- Email: [email protected]
Effects of nicotine on intercellular adhesion molecule expression in endothelial cells and integrin expression in neutrophils in vitro
Effects of nicotine on intercellular adhesion molecule expression in endothelial cells and integrin expression in neutrophils in vitro Paul Speer, MD,a Yanping Zhang, MD,a Yang Gu, MD,a Michael J. Lucas, MD,a and Yuping Wang, MD, PhDa,b Shreveport, La OBJECTIVE: We previously reported that nicotine decreases leukocyte adhesion to uterine vascular endothelial cells in vivo under ischemic conditions in pregnant rabbits. To further investigate the mechanism of decreased leukocyte-endothelial adhesion by nicotine exposure, the effect of nicotine on endothelial cell intercellular adhesion molecule expression and neutrophil integrin expression of CD62L, CD11a, and CD11b were examined. STUDY DESIGN: Endothelial cells were isolated from human umbilical cord veins from normal pregnancies in nonsmoking women immediately after delivery. Neutrophils were isolated from healthy nonpregnant and nonsmoking female volunteers. First passage of endothelial cells and fresh isolated neutrophils were exposed to nicotine at different concentrations. Surface adhesion molecule expression of intercellular adhesion molecule on endothelial cells was determined by colorimetric assay. Neutrophil integrin expressions for CD62L, CD11a, and CD11b were determined by flow cytometry. Messenger RNA expression for intercellular adhesion molecule in endothelial cells was examined by reverse transcription–polymerase chain reaction. RESULTS: Nicotine at a lower concentration of 0.01 µmol/L had no effect on endothelial cell surface intercellular adhesion molecule expression compared with controls (P = .614). Nicotine at a higher concentration of 10 µmol/L completely inhibited endothelial cell surface intercellular adhesion molecule-1 expression (P < .0001). At concentrations between 0.10 and 10 µmol/L, nicotine inhibited intercellular adhesion molecule expression in a dose-dependent manner. Messenger RNA expression of intercellular adhesion molecule in endothelial cells was not changed after exposure to nicotine. Decreased integrin expressions of CD62L, CD11a, and CD11b were observed on neutrophils after exposure to nicotine. CONCLUSION: Nicotine exerts inhibitory effects on both endothelial cell surface intercellular adhesion molecule expression and neutrophil integrin expressions of CD62L, CD11a, and CD11b in vitro. These in vitro effects of nicotine may relate to the clinical observation of reduced incidence of preeclampsia in women that smoke. (Am J Obstet Gynecol 2002;186:551-6.)
Key words: Neutrophil, endothelial cell, intercellular adhesion molecule, integrin expression
Inconsistent effects of cigarette smoking have been observed in outcomes of human pregnancy. For example, cigarette smoking has been associated with low-birthweight infants because of increases in both intrauterine growth retardation and preterm birth.1 Although these outcomes are also associated with preeclampsia, ironi-
From the Departments of Obstetrics and Gynecology,a and Cellular and Molecular Physiology,b Louisiana State University Health Sciences Center. Supported in part by the Board of Regents Support Fund from the State of Louisiana, LEQSF-RD-A13 (Y. W.) and from the National Institutes of Health, grant No. HD36822 (Y. W.). Received for publication June 8, 2001; revised September 24, 2001; accepted October 18, 2001. Presented at the 21st Annual Meeting of the Society for Maternal-Fetal Medicine, Reno, Nevada, February 5-10, 2001. Reprint requests: Yuping Wang, MD, PhD, Louisiana State University Health Sciences Center, Department of Obstetrics and Gynecology, PO Box 33932, Shreveport, LA 71130. E-mail: [email protected] Copyright 2002, Mosby, Inc. All rights reserved. 0002-9378/2002 $35.00 + 0 6/1/121106 doi:10.1067/mob.2002.121106
cally, women who smoke during pregnancy have a lower incidence of preeclampsia than do women who do not smoke.2-5 Nicotine, a major constituent of cigarette smoke, has been the most studied of the various smoke ingredients. One mechanism by which nicotine might interfere with fetal growth may be by the inhibition of acetylcholine-facilitated amino acid transport and by suppression of endothelial muscaric receptor–regulated placental blood flow.6 Nicotine and its major metabolite cotinine can also stimulate endogenous prostaglandin E2 production, and increased prostaglandin E2 can stimulate labor. Such a mechanism might explain the association between smoking and preterm delivery.7 Neither of these possible effects of nicotine on low birth weight is likely protective against preeclampsia. It is not clear why women who smoke have a decreased incidence of preeclampsia. Endothelial cell dysfunction and increased neutrophil activation are considered to play significant roles in the pathophysiologic condition and pathogenesis of preeclampsia.8,9 This concept led us 551
552 Speer et al
Fig 1. A schematic drawing of neutrophil and endothelial cell interaction and adhesion.
to undertake our previous study, which investigated the effect of nicotine on leukocyte adhesion to uterine vascular endothelial cells in vivo under ischemia and reperfusion conditions in pregnant rabbits.10 Accessed by in vivo intravital microscopy under ischemia/reperfusion conditions, we observed a decreased leukocyte adhesion to uterine vascular endothelial cells after the administration of nicotine to pregnant rabbits. Endothelial cells express several adhesion molecules, including intercellular adhesion molecule (ICAM), vascular cell adhesion molecule, P-selectin, and E-selectin on the cell surface. Enhanced expression of these adhesion molecules would argue leukocyte-endothelial adhesion. Among these molecules, ICAM, a ligand for the leukocyte integrins of CD11/CD18, is the crucial one in the process of leukocyteendothelial adhesion (Fig 1). To further investigate possible mechanisms of decreased leukocyte-endothelial adhesion by nicotine exposure, the present study investigated the effect of nicotine on ICAM expression on endothelial cells and CD62L, CD11a, and CD11b expression on neutrophils in our in vitro cell model. Material and methods Sample information. Endothelial cells were isolated from human umbilical cord veins (HUVECs) from normal term pregnancies in nonsmoking women immediately after delivery. Leukocytes were isolated from healthy nonsmoking, nonpregnant female volunteers. This study was approved by the Institutional Review Board for Human Research at Louisiana State University Health and Science Center in Shreveport. HUVEC isolation and culture. HUVECs were isolated from normal pregnancies in nonsmoking women (n = 8) by collagenase treatment immediately after delivery, as previously described.11,12 Isolated endothelial cells were incubated with endothelial cell growth medium (Biowhittaker, Walkersville, Md). Cells used for surface molecule expression were seeded into 48-well/cluster cell culture plates, and cells used for the extraction of total RNA were seeded into 75-cm2 cell culture flasks (Costar Corp, Corning Incorporated, Corning, NY). All cell culture wares were precoated with fibronectin. Only the primary passage of endothelial cells was used in our experiments.
March 2002 Am J Obstet Gynecol
Surface molecule expression assay. Confluent endothelial cells were exposed to nicotine (Sigma Chemical Co, St Louis, Mo) at concentrations of 0.01, 0.1, 1.0, 2, 5, and 10 µmol/L for 1 hour at 37°C. After nicotine exposure, cells were gently washed with 0.5 mL of assay buffer (phosphatebuffered saline solution/Hanks’ balanced salt solution in a ratio of 1:1) and fixed with 1% paraformaldehyde for 10 minutes at room temperature. Cellular surface molecule expression of ICAM was assayed as previously described.12 Cells were incubated with primary antibodies (mouse antihuman) to ICAM (CD54) at a concentration of 200 ng/mL (Southern Biotechnology Associates, Birmingham, Ala) at 37°C for 30 minutes. The second antibody was horseradish peroxidase–goat anti-mouse immunoglobulin G (Sigma Chemical Co). The color reaction was started by incubation with 250 µL of 0.003% hydrogen peroxide plus 0.1 mg/mL 3,3,5,5-tetramethylbenzidine for 1 hour in darkness and stopped by the addition of 75 µL of 8N sulfuric acid to each well. An aliquot of 200 µL sample from each well was transferred to 96-well plates. Blank wells were stained with only secondary antibody. The samples were read at 450 nm in a 96-well Spectra Microplate Antoreader (Cayman Chemical Company, Ann Arbor, Mich). Data are represented as an absorption reading at 450 nm (sample) to 450 nm (blank). Messenger RNA expression of ICAM. Total RNA was isolated from endothelial cells by acid guanidine thiocyanate phenol chloroform extraction (Tri Reagent, Molecular Research Center, Inc, Cincinnati, Ohio). One microgram of total RNA from each endothelial sample was reverse transcripted by the use of SuperScript first-strand synthesis system purchased from Life Technologies (Gibco, Rockville, Md). The product was subjected to polymerase chain reaction (PCR) analysis with primers that were specific for the ICAM genes. ICAM-1 primers 5´-GCTTTCCGGCGCCCAACGTGATTCTGA and 3´-ACTCACACAGGACACGAAGCTCCCGGGTGT yield a 260-bp fragment. ICAM-2 primers 5´-AGCCTGTGTCGGACAGCCAGATGGTCATCA and 3´-AGGGCTAAGTCCAGGTGTTTGTATTCG yield a 335-bp fragment. The final volume of 50 µL of each PCR reaction contains 1 µL of 10 µmol of each primer. The cycles were 94°C for 30 seconds, 65°C for 30 seconds, and 72°C for 1 minute, for a total of 35 cycles. The gene expression of human glyceraldehyde 3phosphate dehydrogenase (GAPDH) was also amplified and used as an internal standard for each endothelial sample. GAPDH primers 5´-TGAAGGTCGGAGTCAACGGATTTGGT and 3´-CATGTGGGCCATGAGGTCCACCAC yield a 983-bp fragment. The products were analyzed on 2.0% agarose gels. Neutrophil integrin expression. Neutrophils were isolated immediately from freshly drawn venous blood by previously described methods12,13: dextran sedimentation and histopaque density gradient centrifugation, followed by lysis of the contaminating red blood cells.
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Fig 3. Messenger RNA expression of ICAM-1 and ICAM-2 in endothelial cells after exposure to nicotine at different concentrations. Messenger RNA expression of GAPDH was used as an internal control for each sample. Fig 2. ICAM expression on the surface of endothelial cells after exposure to nicotine at different concentrations, measured by surface molecule expression assay. Asterisk, P < .05; two asterisks, P < .01.
Freshly isolated neutrophils at a concentration of 1 106 cell/per tube were treated with nicotine at 1.0 and 10.0 µmol/L for 15 minutes in triplicate. Then, neutrophils were triple stained with mouse anti-human monoclonal immunoglobulin G–specific antibodies for CD62L, CD11a, and CD11b (PharMingen, San Diego, Calif). The antibody for CD62L was fluorescein isothiocyanate–conjugated. The antibody for CD11a was R-phycoerythrinconjugated, and the antibody for CD11b was Cy-chromeTM-conjugated. The incubation was on ice for 30 minutes. The cells were washed twice with 2 mL of washing buffer and centrifuged at 400g at 4°C for 5 minutes. The cells were suspended in 0.5 mL of 1% paraformaldehyde. Neutrophil expressions of CD62L, CD11a, and CD11b were analyzed by flow cytometry (FACS Vantage SE; Becton Dickinson, San Jose, Calif). Statistical analysis. Data were expressed as mean ± SE and analyzed by analysis of variance and Student–Newman-Keuls post hoc test. A probability level of <.05 was considered to be statistically significant. Results Fig 2 shows the endothelial cell surface adhesion molecule of ICAM expression after exposure to nicotine at different concentrations. Nicotine at the lowest concentration of 0.01 µmol/L had an insignificant effect on ICAM expression compared with controls (0.167 ± 0.013 µmol/L vs 0.171 ± 0.012 µmol/L; P = .614). Nicotine at a higher concentration of 10 µmol/L completely inhibited ICAM expression. The inhibitory effect by nicotine on ICAM expression was in a dose-dependent manner between doses of 0.01 to 10 µmol/L: 0.167 ± 0.013 µmol/L (0.01 µmol/L); 0.155 ± 0.008 µmol/L (0.1 µmol/L; P = .199); 0.141 ± 0.008 µmol/L (1.0 µmol/L; P < .05); 0.115 ± 0.013 µmol/L (2.0 µmol/L; P < .01); 0.019 ± 0.004 µmol/L (5.0 µmol/L; P < .0001); 0.011 ± 0.003 µmol/L (10.0 µmol/L; P < .0001) versus control.
Endothelial cells express both ICAM-1 and ICAM-2 genes. In general, ICAM-1 is inducible by stimulators such as cytokines, and ICAM-2 is constitutive on endothelial cells. To study whether the decreased endothelial cells surface ICAM expression is correlated with altered ICAM gene expression, messenger RNA expression for ICAM-1 and ICAM-2 on endothelial cells with or without exposure to nicotine were examined. Fig 3 shows the messenger RNA expression of ICAM-1 and ICAM-2 in endothelial cells after exposure to nicotine at different concentrations as determined by reverse transcriptase–PCR. The messenger RNA expression of GAPDH was used as an internal standard for each sample. Neither ICAM-1 nor ICAM-2 messenger RNA expression was found to be affected on endothelial cells after exposure to nicotine, compared with control cells. A representative neutrophil integrin expression of CD62L, CD11a, and CD11b that was evaluated by flow cytometry is shown in Fig 4. Neutrophils were treated with nicotine at 1.0 and 10.0 µmol/L. These concentrations were chosen on the basis of results from endothelial surface ICAM expression because nicotine inhibition of ICAM was directly related to nicotine concentration in this range (Fig 2). Compared with negative control neutrophils and untreated control neutrophils, the intensity of CD62L was diminished in neutrophils that were treated with nicotine at 1.0 µmol/L and 10.0 µmol/L, which indicated that neutrophil CD62L expression was partially down-regulated. However, the intensity of CD62L showed no difference between nicotine treatment at 1.0 µmol/L and 10.0 µmol/L. For CD11a, compared with negative control neutrophils and untreated control neutrophils, the intensity of CD11a expression was diminished with nicotine treatment at 1.0 µmol/L and was completely inhibited with nicotine concentration at 10.0 µmol/L. For CD11b, the intensity of CD11b expression was also progressively diminished with nicotine concentration at 1.0 µmol/L and 10.0 µmol/L, compared with negative control neutrophils and untreated neutrophils.
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Fig 4. A representative neutrophil integrin expression of CD62L (L-selectin), CD11a, and CD11b evaluated by flow cytometry. Neutrophils were treated with different concentrations of nicotine. A, CD62L. B, CD11a. C, CD11b. 1, Negative control; 2, neutrophils with no nicotine exposure; 3, neutrophils exposed to nicotine at 1.0 µmol/L; 4, neutrophils exposed to nicotine at 10.0 µmol/L.
Each experiment was performed in triplicate, and consistent results were obtained from the 3 repeated experiments. Comment Nicotine and its metabolites exert a variety of effects on endothelial cells and neutrophils. In the present study, we present evidence that nicotine exerts inhibitory effects on ICAM expression on endothelial cells and integrin expression of CD62L, CD11a, and CD11b on neutrophils. These inhibitory effects are in a dose-dependent manner. Nicotine concentrations that were used in our experiments are comparable to plasma concentrations that are measurable in active and passive smokers, with peak levels between 10–6 and 10–8 mol/L that occur within 10 minutes of smoking initiation and that decrease with an average elimination half-life of approximately 2 hours.14,15 These concentrations do not cause cell death but do appear to impair endothelial function. For example, exposure of endothelial cells to nicotine at concentrations are similar to concentrations found in the blood plasma of smokers. Cucina et al16 observed significant alterations of endothelial cell cytoskeletal expression of α-actin fibers and vimentin. They also noticed that these changes were
associated with enhanced release of platelet-derived growth factor BB.16 Several studies have used cigarette smoke condensate to study the effects of cigarette smoking on cell function. Snajdar et al17 found that cigarette smoke condensate can inhibit endothelial cell migration in a dose-dependent manner. Kalra et al18 found that the treatment of HUVECs with cigarette smoke condensate resulted in an increase in expression of ICAM-1 and endothelial cell leukocyte adhesion molecule. Because cigarette smoke condensate is composed of numerous active substances, it is not surprising that nicotine and cigarette smoke condensate might exert differential effects on vascular endothelial function. Cigarette smoking has been associated with a decreased incidence of preeclampsia. Several recent meta-analyses have described a lower risk of preeclampsia that is associated with cigarette smoking during pregnancy.2-5 CondeAgudelo et al4 reviewed 28 cohort and 7 case-control studies that included a total of 833,714 women; a retrospective analysis by Zhang et al2 from prenatal care subjects at 12 hospitals from the years 1959 to 1965 included 9651 women. The mechanism of cigarette smoking in reducing the incidence of preeclampsia during pregnancy is not clear, but a study of smoking effects may offer patho-
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physiologic insights into preeclampsia. Endothelial dysfunction and neutrophil activation are important pathophysiologic processes in this pregnancy disorder. Soluble cell surface molecules (SICAM-1, SVCAM-1, SE-selectin, and SPECAM-1) have been reported to be increased in blood serum or plasma obtained from women with preeclampsia.19,20 Increased soluble endothelial adhesion molecule levels along with increased activated leukocytes and the upregulation of neutrophil CD11b expression have been measured in women with preeclampsia,19-23 which suggests the possibility of increased interaction and adhesion of leukocytes to endothelial cells. Increased neutrophil-endothelial interaction that was observed in endothelial cells that were derived from preeclamptic pregnancies12 supports this notion. Endothelial ICAM-1, which belongs to the immunoglobulin superfamily, is a ligand for the β2 integrin molecules present on leukocytes, LFA-1 (CD11a/CD18), and MAC-1 (CD11b/CD18). Down-regulation of ICAM-1 expression would lead to a decrease in neutrophil interaction with endothelium. It seems that the effect of the nicotine-induced inhibition of ICAM-1 expression that was observed in our experiment might be a functional surface alteration to endothelial cells, because messenger RNA expressions of both ICAM-1 and ICAM-2 are not affected by exposure to nicotine. L-selectin is responsible for the attachment of leukocytes to endothelium. We observed that exposure of neutrophils to nicotine resulted in the down-regulation of CD62L (L-selectin). However, it is not known whether the down-regulation of CD62L expression on neutrophils that are induced by nicotine also coexist with the shedding of L-selectin. The extracellular domain of L-selectin has been found to be proteolytically shed from leukocytes after cellular activation.24,25 LFA-1 (CD11a/CD18) has a key role in mediating leukocyte adhesion to endothelium during inflammatory responses through binding to ICAM-1. MAC-1 (CD11b/CD18) mediates neutrophils and monocyte adherence to endothelium and, subsequently, neutrophil extravasation to sites of inflammation. We found that nicotine can inhibit both CD11a and CD11b expression on neutrophils in a dose-dependent manner. However, the inhibitory effects of nicotine on CD11a and CD11b are different. CD11a expression is completely inhibited; CD11b expression is only partially downregulated at the higher dose of nicotine (10 µmol/L) used in our experiment. The reason for this differential inhibitory effect of nicotine on CD11a and CD11b expression is not known, but it could be due to structural differences between αL chain in LFA-1 (CD11a) and αM chain in MAC-1 (CD11b) cross-linking with nicotine receptors on neutrophils. In summary, we have observed that nicotine exerts differential inhibitory effects on endothelial ICAM expression and on the neutrophil integrin expression of
L-selectin, CD11a, and CD11b. Because endothelial dysfunction and neutrophil activation has been proposed to be involved in the pathophysiologic factors of preeclampsia, the inhibitory effects of nicotine on adhesion molecule expression on endothelial cells and on neutrophils may explain partially the protective effect of cigarette smoking against preeclampsia. The differential effects of smoking (protection against preeclampsia although associated with low-birth-weight deliveries) are important to investigate; studies that have considered both preeclampsia and small gestational age birth weight to be products of the same vascular dysfunction may be inherently confounded. The relationship of our results to in vivo changes with preeclampsia is speculative. However, the down-regulation of vascular adhesion molecules and the decreased interaction of leukocytes with endothelial cells induced by nicotine suggest that these cell surface molecule changes may be important in the pathophysiology of preeclampsia. REFERENCES
1. Smoking and women’s health. Washington (DC): American College of Obstetrics and Gynecology; 1997. Technical Bulletin No.: 240. 2. Zhang J, Klebanoff MA, Levine RJ, Puri M, Moyer P. The puzzling association between smoking and hypertension during pregnancy. Am J Obstet Gynecol 1999;181:1407-13. 3. Marcoux S, Brisson J, Fabia J. The effect of cigarette smoking on the risk of preeclampsia and gestational hypertension. Am J Epidemiol 1989;130:950-7. 4. Conde-Agudelo A, Althabe F, Belizan JM, Kafury-Goeta AC. Cigarette smoking during pregnancy and risk of preeclampsia: a systematic review. Am J Obstet Gynecol 1999;181:1026-35. 5. Cnattingius S, Mills J, Yuen, J, Eriksson O, Ros HS. The paradoxical effect of smoking in preeclamptic pregnancies: smoking reduces the incidence but increases the rates of perinatal mortality, obruptio placentae, and intrauterine growth restriction. Am J Obstet Gynecol 1997;177:156-61. 6. Sastry BV. Placental toxicology: tobacco smoke, abused drugs, multiple chemical interactions, and placental function. Reprod Fertil Dev 1991;3:355-72. 7. Sastry BV, Hemontolor ME, Olenick M. Prostaglandin E2 in human placenta: its vascular effects and activation of prostaglandin E2 formation by nicotine and cotinine. Pharmacology 1999;58:70-86. 8. Roberts JM, Taylor RN, Musci TJ, Rodgers GM, Hubel CA, McLaughlin MK. Preeclampsia: an endothelial cell disorder. Am J Obstet Gynecol 1989;161:1200-4. 9. Taylor RN, Roberts JM. Endothelial cell dysfunction. In: Lindheimer MD, Roberts JM, Cunningham FG, editors. Hypertensive disorders in pregnancy. Stamford (CT): Appleton & Lange; 1999. p. 395-429. 10. Speer P, Adair CD, Wang Y. Effects of nicotine on leukocyte adhesion to uterine vasculature in pregnant rabbits as assessed by in vivo intravital microscopy [abstract]. Am J Obstet Gynecol 2000;182(Suppl):S90. 11. Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cells derived from umbilical veins: identification by morphologic and immunologic criteria. J Clin Invest 1973;52:2745-56. 12. Wang Y, Adair CD, Coe L, Weeks JW, Lewis DF, Alexander JS. Activation of endothelial cells in preeclampsia: increased neutrophil-endothelial adhesion correlates with up-regulation of adhesion molecule P-selectin in human umbilical vein endothelial cells isolated from preeclampsia. J Soc Gynecol Invest 1998;5:237-43.
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13. Markert M, Andrews PC, Babior BM. Measurement of O2– production by human neutrophils: the preparation and assay of NADPH oxidase-containing particles from human neutrophils. Methods Enzymol 1984;105:358-65. 14. Benowitz NL, Kuyt F, Jacob P. Influence of nicotine on cardiovascular and hormonal effects of cigarette smoking. Clin Pharmacol Ther 1984;36:74-80. 15. Darby TD, McNamee JE, van Rossum JM. Cigarette smoking pharmacokinetics and its relationship to smoking behavior. Clin Pharmacokinet 1984;9:435-49. 16. Cucina A, Sapienza P, Borrelli V, Corvino V, Foresi G, Randone B, et al. Nicotine reorganizes cytoskeleton of vascular endothelial cell through platelet-derived growth factor BB. J Surg Res 2000;92:233-8. 17. Snajdar RM, Busuttil SJ, Averbook A, Graham DJ. Inhibition of endothelial cell migration by cigarette smoke condensate. J Surg Res 2001:96:10-6. 18. Kalra VK, Ying Y, Deemer K, Natarajan R, Nadler JL, Coates TD. Mechanism of cigarette smoke condensate induced adhesion of human monocytes to cultured endothelial cells. J Cell Physiol 1994;160:154-62. 19. Lyall F, Greer IA, Boswell F, Macara LM, Walker JJ, Kingdom JCP. The cell adhesion molecule, VCAM-1, is selectively elevated in
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20.
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22. 23.
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serum in pre-eclampsia: does this indicate the mechanism of leucocyte activation? Br J Obstet Gynaecol 1994;101:485-7. Krauss T, Kuhn W, Lakoma C, Augustin HG. Circulating endothelial cell adhesion molecules as diagnostic markers for the early identification of pregnant women at risk for development of preeclampsia. Am J Obstet Gynecol 1997;177:443-9. Higgins JR, Papayianni A, Brady HR, Darling MRN, Walshe JJ. Circulating vascular cell adhesion molecule-1 in pre-eclampsia, gestational hypertension, and normal pregnancy: evidence of selective dysregulation of vascular cell adhesion molecule-1 homeostasis in pre-eclampsia. Am J Obstet Gynecol 1998;179: 464-9. Clark P, Boswell F, Greer IA. The neutrophil and preeclampsia. Sem Reprod Endocrinol 1998;16:57-64. Barden A, Graham D, Beilin LJ, Ritchie J, Baker R, Walters BN, et al. Neutrophil CD 11B expression and neutrophil activation in pre-eclampsia. Clin Sci 1997;92:37-44. Schleiffenbaum B, Spertini O, Tedder TF. Soluble L-selectin is present in human plasma at high levels and retains functional activity. J Cell Biol 1992;119:229-38. Spertini O, Schleiffenbaum B, White-Owen C, Ruiz P Jr, Tedder TF. ELISA for quantitation of L-selectin shed from leukocytes in vivo. J Immunol Methods 1992;156:115-23.
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Key words: Neutrophil, endothelial cell, intercellular adhesion molecule, integrin expression
Inconsistent effects of cigarette smoking have been observed in outcomes of human pregnancy. For example, cigarette smoking has been associated with low-birthweight infants because of increases in both intrauterine growth retardation and preterm birth.1 Although these outcomes are also associated with preeclampsia, ironi-
From the Departments of Obstetrics and Gynecology,a and Cellular and Molecular Physiology,b Louisiana State University Health Sciences Center. Supported in part by the Board of Regents Support Fund from the State of Louisiana, LEQSF-RD-A13 (Y. W.) and from the National Institutes of Health, grant No. HD36822 (Y. W.). Received for publication June 8, 2001; revised September 24, 2001; accepted October 18, 2001. Presented at the 21st Annual Meeting of the Society for Maternal-Fetal Medicine, Reno, Nevada, February 5-10, 2001. Reprint requests: Yuping Wang, MD, PhD, Louisiana State University Health Sciences Center, Department of Obstetrics and Gynecology, PO Box 33932, Shreveport, LA 71130. E-mail: [email protected] Copyright 2002, Mosby, Inc. All rights reserved. 0002-9378/2002 $35.00 + 0 6/1/121106 doi:10.1067/mob.2002.121106
cally, women who smoke during pregnancy have a lower incidence of preeclampsia than do women who do not smoke.2-5 Nicotine, a major constituent of cigarette smoke, has been the most studied of the various smoke ingredients. One mechanism by which nicotine might interfere with fetal growth may be by the inhibition of acetylcholine-facilitated amino acid transport and by suppression of endothelial muscaric receptor–regulated placental blood flow.6 Nicotine and its major metabolite cotinine can also stimulate endogenous prostaglandin E2 production, and increased prostaglandin E2 can stimulate labor. Such a mechanism might explain the association between smoking and preterm delivery.7 Neither of these possible effects of nicotine on low birth weight is likely protective against preeclampsia. It is not clear why women who smoke have a decreased incidence of preeclampsia. Endothelial cell dysfunction and increased neutrophil activation are considered to play significant roles in the pathophysiologic condition and pathogenesis of preeclampsia.8,9 This concept led us 551
552 Speer et al
Fig 1. A schematic drawing of neutrophil and endothelial cell interaction and adhesion.
to undertake our previous study, which investigated the effect of nicotine on leukocyte adhesion to uterine vascular endothelial cells in vivo under ischemia and reperfusion conditions in pregnant rabbits.10 Accessed by in vivo intravital microscopy under ischemia/reperfusion conditions, we observed a decreased leukocyte adhesion to uterine vascular endothelial cells after the administration of nicotine to pregnant rabbits. Endothelial cells express several adhesion molecules, including intercellular adhesion molecule (ICAM), vascular cell adhesion molecule, P-selectin, and E-selectin on the cell surface. Enhanced expression of these adhesion molecules would argue leukocyte-endothelial adhesion. Among these molecules, ICAM, a ligand for the leukocyte integrins of CD11/CD18, is the crucial one in the process of leukocyteendothelial adhesion (Fig 1). To further investigate possible mechanisms of decreased leukocyte-endothelial adhesion by nicotine exposure, the present study investigated the effect of nicotine on ICAM expression on endothelial cells and CD62L, CD11a, and CD11b expression on neutrophils in our in vitro cell model. Material and methods Sample information. Endothelial cells were isolated from human umbilical cord veins (HUVECs) from normal term pregnancies in nonsmoking women immediately after delivery. Leukocytes were isolated from healthy nonsmoking, nonpregnant female volunteers. This study was approved by the Institutional Review Board for Human Research at Louisiana State University Health and Science Center in Shreveport. HUVEC isolation and culture. HUVECs were isolated from normal pregnancies in nonsmoking women (n = 8) by collagenase treatment immediately after delivery, as previously described.11,12 Isolated endothelial cells were incubated with endothelial cell growth medium (Biowhittaker, Walkersville, Md). Cells used for surface molecule expression were seeded into 48-well/cluster cell culture plates, and cells used for the extraction of total RNA were seeded into 75-cm2 cell culture flasks (Costar Corp, Corning Incorporated, Corning, NY). All cell culture wares were precoated with fibronectin. Only the primary passage of endothelial cells was used in our experiments.
March 2002 Am J Obstet Gynecol
Surface molecule expression assay. Confluent endothelial cells were exposed to nicotine (Sigma Chemical Co, St Louis, Mo) at concentrations of 0.01, 0.1, 1.0, 2, 5, and 10 µmol/L for 1 hour at 37°C. After nicotine exposure, cells were gently washed with 0.5 mL of assay buffer (phosphatebuffered saline solution/Hanks’ balanced salt solution in a ratio of 1:1) and fixed with 1% paraformaldehyde for 10 minutes at room temperature. Cellular surface molecule expression of ICAM was assayed as previously described.12 Cells were incubated with primary antibodies (mouse antihuman) to ICAM (CD54) at a concentration of 200 ng/mL (Southern Biotechnology Associates, Birmingham, Ala) at 37°C for 30 minutes. The second antibody was horseradish peroxidase–goat anti-mouse immunoglobulin G (Sigma Chemical Co). The color reaction was started by incubation with 250 µL of 0.003% hydrogen peroxide plus 0.1 mg/mL 3,3,5,5-tetramethylbenzidine for 1 hour in darkness and stopped by the addition of 75 µL of 8N sulfuric acid to each well. An aliquot of 200 µL sample from each well was transferred to 96-well plates. Blank wells were stained with only secondary antibody. The samples were read at 450 nm in a 96-well Spectra Microplate Antoreader (Cayman Chemical Company, Ann Arbor, Mich). Data are represented as an absorption reading at 450 nm (sample) to 450 nm (blank). Messenger RNA expression of ICAM. Total RNA was isolated from endothelial cells by acid guanidine thiocyanate phenol chloroform extraction (Tri Reagent, Molecular Research Center, Inc, Cincinnati, Ohio). One microgram of total RNA from each endothelial sample was reverse transcripted by the use of SuperScript first-strand synthesis system purchased from Life Technologies (Gibco, Rockville, Md). The product was subjected to polymerase chain reaction (PCR) analysis with primers that were specific for the ICAM genes. ICAM-1 primers 5´-GCTTTCCGGCGCCCAACGTGATTCTGA and 3´-ACTCACACAGGACACGAAGCTCCCGGGTGT yield a 260-bp fragment. ICAM-2 primers 5´-AGCCTGTGTCGGACAGCCAGATGGTCATCA and 3´-AGGGCTAAGTCCAGGTGTTTGTATTCG yield a 335-bp fragment. The final volume of 50 µL of each PCR reaction contains 1 µL of 10 µmol of each primer. The cycles were 94°C for 30 seconds, 65°C for 30 seconds, and 72°C for 1 minute, for a total of 35 cycles. The gene expression of human glyceraldehyde 3phosphate dehydrogenase (GAPDH) was also amplified and used as an internal standard for each endothelial sample. GAPDH primers 5´-TGAAGGTCGGAGTCAACGGATTTGGT and 3´-CATGTGGGCCATGAGGTCCACCAC yield a 983-bp fragment. The products were analyzed on 2.0% agarose gels. Neutrophil integrin expression. Neutrophils were isolated immediately from freshly drawn venous blood by previously described methods12,13: dextran sedimentation and histopaque density gradient centrifugation, followed by lysis of the contaminating red blood cells.
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Fig 3. Messenger RNA expression of ICAM-1 and ICAM-2 in endothelial cells after exposure to nicotine at different concentrations. Messenger RNA expression of GAPDH was used as an internal control for each sample. Fig 2. ICAM expression on the surface of endothelial cells after exposure to nicotine at different concentrations, measured by surface molecule expression assay. Asterisk, P < .05; two asterisks, P < .01.
Freshly isolated neutrophils at a concentration of 1 106 cell/per tube were treated with nicotine at 1.0 and 10.0 µmol/L for 15 minutes in triplicate. Then, neutrophils were triple stained with mouse anti-human monoclonal immunoglobulin G–specific antibodies for CD62L, CD11a, and CD11b (PharMingen, San Diego, Calif). The antibody for CD62L was fluorescein isothiocyanate–conjugated. The antibody for CD11a was R-phycoerythrinconjugated, and the antibody for CD11b was Cy-chromeTM-conjugated. The incubation was on ice for 30 minutes. The cells were washed twice with 2 mL of washing buffer and centrifuged at 400g at 4°C for 5 minutes. The cells were suspended in 0.5 mL of 1% paraformaldehyde. Neutrophil expressions of CD62L, CD11a, and CD11b were analyzed by flow cytometry (FACS Vantage SE; Becton Dickinson, San Jose, Calif). Statistical analysis. Data were expressed as mean ± SE and analyzed by analysis of variance and Student–Newman-Keuls post hoc test. A probability level of <.05 was considered to be statistically significant. Results Fig 2 shows the endothelial cell surface adhesion molecule of ICAM expression after exposure to nicotine at different concentrations. Nicotine at the lowest concentration of 0.01 µmol/L had an insignificant effect on ICAM expression compared with controls (0.167 ± 0.013 µmol/L vs 0.171 ± 0.012 µmol/L; P = .614). Nicotine at a higher concentration of 10 µmol/L completely inhibited ICAM expression. The inhibitory effect by nicotine on ICAM expression was in a dose-dependent manner between doses of 0.01 to 10 µmol/L: 0.167 ± 0.013 µmol/L (0.01 µmol/L); 0.155 ± 0.008 µmol/L (0.1 µmol/L; P = .199); 0.141 ± 0.008 µmol/L (1.0 µmol/L; P < .05); 0.115 ± 0.013 µmol/L (2.0 µmol/L; P < .01); 0.019 ± 0.004 µmol/L (5.0 µmol/L; P < .0001); 0.011 ± 0.003 µmol/L (10.0 µmol/L; P < .0001) versus control.
Endothelial cells express both ICAM-1 and ICAM-2 genes. In general, ICAM-1 is inducible by stimulators such as cytokines, and ICAM-2 is constitutive on endothelial cells. To study whether the decreased endothelial cells surface ICAM expression is correlated with altered ICAM gene expression, messenger RNA expression for ICAM-1 and ICAM-2 on endothelial cells with or without exposure to nicotine were examined. Fig 3 shows the messenger RNA expression of ICAM-1 and ICAM-2 in endothelial cells after exposure to nicotine at different concentrations as determined by reverse transcriptase–PCR. The messenger RNA expression of GAPDH was used as an internal standard for each sample. Neither ICAM-1 nor ICAM-2 messenger RNA expression was found to be affected on endothelial cells after exposure to nicotine, compared with control cells. A representative neutrophil integrin expression of CD62L, CD11a, and CD11b that was evaluated by flow cytometry is shown in Fig 4. Neutrophils were treated with nicotine at 1.0 and 10.0 µmol/L. These concentrations were chosen on the basis of results from endothelial surface ICAM expression because nicotine inhibition of ICAM was directly related to nicotine concentration in this range (Fig 2). Compared with negative control neutrophils and untreated control neutrophils, the intensity of CD62L was diminished in neutrophils that were treated with nicotine at 1.0 µmol/L and 10.0 µmol/L, which indicated that neutrophil CD62L expression was partially down-regulated. However, the intensity of CD62L showed no difference between nicotine treatment at 1.0 µmol/L and 10.0 µmol/L. For CD11a, compared with negative control neutrophils and untreated control neutrophils, the intensity of CD11a expression was diminished with nicotine treatment at 1.0 µmol/L and was completely inhibited with nicotine concentration at 10.0 µmol/L. For CD11b, the intensity of CD11b expression was also progressively diminished with nicotine concentration at 1.0 µmol/L and 10.0 µmol/L, compared with negative control neutrophils and untreated neutrophils.
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Fig 4. A representative neutrophil integrin expression of CD62L (L-selectin), CD11a, and CD11b evaluated by flow cytometry. Neutrophils were treated with different concentrations of nicotine. A, CD62L. B, CD11a. C, CD11b. 1, Negative control; 2, neutrophils with no nicotine exposure; 3, neutrophils exposed to nicotine at 1.0 µmol/L; 4, neutrophils exposed to nicotine at 10.0 µmol/L.
Each experiment was performed in triplicate, and consistent results were obtained from the 3 repeated experiments. Comment Nicotine and its metabolites exert a variety of effects on endothelial cells and neutrophils. In the present study, we present evidence that nicotine exerts inhibitory effects on ICAM expression on endothelial cells and integrin expression of CD62L, CD11a, and CD11b on neutrophils. These inhibitory effects are in a dose-dependent manner. Nicotine concentrations that were used in our experiments are comparable to plasma concentrations that are measurable in active and passive smokers, with peak levels between 10–6 and 10–8 mol/L that occur within 10 minutes of smoking initiation and that decrease with an average elimination half-life of approximately 2 hours.14,15 These concentrations do not cause cell death but do appear to impair endothelial function. For example, exposure of endothelial cells to nicotine at concentrations are similar to concentrations found in the blood plasma of smokers. Cucina et al16 observed significant alterations of endothelial cell cytoskeletal expression of α-actin fibers and vimentin. They also noticed that these changes were
associated with enhanced release of platelet-derived growth factor BB.16 Several studies have used cigarette smoke condensate to study the effects of cigarette smoking on cell function. Snajdar et al17 found that cigarette smoke condensate can inhibit endothelial cell migration in a dose-dependent manner. Kalra et al18 found that the treatment of HUVECs with cigarette smoke condensate resulted in an increase in expression of ICAM-1 and endothelial cell leukocyte adhesion molecule. Because cigarette smoke condensate is composed of numerous active substances, it is not surprising that nicotine and cigarette smoke condensate might exert differential effects on vascular endothelial function. Cigarette smoking has been associated with a decreased incidence of preeclampsia. Several recent meta-analyses have described a lower risk of preeclampsia that is associated with cigarette smoking during pregnancy.2-5 CondeAgudelo et al4 reviewed 28 cohort and 7 case-control studies that included a total of 833,714 women; a retrospective analysis by Zhang et al2 from prenatal care subjects at 12 hospitals from the years 1959 to 1965 included 9651 women. The mechanism of cigarette smoking in reducing the incidence of preeclampsia during pregnancy is not clear, but a study of smoking effects may offer patho-
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physiologic insights into preeclampsia. Endothelial dysfunction and neutrophil activation are important pathophysiologic processes in this pregnancy disorder. Soluble cell surface molecules (SICAM-1, SVCAM-1, SE-selectin, and SPECAM-1) have been reported to be increased in blood serum or plasma obtained from women with preeclampsia.19,20 Increased soluble endothelial adhesion molecule levels along with increased activated leukocytes and the upregulation of neutrophil CD11b expression have been measured in women with preeclampsia,19-23 which suggests the possibility of increased interaction and adhesion of leukocytes to endothelial cells. Increased neutrophil-endothelial interaction that was observed in endothelial cells that were derived from preeclamptic pregnancies12 supports this notion. Endothelial ICAM-1, which belongs to the immunoglobulin superfamily, is a ligand for the β2 integrin molecules present on leukocytes, LFA-1 (CD11a/CD18), and MAC-1 (CD11b/CD18). Down-regulation of ICAM-1 expression would lead to a decrease in neutrophil interaction with endothelium. It seems that the effect of the nicotine-induced inhibition of ICAM-1 expression that was observed in our experiment might be a functional surface alteration to endothelial cells, because messenger RNA expressions of both ICAM-1 and ICAM-2 are not affected by exposure to nicotine. L-selectin is responsible for the attachment of leukocytes to endothelium. We observed that exposure of neutrophils to nicotine resulted in the down-regulation of CD62L (L-selectin). However, it is not known whether the down-regulation of CD62L expression on neutrophils that are induced by nicotine also coexist with the shedding of L-selectin. The extracellular domain of L-selectin has been found to be proteolytically shed from leukocytes after cellular activation.24,25 LFA-1 (CD11a/CD18) has a key role in mediating leukocyte adhesion to endothelium during inflammatory responses through binding to ICAM-1. MAC-1 (CD11b/CD18) mediates neutrophils and monocyte adherence to endothelium and, subsequently, neutrophil extravasation to sites of inflammation. We found that nicotine can inhibit both CD11a and CD11b expression on neutrophils in a dose-dependent manner. However, the inhibitory effects of nicotine on CD11a and CD11b are different. CD11a expression is completely inhibited; CD11b expression is only partially downregulated at the higher dose of nicotine (10 µmol/L) used in our experiment. The reason for this differential inhibitory effect of nicotine on CD11a and CD11b expression is not known, but it could be due to structural differences between αL chain in LFA-1 (CD11a) and αM chain in MAC-1 (CD11b) cross-linking with nicotine receptors on neutrophils. In summary, we have observed that nicotine exerts differential inhibitory effects on endothelial ICAM expression and on the neutrophil integrin expression of
L-selectin, CD11a, and CD11b. Because endothelial dysfunction and neutrophil activation has been proposed to be involved in the pathophysiologic factors of preeclampsia, the inhibitory effects of nicotine on adhesion molecule expression on endothelial cells and on neutrophils may explain partially the protective effect of cigarette smoking against preeclampsia. The differential effects of smoking (protection against preeclampsia although associated with low-birth-weight deliveries) are important to investigate; studies that have considered both preeclampsia and small gestational age birth weight to be products of the same vascular dysfunction may be inherently confounded. The relationship of our results to in vivo changes with preeclampsia is speculative. However, the down-regulation of vascular adhesion molecules and the decreased interaction of leukocytes with endothelial cells induced by nicotine suggest that these cell surface molecule changes may be important in the pathophysiology of preeclampsia. REFERENCES
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