Cigarette smoking is associated with increased human monocyte adhesion to endothelial cells: reversibility with oral L-arginine but not vitamin C

OBJECTIVES: This study sought to assess the effect of cigarette smoking on adhesion of human monocytes to human endothelial cells and to measure the effect of L-arginine and vitamin C supplementation on this interaction. BACKGROUND: Cigarette smoking has been associated with abnormal endothelial function and increased leukocyte adhesion to endothelium, both key early events in atherogenesis. Supplementation with both oral L-arginine (the physiologic substrate for nitric oxide) and vitamin C (an aqueous phase antioxidant) may improve endothelial function; however, their benefit in cigarette smokers is not known. METHODS: Serum was collected from eight smokers (mean [+/-SD] age 33 +/- 5 years) with no other coronary risk factors and eight age- and gender-matched lifelong nonsmokers. The serum was added to confluent monolayers of human umbilical vein endothelial cells and incubated for 24 h. Human monocytes obtained by counterflow centrifugation elutriation were then added to these monolayers for 1 h, and adhesion then was measured by light microscopy. To assess reversibility, monocyte/ endothelial cell adhesion was then measured for each subject 2 h after 2 g of oral vitamin C and 2 h after 7 g of oral L-arginine. RESULTS: In smokers compared with control subjects, monocyte/ endothelial cell adhesion was increased (46.4 +/- 4.5% vs. 27.0 +/- 5.2%, p < 0.001), endothelial expression of intercellular adhesion molecule (ICAM)-1 was increased (0.31 +/- 0.02 vs. 0.22 +/- 0.03, p = 0.004), and vitamin C levels were reduced (33.7 +/- 24.1 vs. 53.4 +/- 11.5 mumol/liter, p = 0.028). After oral L-arginine, monocyte/ endothelial cell adhesion was reduced in smokers (from 46.4 +/- 4.5% to 35.1 +/- 4.0%, p = 0.002), as was endothelial cell expression of ICAM-1 (from 0.31 +/- 0.02 to 0.27 +/- 0.01, p = 0.001). After vitamin C, there was no significant change in monocyte/ endothelial cell adhesion or ICAM-1 expression from baseline in the smokers despite an increase in vitamin C levels (to 115 +/- 7 mumol/liter). CONCLUSIONS: Cigarette smoking is associated with increased monocyte-endothelial cell adhesion when endothelial cells are exposed to serum from healthy young adults. This abnormality is acutely reversible by oral L-arginine but not by vitamin C.     

Differential regulation of eosinophil adhesion and transmigration by pulmonary microvascular endothelial cells

In bronchial asthma, eosinophils (EOS) adhere to, and migrate across, the lung microvasculature to exert their effector functions in the airways. This study was conducted to determine the effect of cytokines on adhesion molecule expression on human pulmonary microvascular endothelial cells (HPMEC) and the influence of these molecules on EOS adhesion and transmigration in vitro. Unlike ICAM-1 expression (>80% positive cytokine-treated HPMEC by flow cytometry), VCAM-1 expression varied with the cytokine(s) pretreatment; the order of potency was: TNF-alpha + IL-4 (82.2 +/- 4.2% positive cells) > TNF-alpha (41.8 +/- 5.1%) > IL-1beta (20.8 +/- 4.7%). IL-4 alone had no effect on either ICAM-1 or VCAM-1 expression. EOS adhesion to cytokine-treated HPMEC followed the same order as that observed for VCAM-1 expression. Interestingly, EOS migration across cytokine-treated HPMEC varied inversely with VCAM-1 expression on, and EOS adhesion to, HPMEC; IL-1beta (21.2 +/- 1.4% migration) > TNF-alpha (12.6 +/- 2.6%) > TNF-alpha + IL-4 (9.1 +/- 2.0%). EOS adhesion was greatest with TNF-alpha + IL-4-treated HPMEC, was dependent on VCAM-1, and inhibited with anti-alpha4 integrin mAb (67.7 +/- 7.5% inhibition, p < 0.0005). In contrast, the highest EOS migration occurred across IL-1beta-treated HPMEC and was inhibited by anti-beta2 integrin mAb (40.4 +/- 2.5% inhibition, p < 0.005). Viable HPMEC were required for EOS migration but not adhesion. Our results suggest that EOS adhesion and transmigration are differentially regulated by VCAM-1 and ICAM-1 expression and the interaction of these adhesion proteins with their respective counterligands, i.e., alpha4 and beta2 integrins on EOS.     

Enhanced monocyte tissue factor response after experimental balloon angioplasty in hypercholesterolemic rabbit: inhibition with dietary L-arginine

BACKGROUND: There is evidence that tissue factor (TF) is a major contributor to the thrombogenicity of a ruptured atherosclerotic plaque. Nitric oxide (NO) has antiatherogenic and antithrombotic properties. We investigated whether L-arginine (L-arg), the endogenous precursor of NO, might affect the ability of monocytes to produce TF. METHODS AND RESULTS: We studied TF expression in 18 rabbits with atherosclerosis induced by bilateral iliac damage and 10 weeks of a 2% cholesterol diet. Six weeks after the initiation of the diet, an angioplasty was performed. After angioplasty, the surviving rabbits (n=15) were randomized to receive L-arg (2.25%) supplementation in drinking water (L-arg group, n=8) or no treatment (untreated group, n=7). TF expression was evaluated in mononuclear cells from arterial blood in the presence and absence of endotoxin stimulation. Monocyte TF expression, as assessed with an amidolytic assay, did not differ significantly before or after the induction of atherosclerotic lesions (87+/-15 versus 70+/-12 mU of TF/1000 monocytes, P=NS). Endotoxin-stimulated TF activity increased significantly 4 weeks after angioplasty (138+/-22 versus 70+/-12 mU of TF/1000 monocytes, P=0.02). This increase was blunted by L-arg (43+/-16 mU of TF/1000 monocytes, P=0.01). CONCLUSIONS: This study demonstrates that angioplasty-induced plaque rupture is associated with a marked increase in monocyte TF response that is blunted by the oral administration of L-arg. This suggests that the documented antithrombotic properties of NO may be related in part to an inhibitory effect on monocyte TF response.     

Compartmentalized roles for leukocytic adhesion molecules in lung inflammatory injury

By using the model of acute injury caused by intrapulmonary deposition of IgG immune complexes, blocking mAb to CD11a, CD11b, L-selectin, and intercellular adhesion molecule-1 (ICAM-1) were administered either i.v. or intratracheally (i.t.). The effects of these interventions were assessed according to lung injury, lung content of myeloperoxidase (MPO), TNF-alpha, and cellular content in bronchoalveolar lavage (BAL) fluids, and up-regulation of pulmonary vascular ICAM-1. In animals treated i.v. with Abs to CD11a, L-selectin, or ICAM-1 lung injury was significantly attenuated in parallel with reduced lung content of MPO. Under similar conditions, treatment with anti-CD11b had no effect. However, when the same mAb were administered i.t., anti-CD11a and anti-L-selectin were without protective effects, whereas i.t. administered anti-CD11b and anti-ICAM-1 were each highly protective. The protective effects of anti-CD11b were related to profound reductions in BAL levels of TNF-alpha, pulmonary vascular up-regulation of ICAM-1, and lung content of MPO. The protective effects of i.t.-administered anti-ICAM-1 were not associated with reduced BAL levels of TNF-alpha. Protective effects of mAb were also reflected in reductions of retrievable neutrophils in BAL fluids. mAb to rat CD11b and CD18 but not to rat CD11a suppressed in vitro production of TNF-alpha by immune complex-stimulated rat alveolar macrophages. The mAb did not reduce NO2-/NO3- generation in stimulated macrophages but all mAb (except anti-ICAM-1) reduced O2- responses in macrophages. These data suggest a compartmentalized role for adhesion molecules in lung inflammatory injury after intraalveolar deposition of IgG immune complexes, with CD11a, L-selectin, and ICAM-1 being important in the vascular compartment for neutrophil recruitment, whereas in the alveolar compartment CD11b and ICAM-1 (but not CD11a and L-selectin) seem to play key roles.     

Insulin resistance and impaired insulin secretion due to phosphofructo-1-kinase-deficiency in humans

Recent epidemiologic evidence suggests that patients with chronic pancreatitis (CP) have an increased risk of developing pancreatic carcinoma (PCA). In spite of numerous similarities in both diseases, mechanisms for progression from CP to PCA are poorly understood. We hypothesized that enhanced angiogenesis might play a pivotal role in the etiology and histopathology of both CP and PCA, and thus form a possible link between precancer and carcinoma. In surgical specimens of 18 patients with CP, 10 with PCA, and 18 controls, absolute numbers of blood vessels and relative blood vessel density were assessed after immunostaining of endothelial cells for von Willebrand factor and PECAM-1 (platelet/ endothelial cell adhesion molecule-1). Furthermore, the expression of cell adhesion molecules ICAM-1 (intercellular adhesion molecule-1) and VCAM-1 (vascular cell adhesion molecule-1) and of VEGF (vascular endothelial growth factor) was investigated in all specimens. Both CP and PCA exhibited areas of high vascular density ("hot spots"). The mean number of blood vessels in these areas in PCA was 132.2+/-16.8 per mm2, and in CP, 99.2+/-7.4 per mm2. The mean vessel count in controls was 25.1+/-5.1. Relative vessel density was increased in both PCA (41.3+/-3.5%) and CP (30.6+/-2.6%) versus controls (8.0+/-0.8%). Both absolute vessel count and relative vessel density were significantly higher (p<0.05) in PCA than in CP. Enhanced expression of ICAM-1 in CP and PCA was seen in ductal cells in CP and cancer cells. In controls, ICAM-1 and VCAM-1 were expressed only at low levels in endothelial cells. VCAM-1 was strongly expressed in acinar cells as well as in ductal cells. In CP and PCA, VEGF was strongly expressed in ductal cells in CP as well as in cancer cells. We show for the first time that angiogenic activity is increased in both CP and PCA. Based on this study, we suggest that antiangiogenesis might be a novel target for prevention or therapy in chronic pancreatic diseases.     

The role of neutrophils in peritoneal adhesion formation

The most common cause of intraperitoneal adhesions is previous abdominal surgery. Postoperative adhesion formation results from a fibroproliferative inflammatory reaction that begins with an influx of polymorphonuclear leukocytes (PMNs) into the peritoneal cavity. Adherence of the PMNs to the endothelial cells (EC) is necessary for PMN migration into the tissue in response to a stimulus. Several receptor-counterreceptor pairs of ligands such as CD11/CD18 on the PMN and ICAM-1 (CD54) on EC have been identified. Monoclonal antibody against CD11/CD18 (R15.7) inhibits PMN adherence and migration and consequently protects against PMN-induced tissue injuries. We therefore studied the effect of preventing PMN-EC adherence, using anti-CD18 monoclonal antibody, on postoperative adhesion formation in rabbits. Group 1 was a control receiving physiologic saline, and group 2 received anti-CD18 antibody (R15.7, 2 mg/kg). The treatment was administered iv at the end of surgery and repeated on the first and second postoperative days. Peritoneal adhesions were induced at laparotomy by repairing two peritoneal defects, by oversewing the defect (model 1), and by resuturing the removed parietal peritoneum in its place as an ischemic graft (model 2). Adhesions were evaluated blindly at 10 days after operation by measuring the percentage of the suture line covered with adhesions (model 1) or by a scoring system (model 2). All control animals developed intraperitoneal adhesions and the percentage of the suture line covered with adhesions was 25 +/- 5.9% (mean +/- SEM) and the mean score in model 2 was 0.9 +/- 0.2. Anti-CD18 antibody, R15.7, increased the degree of postoperative adhesion formation in both models, but the results were significant only in model 2. Also, anti-CD18 antibody significantly decreased peritoneal neutrophils from 11.1 x 10(7) +/- 1.8 x 10(7) to 2.2 x 10(7) +/- 0.4 x 10(7) (P < 0.001) on the first postoperative day. It is concluded that inhibition of PMN-EC adherence does influence the postoperative adhesion formation. These results might suggest that PMNs have a role in modulating postoperative adhesion formation.     

Involvement of lymphocyte function-associated antigen-1 and intercellular adhesion molecule-1 in osteoclastogenesis: a possible role in direct interaction between osteoclast precursors

In our search for molecules involved in the process of osteoclast differentiation, we examined the surface phenotypes of the preosteoclast-like cells and osteoclast-like multinucleated cells (MNCs) formed in bone marrow cultures, using monoclonal antibodies recognizing different antigen molecules expressed on hematopoietic cells. Among these cell surface antigens, lymphocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) were highly expressed on mononuclear cells in the cultures for forming preosteoclast-like mononuclear cells. The double detection of these two antigen molecules with osteoclast-specific antigen and with calcitonin receptor, using a fluorescence-activated cell sorter or autoradiography technique, revealed that LFA-1 and ICAM-1 were expressed on the preosteoclasts. The expression of ICAM-1 was detected on both preosteoclasts and osteoclast-like MNCs, whereas the expression of LFA-1 was restricted to preosteoclasts. We designed a peptide with the sequence of the binding site of ICAM-1 against the ligand LFA-1. In the whole bone marrow culture system for forming osteoclast-like MNCs, a significant inhibition of MNC formation was observed by the addition of this peptide. These results strongly suggest the involvement of an LFA-1/ICAM-1-interaction in osteoclastogenesis.     

Twenty-four-hour profiles of luteinizing hormone, follicle-stimulating hormone, testosterone, and estradiol levels: a semilongitudinal study throughout puberty in healthy boys

Monocytes and endothelial cells interact at sites of vascular injury during inflammatory response, thrombosis, and development of atherosclerotic lesions. Such interactions result in modulation of several biological functions of the two cell types. Because both cells, on appropriate stimulation, synthesize tissue factor (TF), we examined the effect of human umbilical vein endothelial cell (HUVEC)/monocyte coculture on the expression of TF. We found that the coincubation resulted in TF generation, which was maximal at 4 hours, increased with increasing numbers of monocytes, and required mRNA and protein synthesis. Supernatant from HUVEC/monocyte coculture induced TF activity in HUVECs, but not in monocytes, indicating that HUVEC were the cells responsible for the activity, and that soluble mediators were involved. Interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha), well-known inducers of TF in HUVECs, were found in the supernatant from the coculture, and specific antibodies directed against either cytokine inhibited TF generation. The need of IL-1 beta and TNF-alpha synthesis in order to elicit TF expression was also suggested by the delay observed in TF mRNA formation and TF activity generation when monocytes were incubated with HUVECs. IL-1 beta and TNF-alpha antigen levels in the coculture supernatant, and, consequently, HUVEC TF expression, were inhibited in the presence of anti-CD18 monoclonal antibody. These findings emphasize the role of cell-cell contact and cross-talk in the procoagulant activity, which could be responsible for the thromboembolic complications observed in those vascular disorders in which monocyte infiltration is a common feature.     

Effects of aminoguanidine on adhesion molecule expression of human endothelial cells

The effect of aminoguanidine (AG) on the expression of adhesion molecules on nonactivated human umbilical vein endothelial cells (HUVEC) was investigated in vitro. Nonactivated HUVEC cultivated on long-term glycated fibronectin (FN) as compared to native FN showed a significant upregulation of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and CD31 which could be further promoted by long-term glycated bovine serum albumin. AG, at a concentration of 0.01 mol/l, caused an upregulation of ICAM-1 of 48 +/- 17.4% in HUVEC cultivated on gelatin. In contrast, VCAM-1 and E-selectin remained unaffected. At this concentration, formation of advanced glycation end products (AGE) was inhibited by 57%, as determined immunologically, and by 50%, as verified by AGE-specific fluorescence. A hypothesis concerning the upregulation of ICAM-1 by AG as compared to VCAM-1 is proposed relating to its relative redox insensitivity. Our results demonstrate that the beneficial effect of AG in reducing the risk of accelerated development of atherosclerosis in diabetic patients by inhibiting formation of AGE on matrix proteins such as FN might be hampered by its tendency to upregulate ICAM-1 on endothelial cells.     

Rickettsia conorii infection enhances vascular cell adhesion molecule-1- and intercellular adhesion molecule-1-dependent mononuclear cell adherence to endothelial cells

Leukocyte adherence to the endothelium is an essential component of the inflammatory response during rickettsial infection. In vitro, Rickettsia conorii infection of endothelial cells enhances the expression of adhesive molecules E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) in a time- and dose-dependent manner. Rickettsial lipopolysaccharide does not seem to be involved, because polymyxin B does not reduce their expression. The intracellular presence of the organism and de novo host protein synthesis are required for expression of cell adhesive molecules, since rickettsial inactivation by formol and pretreatment of cells with cycloheximide inhibits an increase in expression. The contribution of interleukin-1alpha (IL-1alpha) to this endothelial adhesive phenotype was shown by inhibitory experiments 8 and 24 h after infection with IL-1 receptor antagonist and IL-1alpha blocking antibodies. Enhanced adherence of mononuclear cells to infected endothelial cells involved VCAM-1- and ICAM-1-dependent mechanisms at the late phase of the inflammatory response. This endothelial adhesive phenotype may constitute a key pathophysiologic mechanism in R. conorii-induced vascular injury.     

Beta2-integrins mediate stable adhesion in collisional interactions between neutrophils and ICAM-1-expressing cells

The aggregation of human neutrophils in suspension has features that are analogous to their attachment to activated endothelium in that both involve selectin and beta2-integrin adhesion receptors. For the collisional interaction that forms neutrophil aggregates in suspension, there is a tethering step in which L-selectin on neutrophils binds PSGL-1. At relatively low shear rates (100-200 s(-1)) firm adhesion is mediated in equal measure by LFA-1 binding to ICAM-3, and Mac-1 binding to an as yet undefined ligand. In this report we used a mouse melanoma cell line expressing an estimated 700,000 ICAM-1 (CD54) to examine the relative roles of LFA-1 and Mac-1 over the kinetics of heterotypic cell adhesion in shear mixed suspensions. Neither heterotypic nor homotypic neutrophil aggregates formed with application of shear alone. However, the rate of aggregation peaked within seconds of chemotactic stimulation. In contrast to homotypic aggregation, neither L-selectin nor its O-glycoprotein ligands on neutrophils contributed to heterotypic adhesion. Adhesion was inhibited in a dose-dependent manner as ICAM-1 was titrated with blocking mAb. A direct interaction between LFA-1 and ICAM-1 was preferred over the first minute of stimulation, whereas at later times adhesion was supported equally by Mac-1. Activation with MnCl2 also favored participation of the constitutively expressed LFA-1. Application of defined shear in a cone and plate viscometer showed that adhesion to the ICAM-1 cells decreased from a maximum level to baseline as shear rate increased up to 400 s(-1) in a manner typical of integrin adhesion alone. In contrast, homotypic aggregation supported by the transition from selectin to integrin binding exhibited an increase in efficiency up to 800 s(-1). The pathophysiological significance of receptor site density and duration of contact in collisional interactions relevant to leukocyte recruitment compared to leukocyte-endothelial cell interactions on surfaces is discussed.     

Talc-induced expression of C-C and C-X-C chemokines and intercellular adhesion molecule-1 in mesothelial cells

Treatment of symptomatic carcinomatous pleural effusions is primarily directed at local palliation with a wide variety of sclerosing agents, of which talc is considered to be the most successful. The mechanism whereby talc achieves this effect is unknown. The objective of this study was to investigate whether talc stimulates pleural mesothelial cells (PMC) to release C-X-C and/or C-C chemokines and express adhesion molecules that initiate and amplify the inflammatory process in the pleural space. When PMC were challenged with talc in vitro, interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) levels were increased (p < 0.001) both at the protein and the mRNA level as compared with unstimulated cultures. Talc-stimulated PMC culture supernatant showed chemotactic activity for neutrophils and monocytes. The chemotactic activity of PMC culture supernatant was blocked by 44.2% with IL-8-specific antibody and by 55.7% with MCP-1-specific antibody, demonstrating that PMC-derived chemokines are bioactive. Talc also enhanced intercellular adhesion molecule-1 (ICAM-1) expression in PMC. The data demonstrate that talc stimulates PMC to release chemokines and express adhesion molecules that may play a critical role in pleurodesis.