Inhibition of leukocyte rolling with polysaccharide fucoidin prevents pleocytosis in experimental meningitis in the rabbit.

Inflammatory recruitment of leukocytes into the cerebrospinal fluid (CSF) during bacterial meningitis has been shown to contribute significantly to the neurological damage commonly associated with this serious disease. In this study we tested whether or not inhibition of leukocyte rolling, a precondition for firm leukocyte adhesion to vascular endothelium in vivo, may reduce CSF leukocyte recruitment and associated inflammatory changes in rabbits with experimental meningitis. As documented by intravital microscopy of small venules in the rabbit mesentery and tenuissimus muscle, leukocyte rolling was rapidly and profoundly reduced by intravenous treatment with the polysaccharide fucoidin, a homopolymer of sulfated L-fucose known to block the function of the leukocytic "rolling receptor" L-selectin. Moreover, fucoidin treatment dramatically reduced the accumulation of both leukocytes and plasma protein in the CSF of rabbits challenged intrathecally with pneumococcal antigen. These main findings thus illustrate that inhibition of leukocyte rolling, an early and obligatory step in the process of leukocyte extravasation, may be an effective therapeutic approach to attenuate leukocyte-dependent central nervous system damage in bacterial meningitis.

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, PGF2 alpha, 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 exogenous 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.

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

1. 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). 2. 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. 3. 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. 4. Intraperitoneal injection of 10(-3) M 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 approximately 90%. Intraperitoneal injection of leukotriene C4, but not prostaglandin E2, caused a significant increase in venular PMNL concentration. 5. 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. 6. 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.

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

1. 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. 2. 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. 3. 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). 4. 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. 5. 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. 6. 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).

Dual inhibitory action of nedocromil sodium on antigen-induced inflammation.

In human lung tissue in vitro, nedocromil sodium inhibited the release of histamine and leukotrienes induced by anti-IgE, as well as the contraction of isolated bronchi which followed this challenge. In the hamster cheek pouch in vivo, nedocromil sodium inhibited the inflammatory response induced by challenge with either antigen or the individual inflammatory mediators, histamine and leukotriene B4. The findings thus indicate that nedocromil sodium has a dual anti-inflammatory action: inhibition of mediator secretion and antagonism of mediator action.

Low molecular weight heparin as adjuvant therapy in active ulcerative colitis.

BACKGROUND: Heparin given intravenously has shown beneficial effects in the treatment of refractory ulcerative colitis in open trials. Low molecular weight heparin (LMWH) offers advantages in the method of administration but have not been evaluated in inflammatory bowel disease conditions. AIM: To assess the tolerability and safety of subcutaneous self-administered LMWH in outpatients with refractory ulcerative colitis and to evaluate any potential adjuvant therapeutic effect. PATIENTS AND METHODS: Twelve patients with mild to moderately active ulcerative colitis were included in the trial. The patients had either responded poorly to treatment with conventional therapy, including oral and/or rectal glucocorticosteroids, or had experienced a rapid relapse during or shortly after GCS therapy. Dalteparin sodium 5000 units s.c. injection was administered twice daily for 12 weeks. Patients were monitored for possible adverse events and changes in clinical symptoms, and endoscopic and histological scores were analysed. Leucocyte scanning was performed at inclusion and at the end of the study. RESULTS: Tolerability and compliance were excellent and no serious adverse events occurred. Eleven patients improved symptomatically and six (50%) attained complete remission after 12 weeks of treatment. Endoscopic, scintigraphic and histological scores were found to be significantly improved. CONCLUSION: Self-administered LMWH given s.c. may be a safe adjuvant therapy for patients with active, glucocorticosteroids-refractory ulcerative colitis. A controlled trial should be undertaken to confirm the positive effects found in this study.

Delayed anti-inflammatory action of nedocromil sodium in the rat paw is dependent on de novo protein synthesis.

Nedocromil sodium is commonly suggested to reduce allergic inflammation by inhibiting mediator release from mast cells. However, nedocromil also exhibits a wide range of additional anti-inflammatory activities, including inhibition of increased vascular permeability induced by individual mediators such as histamine. In the present study, we have further characterized the mode of action of nedocromil in a rat model for hind paw edema. Mast cell-dependent edema was induced with compound 48/80 (edema response mainly due to 5-hydroxytryptamine release), and direct mediator-induced plasma extravasation was evoked by exogenous 5-hydroxytryptamine (both agents injected locally). Local pretreatment with nedocromil for 20 min dose-dependently inhibited the edema evoked by compound 48/80 more effectively than that induced by 5-hydroxytryptamine. However, after 2 h pretreatment, both the 5-hydroxytryptamine-and compound 48/80-induced edema responses were inhibited to approximately the same extent by a range of concentrations of nedocromil, as well as by dexamethasone. Local inhibition of RNA/protein synthesis with actinomycin-D abolished the effects of both dexamethasone and nedocromil (2 h local pretreatment). We thus conclude that nedocromil can produce an 'anti-exudative' effect that is independent of inhibition of mast cell mediator release, is slow in onset, and requires de novo protein synthesis.

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.

Using mothers' and newborns' clinical anthropometric data to analyse birth histories.

The aim of the study was to measure the effect of mothers' and babies' various anthropometric parameters on the deviation from the normal course of pregnancy and labour. For this purpose the clinical anthropometric parameters of 532 parturients (primipara) and their newborns, and some additional indices formed from these data were correlated with the sum in points of all individual deviations from the normal course of anamnesis, pregnancy and childbirth as independent risk factors ("birth index" BI). The analysis showed that mothers' and babies' anthropometric data are essential co-factors in the formation of the total risk for mothers' and babies' health. Our investigation has demonstrated that a two-dimensional classification formed from height and parturient's complex body build index (PCBBI) (3 x 3 SD classes with appropriate statistical data-processing) can form a common methodological basis for using anthropometric characteristics in evaluation of obstetric data. As in the future analogous classifications could be used on obstetric material by different authors, the content of all corresponding classes--the mean values of newborns' birthweight and the birth index--would also be statistically comparable.

Intravital microscopic studies on acute mast cell-dependent inflammation.

Intravital microscopy of the hamster cheek pouch was adopted to serve as a model for quantitative studies of microvascular dynamics and parallel measurements of histamine release during immediate-type mast cell-dependent reactions. Topical challenge with specific antigen in the cheek pouch of immunized hamsters caused an acute inflammatory reaction, including leakage of plasma, vasodilation, and accumulation of leukocytes. Several lines of evidence indicated that the response was due to activation of mast cells: 1) an almost identical inflammatory reaction was seen after challenge with the mast cell secretagogue compound 48/80; 2) both antigen and compound 48/80 evoked distinct mast cell degranulatio and histamine release; 3) blockage of histamine 1-receptors reduced the plasma leakage response (but not leukocyte accumulation) to antigen and compound 48/80 in a very similar manner. In addition, fluorescein-labelled antigen bound specifically to mast cells in cheek pouches of immunized animals, suggesting involvement of mast cell-fixed antigen-specific antibodies, possibly immunoglobulin E. It is suggested that vasodilating prostaglandins exert both pro- and anti-inflammatory actions in vivo, and that they modulate acute allergic inflammation by i) inhibition of inflammatory mediator release, most likely unrelated to prostanoid-induced vasodilation, but caused by cAMP elevation in the mediator-secreting cells, and ii) enhancement of the target action of individual inflammatory mediators (i.e. plasma leakage and leukocyte emigration), most likely as a direct consequence of prostaglandin-induced vasodilation. This view is based on the following observations in the hamster cheek pouch: 1) Inhibition of prostaglandin synthesis with two different nonsteroidal anti-inflammatory drugs (NSAIDs) greatly potentiated plasma leakage, leukocyte emigration and histamine release after challenge with antigen or compound 48/80. The enhanced antigen-induced extravasation of plasma and leukocytes was significantly reduced by 5-lipoxygenase inhibitors, but was unaffected by PAF-receptor antagonism. 2) All aspects of NSAID-induced potentiation, including the increased histamine release, were effectively prevented by topically applied prostaglandin E2 (PGE2, 30 nM), which per se caused a five-fold increase in arteriolar blood flow. Moreover, PGE2 as well as prostaglandin I2 (PGI2) in vasodilating concentrations suppressed the antigen-induced plasma leakage also in the absence of NSAID treatment. 3) In contrast to the mast cell-dependent reactions, the inflammatory effects of individual mediators histamine, leukotrienes B4 and C4) were not influenced by NSAID treatment, and were markedly enhanced by both PGE2 and PGI2.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of local treatment with salmeterol and terbutaline on anti-IgE-induced wheal, flare, and late induration in human skin.

The capacity of terbutaline and the long-acting beta 2-agonist salmeterol to suppress wheal-and-flare reactions (WFRs) to intradermal antihuman IgE and to histamine was evaluated in 36 healthy volunteers. We also examined effects of the two drugs on the subsequent late cutaneous reaction (LCR) to anti-IgE. Salmeterol (10(-10)-10(-6) M) and terbutaline (10(-10)-10(-5) M), injected intradermally 2 or 15 min before anti-IgE challenge, produced a dose-dependent inhibition of the WFRs to anti-IgE (titer 1 : 10000) with a maximal effect of approximately 60% (wheal) and approximately 75% (flare) by both drugs. On a molar basis, salmeterol was approximately 10-100 times more potent than terbutaline in inhibiting the WFRs. Moreover, the wheal response to histamine (4 nmol) was antagonized by approximately 40% after pretreatment with either salmeterol (10(-5) M) or terbutaline (10(-4) M). We found that both salmeterol and terbutaline exhibited anti-WFR activity for up to 24 h, salmeterol being significantly more potent in this regard. When selecting a 15-min pretreatment interval with equieffective anti-WFR doses from the first dose-response experiments (i.e., a salmeterol: terbutaline ratio of 1 : 10), the WFRs to high-dose anti-IgE (titer 1 : 100) were inhibited by terbutaline (10(-5) M) by 25-30%, but not by salmeterol (10(-6)). On the other hand, salmeterol attenuated (by up to approximately 35%) the subsequent LCR more effectively than terbutaline. As compared to the pretreatment procedure, infiltration of the drugs (single doses) into the wheals 30 min after challenge with anti-IgE (titer 1 : 100) proved to be less effective on the development of LCRs. Taken together, salmeterol was found to express higher potency and have longer duration of action than terbutaline in inhibition of IgE-mediated inflammation in human skin.

Cytokine-induced leukocyte rolling in mouse cremaster muscle arterioles in P-selectin dependent.

Leukocyte rolling and adhesion are generally observed in venules but rarely observed in arterioles. With the use of intravital microscopy, we found that a 4-h treatment with interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) dose dependently induced leukocyte rolling and adhesion in arterioles of the mouse cremaster muscle. The rolling response lasted more than 24 h and was completely inhibited by treatment with the sulfated polysaccharide fucoidin. Moreover, we found that costimulation with IL-1beta and TNF-alpha for 4 h synergistically increased arteriolar leukocyte rolling, i.e., threshold doses of IL-1beta and TNF-alpha together caused a more than 10-fold increase of rolling in arterioles compared with the sum of the individual responses. This rolling interaction was abolished by treatment with a monoclonal antibody directed against P-selectin (RB40.34), but it apparently was unaffected by a monoclonal antibody against L-selectin (MEL-14). Taken together, our functional data show that IL-1beta and TNF-alpha separately induce and synergistically increase P-selectin-dependent leukocyte rolling and firm adhesion in mouse cremaster arterioles.