Inhibitory effect of trilinolein on endothelin-1-induced c-fos gene expression in cultured neonatal rat cardiomyocytes

Trilinolein, isolated from the traditional Chinese herb Sanchi (Panax notoginseng), has been shown to have myocardial protective effects via its antioxidant ability. However, the cellular and molecular mechanisms of the protective effect of trilinolein in the heart remain to be elucidated. Oxidative mechanisms have been implicated in neonatal cardiomyocyte hypertrophy. We previously reported that ET-1 induces ROS generation via the ET_A receptor and ROS modulates c-fos gene expression. We have therefore examined whether trilinolein attenuates ROS production and ET-1-induced c-fos gene expression in cardiomyocytes. Cultured neonatal rat cardiomyocytes were stimulated with ET-1 (10 nM), and c-fos gene expression was examined. Trilinolein (1 and 10 μM) inhibited ET-1-induced c-fos gene expression in cardiomyocytes. We also examined the effects of trilinolein on ET-1- increased NADPH oxidase activity and superoxide formation. Trilinolein inhibited ET-1-increased NADPH oxidase activity and superoxide formation in a concentration-dependent manner. This increase in superoxide production by ET-1 was significantly inhibited by trilinolein, diphenyleneiodonium, or N-acetylcysteine. Trilinolein also decreased ET-1- or H₂O₂-induced extracellular signal-regulated kinase (ERK) phosphorylation, c-Jun NH₂-terminal kinase (JNK) phosphorylation, and activator protein-1 activation. These data indicate that trilinolein inhibits ET-1-induced ERK phosphorylation, JNK phosphorylation, and c-fos gene expression via attenuating superoxide production in cardiomyocytes. Hung-Yu Yang and Ju-Chi Liu equally contributed to this work     

Effect of trilinolein on cyclic nucleotide formation in human platelets: relationship with its antiplatelet effect and nitric oxide synthesis.

1. Trilinolein, a triacylglycerol with linoleic acid as the only fatty acid residue in all three esterified positions of glycerol, was recently reported to have an inhibitory effect on adrenaline-induced platelet aggregation. In the present study, we found that trilinolein at concentrations ranging from 0.01 to 1 microM increased cyclic GMP formation and decreased cyclic AMP formation in washed human platelets. Both NG-nitro-L-arginine methyl ester (L-NAME) and methylene blue attenuated the trilinolein-induced increase in cyclic GMP. 2. Adrenaline decreased not only the production of cyclic AMP but also that of cyclic GMP. Trilinolein antagonized the inhibitory effect of adrenaline on cyclic GMP formation, but potentiated the inhibitory effect of adrenaline on cyclic AMP accumulation. 3. Both trilinolein and adrenaline enhanced intracellular calcium but the increment of intracellular calcium induced by them was much less than that produced by thrombin. 4. We propose that the anti-platelet effect of trilinolein is mediated through an increase in cyclic GMP, and that the change in cyclic GMP results from stimulation of nitric oxide synthesis in platelets. 5. We also propose that reduction of both cyclic AMP and cyclic GMP are involved in adrenaline-induced platelet aggregation.     

槲皮素对TNFα诱导的内皮细胞与中性粒细胞粘附的抑制作用

目的:研究槲皮素对TNF_α诱导的内皮细胞与中性粒细胞粘附的抑制作用及作用机制。方法:用髓过氧化酶法测定中性粒细胞与内皮细胞的粘附,ELISA法测定内皮细胞粘附分子的表达。结果:TNF_α通过增加/诱导内皮细胞ICAM-1,VCAM-1及E-selectin的表达而剂量、时间依赖性地增加内皮细胞与中性粒细胞的粘附。槲皮素可剂量依赖性地抑制上述粘附,其作用机制为抑制TNF_α诱导内皮细胞上述3种粘附分子的表达。结论:槲皮素通过抑制ICAM-1,VCAM-1及E-selectin的表达而降低TNF_α诱导的内皮细胞与中性粒细胞的粘附。     

Molecular mechanism of the inhibitory effect of trilinolein on endothelin-1-induced hypertrophy of cultured neonatal rat cardiomyocytes

Trilinolein, isolated from the traditional Chinese herb Sanchi ( Panax notoginseng), has been shown to have myocardial protective effects via its antioxidant ability. However, the cellular and molecular mechanisms of the protective effect of trilinolein in the heart remain to be elucidated. Oxidative mechanisms have been implicated in neonatal cardiomyocyte hypertrophy. We therefore have examined whether trilinolein attenuates reactive oxygen species (ROS) production and thus ET-1-induced hypertrophy of cardiomyocytes. Cultured neonatal rat cardiomyocytes were stimulated with ET-1 (10 nM), [3H]leucine incorporation and the beta-myosin heavy chain (beta-MyHC) promoter activity were examined. Trilinolein (1 and 10 microM) inhibited the ET-1-induced increase of [3H]-leucine incorporation in a concentration-dependent manner. Trilinolein (1 and 10 microM) also inhibited ET-1-induced beta-MyHC promoter activity in cardiomyocytes. We further examined the effects of trilinolein on ET-1-induced intracellular ROS generation by measuring a redox-sensitive fluorescent dye, 2',7'-dichlorofluorescin diacetate, fluorescence intensity. Trilinolein (1 and 10 microM) inhibited ET-1-increased intracellular ROS levels in a concentration-dependent manner. This increase of ROS by ET-1 (10 nM) or H2O2 (25 microM) was significantly inhibited by trilinolein (10 microM) and N-acetylcysteine (10 mM). Moreover, ET-1- or H2O2-induced beta-MyHC promoter activity and protein synthesis were also inhibited by trilinolein (10 microM). These data indicate that trilinolein inhibits ET-1-induced beta-MyHC promoter activity, and subsequent hypertrophy via its antioxidant ability in cardiomyocytes.     

Protective effect of taurine on indomethacin-induced gastric mucosal injury

It has been suggested that oxygen-derived free radicals play an important role in the pathophysiology of acute gastric ulceration induced by NSAIDs and ischemia-reperfusion. Taurine is hypothetized to exert its protective effect on NSAIDs-induced gastric injury by its antioxidant properties. Protective effect of taurine on indomethacin-induced gastric mucosal lesion and its protection mechanism were investigated. Intragastric administration of 25 mg/kg of indomethacin induced hemorrhagic lesions on the glandular stomach in rats. Pretreatment with 0.25 or 0.5 g/kg of taurine one day before or for 3 days significantly reduced the gastric lesion formation and inhibited the elevation of lipid peroxide level, in gastric mucosa. The luminol-dependent chemiluminescence of rat peritoneal neutrophils increased immediately after treatment of FMLP or indomethacin. Taurine (5–20 mM) inhibited chemiluminescence of neutrophils activated by FMLP. Human neutrophils (polymorphonuclear leukocytes) significantly adhered to the confluent monolayer of human umbilical vein endothelial cells (HUVEC) after coincubation with indomethacin. This neutrophil adhesion induced by indomethacin to HUVEC was prevented by taurine in a dose-dependent manner. These results indicate that the protective effect of taurine against NSAIDs-induced gastric mucosal injury is due to its antioxidant effect, which inhibits lipid peroxidation and neutrophil activation.     

Docosahexaenoic acid inhibits cytokine-induced expression of P-selectin and neutrophil adhesion to endothelial cells

Dietary polyunsaturated fatty acids, such as docosahexaenoic acid, may inhibit pathological processes involving endothelial cell activation. Herein, it was found that treatment of endothelial cells with docosahexaenoic acid dose dependently reduced neutrophil adhesion provoked by tumor necrosis factor-alpha (TNF-alpha). In fact, pretreatment with 100 microM of docosahexaenoic acid for 24 h decreased TNF-alpha-induced neutrophil adhesion by 50%. Moreover, this pretreatment with docosahexaenoic acid (100 microM, 24 h) down-regulated TNF-alpha-induced endothelial cell surface expression of P-selection by 75%. Importantly, immunoneutralization of P-selectin reduced neutrophil adhesion to TNF-alpha-activated endothelial cells by more than 50%, indicating a significant role of P-selectin in this model. On the other hand, CXC chemokines, i.e. macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (KC), are also important regulators of neutrophil activation and adhesion. However, pretreatment with docosahexaenoic acid had no effect on TNF-alpha-provoked production of MIP-2 and KC in endothelial cells. Our study provide evidence that docosahexaenoic acid inhibits expression of P-selectin and subsequent adhesion of neutrophils to endothelial cells in response TNF-alpha, which may help explain the anti-inflammatory effects exerted by docosahexaenoic acid.     

The intravenous administration of tumor necrosis factor alpha,interleukin 8 and macrophage-derived neutrophil chemotactic factor inhibits neutrophil migration by stimulating nitric oxide production

1. The i.v. administration of tumor necrosis factor-α (TNF-α), interleukin-8 (IL-8) and the recently described macrophage-derived neutrophil chemotactic factor (MNCF) inhibits the recruitment of neutrophils to the inflammatory site.
2. Pretreatment of mice with the NO synthase antagonist, N^G-monomethyl-L-arginine (L-NMMA, 15–60 mg kg⁻¹), but not the inactive enantiomer D-NMMA (30 mg kg⁻¹), prevented in a dose-dependent manner the TNF-α, IL-8 and MNCF-mediated inhibition of neutrophil migration into thioglycollate-challenged peritoneal cavities.
3. Treatment of the neutrophils with TNFα (10⁻⁷ M), IL-8 (10⁻⁷ M) or MNCF blocked their migration towards FMLP in the chemotaxis assay. The pretreatment of the neutrophils with L-NMMA (50–200 μM) prevented in a dose-dependent manner the inhibition of FMLP-induced chemotaxis by IL-8, but did not alter the inhibition caused by TNF-α or MNCF. Different concentrations of the NO donors, S-nitroso-N-acetylpenicillamine (SNAP) or 3-morpholino-sydnonimine (SIN-1), did not alter this chemotaxis.
4. Preincubating the neutrophils with L-NMMA (200 μM) significantly increased the TNF-α (10⁻⁷ M) and MNCF-mediated neutrophil adhesion to unstimulated endothelial cells, but had no effect on IL-8 (10⁻⁷ M)-mediated adhesion.
5. Although NO donors did not directly affect the mechanisms of neutrophil motility, NO is involved in the in vitro inhibitory action of IL-8 on chemotaxis. The TNF-α and MNCF-mediated inhibition of neutrophil migration seems to be indirect, by affecting the mechanisms of adhesion. It was concluded that TNF-α-, IL-8- and MNCF-mediated inhibition of neutrophil migration is associated with the stimulation of NO production.

     

Reduction of ICAM-1 expression by carbon monoxide via soluble guanylate cyclase activation accounts for modulation of neutrophil migration

Previously, it was demonstrated that the heme/heme oxygenase (HO)/carbon monoxide (CO) pathway inhibits neutrophil recruitment during the inflammatory response. Herein, we addressed whether the inhibitory effect of the HO pathway on neutrophil adhesion and migration involves the reduction of intracellular adhesion molecule type (ICAM)-1 and β₂-integrin expression. Mice pretreated with a specific inhibitor of inducible HO (HO-1), zinc protoporphyrin (ZnPP) IX, exhibit enhanced neutrophil adhesion and migration induced by intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS). These findings are associated with an increase in ICAM-1 expression on mesentery venular endothelium. In accordance, HO-1 inhibition did not enhance LPS-induced neutrophil migration and adhesion in ICAM-1-deficient mice. Furthermore, the treatment with a CO donor (dimanganese decacarbonyl, DMDC) that inhibits adhesion and migration of the neutrophils, reduced LPS-induced ICAM-1 expression. Moreover, neither DMDC nor ZnPP IX treatments changed LPS-induced β₂-integrin expression on neutrophils. The effect of CO on ICAM-1 expression seems to be dependent on soluble guanylate cyclase (sGC) activation, since 1H-(1,2,4)oxadiazolo (4,3-a)quinoxalin-1-one (sGC inhibitor) prevented the observed CO effects. Finally, it was observed that the nitric oxide (NO) anti-inflammatory effects on ICAM-1 expression appear to be indirectly mediated by HO-1 activation, since the inhibition of HO-1 prevented the inhibitory effect of the NO donor (S-nitroso-N-acetylpenicillamine) on LPS-induced ICAM-1 expression. Taken together, these results suggest that CO inhibits ICAM-1 expression on endothelium by a mechanism dependent on sGC activation. Thus, our findings identify the HO-1/CO/guanosine 3′5′-cyclic monophosphate pathway as a potential target for the development of novel pharmacotherapy to control neutrophil migration in inflammatory diseases.     

Inhibitory effect of the water-soluble extract of Salvia miltiorrhiza on neutrophil-endothelial adhesion

The effect of the water-soluble extract (WSE) of Salvia miltiorrhiza on neutrophil-endothelial cell adhesion was investigated. Cell adhesion was evaluated by testing neutrophil myeloperoxidase activity: expression of adhesion molecules in human umbilical vein endothelial cells (HUVEC) was measured by ELISA: the neutrophil activation rate induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) was tested by the method of nitroblue tetrazolium (NBT) reduction. The results showed that the adhesion rate of neutrophils to unstimulated HUVEC was very low. TNFalpha (50 - 800 U/ml) increased the adhesion of neutrophils to TNFalpha-stimulated HUVEC in a concentration- and time-dependent manner. The WSE of Salvia miltiorrhiza (0.01 - 1 mg/ml) dose-dependently inhibited the adhesion of neutrophils. The inhibitory rate of the WSE of Salvia miltiorrhiza at 0.01, 0.1 and 1 mg/ml was 6.2%, 17.0% and 28.0%, respectively. fMLP (10(-9) - 10(-5) M) increased the activation rate of neutrophils concentration-dependently. The WSE of Salvia miltiorrhiza also concentration-dependently inhibited the adhesion of fMLP-activated neutrophils to HUVEC. The inhibitory rate of the WSE of Salvia miltiorrhiza at 0.001, 0.01 and 0.1 mg/ml was 5.3%, 26.3% and 28.9%, respectively. Moreover, TNFalpha upregulated expression of adhesion molecule E-selectin, ICAM-1 and VCAM-1. The WSE of Salvia miltiorrhiza had an inhibitory effect on TNF alpha-induced expression of these molecules. These results indicated that the WSE of Salvia miltiorrhiza inhibited neutrophil-endothelial adhesion. The action mechanism of the WSE of Salvia miltiorrhiza was partly related to suppressing the expression of adhesion molecules.     

Effects of mycophenolic acid on endothelial cells

Mycophenolate mofetil (MMF) is a potent immunosuppressant that inhibits the activity of inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in de novo synthesis of guanosine nucleotides. MMF has been used widely in solid-organ transplantation. Increased evidence indicated that MMF exhibited beneficial effects on various types of vasculitis, for reasons that were not fully understood. Endothelial cells play a pivotal role in the pathogenesis of vasculitis. Endothelium may not only be the main target for injury, but also be able to amplify the inflammatory response by adhesion molecule expression, leukocyte adhesion, cytokine production and angiogenesis. In the present study, the effect of mycophenolic acid (MPA), the active metabolite of MMF, on human umbilical vein endothelial cells (HUVECs) was investigated. MPA markedly inhibited tumor necrosis factor-alpha (TNFalpha)-induced intercellular adhesion molecule-1 (ICAM-1) mRNA and surface expression, suppressed TNFalpha-induced neutrophils adhesion to endothelial cells, and reduced TNFalpha-induced interleukin-6 (IL-6) secretion. The inhibitory effects of MPA on ICAM-1 surface expression and IL-6 secretion were not attenuated by addition of guanosine, implying that inhibition of these processes were not due to intracellular guanosine nucleotides depletion. MPA also decreased angiogenesis of endothelial cells in three-dimensional collagen gel culture system, reduced the migration in a wounded monolayer of endothelial cells, and inhibited the proliferation of endothelial cells. In conclusion, MPA exhibited multifarious effects on endothelial cells including inhibition of ICAM-1 expression, neutrophil attachment, IL-6 secretion, and the process of angiogenesis, which might contribute to the efficacy of MMF in the treatment of vasculitis.     

Inhibitory effect of trilinolein on angiotensin II-induced cardiomyocyte hypertrophy

The myocardial protective effects of trilinolein, isolated from the Chinese herb Sanchi (Panax notoginseng), may be related to its antioxidant effects. In the present study, we investigated the effects of trilinolein on angiotensin II-induced cardiomyocyte hypertrophy. Cultured neonatal rat cardiomyocytes were stimulated with angiotensin II, [3H]leucine incorporation and the beta-myosin heavy chain promoter activity were examined. We also examined the effects of trilinolein on angiotensin II-induced intracellular reactive oxygen species generation. Trilinolein significantly inhibited angiotensin II-increased protein synthesis, beta-myosin heavy chain promoter activity, and intracellular reactive oxygen species generation. Antioxidant N-acetylcysteine also decreased angiotensin II-increased protein synthesis and beta-myosin heavy chain promoter activity. Furthermore, trilinolein and N-acetylcysteine decreased angiotensin II- or hydrogen peroxide (H2O2)-activated mitogen-activated protein kinases (MAPKs) phosphorylation, and activator protein-1 (AP-1)- [or nuclear factor-kappaB (NF-kappaB)]-reporter activities. These data indicate that trilinolein inhibits angiotensin II-induced cardiomyocyte hypertrophy and beta-myosin heavy chain promoter activity via attenuation of reactive oxygen species generation.     

Superoxide anion mediates the L-selectin down-regulation induced by non-steroidal anti-inflammatory drugs in human neutrophils

Non-steroidal anti-inflammatory drugs (NSAIDs) induce the shedding of L-selectin in human neutrophils through a mechanism still not well understood. In this work we studied both the functional effect of NSAIDs on the neutrophils/endothelial cells dynamic interaction, and the potential involvement of reactive oxygen species (ROS) in the NSAIDs-mediated down-regulation of L-selectin. When human neutrophils were incubated with diclofenac, a significant reduction in the number of cells that rolled on activated endothelial cells was observed. Different NSAIDs (flufenamic acid, meclofenamic acid, diclofenac, indomethacin, nimesulide, flurbiprofen, meloxicam, phenylbutazone, piroxicam, ketoprofen and aspirin) caused variable increase in neutrophil intracellular ROS concentration, which was inversely proportional to the change produced in L-selectin surface expression. Pre-incubation of neutrophils with superoxide dismutase, but not with catalase, showed both a significant protective effect on the L-selectin down-regulation induced by several NSAIDs and a diminished effect of diclofenac on neutrophil rolling. Interestingly, diclofenac and flufenamic acid but not piroxicam significantly increased the extracellular superoxide anion production by neutrophils, and inhibition of nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase activity with diphenyleneiodonium prevented the down-regulation of L-selectin by diclofenac. In accordance with these results, neutrophils from patients with chronic granulomatous disease, a hereditary disease in which neutrophils show a reduced capacity to form superoxide radicals, exhibited a lower down-regulation of L-selectin (IC50: 15.3 μg/ml) compared to normal controls (IC50: 5.6 μg/ml) in response to diclofenac.ConclusionA group of NSAIDs is capable of interfering with the ability of neutrophils to interact with endothelial cells by triggering L-selectin-shedding through the NADPH-oxidase-dependent generation of superoxide anion at the plasma membrane.     

Sulphasalazine metabolites in experimental inflammation: Modulation of neutrophil function

Sulphasalazine is widely used in the treatment of inflammatory bowel disease. Its exact mode of action is unclear, but experimental evidence suggests that the active moiety, 5-aminosalicylic acid (5-ASA), modulates all or most of the functions of neutrophils during acute inflammation. These comprise adhesion to vascular endothelium, migration through the vessel wall in response to a chemotactic gradient, and the release of cytotoxic oxidants and proteases. In vitro, 5-ASA has been shown to reduce both neutrophil adhesion and leukotriene B4-mediated chemotaxis. It also provides significant protection against neutrophil-mediated increases in mucosal permeability induced by naturally occurring inflammatory mediators such asN-formyl-methionine-leucine-phenylalanine (fMLP). Which specific neutrophil function is modulated by 5-ASA to provide the latter effect remains unclear, but there is considerable evidence to indicate that it may both scavenge neutrophil-derived oxidants and inhibit their generation. Although such experimental evidence suggests that 5-ASA acts by modulation of neutrophil functions in patients with IBD, this remains speculation. Additional work is needed to define more clearly the therapeutic significance of the neutrophilic actions of 5-ASA.     

Protective effect of -arginine against stress-induced gastric mucosal lesions in rats and its relation to nitric oxide-mediated inhibition of neutrophil infiltration

Pretreatment with -arginine (150–600 mg kg⁻¹, i.p.), but not -arginine (600 mg kg⁻¹, i.p.), protected against gastric mucosal lesions in rats with water immersion restraint stress over a 6-h period. This protective effect occurred in a dose-dependent manner. Increases in the activities of inducible nitric oxide synthase (iNOS) and myeloperoxidase (MPO), an index of tissue neutrophil infiltration, and the concentration of nitrite/nitrate, breakdown products of nitric oxide, and a decrease in the activity of constitutive nitric oxide synthase (cNOS) occurred in the gastric mucosal tissue with the development of gastric mucosal lesions. The -arginine pretreatment attenuated the increases in iNOS and MPO activities and nitrite/nitrate concentration and the decrease in cNOS activity in the gastric mucosal tissue in a dose-dependent manner, while the -arginine pretreatment did not. Both the protective effect of -arginine (300 mg kg⁻¹) against stress-induced gastric mucosal lesions and the attenuating effect of the amino acid on the increases in gastric mucosal iNOS and MPO activities and the decrease in gastric mucosal cNOS activity with the lesion development were counteracted by pretreatment with N^G-monomethyl- -arginine (100 mg kg⁻¹, s.c.), a nitric oxide synthase inhibitor, but not its -isomer (100 mg kg⁻¹, s.c.). These results suggest that the protective effect of exogenously administered -arginine against stress-induced gastric mucosal lesions in rats is, at least in part, due to nitric oxide-mediated inhibition of neutrophil infiltration into the gastric mucosal tissue.     

Purines and neutrophil leukocytes

• 1.1. Adenosine is a normal constituent of all body fluids and its levels are raised, for example, by hypoxia and ischemia. In addition, both adenosine and ATP can be released by endothelial cells and neutrophils in response to physiologic stimulation.
• 2.2. Human neutrophil leukocytes possess multiple adenosine receptors and P₂ purinoceptors.
• 3.3. ATP can increase intracellular Ca²⁺ levels in neutrophils, cause degranulation and enzyme release, potentiate the oxidative burst and enhance their adhesion to the endothelium. ATP is broken down to adenosine by ecto-enzymes. Via A₁ receptors, adenosine can increase neutrophil chemotaxis and, via A_2A receptors, it can decrease the oxidative burst, degranulation and adhesion to endothelium.
• 4.4. Adenosine and adenine nucleotides are important endogenous modulators of neutrophil functions, and drugs may exert important actions via purinoceptors on neutrophil leukocytes.

     

Biological study of naphthalene derivatives with antiinflammatory activities

In this study, 18 synthetic naphthalene derivatives were tested for their inhibitory effects on the activation of neutrophils stimulated with N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP) or phorbol myristic acetate (PMA). Some of these compounds showed significant antiinflammatory activities. In general, esterification of 1‐naphthalene to compound 14 (2‐hydroxymethyl‐1‐naphthol diacetate; TAC) enhanced the antioxidant activity. Compound 15, N,N‐bis(2‐hydroxy‐1‐naphthylmethyl) amine, has moderate inhibitory activity on neutrophils stimulated with fMLP as compared to Mannich bases of naphthylene derivatives. Either substitution at 1 or 2 position, except TAC, disfavored the inhibitory effects evoked by PMA stimulation. The influence of these compounds on the release of granule enzyme lysozyme‐induced by fMLP was measured. TAC had the highest potency on the inhibition of lysozyme release from rat neutrophil degranulation. The effects of TAC on ionic currents in a mouse neuroblastoma and rat glioma hybrid cell line, NG105‐18, were also investigated with the aid of the whole‐cell patch‐clamp technique. TAC caused an inhibitory effect on voltage‐dependent L‐type Ca²⁺ current (I_Ca,L) with an IC₅₀ value of 0.8 µM. The inhibitory effect of TAC on I_Ca,L may not be caused by its inhibition of superoxide formation. Such an effect may, also in part, affect neuronal function. Drug. Dev. Res. 60:261–269, 2003. © 2003 Wiley‐Liss, Inc.     

Effect of activation on adhesion of flowing neutrophils to cultured endothelium: time course and inhibition by a calcium channel blocker (nitrendipine).

1. Adhesion of neutrophils to vascular endothelium plays an important role in inflammation and thrombosis. Modulation of adhesion may be therapeutic in these conditions. 2. A flow model was used to quantify adhesion of neutrophils to human cultured umbilical vein endothelial cells. The time course of the neutrophil response to activation by N-formyl-methionyl-leucylphenylalanine (fMLP, 10(-7) M) was studied and the inhibitory effects of the calcium-channel blockers, nitrendipine and nifedipine, were investigated. 3. Neutrophils adhered firmly to the endothelial cells without rolling, but initial attachment was highly dependent on shear stress; doubling the stress from 0.05 to 0.1Pa decreased the number of neutrophils adhering by over 80%. 4. Adhesion rapidly increased after activation of neutrophils by fMLP, peaking at 1-3 min post-treatment, and then decreased over the next 10-12 min. A monoclonal antibody to the beta 2-integrin component CD18 inhibited adhesion by over 80% for activated or unactivated cells. 5. The Ca-channel blocker, nitrendipine, but not nifedipine, significantly inhibited the fMLP-induced increase of adhesion in a dose-dependent manner (10(-8) to 10(-6) M). Dihydropyridines may be useful agents for modifying neutrophil function.     

Review article: Pathobiology of neutrophil interactions with intestinal epithelia

Neutrophil-epithelial interactions were modelled using polarized T84 cells and ligands were identified through observations of β₂-integrin dependence in patients with chronic granulomatous disease. Interactions between neutrophils and the apical membrane of crypt cells were analysed using HPLC and an in vitro model with T84 monolayers colonized by Salmonella typhimurium was used to assess neutrophil movement across the epithelium. The decline in transepithelial resistance following movement of neutrophils across the epithelial monolayer may have been due to an interaction between neutrophils and ligand ICAM-1 in which the neutrophils move along the paracellular pathway of epithelial cells. Cell surface polarity may influence these neutrophil-epithelial interactions which influence Cl secretion. These studies revealed that only strains produced in vivo were able to induce neutrophil transmigration in the in vitro model and may be indicative of new progressive therapies for inflammatory bowel disease.