Beneficial microvascular and anti-inflammatory effects of pravastatin during sepsis involve nitric oxide synthase III.

BACKGROUND: Sepsis induces microvascular inflammation and production of the vasodilator nitric oxide (NO) via endothelial and inducible nitric oxide synthase (eNOS or NOS III and iNOS or NOS II). Statins are cholesterol-lowering drugs; however, they also attenuate inflammation. This study aimed to determine whether pravastatin protected against sepsis-induced hypotension, loss of vascular tone, and microvascular inflammation via NOS pathways. METHODS: Male Wistar rats (n=18) were anaesthetized and the mesentery prepared for fluorescent intravital microscopy. Animals received either lipopolysaccharide (LPS; n=6); LPS+pravastatin (18 and 3 h before LPS; n=6), or saline as a control, for 4 h. RESULTS: Mean arterial pressure decreased in LPS-treated animals (P<0.05), but not in those also receiving pravastatin. Acetylcholine-induced relaxation of venules was abolished by LPS but improved by pravastatin. Pravastatin also reduced the increase in nitrite concentration and macromolecular leak from venules induced by LPS (P<0.05). The increased leucocyte adhesion seen in LPS-treated rats was also reduced in those also treated with pravastatin. Immunohistochemical analysis showed that pravastatin increased endothelial cell expression of NOS III during sepsis, but had no effect on LPS-induced up-regulation of NOS II. CONCLUSIONS: Pravastatin improved NOS III-mediated vessel relaxation and exerted anti-inflammatory effects within the microcirculation after LPS administration in rats. Pravastatin therefore appears to have beneficial effects during sepsis, as a result of increased microvascular expression and function of NOS III.

Chemokine-induced eosinophil recruitment. Evidence of a role for endogenous eotaxin in an in vivo allergy model in mouse skin.

Selective eosinophil recruitment into tissues is a characteristic feature of allergic diseases. Chemokines are effective leukocyte chemoattractants and may play an important role in mediating eosinophil recruitment in various allergic conditions in man. Here, we describe a novel mouse model of eosinophil recruitment in which we have compared the in vivo chemoattractant activity of different C-C chemokines. Furthermore, we describe the use of antibodies to chemokines and receptor blockade to address the endogenous mechanisms involved in eosinophil recruitment in a late-phase allergic reaction in mouse skin. Intradermal injection of mEotaxin and mMIP-1alpha, but not mMCP-1, mRANTES, mMCP-5, or mMIP-1beta, induced significant 111In-eosinophil recruitment in mouse skin. Significant 111In-eosinophil recruitment was also observed in an active cutaneous anaphylactic reaction. Pretreatment of skin sites with antieotaxin antiserum, but not an antiMIP-1alpha antibody, suppressed 111In-eosinophil recruitment in this delayed-onset allergic reaction. Similarly, desensitization of the eosinophil eotaxin receptor CCR3 with mEotaxin, or blockade of the receptor with metRANTES, significantly inhibited 111In-eosinophil recruitment in the allergic reaction. These results demonstrate an important role for endogenous eotaxin in mediating the 111In-eosinophil recruitment in allergic inflammation, and suggest that blockade of the CCR3 receptor is a valid strategy to inhibit eosinophil migration in vivo.

Phosphodiesterase (PDE)4 inhibitors: anti-inflammatory drugs of the future?

Phosphodiesterase type 4 (PDE4) plays a major role in modulating the activity of virtually all cells involved in the inflammatory process. Inhibitors of this enzyme family display impressive anti-inflammatory and disease-modifying effects in a variety of experimental models. In this review, Mauro Teixeira, Robert Gristwood, Nicola Cooper and Paul Hellewell examine the capacity of PDE4 inhibitors to exert anti-inflammatory actions in vivo and discuss the potential of this class of drugs to take their place as novel therapeutic agents for a variety of inflammatory diseases.

Mechanisms and pharmacological manipulation of eosinophil accumulation in vivo.

The presence of eosinophils and their products in tissues is frequently associated with the pathogenesis of allergic inflammation. A better understanding of how these cells are recruited from the microcirculation will help in the development of therapies targeted at allergic disorders. Here, Mauro Teixeira, Timothy Williams and Paul Hellewell describe the current concepts of eosinophil accumulation, examine the potential ways of modulating this process, and discuss whether antagonists of eosinophil-specific mediators or functional antagonists would be the preferred therapies.

Tumor necrosis factor and steroid metabolism in chronic heart failure: possible relation to muscle wasting.

OBJECTIVES: We sought to assess the possible relations between clinical severity of chronic heart failure and catabolic factors, specifically tumor necrosis factor (TNF), soluble TNF receptors 1 and 2 (sTNFR-1 and sTNFR-2), cortisol, testosterone and dehydroepiandrosterone (DHEA). BACKGROUND: Chronic heart failure is associated with loss of muscle bulk that may be related to alteration of the balance between catabolism and anabolism. METHODS: Sixty-three patients (average age +/- SD 60.4 +/- 11.3 years) with stable chronic heart failure and 20 control subjects aged 52.8 +/- 11.4 years were studied. We measured body mass index (BMI) and obtained maximal incremental exercise testing with metabolic gas exchange measurements and measurements of venous levels of TNF, sTNFR-1 and sTNFR-2, cortisol and DHEA. RESULTS: There was no difference in total TNF-alpha levels between patients and control subjects (9.76 +/- 8.59 vs. 6.84 +/- 2.7 pg/ml). sTNFR-1 (128.9 +/- 84.5 vs. 63.6 +/- 23.3 pg/ml, p < 0.003) and sTNFR-2 (250.1 +/- 109.5 vs. 187.9 +/- 92.2 pg/ml, p = 0.03) were higher in patients. DHEA was lower in patients (9.88 +/- 6.94 vs. 15.64 +/- 8.33 nmol/liter, p = 0.004). The ratio of log cortisol to log DHEA correlated with log TNF level (r = 0.50, p < 0.001 for the patients alone; r = 0.48, p < 0.001 for the group as a whole). Peak oxygen consumption correlated with both sTNFR-1 and sTNFR-2 (r = -0.51, p < 0.001 and r = -0.39, p < 0.001, respectively). There was a negative correlation between BMI and TNF levels (r = -0.43, p < 0.001 for the patients) and the cortisol/DHEA ratio (r = -0.32, p = 0.01 for the patients). CONCLUSIONS: There is an increase in TNF and its soluble receptors in chronic heart failure. This increase is associated with a rise in the cortisol/DHEA (catabolic/anabolic) ratio. These changes correlate with BMI and clinical severity of heart failure, suggesting a possible etiologic link.

Adhesion mechanisms involved in C5a-induced eosinophil homotypic aggregation.

During the process of migration into tissues, leukocytes interact primarily with vascular endothelial cells but they have also been shown to interact with each other. In this study we investigated the adhesion mechanisms involved in guinea pig eosinophil homotypic aggregation as assessed by changes in light transmission. The anti-CD18 monoclonal antibody (mAb) 6.5E, at concentrations in excess of those previously shown to abrogate CD18-dependent eosinophil adherence to serum-coated plastic, inhibited C5a-induced eosinophil aggregation to a maximum of 49-68%. In contrast, the anti-intercellular adhesion molecule-1 (ICAM-1) mAb RR1/1, which binds to guinea pig eosinophils and has been shown to block guinea pig ICAM-1 function, had no effect on C5a-induced responses. Similarly, two functionally active anti-very late antigen-4 (VLA-4) mAbs has no effect on eosinophil aggregation and did not affect the CD18-independent component of the aggregation response. The role of L-selectin in eosinophil aggregation was investigated by using heparin, the selectin-binding polysaccharide fucoidin, and the anti-L-selectin mAb MEL-14. Heparin concentration dependently inhibited C5a- and platelet- activating factor- (PAF) induced aggregation but C5a-induced responses were inhibited more potently. Fucoidin, but not the carbohydrate dermatan sulphate, effectively inhibited C5a-induced eosinophil aggregation. PMA- and PAF- induced responses were also inhibited by fucoidin. Moreover, fucoidin and 6.5E were additive in their ability to inhibit C5a-induced aggregation. Similarly, MEL-14 effectively inhibited C5a-induced eosinophil aggregation. In conclusion, we have demonstrated that guinea pig eosinophil homotypic aggregation is mostly dependent on CD11/CD18 and L-selectin present on the eosinophil surface. In addition, VLA-4 plays no role in mediating this aggregation response.

Eosinophil recruitment into sites of delayed-type hypersensitivity reactions in mice.

The selective accumulation of eosinophils in tissue is a characteristic feature of allergic diseases where there is a predominance of lymphocytes expressing a Th2 phenotype. In an attempt to define factors determining specific eosinophil accumulation in vivo, we have used a radiolabeled technique to assess the occurrence and the mechanisms underlying (111)In-eosinophil recruitment into Th1- and Th2-predominant, delayed-type hypersensitivity (DTH) reactions. Eosinophils were purified from the blood of IL-5 transgenic mice, labeled with (111)In and injected into nontransgenic CBA/Ca mice. Th1- and Th2-predominant, DTH reactions were induced in mice by immunization with methylated bovine serum albumin (MBSA) in Freund's complete adjuvant or with Schistosoma mansoni eggs, respectively. In these animals, (111)In-eosinophils were recruited in skin sites in an antigen-, time-, and concentration-dependent manner. Depletion of CD4+ lymphocytes abrogated (111)In-eosinophil recruitment in both reactions. Pretreatment of animals with anti-IFN-gamma mAb abrogated (111)In-eosinophil recruitment in MBSA-immunized and -challenged animals, whereas anti-IL-4 inhibited (111)In-eosinophil recruitment in both models. Local pretreatment with an anti-eotaxin polyclonal antibody inhibited the MBSA and SEA reactions by 51% and 39%, respectively. These results demonstrate that, although eosinophilia is not a feature of Th1-predominant, DTH reactions, these reactions produce the necessary chemoattractants and express the necessary cell adhesion molecules for eosinophil migration. The control of the circulating levels of eosinophils appears to be a most important strategy in determining tissue eosinophilia.

Characterization of eosinophil homotypic aggregation.

Guinea pig peritoneal eosinophils stimulated by platelet-activating factor (PAF), leukotriene B4 (LTB4), and human recombinant C5a (C5a) undergo a rapid concentration-dependent and partially reversible homotypic aggregation as assessed by changes in light transmission. The phorbol ester phorbol myristate acetate similarly induces a concentration-dependent aggregation, which is, however, slower in onset, takes longer to reach maximal aggregation, and is irreversible. In addition, we confirmed, using light microscopy, that these agonist-induced changes in light transmission do indeed represent true homotypic aggregation. We further characterized the aggregation response and showed that there is homologous but little heterologous desensitization when PAF and LTB4 are used as stimuli. A requirement for both Ca2+ and Mg2+ for full manifestation of agonist-induced aggregation was observed. LTB4- and PAF-induced superoxide anion generation is enhanced by the diacyglycerol kinase inhibitor R59022, whereas aggregation induced by LTB4, but not PAF, is augmented. Lastly, we show that eosinophil aggregation is partially dependent on the adhesion glycoprotein CD18. In summary, therefore, we believe that eosinophil aggregation provides a useful and reliable measure of eosinophil activation.

Role of the mitogen-activated protein kinases and tyrosine kinases during leukotriene B4-induced eosinophil activation.

Exposure of guinea-pig eosinophils to leukotriene B4 (LTB4; 1 microM) resulted in a rapid generation of H2O2 (index of NADPH oxidase activation), stimulated [3H]arachidonic acid (AA) release (index of phospholipase A2 activity), and promoted CD18-dependent homotypic aggregation. Under similar conditions, LTB4 (1 microM) induced a rapid activation of extracellular-regulated kinases-1 and 2 (ERK-1/2) but not c-jun N-terminal kinases 46 and 54 (JNK-46/54) or p38 mitogen-activated protein kinase (p38 MAP kinase). To examine the role of ERK-1/2 in the mechanism of eosinophil activation, a selective inhibitor of MAP kinase kinase-1/2 (MEK-1/2), PD098059, was employed. However, PD 098059 at concentrations that attenuated ERK-1/2 activation had no significant affect on eosinophil activation. In contrast, a role for tyrosine kinases in LTB4-induced eosinophil activation was suggested by studies with the tyrosine kinase inhibitors, herbimycin A and lavendustin A. However, the results of those experiments implied divergent pathways for the control of eosinophil responses because the inhibitors were more effective at attenuating H2O2 generation than [3H]AA release, and had little effect on homotypic aggregation.

Loss of bone mineral in patients with cachexia due to chronic heart failure.

We studied body composition and cytokine levels in 58 patients with heart failure and 16 control patients using dual-energy x-ray absorptiometry. Bone mineral content and density, and lean and fat tissue content, were reduced in cachectic compared with noncachectic patients and control subjects, with negative relations between indexes of bone composition and tumor necrosis factor and tumor necrosis factor receptor 1.

An anti-inflammatory steroid inhibits tissue sensitization by IgE in vivo.

An anti-inflammatory corticosteroid, dexamethasone, was found to be a potent suppressor of cell sensitization induced by IgE antibody in a model of type I allergic inflammation in vivo. Low doses (approximately less than 10(-10) mol) of dexamethasone, administered intradermally to rabbits 90 min before injection of IgE into the same sites, suppressed local oedema induced by a local challenge with antigen 72 h later. The effect was not due to anti-inflammatory activity persisting in the skin site for the 3 day period. This novel potent activity of the corticosteroid may be an important component of its anti-allergic effect in diseases such as asthma.

The effect of the selectin binding polysaccharide fucoidin on eosinophil recruitment in vivo.

1. In order to accumulate at sites of inflammation, leukocytes initially roll on endothelial cells of postcapillary venules before becoming firmly attached. This process of rolling is mediated by selectins which bind to carbohydrate counter-ligands present on the surface of both leukocytes and endothelial cells. The polysaccharide fucoidin has been previously shown to inhibit leukocyte rolling in the mesenteric circulation and to reduce neutrophil accumulation in the skin and meninges in experimental inflammation. 2. In the present study we have assessed the effects of fucoidin on eosinophil function in vitro and eosinophil accumulation at sites of inflammation in guinea-pig skin. 3. At concentrations of up to 1200 micrograms ml-1, fucoidin inhibited phorbol myristate acetate (PMA)-induced eosinophil homotypic aggregation by up to 60% but had no inhibitory effect on PMA-induced eosinophil adhesion to serum-coated plates. 4. Fucoidin effectively reduced the binding of the anti-L-selectin mAb MEL-14 to guinea-pig eosinophils. Binding of a P-selectin-IgG chimera to eosinophils was also partially inhibited by fucoidin, but binding of an anti-CD18 or an anti-VLA-4 mAb were unaffected. 5. When given systemically to guinea-pigs, fucoidin suppressed 111In-labelled eosinophil recruitment to sites of allergic inflammation. 111In-labelled eosinophil accumulation induced by platelet-activating factor (PAF) and zymosan-activated plasma (as a source of C5a des Arg) was also inhibited. 6. These results demonstrate a role for fucoidin-sensitive selectins in mediating eosinophil recruitment in vivo.

Purinoceptor mediated stimulation of prostacyclin release in the porcine pulmonary vasculature.

Prostacyclin (PGI2) release from the piglet isolated perfused lung was measured by radioimmunoassay of 6-keto-PGF1 alpha in the venous effluent. Basal release of PGI2 was transiently stimulated up to 30 fold, in a dose-dependent manner, by bolus injections of ATP (0.03-3 mumol). A continuous infusion of ATP also produced a transient response. Dose-response curves for purinergic stimulation of PGI2 release showed that ADP was equipotent with ATP, while AMP and adenosine were virtually inactive. The non-hydrolyzable ATP analogue, ATP-gamma-S, elicited PGI2 release of similar magnitude and duration to that of ATP, suggesting that pulmonary catabolism of ATP is not required to induce PGI2 release. The results suggest that the porcine pulmonary vasculature possesses P2-purinoceptors through which the synthesis and release of PGI2 can be mediated.

Effect of a 5-lipoxygenase inhibitor, ZM 230487, on cutaneous allergic inflammation in the guinea-pig.

1. Leukotrienes have potent biological effects in vitro and in vivo and are found in tissue and in biological fluids in various pathological conditions including allergic diseases. Leukotriene B4 (LTB4) is a potent stimulus for eosinophil accumulation and activation and there is much interest in determining its importance in mediating the accumulation of eosinophils at sites of allergic inflammation in vivo. In this study, we investigated the effects of a potent 5-lipoxygenase inhibitor, ZM 230487, on the accumulation of eosinophils and on local oedema formation in cutaneous inflammation in the guinea-pig. 2. The i.d. injection of increasing concentrations of arachidonic acid (AA) led to a dose-dependent accumulation of 111In-eosinophils but oedema formation was only significant at the top dose of AA tested (3 x 10(-8) mol per site). Co-injection of ZM 230487 with AA inhibited 111In-eosinophil accumulation up to 99% but the small oedema response to AA was only partially inhibited. AA-induced oedema formation was only effectively inhibited when a combination of a PAF antagonist, an antihistamine and ZM 230487 was used. 3. Local administration of the cyclo-oxygenase inhibitor, ibuprofen, partially inhibited AA-induced oedema formation suggesting that vasodilator prostaglandins may be released following i.d. injection of AA. AA-induced 111In-eosinophil accumulation was also partially inhibited by ibuprofen. 4. PAF-induced 111In-eosinophil accumulation was partially suppressed by local administration of ZM 230487. In contrast, LTB4-induced 111In-eosinophil accumulation was enhanced by ZM 230487. These data suggest that locally-released leukotrienes may modulate mediator-induced eosinophil accumulation. ZM 230487 had no effect on PAF- or LTB4-induced oedema formation. 5. ZM230487 significantly inhibited the accumulation of 111 In-eosinophils, but did not affect local oedema formation, in a passive cutaneous anaphylaxis (PCA) reaction. However, the PAF antagonist WEB 2086 either alone or in combination with ZM 230487 had no effect on "'In-eosinophil accumulation or oedema formation in the PCA reaction.6. In conclusion, it appears that a product of 5-lipoxygenase, probably LTB4, is important for the accumulation of "'In-eosinophils, but not local oedema formation, in the PCA reaction in guinea-pigskin. These data support a major role for LTB4 in allergic inflammation in the guinea-pig and make this animal (and the PCA model) suitable for studying the effects of inhibitors of leukotriene synthesis or action in vivo.

Antagonism of Paf-induced oedema formation in rabbit skin: a comparison of different antagonists.

1. Eight platelet activating factor (Paf) antagonists were evaluated as inhibitors of oedema formation in rabbit skin induced by intradermal injection of Paf plus prostaglandin E2 (PGE2). Antagonists were tested by both intradermal (i.d.) and intravenous (i.v.) routes. 2. Intradermal injection of two antagonists structurally-related to Paf (SRI 63-675 and CV-3988) resulted in a partial inhibition of Paf-induced oedema formation but at high doses of antagonist, marked agonist activities were detected. CV-3988 administered i.v. inhibited Paf-induced plasma leakage by 73-80%; however, oedema responses to a range of other inflammatory mediators were also reduced, albeit to a lesser extent (40-60%). SRI 63-675 administered i.v. did not significantly inhibit Paf-induced oedema. 3. The antagonist 48740 RP administered either i.d. or i.v. showed partial, but selective, inhibition of Paf-induced oedema formation, although the doses required were high when compared with other antagonists. 4. BN 52021 was a weak Paf antagonist when injected i.d., but following i.v. administration the responses to Paf were inhibited by 63-71%. Responses to all other mediators tested were unaffected. 5. Kadsurenone and its synthetic derivatives, L-652,731 and L-659,989 all blocked responses to Paf in the skin. L-659,989 was the most potent, achieving almost total inhibition when injected i.d. and i.v.; moreover, it was selective for Paf. L-652,731 was more potent than kadsurenone. 6. WEB 2086 given i.d. and i.v. showed similar activity to L-659,989 and it was also selective for Paf-induced oedema formation. 7. These results illustrate that in rabbit skin not all Paf antagonists are selective for Paf, some showing agonist-like activity which can mask antagonist properties. It is suggested that before ascribing a role for endogenous Paf in an inflammatory reaction based on results with antagonists, the activity of the antagonists in the model under investigation should be rigorously established.

Tumour necrosis factor-alpha-induced ICAM-1 expression in human vascular endothelial and lung epithelial cells: modulation by tyrosine kinase inhibitors.

1. Tumour necrosis factor-alpha (TNF alpha) increases the expression of the adhesion molecule intercellular adhesion molecule-1 (ICAM-1) on cultured endothelial and epithelial cells and modulation of this may be important in controlling inflammation. Activation of tyrosine kinase(s) is known to be involved in the signal transduction pathways of many cytokines. In this study we have investigated the effects of the tyrosine kinase inhibitors, ST638, tyrphostin AG 1288 and genistein, on TNF alpha-induced ICAM-1 expression in human alveolar epithelial (A549) and vascular endothelial (EAhy926) cell lines and also normal human lung microvascular endothelial cells (HLMVEC). 2. ICAM-1 expression on cultured cells was determined by a sensitive enzyme-linked immunosorbant assay (ELISA). Endothelial or epithelial monolayers were exposed to increasing doses of TNF-alpha (0.01-10 ng ml-1), in the presence or absence of either ST638 (3-100 microM), AG 1288 (3-100 microM) or genistein (100 microM) and ICAM-1 expression was measured at 4 and 24 h. Control experiments examined the effect of ST638 on phorbol 12-myristate 13-acetate (PMA, 20 ng ml-1, 4 h)-stimulated ICAM-1 and compared it to that of a specific protein kinase C inhibitor, R031-8220 (10 microM). Also, functional consequences of changes in ICAM-1 expression were assessed by measuring adhesion of 111 In-labelled human neutrophils to EAhy926 endothelial and A549 epithelial monolayers treated with TNF alpha, in the presence or absence of ST638. 3. ST638 caused a concentration-dependent reduction in TNF alpha- (0.1-10 ng ml-1)-induced ICAM-1 on EAhy926 endothelial (at 4 h) and A549 epithelial monolayers (at 4 and 24 h). In contrast, ST638 caused a concentration-dependent increase in TNF alpha- (0.1-10 ng ml-1)-induced ICAM-1 on EAhy926 endothelial cells at 24 h. Similar effects were seen with AG 1288 or genistein. ST638 (100 microM) significantly (P < 0.01) inhibited ICAM-1 expression on HLMVEC endothelial cells induced by 0.01 ng ml-1 TNF alpha at 4 or 24 h or 0.1 ng ml-1 at 4 h, but increased ICAM-1 expression induced by 0.1 ng ml-1 TNF alpha at 24 h. ST638 did not significantly change the expression of PMA-stimulated ICAM-1 on either A549 epithelial, EAhy926 or HLMVEC endothelial cells. However, PMA-induced ICAM-1 expression was inhibited by Ro31-8220. Also, treatment of epithelial or endothelial monolayers with TNF alpha and ST638 altered adhesion of human neutrophils to A549 epithelial or EAhy926 endothelial cells in a manner that corresponded to the alteration in ICAM-1 expression. 4. These results show that tyrosine kinase inhibitors alter TNF alpha-induced ICAM-1 expression, but that the cell type, concentration of TNF alpha and time of exposure to this cytokine determine whether expression is decreased or increased by the inhibitor. An increased understanding of the signal transduction pathway(s) involved in TNF alpha-induced ICAM-1 expression on lung epithelial and vascular endothelial cells may be of potential therapeutic value in the treatment of inflammatory disease.

Suppression by intradermal administration of heparin of eosinophil accumulation but not oedema formation in inflammatory reactions in guinea-pig skin.

1. Heparin is widely used in the treatment of thrombotic disorders and as an aid in surgery. Anti-inflammatory effects of heparin have also been described. In this study, we have investigated the effects of locally-injected heparin on the oedema formation and eosinophil accumulation induced by various inflammatory stimuli in guinea-pig skin. 2. Heparin dose-dependently suppressed the accumulation of 111In-labelled eosinophils induced by i.d. injection of zymosan-activated plasma (ZAP). The 111In-eosinophil accumulation induced by other inflammatory stimuli (compound 48/80, platelet activating factor, interleukin-8 and in a passive cutaneous anaphylaxis reaction) was also suppressed by locally-injected heparin. 3. Oedema formation in response to these same stimuli was not altered by the local injection of heparin. 4. Fucoidin, a negatively-charged sulphated algal polymer, had no effect on the 111In-eosinophil accumulation or oedema formation induced by ZAP. Nevertheless, fucoidin significantly suppressed the oedema formation induced by i.d. injection of cationic protein-containing extracts of Schistosoma mansoni larvae. Heparin also inhibited oedema induced by the extracts, suggesting that both fucoidin and heparin were effectively neutralizing the cationic protein of the extracts to inhibit their oedema-inducing activity. 5. Thus, heparin significantly inhibited the accumulation of 111In-eosinophils, but not oedema formation, induced by various inflammatory stimuli. This, taken together with the lack of effect of fucoidin, suggests that heparin interferes with the process of eosinophil trafficking by a mechanism that does not depend on neutralisation of the charge of the stimulatory molecules.

Effect of sulphasalazine on pulmonary inactivation of prostaglandin F2 alpha in the pig.

1 The metabolism of prostaglandin F2 alpha (PGF2 alpha) 15 nm in 100,000 g supernatant fractions from piglet lung homogenates was inhibited by sulphasalazine with an IC50 value of 25 micrometers. 2 The piglet isolated lung perfused with Krebs solution, containing either albumin or Ficoll 70 to prevent oedema and vascular damage, efficiently metabolized PGF2 alpha given as a bolus injection (1 ng in 0.1 ml; 30 nm). 3 In Krebs solution containing Ficoll 70, sulphasalazine inhibited the pulmonary inactivation of PGF2 alpha in a dose-dependent manner with an IC50 value of 110 micrometers. No inhibition of inactivation by sulphasalazine was found when the perfusion fluid contained albumin, which is known to bind this drug effectively. 4 Analysis of the separated efflux profiles for PGF2 alpha and its metabolites with reference to the dilution curve for an extracellular marker provided evidence that sulphasalazine inhibited PGF2 alpha uptake into lung cells. 5 We conclude that the effect of sulphasalazine on pulmonary prostaglandin inactivation is primarily due to inhibition of prostaglandin transport, and not to inhibition of prostaglandin metabolism.

Characteristics of oedema formation induced by N-formyl-methionyl-leucyl-phenylalanine in rabbit skin.

1. The characteristics of N-formyl-methionyl-leucyl-phenylalanine (FMLP)-induced oedema formation were investigated in vivo in rabbit skin. 2. FMLP injected intradermally alone induced a small increase in plasma leakage, but marked synergism with prostaglandin E2 (PGE2) in producing oedema responses was observed. In the presence of PGE2, FMLP was equiactive with C5a des Arg and 100-1000 times more active than histamine in terms of permeability-increasing activity. The response to FMLP was not dependent on endogenous histamine release. 3. FMLP-induced responses were of long duration (t1/2 approximately 40-50 min) when compared with bradykinin (t1/2 approximately 4-5 min). 4. The activity of a range of N-formyl peptides in increasing vascular permeability in skin correlated well with their activity as neutrophil stimulants in vitro. 5. Intravenous infusion of zymosan-activated plasma (ZAP) resulted in transient neutropenia and inhibition of oedema formation induced by FMLP and C5a des Arg in the skin. Responses to bradykinin were unaffected by the infusion of ZAP. 6. Intravenous injection of the non-steroidal antiinflammatory drug, ibuprofen, resulted in an inhibition of FMLP-induced, but not histamine-induced, oedema formation. This effect was independent of cyclo-oxygenase inhibition and the drug did not induced neutropenia. 7. Intravenous injection of the microtubule blocking agent colchicine inhibited FMLP-induced oedema formation. Responses to bradykinin were unaffected. When colchicine was administered after intradermal FMLP, subsequent plasma leakage was abolished. 8. The inference that receptors have evolved to bacterial secretions (i.e. FMLP) and products of the interaction of bacterial cell walls with tissue fluid (i.e. C5a des Arg), is consistent with the hypothesis that oedema formation is fundamentally a functional process concerned with regulating microbial lysis and opsonisation in an infected tissue.

Modulation of cell adhesion molecule expression and function on human lung microvascular endothelial cells by inhibition of phosphodiesterases 3 and 4.

1. Expression of cell adhesion molecules (CAM) on the lung microvascular endothelium is believed to play a key role in the recruitment of leukocytes in pulmonary inflammation. Moreover, regulation of CAM expression may be an important mechanism through which this inflammation may be controlled. Experimental evidence has suggested that combined phosphodiesterase (PDE) 3 and 4 inhibitors increase cyclic AMP levels within cells greater than inhibition of either isoenzyme alone. In the present study we assessed the effect of combinations of rolipram (PDE4 inhibitor), ORG 9935 (PDE3 inhibitor) and salbutamol (beta-agonist) on CAM expression and neutrophil or eosinophil adhesion to human lung microvascular endothelial cells (HLMVEC). 2. Tumour necrosis factor-alpha (TNF-alpha)-induced intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1 and E-selectin expression were measured on HLMVEC monolayers at 6 h by a specific ELISA technique in the presence of different combinations of medium, rolipram, ORG 9935 and salbutamol. 3. Rolipram in combination with salbutamol, but neither agent alone, inhibited TNF-alpha-induced E-selectin expression, whilst ICAM-1 and VCAM-1 expression were not affected. ORG 9935 had no significant effect on CAM expression alone. However, in combination with rolipram a syngergistic inhibition of VCAM-1 and E-selectin, but not ICAM-1, expression was observed. No further inhibition was seen in the additional presence of salbutamol. 4. Neutrophil adhesion to TNF-alpha-stimulated (6 h) HLMVEC was mainly E-selectin dependent in this model, as ENA2 an anti-E-selectin monoclonal antibody (mAb) abrogated neutrophil adhesion. Eosinophil adhesion was E-selectin-, ICAM-1- and VCAM-1-dependent, as assessed by the inhibitory activity of ENA2 and the ability of a mAb to the ICAM-1 ligand, CD18, and a mAb to the VCAM-1 ligand, VLA4, to attenuate adhesion. 5. Rolipram in the presence of salbutamol or ORG 9935 significantly inhibited neutrophil adherence to TNF-alpha-stimulated HLMVEC. Eosinophil adherence to monolayers was inhibited only when HLMVEC were activated in the presence of rolipram and ORG 9935. 6. Collectively, the findings presented in this manuscript suggest that inhibition of PDE4 with appropriate activation of adenylate cyclase is sufficient to inhibit induction of E-selectin expression on HLMVEC to a level that has functional consequences for neutrophil adhesion. In contrast, combined inhibition of PDE3 and 4 isoenzymes is necessary to inhibit VCAM-1 and to have inhibitory effects on eosinophil adhesion to activated HLMVEC. Upregulation of ICAM-1 expression on HLMVEC does not appear to be modulated by PDE3 and 4 inhibition. These data may have implications for the use of selective PDE4 inhibitors in lung inflammation.