Sérgio H. Ferreira

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.

Central and peripheral antialgesic action of aspirin-like drugs

The peripheral and central effects of some non-steroid anti-inflammatory drugs, aspirin, indomethacin, paracetamol and phenacetin were studied by comparing their intraplantar and intracerebroventricular effects on hyperalgesia induced by carrageenin injected into the rat paw. Hyperalgesia was measured by a modification of the Randall-Selitto test. The agents tested had antialgesic effects when given by any route. Their intraventricular administration enhanced the antialgesic effect of anti-inflammatory drugs administered into the paw. Previous treatment of one paw with carrageenin reduced the oedema caused by a second injection of carrageenin in the contralateral paw. In contrast, it had no effect on the intensity of hyperalgesia but shortened the time necessary for it to reach a plateau. Administration of a prostaglandin antagonist (SC-19220) in the cerebral ventricles, in the rat paw or in both sites, significantly inhibited the hyperalgesia evoked by carrageenin. The maximal hyperalgesic effect of intraplantar injections of prostaglandin E2 could be further enhanced by its cerebroventricular administration. It was suggested that carrageenin hyperalgesia has a peripheral and a central component and that the cyclo-oxygenase inhibitors used may exert an antialgesic effect by preventing the hyperalgesia induced by a peripheral and/or central release of prostaglandins.

Peripheral analgesia and activation of the nitric oxide-cyclic GMP pathway

We have previously described the analgesic effect of dibutyryl cyclic GMP or acetylcholine (ACh) injected into rat paws. Since ACh induces nitric oxide (NO) release from endothelial cells, we investigated the possible involvement of the NO-cyclic GMP pathway in ACh-induced analgesia, using a modification of the Randall-Selitto rat paw test. We found that sodium nitroprusside, which releases NO non-enzymatically, caused antinociception in the rat paw made hyperalgesic with prostaglandin E2. The analgesic effect of sodium nitroprusside and ACh was enhanced by intraplantar injection of an inhibitor of cyclic GMP phosphodiesterase (MY 5445) and was blocked by a guanylate cyclase inhibitor, methylene blue (MB). The analgesia induced by ACh, but not by sodium nitroprusside, was blocked by NG-monomethyl-L-arginine (L-NMMA), an inhibitor of the formation of NO from L-arginine. L-arginine itself had little or no effect upon prostaglandin-induced hyperalgesia but caused significant analgesia in paws inflamed with carrageenin. This analgesia was blocked by MB, as well as by L-NMMA, and was potentiated by MY 5445. These results suggest that ACh-induced analgesia was mediated via the release of NO. The results also indicate that the guanylate cyclase system is stimulated in the inflammatory reaction. The analgesia resulting from activation of this system is possibly overshadowed by substances that concomitantly stimulate nociceptor hyperalgesic mechanisms.

The molecular mechanism of action of peripheral morphine analgesia: stimulation of the cGMP system via nitric oxide release

In addition to the central and spinal sites of action of morphine both our laboratory and others have demonstrated that opiates can also cause analgesia through a peripheral mechanism (Ferreira, 1983). However, the molecular basis of the mechanism of the analgesic actions of opiates remains unknown. In vitro studies show that morphine is able to inhibit activation of adenylate cyclase (Collier and Roy, I974) as well as to stimulate formation of cGMP (Minneman and Iversen, I976). Recently, using the rat paw hyperalgesia test, we showed that in the periphery, acetylcholine-induced analgesia was mediated via the activation of the nitric oxide/cGMP pathwny (Duarte et al., I990). This conclusion was based upon the fact that intraplantar injection of sodium nitroprusside, a substance which nonenzymatically releases nitric oxide (NO), caused analgesia. Furthermore, the analgesic effects of acetylcholine (ACh) and sodium nitroprusside were enhanced by intraplantar injection of an inhibitor of cyclic GMP phosphodiesterase, My5445. In addition, methylene bk:e (MB), an inhibitor of guanylate cyclase, blo1.:ked the analgesia induced by acetylcholine and sodium nitroprusside. On the other hand, the analgesia induced by acetylcholine, but not by sodium nitroprusside, was blocked by N°-monomethyl-L-arginine (LNMMA), an inhibitor of the formation of NO from L-arginine. Due to the similarity of the local action of ACh and opiates we investigated whether agents which affect the arginine/NO-cGMP pathway also interfere with morphine-induced peripheral analgesia as tested with our modification of the Randall-Selitto rat paw pressure test (see Duarte et al., I990). In this test a constant pressure of 20 mmHg is applied to the hind paw of rats

Bradykinin initiates cytokine-mediated inflammatory hyperalgesia

1 The hyperalgesic activities in rats of bradykinin, carrageenin and lipopolysaccharide (LPS) were investigated in a model of mechanical hyperalgesia. 2 Bradykinin and carrageenin evoked dose‐dependent hyperalgesia with maximum responses of similar magnitude to responses to LPS (1 and 5 μg). 3 Hoe 140, an antagonist of BK2 receptors, inhibited in a dose‐dependent manner hyperalgesic responses to bradykinin, carrageenin and LPS (1 μg) but not responses to LPS (5 μg), prostaglandin E2, dopamine, tumour necrosis factor α (TNFα), IL‐1, IL‐6 and IL‐8. 4 Responses to bradykinin and LPS (1 and 5 μg) were inhibited by the cyclo‐oxygenase inhibitor, indomethacin and by the β‐adrenoceptor antagonist, atenolol. The effects of indomethacin and atenolol were additive: their combination abolished responses to bradykinin and LPS (1 μg) and markedly attenuated the response to LPS (5 μg). 5 Antiserum neutralizing endogenous TNFα abolished the response to bradykinin whereas antisera neutralizing endogenous IL‐1β, IL‐6 and IL‐8 each partially inhibited the response. The combination of antisera neutralizing endogenous IL‐1β + IL‐8 or IL‐6 + IL‐8 abolished the response to bradykinin. 6 Antisera neutralizing endogenous TNFα, IL‐1β, IL‐6 and IL‐8 each partially inhibited responses to LPS (1 and 5 μg). Increasing the dose of antiserum to TNFα or giving a combination of antisera to IL‐1β + IL‐8 or IL‐6 + IL‐8 further inhibited responses to LPS (1 and 5 μg). 7 These data show that bradykinin can initiate the cascade of cytokine release that mediates hyperalgesic responses to carrageenin and endotoxin (1 μg). The lack of effect of Hoe 140 on hyperalgesic responses to LPS (5 μg) suggests that the release of hyperalgesic cytokines can be initiated independently of bradykinin BK2 receptors.

Inhibition of prostaglandin biosynthesis as the mechanism of analgesia of aspirin-like drugs in the dog knee joint

A method has been developed to measure the analgesic action of aspirin-like drugs in knee joints of anaesthetized dogs. Bradykinin, injected into the joint cavity, induced a reflex rise in blood pressure which was dose-dependent; this was used as a measure of nociceptive activity. The joint cavity became more sensitive to bradykinin as the experiment proceeded, or when a low concentration of prostaglandin E1 or E2 was infused locally. The increase in sensitivity with time was prevented by local injection of aspirin or indomethacin, but that induced by exogenous prostaglandin infusion was not. Injections of carrageenin into dog knee joints increased the prostaglandin E2 content of synovial fluid by up to 160 ng per joint; indomethacin prevented this increase. These experiments support our previous conclusion that local biosynthesis of a prostaglandin (induced by mild trauma) sensitizes pain receptors to mechanical or chemical stimuli. Aspirin-like drugs are analgesic because they prevent prostaglandin biosynthesis, thereby preventing this sensitization.

Analgesia by direct antagonism of nociceptor sensitization involves the arginine-nitric oxide-cGMP pathway

We tested the hypothesis that activation of the nitric oxide (NO)-cGMP pathway is involved in the mechanism of two directly acting non-opiate peripheral analgesics, myrcene and dipyrone, using our modification of the Randall-Selitto test. The NO inhibitor, NG-monomethyl-L-arginine (50 micrograms/paw) and methylene blue (500 micrograms/paw) abolished the analgesic effect of dipyrone and myrcene. Dibutyryl cyclic adenosine monophosphate (DbcAMP) caused a dose-dependent hyperalgesia (20, 50 and 100 micrograms/paw). Only responses to low doses of DbcAMP were inhibited by the two analgesics. Pretreatment with MY5445 (50 micrograms/paw) resulted in potentiation of the effects of both analgesics. These results support our hypothesis that the sensitivity of nociceptors may be controlled by the balance between the levels of cAMP and cGMP. Stimulation of the NO-cGMP pathway is probably the common denominator for the mode of action of peripheral analgesics which block hyperalgesia directly.

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.

IL-33 induces neutrophil migration in rheumatoid arthritis and is a target of anti-TNF therapy

Objectives Interleukin 33 (IL-33) is a new member of the IL-1 family of cytokines which signals via its receptor, ST2 (IL-33R), and has an important role in Th2 and mast cell responses. This study shows that IL-33 orchestrates neutrophil migration in arthritis. Methods and results Methylated bovine serum albumin (mBSA) challenge in the knee joint of mBSA-immunised mice induced local neutrophil migration accompanied by increased IL-33R and IL-33 mRNA expression. Cell migration was inhibited by systemic and local treatments with soluble (s)IL-33R, an IL-33 decoy receptor, and was not evident in IL-33R-deficient mice. IL-33 injection also induced IL-33R-dependent neutrophil migration. Antigen- and IL-33-induced neutrophil migration in the joint was dependent on CXCL1, CCL3, tumour necrosis factor α (TNFα) and IL-1β synthesis. Synovial tissue, macrophages and activated neutrophils expressed IL-33R. IL-33 induces neutrophil migration by activating macrophages to produce chemokines and cytokines and by directly acting on neutrophils. Importantly, neutrophils from patients with rheumatoid arthritis successfully treated with anti-TNFα antibody (infliximab) expressed significantly lower levels of IL-33R than patients treated with methotrexate alone. Only neutrophils from patients treated with methotrexate alone or from normal donors stimulated with TNFα responded to IL-33 in chemotaxis. Conclusions These results suggest that suppression of IL-33R expression in neutrophils, preventing IL-33-induced neutrophil migration, may be an important mechanism of anti-TNFα therapy of inflammation.

Regulation of chemokine receptor by Toll-like receptor 2 is critical to neutrophil migration and resistance to polymicrobial sepsis

Patients with sepsis have a marked defect in neutrophil migration. Here we identify a key role of Toll-like receptor 2 (TLR2) in the regulation of neutrophil migration and resistance during polymicrobial sepsis. We found that the expression of the chemokine receptor CXCR2 was dramatically down-regulated in circulating neutrophils from WT mice with severe sepsis, which correlates with reduced chemotaxis to CXCL2 in vitro and impaired migration into an infectious focus in vivo. TLR2 deficiency prevented the down-regulation of CXCR2 and failure of neutrophil migration. Moreover, TLR2−/− mice exhibited higher bacterial clearance, lower serum inflammatory cytokines, and improved survival rate during severe sepsis compared with WT mice. In vitro, the TLR2 agonist lipoteichoic acid (LTA) down-regulated CXCR2 expression and markedly inhibited the neutrophil chemotaxis and actin polymerization induced by CXCL2. Moreover, neutrophils activated ex vivo by LTA and adoptively transferred into naïve WT recipient mice displayed a significantly reduced competence to migrate toward thioglycolate-induced peritonitis. Finally, LTA enhanced the expression of G protein–coupled receptor kinases 2 (GRK2) in neutrophils; increased expression of GRK2 was seen in blood neutrophils from WT mice, but not TLR2−/− mice, with severe sepsis. Our findings identify an unexpected detrimental role of TLR2 in polymicrobial sepsis and suggest that inhibition of TLR2 signaling may improve survival from sepsis.