2, SP and 5‐HT, respectively. 8 These results demonstrate that DABK‐induced increase in paw volume in LPS‐treated rats is probably mediated by induction of B1 receptors, associated with downregulation of B2 receptors. The induction of B1 receptors by LPS is sensitive to dexamethasone and cycloheximide treatment and requires activation of cyclo‐oxygenase pathway. In addition, B1 receptors, when upregulated following LPS treatment, can interact in a synergistic manner with several inflammatory mediators such as PGI2, PGE2, CGRP, PAF and 5‐HT. Such results indicate that induction of the B1 receptor might have a significant pathophysiological role in modulating chronic inflammatory diseases.

Orofacial cold hyperalgesia due to infraorbital nerve constriction injury in rats: reversal by endothelin receptor antagonists but not non-steroidal anti-inflammatory drugs.

Abstract The susceptibility of changes in responsiveness to noxious cold stimulation of rats submitted to chronic constriction of the infraorbital nerve (CION) or carrageenan to drug inhibition was compared. Nocifensive responses were measured as total time rats engaged in bilateral facial grooming with both forepaws over the first 2 min following tetrafluoroethane spray application to the snout. Carrageenan (50 &mgr;g, s.c. into upper lip) caused short‐lived ipsilateral cold hyperalgesia (peak at 3 h: vehicle 8.4 ± 1.3, carrageenan 21.2 ± 3.0 s) which was markedly suppressed by i.p. indomethacin (4 mg/kg), celecoxib (10 mg/kg) or s.c. dexamethasone (0.5 mg/kg), endothelin ETA or ETB receptor antagonists (BQ‐123 and BQ‐788, respectively; 10 nmol/lip). CION caused ipsilateral cold hyperalgesia between Days 2 and 12, which peaked on Days 4 (sham 15.3 ± 1.8, CION 32.4 ± 5.3 s) to 6. Established peak CION‐induced cold hyperalgesia was unaffected by indomethacin and celecoxib, whereas dexamethasone, BQ‐123, BQ‐788, and i.v. injections of selective antagonists of ETA (atrasentan, 3–10 mg/kg) or ETB (A‐192621, 5–20 mg/kg) receptors caused significant inhibitions lasting 1–2.5 h (peaks ˜65–90%). Bosentan (dual ETA/ETB receptor antagonist, 10 mg/kg, i.v.) abolished CION‐induced cold hyperalgesia for up to 6 h. Thus, once established, CION‐induced orofacial hyperalgesia to cold stimuli appears to lack an inflammatory component, but is alleviated by endothelin ETA and/or ETB receptor antagonists. If this CION injury model bears predictive value to trigeminal neuralgia (i.e., paroxysmal orofacial pain triggered by various stimuli), endothelin receptors might constitute new targets for treatment of this disorder.

Anti-inflammatory, analgesic and anti-oedematous effects of Lafoensia pacari extract and ellagic acid.

Lafoensia pacari St. Hil. (Lythraceae) is used in traditional medicine to treat inflammation. Previously, we demonstrated the anti‐inflammatory effect that the ethanolic extract of L. pacari has in Toxocara canis infection (a model of systemic eosinophilia). In this study, we tested the antiinflammatory activity of the same L. pacari extract in mice injected intraperitoneally with β‐glucan present in fraction 1 (F1) of the Histoplasma capsulatum cell wall (a model of acute eosinophilic inflammation). We also determined the anti‐oedematous, analgesic and anti‐pyretic effects of L. pacari extract in carrageenan‐induced paw oedema, acetic acid writhing and LPS‐induced fever, respectively. L. pacari extract significantly inhibited leucocyte recruitment into the peritoneal cavity induced by β‐glucan. In addition, the L. pacari extract presented significant analgesic, anti‐oedematous and anti‐pyretic effects. Bioassay‐guided fractionation of the L. pacari extract in the F1 model led us to identify ellagic acid. As did the extract, ellagic acid presented anti‐inflammatory, anti‐oedematous and analgesic effects. However, ellagic acid had no anti‐pyretic effect, suggesting that other compounds present in the plant stem are responsible for this effect. Nevertheless, our results demonstrate potential therapeutic effects of L. pacari extract and ellagic acid, providing new prospects for the development of drugs to treat pain, oedema and inflammation.

Essential role for endothelin ETB receptors in fever induced by LPS (E. coli) in rats

1 The influence of endothelin receptor antagonists on febrile responses to E. coli lipopolysaccharide (LPS), interleukin‐1β (IL‐1β), tumour necrosis factor‐α (TNF‐α) and endothelin‐1 (ET‐1) was assessed in conscious rats. 2 Intravenous (i.v.) LPS (5.0 μg kg−1) markedly increased rectal temperature to a peak of 1.30°C over baseline at 2.5 h. Pretreatment with the mixed endothelin ETA/ETB receptor antagonist bosentan (10 mg kg−1, i.v.) or the selective endothelin ETB receptor antagonist BQ‐788 (N‐cis‐2,6‐dimethyl‐piperidinocarbonyl‐L‐γ‐methylleucyl‐D‐1‐methoxycarboyl‐D‐norleucine; 3 pmol, into a lateral cerebral ventricle–i.c.v.) reduced the peak response to LPS to 0.90 and 0.75°C, respectively. The selective endothelin ETA receptor antagonist BQ‐123 (cyclo[D‐Trp‐D‐Asp‐Pro‐D‐Val‐Leu]; 3 pmol, i.c.v.) was ineffective. 3 Increases in temperature caused by IL‐1β (180 fmol, i.c.v.), TNF‐α (14.4 pmol, i.c.v.) or IL‐1β (150 pmol kg−1, i.v.) were unaffected by BQ‐788 (3 pmol, i.c.v.). 4 Central injection of endothelin‐1 (0.1 to 3 fmol, i.c.v.) caused slowly‐developing and long‐lasting increases in rectal temperature (starting 2 h after administration and peaking at 4–6 h between 0.90 and 1.15°C) which were not clearly dose‐dependent. The response to endothelin‐1 (1 fmol, i.c.v.) was prevented by BQ‐788, but not by BQ‐123 (each at 3 pmol, i.c.v.). Intraperitoneal pretreatment with the cyclo‐oxygenase inhibitor indomethacin (2 mg kg−1), which partially reduced LPS‐induced fever, did not modify the hyperthermic response to endothelin‐1 (3 fmol, i.c.v.). 5 Therefore, central endothelin(s) participates importantly in the development of LPS‐induced fever, via activation of a prostanoid‐independent endothelin ETB receptor‐mediated mechanism possibly not situated downstream from IL‐1β or TNF‐α in the fever cascade.