Blockade of proteinase-activated receptor 4 inhibits neutrophil recruitment in experimental inflammation in mice

Objective and design

The activation of proteinase-activated receptors (PARs) has been implicated in the development of important hallmarks of inflammation, including in vivo leukocyte recruitment; however, its role in the regulation of leukocyte migration in response to inflammatory stimuli has not been elucidated until now. Here, we examined the effects of the PAR4 antagonist YPGKF-NH 2 (tcY-NH₂) on neutrophil recruitment in experimentally induced inflammation.

Methods

BALB/c mice were intrapleurally injected with tcY-NH₂ (40 ng/kg) prior to intrapleural injection of carrageenan (Cg) or neutrophil chemoattractant CXCL8; the number of infiltrating neutrophils was evaluated after 4 h, and KC production was assessed at different times after Cg injection. Neutrophil adhesion and rolling cells were studied using a brain circulation preparation 4 h after the Cg or CXCL8 challenge in tcY-NH₂-treated mice.

Results

PAR4 blockade inhibited CXCL8- and Cg-induced neutrophil migration into the pleural cavity of BALB/c mice and reduced neutrophil rolling and adherence. Surprisingly, PAR4 blockade increased the level of KC in response to carrageenan.

Conclusion

These results demonstrated that PAR4 blockade impairs neutrophil migration in vivo, suggesting that PAR4 plays an important role in the regulation of inflammation, at least in part because of its ability to inhibit the actions of the neutrophil chemoattractant CXCL8.     

Evidence for participation of GABA(A) receptors in a rat model of secondary hyperalgesia

We investigated the involvement of endogenous gamma-aminobutyric acid (GABA) in the modulation of secondary hyperalgesia induced by intraplantar (i.pl.) injection of 5% formalin in the rat tail-flick test. Intraplantar injection of gabamimetic drugs such as gabapentin (150-600 microg/site) or phenobarbital (20-80 microg/site) reversed secondary hyperalgesia, as measured by an increase in the tail-flick latency, thus displaying a peripheral antihyperalgesic effect. Central inhibition of the secondary hyperalgesia response by gabapentin was obtained following injection of either 200 microg intrathecally (i.t.) or 50 mg intraperitoneally (i.p.). The effects induced by gabamimetics were blocked locally or centrally by prior treatment with the specific GABA(A) receptor antagonist, bicuculline (80 ng/paw or 20 ng, i.t.). These data indicate the participation of endogenous GABA in the modulation of secondary hyperalgesia, through either a peripheral and/or a central action. They also indicate that GABA(A) receptors might be involved since a specific antagonist of these receptors (bicuculline) blocked this response.     

Analgesic and antiinflammatory effects of dipyrone in rat adjuvant arthritis model

Dipyrone, a pirazolone derivative, is a known analgesic drag with minor toxic effects associated with its administration. The aim of the present study was to determine the analgesic and antiinflammatory effects of dipyrone in a model of chronic inflammation (adjuvant-induced arthritis in rats). Hind-paw hyperalgesia was detected in arthritic rats from the 10th to the 16th day of observation. Edema development was maximum (twofold increase) at the 14th day of observation compared to control animals and reduced at the 16th day of observation. Dipyrone (1–50 mg/kg) dose-dependently reduced both hind-paw hyperalgesia and edema from arthritic rats. However, it was shown to be more potent as analgesic than antiinflammatory in the present model. In contrast, indomethacin (2 mg/kg) and dexamethasone (0.4 mg/kg) completely inhibited hind-paw hyperalgesia and edema development. Our results indicate that dipyrone reduced the hyperalgesia and edema in arthritic rats by a mechanism not involving release of prostaglandin-like substances. The possibility of dipyrone inducing analgesia in arthritic rats through a peripheral action supports the use of dipyrone as an alternative choice drug for the treatment of pain associated with arthritislike diseases in selected cases.     

Tissue-selective inflammation in the oral cavity of the rat

In the current study, carrageenan (CG; 100–1000 μg/site) was injected intraorally in the cheeks of Holtzman or Wistar rats to evaluate the consequences of administration of a non-immunogenic stimulus in the orofacial region. Subsequent inflammation was measured as oedema (increased thickness of the cheek wall using digital calipers), relative to the other cheek injected with saline. Oedema formation and tissue collection for histopathological studies were assessed at 0.5, 1, 2, 3, 4, 6, 24, 48, 72, 96, 120 and 144 h after injection. In parallel, other groups of rats were injected with CG in the hind paw, to provide a reference response. The inhibitor of prostaglandin biosynthesis, indomethacin, and antagonists of histamine, serotonin and NK₁ receptors were injected s.c., 0.5 h before CG. CG induced a dose-related oedema more rapidly from 0 to 2 h which lasted for at least 72 h, showing a biphasic profile (peak at 2 and 24 h), compared with the monophasic oedema induced in rat paws (maximal duration of 24 h). Histopathological analysis of the CG-injected cheek revealed oedema formation with little leukocyte recruitment at 1–3 h, mast cell degranulation at 6 h, and a mixed polymorphonuclear and mononuclear cell infiltrate by 24 h. Histamine and serotonin antagonists and indomethacin, but not the NK₁ antagonist, decreased cheek oedema in the first 4 h following carrageenan. Taken together, our data indicated important differences in the pattern of inflammation between the oral cavity and the paw which will determine the therapeutic approach to the treatment of inflammatory conditions in the oral cavity.     

Inflammation mobilizes local resources to control hyperalgesia: the role of endogenous opioid peptides

The aim of the present study was to investigate the mechanisms underlying the endogenous control of nociception at a peripheral level during inflammation. Using a pharmacological approach and the rat paw pressure test, we assessed the effect of an intraplantar injection of naloxone, an opioid receptor antagonist, and bestatin, an aminopeptidase inhibitor, on hyperalgesia induced by carrageenan, which mimics an inflammatory process, or prostaglandin E(2) (PGE(2)), which directly sensitizes nociceptors. Naloxone induced a significant and dose-dependent (25, 50 or 100 μg) increase in carrageenan-induced hyperalgesia, but not PGE(2)-induced hyperalgesia. Bestatin (400 μg/paw) significantly counteracted carrageenan-induced hyperalgesia, inducing an increase in the nociceptive threshold compared to control, but it did not modify hyperalgesia induced by PGE(2) injection into the rat paw. Positive β-endorphin immunoreactivity was increased in paw inflammation induced by carrageenan in comparison with the control group. However, PGE(2) did not significantly alter the immunostained area. These results provide evidence for activation of the endogenous opioidergic system during inflammation and indicate that this system regulates hyperalgesia through a negative feedback mechanism, modulating it at a peripheral level.     

Crucial role of peripheral kappa-opioid receptors in a model of periodontal disease in rats

Background and Objective: Periodontal disease is a chronic inflammatory condition of the tooth supporting tissues, the periodontium. Opioids have been shown to account for the relief of various chronic and acute inflammatory conditions. The aim of the present study was to investigate the participation of peripheral opioid receptors in development of periodontal disease. Material and Methods: Morphine and selective agonists and antagonists of opioid receptors were used in an experimental model of ligature‐induced periodontal disease in rats. To evaluate the development of disease, the loss of fiber attachment, alveolar bone and number of cells in periodontal tissues were assessed. Measurements of these indicators were obtained by morphometric analysis of histological sections of periodontal‐diseased tissues stained with hematoxylin and eosin. Results: Local administration of either morphine or a selective κ‐opioid agonist for three consecutive days from the onset of periodontal disease reduced the loss of periodontal tissues, without changing the number of leukocytes in inflamed periodontium. Nor‐binaltorphimine, a selective κ‐antagonist, reversed the beneficial effects of both morphine and the compound U‐50,488 in this model. The use of either an agonist or an antagonist of δ‐opioid receptors, however, did not affect disease progression. Conclusion: Our results showed that the beneficial effect of opioids in periodontal disease depended mainly on the activation of specific κ‐opioid receptors located in the periphery. Activation of such receptors could be considered in the management of periodontal disease, since it would not present the classical central side‐effects associated with opioid use.     

Vascular and cellular responses to pro-inflammatory stimuli in rat dental pulp

Dental pulp reactivity to various pro-inflammatory stimuli was independently evaluated in rats in terms of a vascular permeability increase and leukocyte recruitment. Substance P, calcitonin-gene related peptide (CGRP) and prostaglandin E(2) (in the picomol range) were applied to the exposed pulp from anesthetised animals and the plasma extravasation measured by the Evans blue content in the tissue following 10 min of administration. Leukocyte recruitment was evaluated morphometrically by counting the cell number present in serial sections of 1:3 4 microm pulp tissue 6 h after bacterial endotoxin (LPS; 0.06-1.2 microg/site) administration. Increase in pulp vascular permeability and cellular recruitment due to the injection of mentioned mediators in the skin or LPS in the peritoneal cavity were used as positive controls. Increase in vascular permeability in the pulp occurred in the same dose-range as observed in the skin, being CGRP the most active substance in both cases. However, it was necessary a higher dose of LPS (1.2 microg) to induce a similar cell recruitment in the pulp as that observed in the rat peritoneal cavity (0.3 microg). These data indicate that dental pulp reactivity presents the same pattern of increase in vascular permeability to other tissues in the rat, being CGRP the most potent mediator in this respect. In addition, they suggest the presence of CGRP receptors in the dental pulp. However, an adequate leukocyte recruitment response to bacterial endotoxin was not mounted, suggesting a deficiency in adhesion molecules in blood vessels in the rat dental pulp.     

Effect of tumor necrosis factor receptor binding protein on cell infiltration induced by lipopolysaccharide and sephadex beads in guinea pig lung

In the present study, the effect of a tumor necrosis factor receptor binding protein (TNFbp) on the cell infiltration induced by lipopolysaccharide (LPS) and Sephadex beads in guinea pig lung was examined. The intratracheal injection of LPS (2.5g) induced a six-fold increase in total cell number recovered in bronchoalveolar lavage (BAL) fluid at 24 hr. This increase in bronchopulmonary inflammation was mainly due to a neutrophil and macrophage infiltration, representing 60% and 35% of the total cells, respectively. The intravenous or intratracheal injection of Sephadex beads to guinea pigs induced a three-fold increase in total cell number recovered in BAL at 24 h and was characterized by a prominent eosinophil, macrophage, and neutrophil infiltration representing 36%, 42%, and 16% of the total cells, respectively. In addition, bronchial tissues isolated from Sephadex-treated guinea pigs showed an increased in vitro reactivity to both histamine and acetylcholine. TNFbp (1–50g) induced a dose-dependent inhibition of cell infiltration induced by LPS. In contrast TNFbp neither attenuated the bronchopulmonary cell infiltration observed 24 h following intravenous or intratracheal administration of Sephadex beads nor inhibited the increase in bronchial reactivity. These results show that TNF plays an important role in cell infiltration induced by LPS, but not that induced by Sephadex, in the guinea pig lung.