Johan Raud

Potent anti-inflammatory action of calcitonin gene-related peptide

Calcitonin gene-related peptide (CGRP), but not substance P (SP), was found to inhibit edema-promoting actions of inflammatory mediators (histamine, leukotrine B4, 5-hydroxytryptamine) in vivo in the hamster cheek pouch, human skin, and rat paw. The effect of CGRP was present in the low nanomolar dose range, and it was mimicked by activation of sensory nerves with capsaicin which caused release of endogenous CGRP-like immunoreactivity (IR). The findings provide new information on the potential impact of sensory nerve activation during inflammatory processes by indicating that sensory nerves may play an anti-inflammatory role.

Characteristics of histamine‐induced leukocyte rolling in the undisturbed microcirculation of the rat mesentery

The main objective of this study was to analyse the role and mode of action of the mast cell mediator histamine in leukocyte‐endothelium interactions in small venules in vivo. For this purpose, we used a histological approach (combined with intravital microscopy) that allows studies of rapid mediator‐induced venular leukocyte accumulation, reflecting leukocyte rolling, in the undisturbed microcirculation of the rat mesentery where rolling is normally absent. We first examined the relative importance of histamine and 5‐hydroxytryptamine (5‐HT) in acute mast cell‐dependent leukocyte recruitment. The mast cell secretagogue compound 48/80 (i.p. for 15 min) induced a marked venular accumulation of polymorphonuclear leukocytes (PMNL) which was almost abolished by combined histamine1 (H1)‐ and histamine2 (H2)‐receptor blockade. In contrast, the 5‐HT‐receptor antagonist methysergide was inactive in this regard. Moreover, exogenous 5‐HT was less active than exogenous histamine in evoking venular PMNL accumulation (histamine response dose‐dependent; 5‐HT response bell shaped). Prostaglandin D2 did not cause PMNL accumulation. The venular PMNL response to exogenous histamine peaked between 15 min and 1 h, was still significantly elevated at 2 h, and then returned to prechallenge values after 3 h. At all time points, the histamine‐induced PMNL accumulation was nearly abolished by i.v. treatment with the polysaccharide fucoidin (which blocks rolling but not firm adhesion per se), suggesting that the PMNL response to histamine was due to rolling rather than firm adhesion over the entire 3 h period. At no time point did histamine trigger accumulation of mononuclear leukocytes (MNL). To examine the role of histamine‐receptors in the histamine‐induced PMNL accumulation (i.e. rolling), the animals were pretreated with diphenhydramine (H1‐receptor antagonist), cimetidine, or ranitidine (H2‐receptor antagonists). Diphenhydramine alone inhibited the venular PMNL response to histamine by 52%, while both H2‐receptor antagonists were completely inactive. However, the combination of cimetidine and diphenhydramine reduced the histamine‐induced PMNL rolling by 82%. Furthermore, in contrast to an H3‐receptor agonist, challenge with either the H1‐receptor agonist 2‐thiazolylethylamine or two different H2‐receptor agonists (impromidine, dimaprit) was sufficient to provoke significant venular PMNL accumulation. Treatment with the nitric oxide‐synthase inhibitor L‐NAME did not affect the histamine‐induced PMNL rolling. On the other hand, 3 h pretreatment with dexamethasone reduced the PMNL response to histamine by 73%, and flow cytometric analysis showed that the dexamethasone treatment almost completely inhibited binding of soluble P‐selectin to rat isolated PMNLs. We conclude that initial leukocyte recruitment after mast cell activation in the rat mesentery is critically dependent on histamine release. The cellular response to histamine was specifically due to PMNL rolling, involved activation of both H1‐ and H2‐receptors, and lasted for 2–3 h. Moreover, the histamine‐induced PMNL rolling was not dependent on nitric oxide synthesis, but was sensitive to glucocorticoid treatment, possibly via inhibition of expression or function of leukocytic P‐selectin ligand(s).

Actions of Lipoxin A4 and Related Compounds in Smooth Muscle Preparations and on the Microcirculation in Vivo

The present chapter summarizes our findings with lipoxins (LX) in spasmogenic assays and in the intact microvasculature of the hamster cheek pouch. The initial observations1,2 were made in experiments using lipoxins isolated from human leukocytes. With the aid of synthetic compounds3, it has been possible to further explore the pharmacodynamics and structure activity relationships for lipoxins in smooth muscle preparations.

Vasodilatation and inhibition of mediator release represent two distinct mechanisms for prostaglandin modulation of acute mast cell-dependent inflammation.

1 Intravital microscopy of the hamster cheek pouch was used to examine the influence of vasodilator prostanoids (prostaglandin E2 (PGE2), PGI2), forskolin, and nitroprusside on the microvascular changes during acute inflammation induced by antigen or histamine. The results extend our previous finding that PGE2 modulates allergic inflammation and histamine release in the cheek pouch model. 2 The microvascular actions of arachidonic acid and different cyclo‐oxygenase products (PGE2, PGD2, PGI2, PGF2α, and the thromboxane A2 (TXA2)‐analogue U‐44069) were first compared with respect to their effects on arteriolar tone. Of the prostaglandins, only PGE2 and PGI2 were potent vasodilators and markedly increased local blood flow. Nitroprusside and forskolin also caused vasodilatation and increased blood flow, but were somewhat less potent than PGE2 and PGI2. 3 Topically applied PGE2 and PGI2 in vasodilator concentrations suppressed the antigen‐induced plasma leakage. On the other hand, although the antigen response was predominantly mediated by histamine, both prostaglandins enhanced the plasma leakage evoked by exogeneous histamine. 4 In contrast, the vasodilator nitroprusside, in a dose causing an increase in blood flow equal to that of PGE2 and PGI2, potentiated both the histamine‐induced plasma leakage, as well as the plasma and leukocyte extravasation after antigen challenge, indicating that the anti‐inflammatory actions of the prostaglandins were unrelated to their vasodilator properties per se. 5 Because forskolin, a specific activator of adenylate cyclase, mimicked the actions of PGE2 and PGI2, i.e. inhibition of the antigen‐induced plasma extravasation and enhancement of the histamine response, it is possible that the observed antiallergic effects of the prostaglandins were related to accumulation of intracellular adenosine 3′: 5′‐cyclic monophosphate (cyclic AMP). 6 Taken together, there appears to be a competition between pro‐ and anti‐inflammatory effects of PGE2 and PGI2 in reactions involving release of endogeneous inflammatory mediators in vivo, i.e. enhancement of inflammatory mediator target action on one hand (‘two mediator synergism’), and suppression of mediator release on the other. Moreover, the observations indicate that vasodilatation and inhibition of mediator release are two distinct actions of PGE2 and PGI2.

Inhibitory effect of locally administered heparin on leukocyte rolling and chemoattractant‐induced firm adhesion in rat mesenteric venules in vivo

1 Anti‐inflammatory actions of heparin and related glycosaminoglycans have been described in the literature. Here, we used intravital microscopy of the rat mesentery microcirculation to examine effects of locally administered heparin on leukocyte rolling and chemoattractant‐induced firm adhesion. 2 It was found that topical application of heparin reduced N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP)‐induced leukocyte adhesion. Notably, the inhibitory action of heparin was not dose‐dependent, but rather a biphasic dose‐response was found, i.e. low (2 and 20 iu ml−1) and high (1000 iu ml−1) concentrations of heparin significantly reduced adhesion, whereas an intermediate dose (200 iu ml−1) was less effective. 3 Heparin, 2 and 20 iu ml−1, decreased rolling leukocyte flux, while having no effect on blood flow or total leukocyte flux. By contrast, heparin, 200 and 1000 iu ml−1, increased total leukocyte flux in parallel with a rise in volume blood flow resulting in recovery of the rolling leukocyte flux at these doses. Thus, the biphasic inhibitory action of heparin on fMLP‐induced firm adhesion could in part be attributed to changes in leukocyte delivery (i.e. blood flow) and rolling leukocyte flux induced by heparin. 4 When compensating for the influence of different rolling levels on fMLP‐evoked adhesion, a dose‐related inhibitory effect of heparin on the firm adhesive response per se was revealed. Similar results were obtained in a static adhesion assay in vitro where heparin 200 and 1000 iu ml−1 (but not 2 and 20 iu ml−1) significantly inhibited fMLP‐induced leukocyte adhesion in the absence of any modulatory influence on changes in rolling. 5 Our data show that locally administered heparin inhibits leukocyte rolling as well as chemoattractant‐induced firm adhesion in vivo which thus may contribute to the postulated anti‐inflammatory activity of this compound. However, because of interference with several microvascular functions, strict dose‐dependent responses to heparin treatment were not found, which illustrates the complex interplay between local blood flow, leukocyte rolling and chemoattractant‐induced adhesion as determinants of leukocyte recruitment to tissues in inflammation.

Microvascular mechanisms of histamine-induced potentiation of leukocyte adhesion evoked by chemoattractants

1 Intravital microscopy of the rat mesentery was used to examine interactions between histamine and the chemoattractant leukotriene B4 (LTB4) with regard to leukocyte adhesion in postcapillary venules. 2 Topical administration of histamine caused a four fold potentiation of LTB4‐induced leukocyte adhesion. 3 Histamine significantly increased the rolling leukocyte flux by 25%, and this effect of histamine on rolling was strictly blood flow‐dependent, i.e. we found significant positive correlations between both blood flow and total leukocyte flux and between total and rolling leukocyte flux, while no changes in leukocyte rolling fraction or rolling velocity were observed. Furthermore, histamine caused a clear‐cut increase in venular plasma protein leakage. 4 The platelet‐activating factor (PAF) receptor antagonist WEB 2086, which effectively inhibited adhesion of leukocytes evoked by exogenous PAF, did not reduce the potentiating effect of histamine on LTB4‐induced leukocyte adhesion. 5 The vasodilator acetylcholine (ACh) caused a moderate enhancement of LTB4‐induced leukocyte adhesion in proportion to its blood flow‐dependent 40% increase in rolling leukocyte flux. In contrast to histamine, ACh did not provoke vascular leakage of plasma proteins. 6 Taken together, our findings suggest that histamine plays an important pro‐inflammatory role in tissues where leukocyte rolling is already present, by potentiating chemoattractant‐induced firm leukocyte adhesion through a combination of microcirculatory changes such as increased rolling leukocyte flux and vascular permeability.

Rabbit leukocyte adhesion molecules CD11CD18 and their participation in acute and delayed inflammatory responses and leukocyte distribution in vivo

In humans the glycoprotein complexes CD11/CD18 mediate leukocyte adhesion to cells. Mouse monoclonal antibodies (mAb) 60.3, 7E4, and IB4 to human CD18, found to cross-react with rabbit white blood cells, were used to identify the antigen in rabbit cells and to study adherence of rabbit leukocytes in vitro and in vivo. These antibodies labeled almost all unfractionated rabbit blood leukocytes and immunoprecipitated surface glycopolypeptides with apparent molecular weights of 85,000 and 150,000 from these cells. Adhesion of purified rabbit polymorphonuclear cells (PMNs) to cultured vascular endothelial cells in the presence of phorbol ester was blocked by the antibodies in a dose-dependent manner. The acute inflammatory response characterized by local accumulation of PMNs and concomitant plasma extravasation following intradermal injections of zymosan-activated serum (ZAS) in rabbits was inhibited in animals pretreated intravenously with anti-CD18 mAb. Intravital microscopy of the rabbit tenuissimus muscle demonstrated that anti-CD18 mAb. Intravital microscopy of the rabbit tenuissimus muscle demonstrated that anti-CD18 treatment specifically blocked the adhesion of activated leukocytes to the venular endothelium and thereby the subsequent diapedesis of these cells into the extravascular space. The lymphocyte-dependent tissue swelling resulting from a delayed-type hypersensitivity reaction in the rabbit ear was partially inhibited by anti-CD18 mAb. Systemic anti-CD18 treatment induced a pronounced increase in the number of circulating mononuclear and polymorphonuclear cells with a maximum at 24 hr after injection of the antibody. It is concluded that GP150/GP85 is the rabbit homologue of human CD11/CD18, and that leukocyte-cell adhesion mediated by these glycoprotein complexes participates in acute and delayed inflammatory responses and leukocyte distribution in vivo.

Lipoxins inhibit microvascular inflammatory actions of leukotriene B4.

The lipoxins constitute a new class of arachidonic acid derivatives which may be produced by for example leukocytes, or by leukocytes interacting with platelets (cf. Dahlen and Serhan 1990). The biosynthesis of lipoxins is the result of interactions between different lipoxygenases, and the lipoxins characteristically possess a conjugated tetraene structure (Serhan et al., 1984; cf. Dahlen and Serhan 1990). The two major compounds, lipoxin A4 and B4 (LXA4, 5S, 6R, 15S-trihydroxy-7, 9, 13-trans-ll-cis-eicosatetraenoic acid; LXB4, 5S,14R,15S-trihydroxy-6, 10, 12-trans-8-cis-eicosatetraenoic acid), display a number of biological actions, indicating their involvement as modulators of events such as intracellular signal transduction, immunological defence and inflammation (cf. Dahlen and Serhan 1990).

An approach for studies of mediator-induced leukocyte rolling in the undisturbed microcirculation of the rat mesentery.

Although intravital microscopy is the method of choice for observation of inflammatory leukocyte rolling and adhesion in small venules in vivo, a problem with this technique is that surgical exposure of suitable tissues per se triggers the rolling mechanism. In this study, we describe an approach to investigate induction of rolling in undisturbed microvessels. For this purpose, intravital microscopic observation of leukocyte rolling and adhesion in the rat mesentery was combined with histological determination of the intravascular concentrations of polymorphonuclear and mononuclear leukocytes (PMNL and MNL). By relating the histologically determined number of intravascular leukocytes to either microvessel volume or to the erythrocyte concentration, the baseline MNL and PMNL content was found to be 3–6 fold higher in venules than in systemic blood. This increase in microvessel leukocyte concentration did not seem to be related to leukocyte‐endothelium interactions, because the leukocyte concentration was similarly elevated in arterioles where rolling and adhesion did not take place. Preparation of the rat mesentery for intravital microscopy time‐dependently increased the venular PMNL concentration to over 100 fold the systemic PMNL concentration 45 min after exteriorization of the small intestine. The MNLs were much less responsive to the preparative manipulation. By treatment with the polysaccharide fucoidin (inhibits rolling but not firm adhesion per se), or by use of intravital microscopy immediately before tissue fixation, approximately 90% of the accumulated venular PMNLs were found to represent rolling cells. Intraperitoneal injection of 10−3M histamine increased the venular PMNL (but not the MNL) concentration to almost 50 fold the systemic PMNL value. The histamine response did not vary with venular diameter, and the relative contribution of rolling vs firmly adherent cells to the PMNL, accumulation was again ≈amp;90%. Intraperitoneal injection of leukotriene C4, but not prostaglandin E2, caused a significant increase in venular PMNL concentration. Systemic treatment with the anti‐P‐selectin monoclonal antibody PB1.3 had no effect on the histamine‐induced venular PMNL accumulation (i.e. rolling) in female Wistar or male Sprague‐Dawley rats. On the other hand, identical treatment with PB1.3 very effectively inhibited the histamine‐induced PMNL response in the mesentery of rabbits. In conclusion, we have shown that a histologically determined increase in leukocyte concentration in rat mesenteric venules may be used as an index of mediator‐induced leukocyte rolling if the relative contribution of rolling and firm leukocyte adhesion is first determined, for example by the means described in this study. This relatively simple approach may be very useful for studying various aspects of leukocyte rolling when the ‘spontaneous’ rolling triggered by preparation of tissues for intravital microscopy is undesirable.

Propentofylline inhibits polymorphonuclear leukocyte recruitment in vivo by a mechanism involving adenosine A2A receptors

Propentofylline is an atypical xanthine derivative that blocks adenosine uptake and has been shown to protect against ischemia-induced cerebral damage. We have studied the effect of propentofylline on recruitment of polymorphonuclear leukocytes during acute peritonitis induced by zymosan in mice. Following i.p. injection of zymosan, recruitment of polymorphonuclear leukocytes, reflected by myeloperoxidase activity in the peritoneal cavity, increased from 2 h onwards, peaked at 4 h and then decreased gradually. Propentofylline antagonized the zymosan-induced peritoneal myeloperoxidase accumulation in a concentration-dependent manner. This effect of propentofylline was counteracted by the non-selective adenosine receptor antagonist theophylline (50 mg/kg), and by the selective adenosine A2A receptor antagonists, 4-amino-8-chloro-1-phenyl-[1,2,4]-triazolo[4,3-a]quinoxaline (CP 66713) and 1,3-dipropyl-8-[3,4-dimethoxystyryl]-7-methylxanthine (KF 17387) (both at 2 mg/kg). The results indicate that propentofylline can reduce polymorphonuclear leukocyte recruitment in vivo and that this effect is related to an action on adenosine A2A receptors.