Published online before print January 27, 2003, doi: 10.1161/01.CIR.0000047279.42427.6D
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(Circulation. 2003;107:604.)
© 2003 American Heart Association, Inc.
Basic Science Reports |
Nicotine Strongly Activates Dendritic Cell–Mediated Adaptive Immunity
Potential Role for Progression of Atherosclerotic Lesions
From the Department of Internal Medicine IV, Molecular Cardiology, University of Frankfurt, Germany (A.A., C.H., A.M.Z., S.D.); the Department of Molecular Immunology and Gene Therapy, Max-Delbrück-Center for Molecular Medicine, Berlin-Buch, Germany (M.M.); and the Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, Calif (J.P.C.).
Correspondence to Stefanie Dimmeler, PhD, Department of Molecular Cardiology, University of Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany. E-mail Dimmeler{at}em.uni-frankfurt.de
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Abstract |
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Background— Antigen-presenting cells (APCs) such as monocytes and dendritic cells (DCs) stimulate T-cell proliferation and activation in the course of adaptive immunity. This cellular interaction plays a role in the growth of atherosclerotic plaques. Nicotine has been shown to increase the growth of atherosclerotic lesions. Therefore, we investigated whether nicotine can stimulate APCs and their T cell–stimulatory capacity using human monocyte–derived DCs and murine bone marrow–derived DCs as APCs.
Methods and Results— Nicotine dose-dependently (10-8 to 10-4 mol/L) induced DC expression of costimulatory molecules (ie, CD86, CD40), MHC class II, and adhesion molecules (ie, LFA-1, CD54). Moreover, nicotine induced a 7.0-fold increase in secretion of the proinflammatory TH1 cytokine interleukin-12 by human DCs. These effects were abrogated by the nicotinic receptor antagonist 
-bungarotoxin and mecamylamine, respectively. The effects of nicotine were mediated in part by the phosphorylation of the PI3 kinase downstream target Akt and the mitogen-activated kinases ERK and p38 MAPK. Nicotine-stimulated APCs had a greater capacity to stimulate T-cell proliferation and cytokine secretion, as documented by mixed lymphocyte reactions and ovalbumin-specific assays with ovalbumin-transgenic DO10.11 mice. In a murine model of atherosclerosis, nicotine significantly enhanced the recruitment of DCs to atherosclerotic lesions in vivo.
Conclusions— Nicotine activates DCs and augments their capacity to stimulate T-cell proliferation and cytokine secretion. These effects of nicotine may contribute to its influence on the progression of atherosclerotic lesions.
Key Words: cells • immunity • atherosclerosis
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Introduction |
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Nicotine is a major component of cigarette smoke, a widely accepted risk factor for atherosclerosis. It is not known how and to what extent nicotine contributes to the adverse effects of chronic tobacco use, apart from its psychoactive actions and addictive properties. Nicotine activates nicotinic acetylcholine (nACh) receptors that belong to a family of ionotropic receptors and consist of 5 transmembrane subunits building up ion channels. The nACh receptors are widely distributed throughout the central and peripheral nervous system and are involved in signal transmission at the skeletal neuromuscular junction, in autonomic ganglia, and in the brain. In addition, nonneuronal cells such as monocytes, endothelial cells, and epithelial cells also express nACh receptors.1–4 However, the effect of nicotine on immune cells is incompletely characterized and controversial. Some investigators have provided evidence that nicotine promotes inflammation.5,6 By contrast, other studies indicate that nicotine may have immunosuppressive effects, although these results were achieved with relatively high doses of nicotine.7 Moreover, nicotine was recently demonstrated to promote progression of advanced atherosclerotic plaques.8 This may result in ischemia and infarction of the heart. Innate and adaptive immune responses play a crucial role for the growth and instability of atherosclerotic plaques.9 Recently, dendritic cells (DCs) were detected in the arterial wall.10,11 DCs are potent antigen-presenting cells required for the initiation of adaptive immune responses.12 The number of DCs is increased in atherosclerotic lesions, suggesting that DCs might contribute to T-cell activation in atherosclerosis.13 Proinflammatory factors can induce maturation of DCs corresponding to a switch from a phagocytic stage to a stage of strong T cell–stimulatory capacity. Accordingly, we examined the effect of nicotine in activation of DCs and on their ability to activate T-cell proliferation and cytokine secretion.
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Methods |
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Reagents
Nicotine [1-methyl-2-(3-pyridyl)pyrrolidine] was obtained from Sigma and tested for potential endotoxin contamination by use of the Endotox assay (Sigma). In addition, to further exclude a contribution of endotoxin to our results, we performed some experiments in the presence of 5 µg/mL polymyxin B (Sigma) that binds and neutralizes lipid A, the active moiety of lipopolysaccharide (LPS). LPS from Salmonella minnesota Re595 as well as the nicotinic receptor antagonists
-bungarotoxin and mecamylamine were purchased from Sigma. The p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 was obtained from Alexis, the MEK inhibitor PD98059 from Biomol, and the phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002 from Calbiochem. Human granulocyte-macrophage colony stimulating factor (GM-CSF; Leucomax) was from Novartis, and interleukin-4 (IL-4) was bought from R&D. Murine GM-CSF was obtained from supernatants of NIH-3T3 cells transfected with murine GM-CSF (provided by Thomas Blankenstein). FITC-dextran (40 000 kDa) and carboxyfluorescein diacetate succinimidyl ester (CFSE) were purchased from Molecular Probes. Synthetic ovalbumin (OVA) peptide representing amino acids 323 to 339 of chicken OVA was obtained from Biosynthan.
Animals
OVA-specific DO11.10 transgenic mice (BALB/c background, I-Ad) to obtain OVA-specific T lymphocytes were purchased from Charles River (Sulzbach, Germany). Murine bone marrow–derived DCs as stimulator cells were prepared from sex-matched BALB/c mice (Charles River). Annexin V, 7-aminoactinomycin D (AAD), and CD3-FITC were purchased from BD Pharmingen. CD40L (CD154) was from Beckman Coulter.
Preparation of Human Monocytes and Monocyte-Derived Human DCs
Human monocytes and DCs were prepared as previously described14 and were stimulated with nicotine for 12 hours.
Bone Marrow–Derived Murine DCs
Bone marrow–derived DCs were prepared as previously decribed.15
Mixed Lymphocyte Reactions
For the mixed lymphocyte reactions (MLRs), allogeneic CD3+ T lymphocytes were isolated from peripheral blood by immunomagnetic beads (Milteny Biotech). To measure their proliferation, CD3+ T cells were labeled with the fluorescent dye CFSE. With each cell division, the fluorescence intensity of the cells is reduced by half.16 After thorough washing to remove unbound fluorescent dye, 200 000 CD3+ T cells were mixed with human DCs and monocytes as stimulators (to ratios of 1:10 and 1:50, DCs/monocytes:T cells). Antigen-presenting cells were prestimulated with nicotine (10-7 mol/L) for 12 hours before use in MLRs. Experiments were performed in 96-well plates in triplicate. After 3 days of coculture of DCs or monocytes together with T cells in 200 µL RPMI containing 5% FCS, supernatants were collected, stored at -70°C, and analyzed by ELISA for production of human IL-2, a marker of T-cell activation. Cell pellets were washed with PBS and stained with mouse anti-human CD4-allophycocyanin (BD Pharmingen). Finally, costaining of CD4-allophycocyanin with CFSE labeling was determined by FACS analysis.
OVA-Specific Proliferation Assay
Lymph nodes from OVA-specific DO11.10 transgenic BALB/c (H-2d) mice were homogenized to obtain single-cell suspensions. Lymphocytes from total lymph nodes contained more than 50% CD4+ T cells and were labeled with CFSE dye. Bone marrow–derived DCs from BALB/c mice were prestimulated with nicotine (10-7 mol/L) for 12 hours. To assess their T cell–stimulatory capacity, 104 DCs were first loaded with 0.1 and 1 µg/mL OVA for 2 hours, washed twice with PBS, and then mixed with 105 CD4+ DO11.10 transgenic T cells. After 3 days of coculture, supernatants were examined for murine IL-2 secretion. Cell pellets were washed, stained with CD4-allopyocyanine, and prepared for FACS analysis of CFSE fluorescence.
Flow Cytometric Measurement
Human DCs were blocked with 5% human hyperimmune globulin (Flebogamma, Grifols) for human cells and rat anti-mouse CD32 F(ab')2 antibody (BD Pharmingen) at 5 µg/mL in PBS for murine cells at room temperature for 10 minutes. Then, cells were incubated with mouse anti-human CD11a-FITC, CD18-FITC, CD54-FITC, CD83-FITC, CD86-FITC, HLA-DR-FITC (all from BD Pharmingen), and CD40-FITC (Serotec). Surface expression of the nicotinic receptor was detected with mouse anti-human nicotinic receptor (Sigma) followed by goat anti-mouse F(ab')2 antibody (Dako). To characterize murine DCs, rat anti-mouse CD40-FITC, CD54-FITC, CD86-FITC, and I-Ad-FITC (MHC class II) were used (all from BD Pharmingen). Finally, cells were fixed in 1% formaldehyde/PBS and analyzed by FACSCalibur (Becton Dickinson) using CellQuest software (BD Pharmingen). Cell fluorescence intensity obtained with a specific antibody was in each case compared with that obtained with isotype-matched control antibodies purchased from BD Pharmingen. All FACS data were analyzed on 10 000 cells per condition.
Endocytosis Assays
To assess endocytosis of the DC preparations, FITC-dextran was used according to a method described previously.17 Briefly, cells stimulated with nicotine or unstimulated were incubated in complete RPMI medium with FITC-dextran at a final concentration of 1 mg/mL at 37°C for 30 minutes. Background staining at 4°C was used as reference. After the cells had been washed, FITC-dextran uptake of immature DCs was determined by FACS.
Quantification of Cytokine Production
Human IL-12p40 and human and murine IL-2 were measured by ELISA (OptEIA Set, BD Pharmingen). Human IL-10 was measured with a high-sensitivity ELISA (R&D). To detect cytokines, supernatants of 106 APCs/mL were used.
Western Blot Analyses
For analysis of ERK 1/2, p38 MAPK, and Akt phosphorylation, proteins were obtained in lysis buffer as previously described.18 Protein lysates (30 µg/mL) were electrophoresed on 10% SDS-PAGE gels, transferred to PVDF membranes (Millipore), and blotted with polyclonal phospho-ERK antibodies (New England BioLabs), followed by anti-rabbit horseradish peroxidase (Santa Cruz Biotechnology) and detection by chemiluminescence ECL (Amersham). Antibodies to phospho-p38 MAPK or phospho-Akt (Santa Cruz) were used to analyze phospho-p38 MAPK and phospho-Akt expression. As loading controls, antibodies against tubulin (Labvision Corp) were used.
DC Recruitment Assay
Apolipoprotein (apo)E–knockout mice were purchased from Jackson Laboratories as retired breeders (males with C57BL/6J background). Fifty-week-old animals fed a normal chow diet were used for experiments. Bone marrow–derived murine DCs from wild-type C57BL/6J were labeled with CSFE (1 µmol/L) for 10 minutes at 37°C. After 3 washing steps, 106 DCs were resuspended in 200 µL RPMI containing 0.5% FCS and injected intravenously via the tail vein. Starting 3 days before the injection of the DCs, animals were treated with vehicle or nicotine (1 µg per 10 g body weight twice daily) via subcutaneous injection. Forty-eight hours after the injection of the DCs, mice were killed and perfused with 4% formaldehyde with 20 µmol/L EDTA by injection through the apex of the left ventricle. The base of the heart and the ascending aorta were isolated and mounted in TissueTek (Sakura) freezing medium. We analyzed the atheroma of the aortic root using native cryostat sections (10 µm). Serial sections spanning 1.0 mm of the ascending aorta at the level of the sinus of Valsalva were examined under light and fluorescence microscopy. The number of CSFE-labeled DCs attached to the intimal surface or in atheromatous plaques was quantified for each aorta.19,20
Statistical Analysis
All results for continuous variables are expressed as median±SEM intervals. Comparisons between groups were analyzed by t test (2-sided) or ANOVA for experiments with more than 2 subgroups. Post hoc range tests and pairwise multiple comparisons were performed with the t test (2-sided) with Bonferroni adjustment. Comparison of categorical variables was generated by the Pearson 
2 test. All analyses were performed with SPSS 11.0 (SPSS Inc). Probability values of P<0.05 were considered statistically significant.
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Results |
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Expression of the Nicotinic Receptor on Human Monocyte–Derived DCs
To explore the role of nicotine and its receptors in DCs, we first tested whether nicotinic receptors are expressed on monocyte-derived DCs and their precursors, the monocytes. One of the most abundant nicotinic receptors is the
7-nicotinic receptor, whose constitutive expression was demonstrated on both DCs (Figure 1) and monocytes (data not shown). Moreover, stimulation with its ligand nicotine upregulated the expression of the 7-nicotinic receptor in a dose-dependent manner.
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Nicotine-Induced Upregulation of Proinflammatory Surface Molecules and Cytokines in Human and Murine DCs
CD86 is an essential costimulatory molecule on DCs required for efficient T-cell stimulation. Without sufficient costimulation, stimulation of T cells will induce anergy and apoptosis of T cells. Using increasing doses of nicotine (10-8 to 10-6 mol/L), we;F2> observed an increase in CD86 expression that peaked at 10-7 mol/L (Figure 2A; also shown in Figure 3A). Higher concentrations (10-4 mol/L) resulted in decreased expression of CD86 compared with control cells. This reflects the increasing toxicity of nicotine concentrations higher than 10-5 mol/L, as shown in Figure 2B. In contrast, lower doses of nicotine tended to reduce the number of dead cells (P=0.07). To confirm that upregulation of CD86 is a result of stimulation of nACh receptors (using the optimal nicotine dose of 10-7 mol/L), we preincubated DCs with the broad-spectrum nicotinic antagonist mecamylamine (10-7 mol/L). Mecamylamine abrogated the nicotine-induced upregulation of CD86 (Figure 2A).
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Moreover, nicotine (10-7 mol/L) increased the DC expression of adhesion molecules such as the ß2-integrin LFA-1 (CD11a/CD18) and its ligand CD54 (ICAM-1) in human DCs (Figure 3, A and B). In addition, nicotine also increased the expression of the MHC class II molecule HLA-DR and induced the expression of the DC maturation marker CD83 in human DCs (Figure 3B). Nicotine had similar effects on murine DCs. In murine DCs, nicotine increased the expression of CD54, the MHC class II molecule I-Ad, and the costimulatory molecule CD40 (Figure 3C).
Nicotine induced a marked increase in the secretion of the proinflammatory cytokine IL-12 p40 by DCs as detected by ELISA (Figure 4A). These effects of nicotine were blocked by mecamylamine and the selective 7-nACh receptor antagonist -bungarotoxin. The proinflammatory effect of nicotine caused a compensatory upregulation of the antiinflammatory cytokine IL-10 (Figure 4B).
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Nicotine Augments the Ability of DCs to Stimulate T Cells
We next determined whether nicotine could enhance the ability of DCs to stimulate T cells by use of allogeneic MLRs (Figure 5A). Nicotine-stimulated DCs markedly increased the T-cell secretion of IL-2, consistent with T-cell activation. The effect of nicotine (10-7 mol/L) was similar to that of the potent activator LPS (100 ng/mL). Nicotine-stimulated monocytes also induced IL-2 production in allogeneic T lymphocytes but less efficiently than DCs (Figure 5B). To determine whether nicotine stimulation is also effective in promoting antigen-specific responses, we studied its effects on the ability of OVA-exposed murine DCs to stimulate proliferation in DO11.10 OVA-transgenic T cells (Figure 5C). Nicotine significantly enhanced the T cell–stimulatory capacity of syngeneic murine DCs presenting OVA peptide 323 to 339 to OVA-transgenic T lymphocytes. Compared with vehicle-treated cells, nicotine-treated human DCs increased the proliferation of allogeneic CD4+ T cells by more than 3-fold; a similar effect was observed in nicotine-treated monocytes (2.2-fold induction) (Figure 5D). Similarly, nicotine stimulation led to a 2.3-fold increase in proliferation of syngeneic OVA-specific DO11.10 transgenic T cells. Similarly, nicotine stimulation led to a 2.3-fold increase in proliferation of syngeneic OVA-specific DO11.10 transgenic T cells. In addition, nicotine-stimulated DCs significantly increased expression of CD40L (CD154) on T cells in DC–T-cell cocultures (Figure 5E). Acquisition of T-cell–stimulatory capacity in maturing DCs is inversely correlated to their capacity for endocytosis of antigens such as FITC-dextran. Consistent with its effect to activate DCs, nicotine reduced their capacity for endocytosis (Figure 5F).
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Nicotine Stimulation Activates MAPK and Akt Pathways in Human DCs
To gain insight into nicotine-induced signaling, we determined whether MAPKs and Akt might be involved in nicotine-induced signaling pathways in human DCs. Nicotine rapidly increased the phosphorylation of p38 MAPK, an effect that was visible within 5 minutes (Figure 6A). Maximal activation of p38 MAPK was achieved by 15 minutes (547±86% increase), declining after 30 minutes. Phosphorylation of the MAPK ERK reached a maximal induction (332±34% increase) after 30 minutes of nicotine stimulation (Figure 6B). Activation of the Akt pathway, however, peaked relatively late, at 60 minutes, with a 418±78% increase (Figure 6C).
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Role for MAPK and Akt in the Nicotine-Induced Upregulation of the Costimulatory Molecule CD86 in Human DCs
Having defined signaling pathways that are activated in nicotine-stimulated human DCs, we examined the role of these signaling pathways in nicotine-induced expression of the costimulatory molecule CD86. The MAP kinase (MEK) inhibitor PD98059 and the inhibitor of p38 MAPK SB203580 each abolished the nicotine-induced increase in CD86 expression (Figure 7). Likewise, nicotine-induced CD86 upregulation was completely dependent on activation of PI3K/Akt pathways, because it was abrogated by the PI3K inhibitor LY294002 (Figure 7).
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DC Homing to Atherosclerotic Lesions
To investigate whether nicotine affects DCs in vivo, DCs were labeled with CSFE and injected into apoE-deficient mice. Atherosclerotic plaques above the aortic valve at the level of the sinus of Valsalva are the most advanced lesions in apoE-deficient mice. Sections of the aortic root were analyzed with fluorescent microscopy for CSFE-labeled DCs that were adherent to or within the atherosclerotic plaques. The majority of the labeled DCs were detected in regions inside the plaque, suggesting transmigration of the labeled DCs across the endothelial cell layer (Figure 8, A and B). Forty-eight hours after intravenous injection, DC homing to atherosclerotic lesions was significantly enhanced by nicotine. Adjusting for differences in plaque area, we found significantly more DCs in the nicotine group (34.2±10.9 DCs/lesion) than the control group (11.0±5.1; P<0.001). In contrast, atherosclerotic lesions in apoE-deficient mice without DC injection showed only background fluorescence (data not shown).
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Discussion |
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The present study suggests an important role for nicotine in DC activation. This was evidenced by the upregulation of nACh receptors, costimulatory molecules such as CD86 and CD40, MHC class molecules such as HLA-DR, and adhesion molecules such as CD54. Moreover, nicotine enhances the ability of DCs and monocytes to stimulate T-cell proliferation and cytokine production in allogeneic MLRs and antigen-specific assays. This indicates that nicotine enhances adaptive immunity and pathophysiological processes that involve adaptive immunity, such as atherosclerosis. Indeed, we demonstrate that nicotine significantly enhanced DC homing to atherosclerotic lesions.
DCs act as sentinels, which are highly specialized in sampling antigens from sites of inflammation.12,21 In atherosclerosis, possible antigens relevant for the initiation and progression of atherosclerotic lesions might be oxidized LDL, ß2-glycoprotein I, and heat-shock proteins.22 These antigens are actually self-antigens, modified by oxidation in the case of LDL. Prolonged presentation of self-antigens by DCs is crucial for the development of destructive autoimmune responses.23 In autoimmune disease, antigen-specific activation of autoimmune T cells is based on the interactions of T-cell receptors with self-antigens presented by MHC molecules on DCs. The capacity of DCs to stimulate T cells depends on their stage of maturation: after DCs picked up antigens at their immature phagocytic stage, DCs move to the regional lymph nodes while undergoing maturation.12 Maturation boosts their capacity to efficiently present antigens to T cells and is mediated by upregulation of costimulatory molecules such as CD86 (B7.2) and CD40. Costimulation by the ligands CD80/86 and its receptor CD28 on T cells is required for efficient T-cell stimulation.24 Lack of costimulation leads to T-cell anergy and apoptosis. Moreover, MHC class II molecules are upregulated and the possibility of DC–T-cell encounters is favored by enhanced expression of adhesion molecules. In parallel, activated DCs secrete cytokines and chemokines to recruit more immune cells to the site of inflammation. Thus, T cells present in atherosclerotic lesions may be nonspecifically activated T cells or those that have been expanded within lymph nodes in recognition of the presented antigen.
Nicotine at physiologically relevant concentrations promotes progression of advanced atherosclerotic lesions, an effect that was inhibited in part by the specific COX-2 inhibitor rofecoxib.8 Rofecoxib has powerful antiinflammatory properties that, together with its antiangiogenic effect, may have contributed to the inhibition of lesion progression. In our experiments, nicotine had a maximal immunostimulatory effect at concentrations between 10-8 and 10-7 mol/L, which can be found in the plasma of smokers.25 Because atherogenesis is known to rely on both innate and adaptive immune responses, it is tempting to speculate that nicotine-enhanced activation of DCs might contribute to T-cell recruitment and acceleration of inflammation in the atherosclerotic lesions. Indeed, soluble factors such as IL-12 released by activated DCs are well established to contribute to the formation of atherosclerotic lesions and plaque destabilization.26 In addition, nicotine stimulated DC expression of CD40. Ligation of CD40 can induce IL-12 production in DCs.14 Moreover, genetic disruption of the CD40/CD40 ligand system has been described to prevent the initiation and progression of atherosclerosis.27
Antagonists of the 7-nicotinic receptors blocked the effects of nicotine on DC maturation. The nACh receptors are ligand-gated ionotropic channels consisting of -subunits or a combination of - and ß-subunits, each of which has a unique pharmacological profile.28 Intriguingly, we observed a marked expression of the homopentamer 7-nACh receptor in DCs. The 7-nACh receptor appears to play a crucial role in the observed effects of nicotine on DC maturation. The specific 7-nACh receptor antagonist -bungarotoxin abrogated the stimulatory effects of nicotine, an effect similar to what we observed for the nonselective nACh receptor antagonist mecamylamine.
The downstream effector pathways by which nicotine activated DCs appear to rely on the activation of several kinases. Here, we demonstrated activation of ERK1/2, p38 MAPK, and Akt in DCs by nicotine. Activation of ERK 1/2 and Akt is consistent with the effect of nicotine in other cell types, which is mediated via the 7-nACh receptor.29–31 However, p38 MAPK has not yet been involved in activation by nicotine.32 Notably, the p38 MAPK pathway is involved in the LPS-induced upregulation of costimulatory molecules via stimulation of transcription factors such as nuclear factor-
B.33
Finally, nicotine profoundly enhanced homing of DCs to atherosclerotic lesions in vivo, which supports the hypothesis that the proatherogenic effect of nicotine may be mediated in part by recruiting DCs to sites of chronic inflammation such as atherosclerotic plaques. DCs clustering with T cells have been identified in atherosclerotic lesions of apoE-deficient mice and form clusters with T cells.34
Because tobacco smoke consists of more than 4000 chemical constituents, it is impossible to predict the effect of nicotine within this complex mixture of components. Although we show that nicotine has a potent immunostimulatory effect, there are several molecules in cigarette smoke that may be toxic to DCs (eg, cadmium, reactive oxygen species; see Powell35 for review). The net effect of cigarette smoke on endothelial function may be quite different from that of nicotine alone. In addition, our results are in contrast to several studies showing a decrease in immune responses in the presence of nicotine.7 However, these studies used much higher doses of nicotine that, in our experiments, showed cytotoxic effects, which cannot be reversed by nACh receptor antagonists (Figure 2).
In conclusion, the present study indicates that nicotine enhances adaptive immunity, an effect that is mediated by nicotinic ACh receptors on DCs and monocytes. Nicotine activates these antigen-presenting cells, inducing the expression of surface molecules involved in inflammation and augmenting T-cell proliferation and cytokine secretion. These effects of nicotine together with the enhanced recruitment of DCs to atherosclerotic lesions may contribute to pathophysiological processes involved in the growth and destabilization of atherosclerotic plaques.
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Acknowledgments |
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Dr Aicher was supported by a Young Investigator Grant of the University of Frankfurt. We thank Christiane Mildner-Rihm and Barbara Lafferton for their excellent technical assistance. We are grateful to Dr Thomas Blankenstein for supporting this work. Results from experiments depicted in Figures 3C and 5C were performed in his laboratory.
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Footnotes |
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*The first 2 authors contributed equally to this work.
Received September 3, 2002; revision received October 17, 2002; accepted October 28, 2002.
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