Collagenases are thought to play a major role in the pathology of gas gangrene caused by Clostridium histolyticum, because they can destroy the connective tissue barriers. We investigated possible mediators involved in the oedema formation and plasma protein extravasation which follow the injection of a collagenase (EC 3.4.24.3) from Clostridium histolyticum into one hind paw of anaesthetized rats. 2. The magnitude of the oedema following a subplantar injection was dependent on the dose of collagenase (30, 100 and 300 micrograms) injected. It reached its maximum within 30 min and remained unchanged for at least 5 h. Plasma protein extravasation into the paw was most pronounced within 20 min of the injection. Heat-inactivated collagenase was ineffective. 3. The B2 bradykinin (BK) antagonist icatibant (D-Arg-[Hyp3-Thi5-D-Tic7- Oic8] bradykinin, formerly named Hoe-140) reduced oedema formation in a dose-dependent manner with a maximal reduction of around 65% at a dose of 100 nmol kg-1 (s.c.). A significant effect could already be observed at a dose of 10 nmol kg-1. The duration of the effect of icatibant (100 nmol kg-1) was found to be at least 3 h. These results demonstrate the high potency and long duration of action of icatibant. Pretreatment of rats with the bradykinin B1 antagonist, des-Arg9-[Leu8]-BK did not affect collagenase-induced paw oedema. Thus, the observed collagenase-induced effects are mainly mediated by BK through activation of B2 receptors. 4. Pretreatment of adult rats with capsaicin (125 mg kg-1, s.c.) three weeks before the collagenase injection caused a significant attenuation of the paw oedema and of plasma extravasation but was significantly less effective than icatibant (100 nmol kg-1, s.c.). The non-peptide substance P antagonist,CP-96,345 (l0 micromol kg-1, i.v.) significantly reduced collagenase-induced oedema formation to a degree comparable with that seen after capsaicin pretreatment. The inhibition by the substance P antagonist was significantly smaller than that seen after icatibant. The inhibitory effect of icatibant in capsaicin pretreated rats, or of icatibant together with CP-96,345 in untreated rats, was not greater than that oficatibant alone in rats treated with the vehicle for either capsaicin or CP-96,345. CP-96,344(10 micromol kg-1, i.v.), the inactive enantiomer of CP-96,345, did not affect collagenase-induced paw oedema. In capsaicin-pretreated rats, CP-96,345 (10 micromol kg-1, i.v.) did not reduce collagenase-induced paw oedema.The subplantar injection of bradykinin (30 nmol) induced a paw oedema comparable with that induced by collagenase (100 microg). CP-96,345 (10 micromol kg-1, i.v.), but not CP-96,344 (1O micromol kg-1, i.v.),significantly reduced the bradykinin-induced paw oedema. These findings indicate that collagenase leads to the release of bradykinin; bradykinin then stimulates afferent C-fibre terminals and causes the release of substance P and probably also neurokinin A, which augment the oedema-inducing effect of bradykinin.5. Indomethacin or mepyramine plus cimetidine failed to inhibit collagenase-induced paw oedema.Thus, prostaglandins and histamine do not seem to be involved in collagenase-induced paw oedema.6. After subplantar injection of collagenase, the sensitivity scores in a modified formalin-test rapidly increased during the first 10 min. This increase was abolished by pretreatment with icatibant(100 nmol kg-1, s.c.) indicating that the stimulation of nociceptive afferent neurones following injection of collagenase is due to the action of released kinins.7. In conclusion, bradykinin appears to be the main mediator of inflammation induced by a collagenase from Clostridium histolyticum. As well as having direct relevance to a known pathological condition,collagenase-induced paw oedema could prove to be a useful model in inflammation research and in the investigation of bradykinin antagonists. The present results might provide an experimental basis for clinical investigations of the effects of icatibant in infectious diseases where the release of collagenases from bacteria causes rapid spreading of inflammation.
