Background Thrombin downregulates endothelial ectonucleotidase activity resulting in high levels of adenosine diphosphate (ADP) and adenosine triphosphate (ATP) which lead to platelet, leukocyte and endothelial activation. Depending on adenosine nucleotide levels, resting platelets inhibit and thrombin-activated platelets increase respiratory burst of neutrophils. Whether the red wine polyphenols quercetin and resveratrol affect thrombin-dependent adenosine nucleotide, metabolism and thrombin-induced signaling is unknown. Materials and methods ATP and ADP secretion by platelets, the impact on subsequent oxidative burst activity in neutrophils and CD39/ATPdase function in endothelial cells (ECs)was studied. Cell migration was measured in modified Boyden chambers; adenosine metabolites were quantified by high-performance liquid chromatography (HPLC). Signal transduction was studied by Western blotting. Results Quercetin and resveratrol inhibited thrombin-induced ADP and ATP secretion from platelets in a concentration-dependent manner. Augmented respiratory burst of neutrophils in response to thrombin-activated platelets was also inhibited by the two polyphenols as was neutrophil migration toward thrombin-induced supernatants of platelets. Quercetin and resveratrol restored the decreased CD39/ATPdase activity in human umbilical vein endothelial cells, in response to thrombin as demonstrated by adenosine monophosphate (AMP) and adenosine increases in endothelial culture supernatants. Both polyphenols inhibited thrombin-induced MAPK, JNK and focal adhesion kinase activities in endothelial cells. Conclusion Quercetin and resveratrol interfere with the proinflammatory signaling of thrombin resulting in the inhibition of adenosine nucleotide secretion from activated platelets and decreased neutrophil function. Moreover, the polyphenols protect endothelial adenosine nucleotide metabolism when downregulated by thrombin. These observations may explain cardioprotective effects of grape products.

ABSTRACT Ethanol inhibits the respiratory burst of neutrophils. Therefore, the effects of alcohol-based skin disinfection on oxygen metabolism in neutrophils were tested using 70% ethanol or an ethanol–isopropanol– n -propanol mixture. Neutrophil respiratory burst activity as assessed fluorometrically by oxidation of 2′,7′-dichlorofluorescein diacetate increased at 10 min after disinfection with 70% ethanol compared to the activity at 30 s. The increase was significant for triggering oxidative burst with formylpeptide but not with phorbol myristate acetate.

Secretoneurin, derived from the chromogranin secretogranin II, triggers the selective migration of human monocytes, eosinophils, fibroblasts, endothelial and smooth muscle cells. More recently, we located specific binding sites on the human monocytic cell line MonoMac-6. Differentiated U937 transendothelial diapedesis was evaluated using an in vitro model of the vascular wall and specific monoclonal antibodies against CD11/CD18 and the α-chains of the very late activation antigen (VLA)-4 were used to evaluate involved adhesion molecules. Results showed a significant migratory response to secretoneurin between 10−8 to 10−10 M. Migration was comparable to a maximal effect induced by the monocyte chemotactic agent N-formyl-Met–Leu–Phe (fMLP, 10−8 M). Rabbit anti-secretoneurin antibodies were able to block the neuropeptide effect but not of fMLP and a trypsinized secretoneurin preparation as well as the secretogranin II-fragment EL-17 were ineffective in eliciting migration. Transmigration of U937 across endothelial-layers toward secretoneurin is inhibited by antibodies to CD11/CD18 adhesion molecules. The novel neuropeptide secretoneurin may play a role in regulating migration of monocytes into the subendothelial space, supposing a role in inflammatory responses.

AIM: Review of evidence that the 33-amino-acid polypeptide secretoneurin, which is generated by proteolytic cleavage of secretogranin II, plays a role in neurogenic inflammation. METHODS: Survey of the literature using a MEDLINE search database. RESULTS: Secretoneurin is synthesized in spinal ganglia, transported through the dorsal roots and stored in the axon terminals of primary afferent neurons. Investigations using capsaicin suggest that secretoneurin functions as an excitatory transmitter. Secretoneurin specifically activates various cell functions including the chemotactic migration of monocytes, eosinophils, fibroblasts, smooth muscle cells, and endothelial cells, which suggests that the peptide may modulate inflammatory reactions. Secretoneurin receptors have been functionally characterized. They are G-proteins linked and effects are abrogated by inhibition of protein kinase C. CONCLUSION: With actions as diverse as those seen with other mediators such as tachykinins, secretoneurin may be considered another sensory neuropeptide with modulatory potential in neurogenic inflammation.

OBJECTIVETo define the respiratory burst activity of neutrophils, the total anti-oxidative status of plasma, and the parameters of systemic inflammation in patients with ankylosing spondylitis (AS) before and after a combined radon-hyperthermia treatment in the thermal tunnels of Böckstein-Bad Gastein in Austria.METHODSIn 20 patients with AS the effects of a total of 15 hours of radon-hyperthermia-treatment spread over a period of three weeks were studied. The respiratory burst activity of neutrophils was measured fluorometrically using dichlorofluorescein diacetate, the total anti-oxidant status was measured using azinodiethyl-benzthiazoline-sulphonate, and inflammation parameters were determined by routine laboratory assays.RESULTSBefore treatment, the basal neutrophil respiratory burst in patients (n = 20) was 409 +/- 62 fluorescence arbitrary units (AU; mean +/- SEM) and 359 +/- 37 AU in controls (n = 9; p > 0.5); the stimulated respiratory burst (fMet-Leu-Phe, 10(-6) M) was 1,027 +/- 133 AU in patients and 1,152 +/- 218 AU in controls (p > 0.5). After treatment, the basal neutrophil respiratory burst in patients (n = 19) was 137 +/- 16 and in controls it was 174 +/- 35 AU (n = 8; p > 0.1); the stimulated respiratory burst was 670 +/- 66 and 1,305 +/- 82 AU, in patients and controls respectively (p < 0.001). No effects of treatment on the total anti-oxidant status of the plasma or on the parameters of inflammation were detected.CONCLUSIONCombined radon-hyperthermia treatment reduces the respiratory burst activity of the blood circulating neutrophils in patients with AS. If respiratory burst activity from the neutrophils plays a role in the pathophysiology of ankylosing spondylitis, the observed reduction may be related to the beneficial effects of radon-hyperthermia treatment.

In the present study, we investigated the effects of the anti-inflammatory drug pentoxifylline (PTX) on activation of endothelial cells for enhanced adhesion and transmigration of neutrophils by lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF), interleukin-1 (IL-1) and granulocyte colony-stimulating factor (G-CSF). To evaluate the mechanism by which PTX exerts its effect, human umbilical vein endothelial cells (HUVEC) were pretreated with theophylline, 2′-O-dibutyryl-3′, 5′-cyclic adenosine monophosphate (db cAMP), and 3-isobutyl-1-methylxanthine, respectively, prior to stimulation. Pretreatment of HUVEC with PTX significantly antagonized TNF-, IL-1-, and G-CSF-activated transmigration of neutrophils. Additive stimulatory effects of PTX were seen with LPS. With the exception of theophylline, all other test cAMP-raising agents stimulated transmigration in similar fashion to PTX. Upon stimulation with TNF or LPS, HUVEC produced IL-8 and PTX affected this process in opposing fashions, with inhibition of the effects of TNF and augmentation of those of LPS. These results demonstrate that PTX differentially affects mediator-induced activation of HUVEC. The present IL-8 dependent and cAMP-regulated augmentation of LPS-induced stimulation of transmigration is the first description of an additive effect of PTX with a pro-inflammatory agent.

Effects of vasoactive intestinal peptide (VIP) on T cell migration are mediated by structurally distinct types I (VIPR1) and II (VIPR2) G protein-associated receptors. The two receptor types were proposed to transduce opposite effects on human T cells, since cytokine-induced chemotaxis of VIPR1-bearing HuT 78 human T cells, in contrast to T cells that express VIPR2, was inhibited by VIP. We studied chemotactic effects of VIP and related agonists with different affinities for VIP- and peptide histidine-isoleucine (PHI)-related receptors. All, VIP, secretin (SEC), a specific ligand for VIPR1, helodermin (HEL), an activator of helodermin-preferring VIPR2, as well as PHI, stimulated chemotaxis into micropore filters of both normal human peripheral blood T and B cells. Involvement of VIPRs was supported by inhibition of VIP-related agonist-induced migration of T and B cells with a VIPR antagonist. Peripheral blood lymphocyte (PBL) chemotaxis to VIP, SEC, HEL and PHI was reduced by inhibition of tyrosine kinase and pertussis or cholera toxin, whereas inhibition of protein kinase C only affected SEC-induced chemotaxis of PBL significantly. VIP-related agonists induced deactivation of migration at high concentrations. Findings in PBL suggest that VIPR1 activation can stimulate normal T and B cell chemotaxis. Different signaling mechanisms may be involved in mediating chemotactic activation of VIPRs and PHIRs, which may allow further exploration of receptor-dependent mechanisms and signaling pathways of VIP as mediator of PBL functions.

The identification of opioid receptors on peripheral sensory nerves has prompted investigations evaluating the role of opioids in peripheral antinociception [1]. The presence of endogenous ligands of these receptors, the opioid peptides, has been demonstrated in lymphocytes and monocytes/macrophages within the inflamed tissue [2,3]. Release of opioid peptides from these cells may activate opioid receptors of leukocytes, and in vitro studies suggest that immune cells themselves are responsive to opioids [1]. Because chemotactic activation of leukocytes regulates the presence of the various immune cell types at sites of inflammation, chemoattractants may play a role in peripheral antinociception [1]. Activation of opioid receptors on leukocytes by beta-endorphin (END) induces in vitro chemotaxis of monocytes and T lymphocytes [4]. We investigated whether END is a chemoattractant for B lymphocytes. Methods Mononuclear leukocytes (MNL) were isolated from EDTA-treated blood of healthy volunteers by using Lymphoprep[registered sign] density-gradient centrifugation (Nycomed Pharma AS, Oslo, Norway) as previously described [5]. The MNL preparations were resuspended in RPMI-1640/10% fetal calf serum (Biologic Industries, Kibbutz Beit Haemek, Israel) and incubated at 106 cells/mL at 38[degree sign]C for 30 min. Nonadherent MNL (peripheral blood lymphocytes; PBL) were placed into new tissue culture dishes and incubated for another 90 min. PBL were then washed and resuspended in RPMI-1640/0.5% bovine serum albumin (BSA; Behringwerke AG, Marburg, Germany). Isolation of B lymphocytes was performed by using magnetic cell sorting colloidal superparamagnetic microbeads conjugated with monoclonal anti-human CD19 antibodies (Miltenyi Biotec, Bergisch Gladbach, Germany) as previously described [5]. The CD19+ PBL were resuspended in RPMI-1640 containing 0.5% BSA. Purity of the sorted CD19+ cells was >95% by fluorescence-activated cell sorter analysis. The CD19 fraction of PBL was used for T lymphocyte studies. Cell migration was measured using a modification of the Boyden chamber assay [5,6]. Cells were resuspended at 106 cells/mL in RPMI-1640/0.5% BSA before being placed in the upper well of a 48-well chemotaxis chamber (Neuroprobe, Cabin John, MD). The lower wells of the chemotaxis chamber contained chemoattractants. In checkerboard experiments, chemoattractant was put in upper and lower wells. Upper and lower wells were separated by a 5-[micro sign]m pore cellulose nitrate filter (Sartorius, Vienna, Austria). After incubation for 2 h at 37[degree sign]C, migration into the filter was quantitated by the leading front technique [5,6]. Data are expressed as the chemotaxis index, which is the ratio between the distance cells migrate toward test substances and the distance they migrate toward the control medium. Migration toward medium reflects spontaneous migration. END, fMet-Leu-Phe (FMLP), substance P (SP), and naloxone were purchased from Sigma Chemical Company (St. Louis, MO). Interleukin-8 (IL-8) was obtained from Boehringer-Mannheim (Mannheim, Germany). Lyophilized END and SP were reconstituted in 0.2N acetic acid, FMLP was dissolved in dimethylsulfoxide (10 mM), and IL-8 was dissolved in assay medium (1 mM). All these stock solutions were stored at -20[degree sign]C. Before use, END was diluted into assay medium to concentrations of 1 [micro sign]M-1 fM. For positive controls, IL-8 and SP were used at optimal concentrations of 1 nM and 0.1 [micro sign]M, respectively [5]. FMLP was used at 10 nM, which is the optimal chemotactic concentration for monocytes [5]. We used Mann-Whitney U-tests and Kruskal-Wallis tests for nonparametric analyses of results (StatView Software Package; Abacus, Berkeley, CA). A P value of < 0.05 was considered significant. Results END stimulated chemotactic migration of PBL, T lymphocytes, and B lymphocytes in a dose-dependent manner, with two peaks: one occurring at 0.1 pM and another at 0.1 [micro sign]M, the latter being the highest concentration tested (Figure 1).Figure 1: Effects of various concentrations of beta-endorphin on migration of human peripheral blood lymphocytes (PBL), T lymphocytes, and B lymphocytes into nitrocellulose micropore filters. The cell populations were incubated in the upper wells of modified multiwell Boyden chambers and exposed to concentration gradients of beta-endorphin added to the lower wells (n = 7). fMet-Leu-Phe (FMLP) and interleukin 8 (IL-8) were used as negative and positive controls, respectively (n = 2). The chemotaxis index represents the ratio between the distance cells migrate toward test substances and the distance they migrate toward control medium (mean +/- SEM, independent experiments each performed in replicates of four). *P < 0.05, **P < 0.01 (Mann-Whitney U-test) versus medium control calculated from raw data before normalization.The migratory response of B lymphocytes to END could be chemotaxis (directed movement of cells along a chemotactic gradient) or chemokinesis (enhanced speed or frequency of migration). In a checkerboard analysis, the addition of END exclusively to the upper compartment resulted in enhanced migratory responses of B lymphocytes (chemokinesis); a gradually increasing concentration gradient of END between the lower and upper compartment also led to increased migration of PBL toward the lower compartment, and this effect was significantly stronger than that for upper chamber chemokinesis (chemotaxis) (Table 1).Table 1: Effect of Various Concentration Gradients of beta-Endorphin on B Lymphocyte MigrationNaloxone itself has a weak but significant stimulatory effect on the migratory behaviors of B lymphocytes as well as T lymphocytes, and is at the same time an opiate receptor antagonist. SP-stimulated migration of cells was not affected by naloxone (0.1 mM). However, naloxone prevented the migration of lymphocytes to 0.1 [micro sign]M but not to 0.1 pM END, thus confirming that the migration of lymphocytes is mediated by both opiate- and nonopiate-dependent mechanisms (Figure 2).Figure 2: Effects of naloxone on migration of human T lymphocytes and B lymphocytes into nitrocellulose micropore filters toward beta-endorphin (END) or substance P (SP). The cell populations were incubated in the upper wells of modified multiwell Boyden chambers and exposed to concentration gradients at 0.1 [micro sign]M substance P or 0.1 [micro sign]M or 0.1 nM beta-endorphin added to the lower wells, with and without 10 [micro sign]M naloxone (n = 7). The chemotaxis index represents the ratio between the distance cells migrate toward test substances and the distance they migrate toward control medium (mean +/- SEM, independent experiments each performed in replicates of four). n.s. = not significant (P > 0.05). *P < 0.05 (Mann-Whitney U-test) versus medium control calculated from raw data before normalization.Discussion Previous studies have demonstrated that END is chemotactic for neutrophils, monocytes, and T lymphocytes via classical (naloxone-sensitive) [4,7,8] and nonclassical (naloxone-insensitive) mechanisms [9]. END-induced directed migration of PBL and T lymphocytes was confirmed in the present study and the two optimal doses were 0.1 [micro sign]M and 0.1 pM, respectively [4]. We have extended the previous work by showing that END has a chemotactic effect on B lymphocytes as well as on T lymphocytes. The two optimal dose-responses were also seen for B lymphocytes. Our data allow the conclusion that migration of B lymphocytes toward END is a result of both chemokinesis and chemotaxis, as positive concentration gradients of END were required to induce maximal migration. In inflammation, the functions of endogenous opioids may involve all or any of micro-, kappa-, and delta-opiate receptors. The effects of END are preferentially mediated by micro- and delta-opiate receptors. [9]. The analysis of human cell lines revealed delta-opioid receptor transcripts in T and B lymphocyte and monocyte cell lines, whereas the kappa transcript was found in B cell lines only [3]. Production of immunoglobulin by B lymphocytes was inhibited by opioid receptor class-selective agonists [10]. Naloxone, a nonpeptide antagonist with high affinity and well established specificity for opiate receptors, abolished opioid-induced migration and adhesion molecule expression in human pre-B leukemia cell lines [11]. Our data also clearly indicate that human peripheral blood B lymphocyte migration to END at doses of approximately 0.1 [micro sign]M is opiate receptor-mediated. Migration of B lymphocytes stimulated by low concentrations of END is not antagonized by naloxone, which suggests the involvement of a nonopiate-dependent mechanism, as was previously reported for monocytes and T lymphocytes [12]. Naloxone alone was able to induce lymphocyte migration, which suggests a novel role as a partial lymphocyte chemotaxis receptor agonist. The mechanism of this action is unknown. Chemotaxis in response to sensory neuropeptides is a critical step in neurogenic inflammation. Under identical conditions, we have previously compared the effects of four major neuropeptides from primary afferent sensory nerves-namely SP, vasoactive intestinal peptide, calcitonin gene-related peptide, and secretoneurin-on the migration of monocytes and lymphocytes [5]. Using this approach, SN was identified as a neuropeptide eliciting only chemotactic migration of monocytes, whereas lymphocyte migration remained unaffected. In contrast to the sensory neuropeptides, END is a neuroendocrine and immune hormone [1,2]. This antinociceptive opioid peptide may also contribute to the local accumulation of inflammatory cells, including neutrophils, monocytes, and T lymphocytes [7-9,12]. Our data ascribing a B lymphocyte chemotactic activity to this peptide strengthen the argument that this peptide may be involved in region-specific attraction of immune cells in pain conditions.

Proliferative vitreoretinopathy is a severe ocular disorder characterized by unwanted proliferation of cells and excessive production of fibrous tissue, which leads to the formation of cellular membranes on the surface of the retina and in the vitreous. Proliferative vitreoretinopathy is the most common cause of failure in retinal reattachment surgery, approximately occurring in one out of ten operated eyes. Proliferation of retinal pigment epithelial cells and fibroblasts is a cornerstone in the pathogenesis of proliferative vitreoretinopathy. An in vitro-proliferation assay showed previously that intraocular fluid from patients with proliferative vitreoretinopathy is potently effective in stimulating proliferation of human fibroblasts. Here we show that exposure of human fibroblasts to vitreous fluids from patients with proliferative vitreoretinopathy causes a rapid and sustained increase in arachidonic acid metabolite release as measured by competitive enzyme-immunoassay. The findings implicate prostaglandin E2 as a contributor to enhanced intraocular fibrosis in proliferative vitreoretinopathy. As prostaglandin E2 is a mediator of continuous aqueous-blood retinal barrier breakdown in this severe disease, cycclooxygenase inhibitors such as acetylsalicylic acid, which was successfully used in this study for blocking the effect of intraocular fluid, may be useful agents in targeting the progression of intraocular fibrosis.

Secretoneurin (SN), a 33-amino acid neuropeptide, is derived from secretogranin II that is released from sensory afferent C-fibers by capsaicin. Described functions of secretoneurin include chemotaxis of monocytes and endothelial cells, and inhibition of endothelial cell proliferation. Inhibition of monocyte chemotaxis by staurosporine indicated involvement of specific signaling pathways. We have tested effects of SN, substance P (SP), and interleukin-8 (IL-8) on eosinophil migration in modified Boyden chambers including signaling mechanisms of neuropeptide and cytokine stimulation of human eosinophils. Experiments showed SN as eosinophil chemoattractant comparable in its potency to IL-8. Checkerboard analysis, usage of a specific anti–SN-antibody, and receptor desensitization experiments confirmed the chemotactic activity. Preincubation of the cells with effective concentrations of staurosporine or tyrphostin-23 showed no effect, whereas treatment with wortmannin (WTN) or 3-isobutyl-1-methylxantin (IBMX) completely blocked SN-induced migration. Additionally, experiments ruled out tyrphostin-23- and WTN-sensitive signaling pathways for SP-induced chemotaxis of eosinophils. We conclude that SN-stimulated human eosinophil chemotaxis is mediated via a unique and specific signal transduction pathway that involves activation of phosphodiesterases and WTN-sensitive enzymes, ie, phospholipase D and phosphatidylinositol-3-kinase. In contrast, we report that activation of the latter and tyrosine kinases is required for SP-induced chemotaxis of eosinophils.

1 Stimulation of chemotaxis of human polymorphonuclear leucocytes (PMNs) with the chemoattractive peptide fMLP (N-formyl-Met-Leu-Phe) is paralleled by profound morphological and metabolic alterations like changes of intracellular pH (pH(i)) and cell shape. The present study was performed to investigate the interrelation of cell volume (CV) regulatory ion transport, pH(i) and migration of fMLP stimulated PMNs.2 Addition of fMLP to PMNs stimulated directed migration in Boyden chamber assays and was accompanied by rapid initial intracellular acidification and cell swelling.3 Inhibition of the Na+/H+ exchanger suppressed fMLP stimulated cell migration, accelerated the intracellular acidification and inhibited the fMLP-induced cell swelling.4 Step omission of extracellular Na+ caused intracellular acidification, which was accelerated by subsequent addition of gastric H+/K+ ATPase inhibitor SCH 28080, or by omission of extracellular K+ ions. In addition Na+ removal caused cell swelling, which was further enhanced by fMLP.5 H+/K+ ATPase inhibitors omeprazole and SCH 28080 inhibited stimulated migration and blunted the fMLP-induced increase in CV.6 Increasing extracellular osmolarity by addition of mannitol to the extracellular solution caused cell shrinkage followed by regulatory volume increase, partially due to activation of the Na+/H(+ )exchanger. In fMLP-stimulated cells the CV increase was counteracted by simultaneous addition of mannitol. Under these conditions the fMLP stimulated migration was inhibited.7 The antibacterial activity of PMNs was not modified by Hoe 694 or omeprazole.8 Western analysis with a monoclonal anti gastric H+/K+-ATPase beta-subunit antibody detected a glycosylated 35 kD core protein in lysates of mouse and human gastric mucosa as well as in human PMNs.9 The results indicate that fMLP leads to cell swelling of PMNs due to activation of the Na+/H+ exchanger and a K+-dependent H+-extruding mechanism, presumably an H+/K+ ATPase. Inhibition of these ion transporters suppresses the increase in CV and precludes PMNs from stimulated migration.

Objectives: The release of monocyte chemoattractant protein-1 (MCP-I) in the vessel wall may lend to accumulation of monocytes in the subendothelial space. The role of neutrophils (PMNL) in the initiation of this process is unknown. We tested whether PMNL are able to induce the production and release of MCP-1 in endothelial cells. Methods: PMNL were allowed to interact with human umbilical vein endothelial cell (HUVEC) monolayers in culture. Culture media were collected and assessed for chemotactic activity on mononuclear leukocytes (MNC) or purified monocytes in a modified Boyden chamber assay. Additionally, MCP-I levels in supernatants were quantified by ELISA. Results: Media from unstimulated HUVEC culture supernatants induced a slight increase (1.2-fold) of MNC and purified monocyte chemotaxis, which was significantly augmented by addition of PMNL for 1 h (1.4-fold; P < 0,05). The increase in chemotaxis was time- and dose-dependent and could be blocked by an anti-MCP-1 monoclonal antibody. Media obtained after coculture of PMNL and HUVEC for 1-5 h contained increased amounts of MCP-1 as measured by ELISA; addition of cycloheximide abolished this response. Conclusions: Interaction of PMNL with endothelium induces the release of functionally active MCP-I suggesting, that in the vascular wall, PMNL may play a role in the recruitment of MNC. (C) 1998 Elsevier Science B.V. All rights reserved.

Background In atherosclerosis, both reductions and elevations in plasma levels of antioxidants have been reported. This study investigated total antioxidant capacity of plasma from subjects with atherosclerotic disease. Materials and methods The study population consisted of 48 men with or without carotid atherosclerosis. At baseline (1990) carotid arteries were evaluated by duplex sonography and plasma samples were obtained for testing antioxidant capacity by two different test systems. One assay system used neutrophils from healthy volunteers as a source of oxygen free radicals activating the non‐fluorescent dichlorofluorescin diacetate in the presence of antioxidant containing plasma from study subjects. In the other test system, total plasma antioxidants were detected colorimetrically by using 2,2′‐azino‐di‐(3‐ethylbenzthiazoline sulphonate), metmyoglobin and superoxide in the presence of plasma. Carotid arteries were re‐evaluated for the development of new plaques 5 years later (1995). Results Increased baseline total antioxidant capacity of plasma was significantly associated with the development of new atherosclerotic lesions during a period of 5 years. Conclusions Endogenous antioxidant capacity of plasma is increased in patients with active atherosclerotic disease. As scavenging of oxygen free radicals is thought to protect from atherogenesis, elevated antioxidative capacity may represent an adaptive mechanism.

Vascular endothelium activated by endotoxin and cytokines plays an important role in organ inflammation and blood leukocyte recruitment. Neutrophils, which are a homogeneous population of effector cells, are rapidly attracted in large numbers to sites of inflammation where they form an early response to infection or injury. Excessive production of various interleukins, TNF, arachidonic acid metabolites, and other substances by neutrophils and macrophages results in systemic endothelial cell injury, a fundamental problem. In the present study, we investigated in vitro the effects of thalidomide (THD) on activation of endothelial cells for enhanced transmigration of neutrophils by lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF), and interleukin-1 (IL-1). Modulation of endotoxin- and cytokine-induced neutrophil chemotaxis and respiratory burst by THD were also studied. Treatment of HUVEC with THD in combination with LPS, TNF, and IL-1, respectively, antagonized LPS-activated transmigration of neutrophils but stimulated the effects of TNF and IL-1. All of the agents used – THD, LPS, TNF, and IL-1 – inhibited neutrophil chemotaxis. Addition of THD to the neutrophils had no effect on LPS-inhibited chemotaxis whereas the TNF- and IL-1-induced chemotaxis was modulated in a bimodal manner. However, THD failed to influence neutrophil respiratory burst activity. Results demonstrate that THD differentially affects mediator-induced activation of HUVEC and neutrophils.

Vascular cell responses in inflammation are affected by several neuropeptides of perivascular nerve fibers. Secretoneurin is a 33-amino acid peptide that is coreleased from these nerve endings with other proinflammatory neuropeptides, eg, substance P and calcitonin gene-related peptide. Furthermore, secretoneurin has been shown to be chemotactic for human skin fibroblasts and human blood monocytes in vitro and in vivo. An action on cellular components of the vascular wall is not yet reported. We therefore investigated in vitro effects of this novel sensory neuropeptide on endothelial cells. Secretoneurin exerted a potent and reversible inhibitory effect both on endothelial cell growth under low serum conditions (1% fetal calf serum) and endothelial cell growth factor-activated endothelial cell proliferation. We show in the present study that secretoneurin exerts this effect on aortic (rat) and pulmonary artery (bovine) endothelial cells, as well as venous (human umbilical vein) endothelium. Endothelial cell chemotaxis was tested by means of three different migration assays employing nitrocellulose and polycarbonate micropore filters. Secretoneurin consistently exhibited potent chemoattractant activity. The effective concentrations for the observed effects were in the picomolar range. The combination of chemotactic and antiproliferative effects on endothelial cells suggests that secretoneurin may act as a regulatory factor of vascular cell functions.

Pravastatin, a hydrophilic inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, has been reported to beneficially affect atherogenesis, plaque stability, and transient myocardial ischemia in significant coronary artery disease by influencing lipid metabolism and by intracellular signaling via mevalonate pathway products other than cholesterol. Leukocytes are implicated to play a pathophysiological role in these events. We were interested in finding out whether pravastatin could affect transendothelial migration (TEM), chemotaxis, and respiratory burst activity of the neutrophil ex vivo. In addition, effects on monocyte and T-lymphocyte chemotaxis were tested. For TEM assays, monolayers of human umbilical vein endothelial cells (HUVECs) were grown to confluence on polycarbonate filters bearing 5-microns pores in Transwell (Costar) culture plate inserts. Chemotaxis experiments were performed using modified Boyden chambers with cellulose nitrate micropore filters. Respiratory burst activity was measured fluorometrically. Treatment of neutrophils and monocytes with pravastatin at 2 to 200 mumol/L and 10 to 1000 mumol/L, respectively, significantly decreased chemotaxis triggered by fMet-Leu-Phe. This effect was abolished in the presence of mevalonic acid (500 mumol/L); no effect of pravastatin was seen on T-lymphocyte chemotaxis triggered by interleukin-8. Preincubation of neutrophils with pravastatin (200 mumol/L) also resulted in a significant reduction in the number of neutrophils that transmigrated a tumor necrosis factor-stimulated or lipopolysaccharide-stimulated HUVEC monolayer. At none of the concentrations tested (2 pmol/L to 200 mumol/L) did pravastatin affect neutrophil respiratory burst activity. We conclude that pravastatin may alter monocyte chemotaxis and neutrophil-endothelial interactions in migratory responses at concentrations obtained in vivo with cholesterol-lowering doses.

We studied the chemotactic effects of calcitonin gene-related peptide, vasoactive intestinal peptide, substance P (SP), and secretoneurin on PBMC and PBL using micropore filter assays. All four peptides induced migration of PBMC, whereas only calcitonin gene-related peptide, vasoactive intestinal peptide, and SP were chemotactic for PBL. Secretoneurin, known to induce monocyte chemotaxis, was unable to affect lymphocyte migration. Effects of SP on PBL were characterized by checkerboard analyses and represented true chemotaxis. Both T and B cells responded chemotactically to SP, the functional activity of SP residing in its C-terminal amino acid sequence. Involvement of neurokinin (NK) receptors was supported by inhibition of SP-induced migration of PBL with an NK1 receptor antagonist and induction of migration with [Sar9, Met(O2)11]SP and [PyrGlu6, Pro9]SP(6-11), two specific agonists for NK1 receptors, but not with [beta-Ala8]NK A(4-10), an agonist for NK2 receptors. PBL chemotaxis to SP was abolished by inhibition of tyrosin kinase but not by that of protein kinase C. Preincubation of PBL with pertussis or cholera toxin inhibited SP chemotaxis, indicating that in PBL, NK receptors for chemotaxis probably are coupled with G protein and involve a tyrosin kinase signaling pathway. We conclude that, together with calcitonin gene-related peptide and vasoactive intestinal peptide, SP is a lymphocyte chemoattractant, whereas secretoneurin, which is coreleased from sensory nerve endings, is not.

TNF is produced by monocytes/macrophages in response to endotoxin, which may lead to septic shock. TNF stimulates neutrophil adherence, degranulation, and superoxide production, but inhibits neutrophil migration. A mitigating anti-inflammatory effect can be experimentally induced in septic shock by TNF blockers, such as pentoxifylline, and is also suggested for treatment with hrG-CSF. With regard to the combination of pentoxifylline and hrG-CSF, the purpose of this investigation was to explore whether and in what way the effects of hrG-CSF and pentoxifylline interact with each other in neutrophils. To this end, we studied the effects of pentoxifylline on TNF- and G-CSF-induced modulation of neutrophil chemotaxis and O2 release. TNF and G-CSF decreased directed migration of neutrophils to FMLP or IL-8. High-dose pentoxifylline (1 mM) was able to counteract the effect of TNF but not that of G-CSF on neutrophil migration. In the presence of pentoxifylline, TNF and G-CSF were unable to stimulate respiratory burst. In contrast, pre-exposure of cells to pentoxifylline followed by washing increased the priming effect of TNF or hrG-CSF on neutrophil respiratory burst activity. The methylxanthine derivative by itself showed no effect on spontaneous and fMLP-stimulated O2 release by neutrophils. Stimulation of neutrophil respiratory burst by pentoxifylline may not be detectable in the presence of pentoxifylline due to its known oxygen-radical scavenging function. Results suggest that by blocking the inflammatory action of TNF on neutrophils, pentoxifylline may diminish endothelial cell damage caused by inhibited neutrophil chemotaxis. On the other hand, since transiently present pentoxifylline may enhance the respiratory burst activity of TNF- or hrG-CSF-primed neutrophils, concomitant administration of pentoxifylline and hrG-CSF to patients with SIRS/sepsis might diminish beneficial effects of the latter and additional deleterious effects might occur.

Endothelial cells used in transendothelial migration assays have been derived from different sources including human umbilical vein and bovine pulmonary artery. As plasma-induced activation of endothelial cells for enhanced transmigration of leukocytes may be pathophysiologically relevant, the role of species differences between plasma and endothelial cells was studied. The effects of human plasma on human umbilical vein and bovine pulmonary artery endothelial cells with regard to transmigration of neutrophils were compared. Transendothelial migration of neutrophils was tested employing endothelial cell-covered Transwell cell culture chamber inserts with micropore filters of 5 microns pore size. Results showed that human plasma induces neutrophil transmigration of both human umbilical vein and bovine pulmonary artery endothelial cell monolayers. Plasma samples from 50 to 69 year old men with and without carotid arteriosclerosis (N = 152) stimulated endothelial cells of human origin significantly stronger for transmigration of neutrophils than endothelial cells of bovine origin. Analyses of limits of agreement and of correlation of the two methods indicated that bovine pulmonary artery and human umbilical vein endothelial cells cannot replace each other in assays of plasma-induced activation of transmigration. Plasma levels of soluble adhesion molecule P-selectin, which are elevated in patients with arteriosclerotic diseases, are inversely related to plasma-induced activation of neutrophil transmigration of endothelial monolayers of human origin but not of bovine origin (N = 65). As rates of transmigration that were induced by plasma from subjects without carotid arteriosclerosis (N = 73) were significantly related between human and bovine endothelium, elevated plasma levels of soluble P-selectin may thus affect results of assays for plasma-induced activation of neutrophil transmigration of endothelial monolayers in a species-specific manner.

Tumor necrosis factor-α (TNF) is reported to cause tissue damage via enhanced neutrophil (PMNL) degranulation, superoxide production and altered PMNL migration. We investigated the effect of pentoxifylline (PTX) on TNF-induced respiratory burst activity of the PMNL. Since PTX has been reported to influence TNF-induced PMNL functions, our studies focused on the effects of timing and duration of the presence of PTX on superoxide anion production by PMNL exposed to TNF. When PMNL are exposed to PTX prior to priming with TNF and triggering with fMLP, respiratory burst activity was significantly enhanced by PTX, whereas the stimulatory effect of TNF was completely blunted by the continuous presence of PTX. Since free radical-scavenging properties of PTX have only recently been identified and may explain inhibitory effects on TNF-induced respiratory burst reported in the literature, our data for the first time suggests additive priming actions of PTX and TNF on PMNL respiratory burst activity.