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Expansion of Circulating Toll-Like Receptor 4–Positive Monocytes in Patients With Acute Coronary Syndrome
http://www.100md.com 《循环学杂志》
     the Department of Cardiology, University Hospital Grosshadern, Ludwig-Maximilians University, Munich, Germany.

    Abstract

    Background— Atherosclerosis is an inflammatory disease in which monocytes and macrophages have been suggested to play an essential role. The underlying signaling mechanisms are unknown thus far. We hypothesized that the human isoform of Toll-like receptor (hTLR)-4 is involved in monocyte activation of patients with accelerated forms of atherosclerosis.

    Methods and Results— Expression of hTLR4 on circulating monocytes from 30 controls, 20 patients with stable angina (SA), 40 patients with unstable angina (UA), and 28 patients with acute myocardial infarction (AMI) was compared with the use of flow-cytometry and reverse transcription–polymerase chain reaction. Regulation of interleukin (IL)-12 and B7-1 as downstream events of TLR4 activation was analyzed after lipopolysaccharide stimulation of monocytes. TLR4-transfected Chinese hamster ovary (CHO) cells were used to identify potential hTLR4 ligands in the serum of patients with UA or AMI. Circulating hTLR4+/CD14+ monocytes were 2.5-fold increased above controls and patients with SA in the UA and AMI groups (P<0.0001). This was paralleled by enhanced transcript levels of TLR4 and Myd88 in patients with UA and AMI (P<0.0001) and increased expression of IL-12 (UA 35.5±7.8, AMI 31.8±7.7 versus SA 2.2±0.5, controls 2.1±0.3 pg/mL; P<0.0002) and B7-1 (UA 27.3±14.4, AMI 22.6±11.1 versus SA 3.4±2.5, controls 2.4±2.3%; P<0.0001). Compared with serum from patients with UA and AMI, challenging TLR4-transfected CHO cells with serum from SA patients yielded only a weak response (P<0.0001). Coincubation with anti–heat shock protein 60 inhibited CHO cell activation.

    Conclusions— UA and AMI are associated with enhanced expression and signaling events downstream of hTLR4 in circulating monocytes. These observations suggest hTLR4 activation as a signaling mechanism in immune-mediated progression of atherosclerosis.

    Key Words: coronary disease ; monocytes ; immune system ; receptors, cell surface

    Introduction

    Atherosclerosis resembles many features of a chronic inflammatory disease.1 Atherosclerotic lesions are enriched for macrophages and T cells that play important roles in early (innate) and advanced (acquired) immune responses. In general, the immune system responds effectively to danger signals by close interplay between innate and adaptive immune recognition systems. Progression of atherosclerosis has been associated with clonal expansion of differentiated T cells as a feature common to all adaptive immune responses.2 Efficient priming of adaptive immune responses requires not only presentation of antigen but also the induction of accessory signals (costimulators and cytokines) on antigen-presenting cells. The precise signaling mechanisms responsible for activation of these cells in the pathogenesis of atherosclerosis remain ill defined. Recently, Toll-like receptors (TLRs) have been identified as key recognition components of the innate immune system in mammals.3,4 Of 11 different human TLR family members, TLR4 is the best characterized. Several exogenous and endogenous ligands such as lipopolysaccharide (LPS),5,6 fibrinogen,7 minimally modified LDL,8 and heat shock protein (HSP)609 have been identified. Enhanced monocyte/macrophage expression of costimulatory molecules (such as B7-1 and B7-2) and proinflammatory cytokines (such as interleukin [IL]-1;, IL-6, IL-12, and tumor necrosis factor- [TNF-]) have been demonstrated as downstream effects of TLR activation.3,10

    Although the importance of TLRs in antimicrobial responses is well established, their role in atherosclerotic disease processes as early triggers of immune-mediated initiation or progression is not well understood. Immunohistochemical staining of murine and human atherosclerotic tissue revealed prominent expression of TLR4, especially at the lipid-rich, macrophage-infiltrated shoulder region of plaques.11 Edfeldt et al12 provided evidence that TLR-expressing cells in the vessel wall are activated. Furthermore, TLR4 has been suggested to play a role in outward remodeling,13 and an enhanced in vitro response of monocytes to LPS has been demonstrated in patients with recurrent unstable angina (UA).14 However, the nature of potential ligands responsible for activation of TLR4 and downstream effects in this setting is incompletely understood. HSP60 has been suggested as an endogenous trigger on the basis of its abundance in atherosclerotic plaques15 and previous evidence in various animal and human models implying its potency to activate TLR4.2,9,15

    The purpose of this study was to compare the expression of the human isoform of TLR4 (hTLR4) on circulating CD14+ monocytes in patients with various clinical stages of atherosclerosis (patients with stable angina [SA], UA, and AMI) to better understand the association of hTLR4 with the progression and deterioration of atherosclerosis. Changes in expression patterns for hTLR4 were correlated with changes in monocyte-specific expression of the costimulatory molecule B7-1 as well as IL-12 as typical hTLR4 downstream effects. In trying to prove and identify a serum factor that might be responsible for the hypothesized atherosclerosis-associated activation of hTLR4, stably transfected Chinese hamster ovary (CHO)–TLR4+ cells were incubated with serum from patients with various clinical stages of atherosclerosis.

    Methods

    Study Patients

    This investigation was performed with approval by the institutional ethics committee. Informed consent was obtained from all subjects. Eighty-eight of 143 consecutive patients admitted to our hospital with a diagnosis of coronary artery disease (CAD) were included in the study. Four groups of subjects were studied (Table 1). Blood samples were obtained from all patients in the recumbent position with a 21-gauge needle via antecubital venepuncture as soon as possible after admission. Concentrations of C- reactive protein, white blood cell count, cholesterol, glucose, troponin I, and creatine kinase (CK) were measured according to routine protocols.

    Group I included 20 patients with clinical evidence of Canadian Cardiovascular Society class II and III SA and at least 1 coronary artery stenosis detected at angiography (>50% reduction of lumen diameter). Group II included 40 patients admitted to the Coronary Care Unit of our hospital with a diagnosis of UA characterized as class II or III within the Braunwald classification. All patients with UA had experienced ischemic chest pain at rest within the preceding 6 hours but no evidence of myocardial infarction by ST elevation or significant rise in CK or CK-MB (34±13 U/L). Troponin I was detected in 18 of 40 patients but in all was <8 ng/mL (average, 4.8±2.7 ng/mL). All UA patients exhibited transient ST-T–segment depression and/or T-wave inversion. Group III included 28 patients with an AMI who presented within 6 hours of the onset of pain. Identification of AMI followed the recent consensus document of the American Heart Association and the American College of Cardiology.16 All patients showed elevated ST segments and a characteristic pattern of myocardial serum enzymes in which the total CK was at least twice the upper reference limit for CK accompanied by a rise in CK-MB mass (593±218 U/L) and troponin I (104±29 ng/mL). All patients underwent coronary angiography during hospitalization.

    Exclusion criteria were as follows: previous myocardial infarction within 6 months (13 patients); admission >6 hours from onset of symptoms for group II and III (21 patients); inflammatory conditions likely to be associated with an acute phase response (8 patients); autoimmune disease (5 patients); and neoplastic disease (8 patients). None of the included patients had advanced liver disease, renal failure, or valvular heart disease.

    Group IV included 30 healthy volunteers with no clinical signs of CAD and without coronary risk factors. Controls were recruited from the staff of our hospital and from visitors. All of these controls had normal ECG and echocardiogram and no evidence of atherosclerosis by carotid artery sonography.

    Isolation of Peripheral Blood Mononuclear Cells, mRNA Isolation, and Semiquantitative Reverse Transcription-Polymerase Chain Reaction

    Isolation of mononuclear cells from heparinized blood was performed by Ficoll density gradient centrifugation (Becton Dickinson). Serum was stored at –70°C. The cell fraction was processed immediately after separation.

    CD14+ cells were further isolated with the use of MACS CD14+ MicroBeads according to the manufacturer’s instructions (Miltenyi Biotec). Dynabeads oligo(dT)25 were used to isolate mRNA according to the manufacturer’s instructions (Dynal). First-strand cDNA synthesis was performed with the use of oligo(dT) 12-18 and a superscript preamplification kit (Life Technologies-Invitrogen).

    Polymerase chain reaction (PCR) primers were designed with MacVector 5.0 (Oxford Molecular Scientific). For each individual primer pair, specific annealing temperature and cycle number were optimized by serial annealing studies, PCR cycle studies, and cDNA dilution studies (Table 2).

    Duplicate samples were amplified with AmpliTaq Gold DNA polymerase (Applied Biosystems). After electrophoresis, agarose gels were incubated for 20 minutes at room temperature with SYBRGold (Molecular Probes). Fluorescence was induced by UV transillumination (300 nm), and samples were quantified with a BioRad FluorS-Multiimager. Fluorescence was measured by densitometric analysis with the use of Quantity One Software (BioRad). A negative control and an internal standard were run on each gel. Corrected levels of the specific transcript were derived by dividing the amplified product value by the mean value for the control gene GAPDH in the respective sample.

    Flow Cytometry Analysis

    Cells were incubated with hTLR4 antibody (mouse anti-human monoclonal antibody, clone HTA125, IgG2a, HyCult Biotechnology, Uden, Netherlands) and CD14-PE antibody (mouse anti-human monoclonal antibody, clone 116 [Mo2], IgM, Beckman-Coulter, Krefeld, Germany) or with mouse IgG2a/IgM isotype controls (DakoCytomation, Hamburg, Germany). After cells were washed with staining buffer (PBS containing 0.1% BSA and 0.1% sodium azide), a monoclonal goat anti-mouse IgG2a-FITC antibody (Southern Biotechnology, Birmingham, Ala) was added.

    For measurement of B7-1 expression, monocytes were stimulated with 1 μg/mL LPS (from Escherichia coli 0111:B4, Sigma-Aldrich Chemical Co, Munich, Germany) for 12 hours and stained with a B7-1/FITC antibody (mouse anti-human monoclonal antibody, clone BB1, Southern Biotech) or with mouse IgG2a isotype controls. Ten thousand CD14+ cells were analyzed on a FACSCalibur flow cytometer with CellQuest acquisition analysis software (Becton Dickinson Immunocytometry Systems).

    Serum HSP60 Assay

    A sandwich enzyme-linked immunosorbent assay (ELISA) was performed with the use of a monoclonal antibody against HSP60 (StressGen Biotechnologies Corporation, Victoria, British Columbia, Canada). In short, 96-well microtiter plates were coated through overnight incubation at 4°C with a murine monoclonal antibody to HSP60 (2 μg/mL; clone LK.1, StressGen). Plates were washed with PBS containing 1% Tween 20 and blocked by incubation with 1% BSA in PBS/Tween. Serum samples (diluted 1:10 in PBS) were added, and bound HSP was detected by addition of goat polyclonal anti-HSP60 antibody (1:1000; StressGen). Bound polyclonal antibody was detected with alkaline phosphatase–conjugated rabbit anti-goat IgG (minimum cross-reactivity to human serum proteins; Rockland Immunochemicals, Gilbertsville, Pa), followed by p-nitrophenyl phosphate substrate (Sigma-Aldrich). The resultant absorbance was measured at 405 nm with a Titertek Multiscan MCC/340 plate reader. Standard dose-response curves were generated in parallel with recombinant human HSP60 (0 to 2500 ng/mL; StressGen), and concentrations of HSP60 were determined by reference to these standard curves.

    CD25 Expression Analysis

    The CHO/CD14/TLR4 reporter line is a stably transfected human CD14+ and TLR4+-CHO cell line that expresses inducible membrane CD25 under the transcriptional control of the human E-selectin promoter, which has nuclear factor (NF)-B binding sites (kindly provided by Douglas T. Golenbock, University of Massachusetts Medical School, Worcester). Cells were grown in Ham’s F-12 medium (GIBCO-BRL) supplemented with 10% fetal bovine serum (Life Technologies-Invitrogen), 1 mg/mL G418, and 400 U/mL hygromycin B (both Calbiochem-Novabiochem GmbH) at 37°C, 5% CO2.

    When cells were 90% confluent, serum from patients and controls (diluted 1:3 with Ham’s F-12 medium) with or without anti-HSP60 (1:100; StressGen) was added for 16 hours. Stimulation of CHO cells with 1 μg/mL LPS was used as a positive control. As a control for the specificity of the HSP60 effect on CD25 expression, CHO cells were coincubated with serum and an irrelevant antibody (mouse anti-human TLR9, clone 5G5, IgG2a, HyCult Biotechnology) or with recombinant HSP60 (StressGen) and anti-human TLR9. After 16 hours, cells were washed with PBS and detached with 2 mmol/L EDTA in PBS. CHO cells were then labeled with FITC-conjugated mouse anti-human CD25 (clone M-A251, Becton Dickinson), and 104 cells were analyzed on a FACSCalibur flow cytometer with CellQuest acquisition analysis software. Binding of anti-CD25 antibody to its epitope was expressed as percentage of the fold increase in mean fluorescence intensity compared with LPS-treated CHO cells.

    To further test specificity of the used HSP60 antibody, a Western blot with serum samples and recombinant human HSP60 as a positive control was performed. Protein concentrations in the samples were measured with a BCA protein assay kit (Pierce) based on the Lowry method, and 0.45 μg protein was analyzed on a 4% to 12% polyacrylamide gel (Life Technologies-Invitrogen). The proteins were subjected to electrophoresis and transferred to nitrocellulose membranes. The membranes were blocked with MOPS buffer (Life Technologies-Invitrogen) and reacted with anti-HSP60, followed by incubation with alkaline phosphatase–conjugated goat anti-mouse IgG (minimum cross-reactivity to human serum proteins; Rockland Immunochemicals) and by p-nitrophenyl phosphate substrate (Sigma).

    Measurement of IL-12 Concentration by ELISA

    CD14+ monocytes (2x106) were incubated for 24 hours in siliconized glass with 1 μg/mL LPS in a total volume of 2 mL complete RPMI-1640 medium. Supernatants of monocytes were separated by centrifugation, and IL-12 concentration was quantified by ELISA (R&D Systems; detection limit <0.5 pg/mL). Supernatants for each individual were stored at –70°C and measured at the same time by the same ELISA to avoid variations of assay conditions.

    Statistical Analysis

    Data are presented as mean and median±SD (mean; median±SD) or number (percentage). Continuous variables between 2 groups were compared by Wilcoxon test and between >2 groups by Kruskal-Wallis test. Categorical variables were compared by the 2 test. Comparisons between B7-1 and IL-12 expression levels and frequencies of hTLR4+/CD14+ monocytes were performed with the use of Spearman’s rank correlation test. Statistical analysis was performed with JMP statistical software (SAS Institute Inc). A probability value <0.05 was considered statistically significant.

    Results

    Characteristics of Patients

    Assessment of Frequencies of hTLR4-Expressing CD14+ Monocytes

    Protein levels of hTLR4 on the surface of peripheral circulating CD14+ monocytes were quantified by flow cytometry analysis. A mean of 30.4% (32.4±6.3%) of peripheral circulating CD14+ monocytes in normal controls and of 32.7% (32.5±5.4%) in patients with SA showed surface expression for hTLR4 compared with 77.9% (77.3±9.9%) in patients with UA and 78.3% (76.7±14.0%) in patients with AMI. The frequencies of hTLR4+/CD14+ monocytes were significantly higher in patients with UA and AMI than in SA and control patients (P<0.0001; Figure 1). Differences between patients with UA and with AMI as well as between controls and patients with SA were not significant.

    Assessment of hTLR4 and Myd88 Transcript Levels

    mRNA expression by reverse transcription (RT)–PCR followed protein detection by FACS analysis. Transcript levels for hTLR4 were significantly higher in the UA (1.78; 1.83±0.26 relative units [RU]; P<0.0001) and the AMI groups (1.82; 1.83±0.33 RU; P<0.0001) than in controls (0.92; 0.93±0.11 RU) and in SA patients (0.96; 0.96±0.19 RU; Figure 2). There were no significant differences between the UA and AMI groups (P=0.46).

    Similar results could be collected for the gene expression of the adaptor protein Myd88. UA and AMI patients had significantly increased transcript levels for Myd88 (0.96; 0.97±0.07 RU and 1.06; 1.04±0.24 RU) compared with controls (0.37; 0.42±0.18 RU; P<0.0001) and SA patients (0.42; 0.44±0.13 RU; P<0.0001; Figure 2). AMI patients had significantly higher Myd88 mRNA transcript levels than UA patients (P<0.05).

    Serum HSP60

    Circulating serum HSP60 levels were significantly lower in controls than in patients with SA (46; 21±55 versus 263; 275±96 ng/mL; P<0.0001), whereas only 12 of the controls showed any circulating HSP60. Patients with UA (1243; 1075±403 ng/mL; P<0.0001 versus SA) and with AMI (2342; 2298±687 ng/mL; P<0.0001; Figure 3) exhibited significantly higher levels of circulating HSP60 than controls and the SA group.

    Serum HSP60 Activates CHO Cells

    On the basis of enhanced expression of TLR4 in patients with acute coronary syndromes (ACS), we hypothesized the presence of circulating ligands for TLR4 in this clinical setting. Therefore, we evaluated whether circulating HSP60 could activate TLR4-transfected CHO cells by challenging them with serum from patients. Sera from 2 patients with HSP60 serum levels in the highest tertile, from 4 patients with HSP60 serum levels in the middle tertile, and from 2 patients with HSP60 serum levels in the lowest tertile of all HSP60 measurements generated in each group were used. Western blot of different serum samples with the applied HSP60 antibody revealed its specificity (data not shown). As shown in Figure 4, serum from patients with ACS induced a significant CD25 expression on CHO cells that was 77% for the UA group and 79% for the AMI group compared with the positive control (1 μg/mL LPS). In comparison, serum from controls showed almost no CD25 expression ability (P<0.0001 versus UA and AMI), and challenging CHO cells with serum from SA patients yielded only a weak response (P<0.001 versus control; P<0.0001 versus UA and AMI). Coincubation of cells with serum and anti-HSP60 nearly rescinded CD25 expression (SA 10%, UA 10%, AMI 15% compared with the LPS response; P<0.0001; Figure 4), whereas coincubation with LPS and anti-HSP60 had no effect on CD25 expression by CHO cells. In addition, coincubation with an irrelevant antibody failed to inhibit the effect of serum or recombinant HSP60 on CD25 expression, respectively (data not shown).

    Upregulation of B7-1 and IL-12 in Patients With UA and AMI

    To investigate expression patterns of TLR4-dependent molecules in LPS-stimulated monocytes, we assessed B7-1 expression via FACS analysis and IL-12 secretion via ELISA. Whereas in controls and in SA patients only few CD14+ monocytes stained positive for B7-1 (control: 2.4%; 1.4±2.3%, SA: 3.4%; 4.2±2.5%), frequencies of B7-1+/CD14+ monocytes were significantly higher in the UA (27.3%; 24.3±14.4%) and AMI groups (22.6%; 21.0±11.1%; P<0.0001 versus control and SA; Figure 5A). Compared with controls and SA patients, IL-12 secretion from LPS-treated monocytes was significantly higher in patients with UA and AMI (control: 2.1; 2.1±0.3 pg/mL; SA: 2.2; 2.0±0.5 pg/mL; UA: 35.5; 35.4±7.8 pg/mL; AMI: 31.8; 32.4±7.7 pg/mL; P<0.0002 versus control and SA; Figure 5B).

    A strong correlation was noted between the frequency of peripheral circulating hTLR4+/CD14+ monocytes and expression levels of B7-1 (r=0.71; P<0.0001) and secretion of IL-12 (r=0.76; P<0.0001) across all patient groups (data not shown).

    Comparable patterns could be seen on mRNA transcript levels with upregulation of B7-1 and IL-12 mRNA transcript levels in patients with UA and AMI (Figure 6).

    Discussion

    In this study we used flow cytometry analysis, RT-PCR, and ELISA to characterize expression levels of hTLR4 and hTLR4 downstream signaling events in circulating monocytes from patients with different stages of CAD. To the best of our knowledge, this is the first study to show enhanced expression of hTLR4 on circulating monocytes in patients with ACS. Enhanced expression of hTLR4 in patients with ACS was associated with elevations of IL-12 and B7-1 expression. Moreover, using a CHO cell line transfected with expression plasmids for TLR4 and CD14, we found circulating HSP60 as a potential endogenous ligand activating TLR4 in this clinical setting.

    Evidence is accumulating that TLR4 and its endogenous ligand HSP60 are important players in the initiation and acceleration of atherosclerotic disease.11,15,17 Animal experiments as well as human studies point to a combination of HSP60-specific humoral and cellular immune reaction in this disease.15 Quite recently, Zal et al18 proved the presence of HSP60-reactive T cells in patients with ACS, and Buono et al19 reported in a mouse model that T cell reactivity to HSP60 as well as development of atherosclerosis depends on costimulatory signaling (B7-1 and B7-2) provided by antigen-presenting cells. Furthermore, outward arterial remodeling, which is believed to be associated with a vulnerable plaque phenotype,20 has been linked with an increase in TLR4 expression and mRNA levels of endogenous ligands (HSP60 and extra domain A of fibronectin).13 This might further indicate that no exogenous ligands are necessary for atherogenesis, as Wright et al21 demonstrated in a murine model. However, it should be recognized that the knowledge about HSP60 as a potential endogenous ligand of TLR4 has been generated in in vitro and artificial in vivo experiments; as revealed in recent publications, it might be possible that the ligand preparations used were contaminated with LPS and/or other microbial components.22–24

    It has been shown that upregulation of TLR4 contributes to sensitization of monocytes, whereas downregulation decreases inducibility of a proinflammatory response.25–27 Elevated expression of IL-12 and B7-1, as typical downstream effects of TLR4 activation, in concert with antigen presentation has been shown to activate T cells and drive their differentiation into T-helper 1 cells,10,28 for which a role in increasing plaque instability has been shown (H. Methe, MD, unpublished data, 2004).29,30

    Furthermore, upregulation of hTLR4 expression in patients with ACS was paralleled by enhanced Myd88 transcript levels in circulating monocytes. Myd88, which is an intracellular adaptor protein mediating TLR signal transduction, is subject to regulation by inflammatory stimuli in monocytes.31,32 Bjorkbacka et al,33 however, demonstrated that Myd88-deficient but not CD14-deficient mice showed a marked reduction in early stages of atherosclerosis. CD14 deficiency would argue against a role of TLR4 and HSP60 in initiating atherosclerosis. However, recent epidemiological evaluations in the Bruneck Study revealed a significantly lower risk of early plaque development and progression of atherosclerotic disease in carotid and femoral arteries in subjects with the Asp299Gly hTLR4 polymorphism.34,35 Furthermore, Michelsen et al36 showed that atherosclerosis-prone hypercholesterolemic mice that also harbor a null mutation in either the adaptor molecule TLR4 or MyD88 exhibit reduced aortic atherosclerosis, plaque lipid content, and plaque macrophage infiltration. Moreover, MyD88 deficiency led to decreased levels of circulating IL-12. However, the role of innate immune mechanisms in advanced atherosclerotic lesions (plaque rupture, thrombus formation, or myocardial infarction) needs further analysis.

    Taken together, the findings of the present study may directly link activation of TLR4 by circulating HSP60 with clinical instability of CAD. This coherence had been already supposed by Pasterkamp et al because endogenous TLR4 ligands, HSP60, and extra domain A of fibronectin have been observed in arthritic and oncological specimens in which matrix turnover is an important feature.37–39 Because we only studied the effect of circulating HSP60 on activation of TLR4-transfected CHO cells, we cannot exclude that other endogenous ligands for TLR4 (eg, minimally modified LDL, extra domain A of fibronectin8,11,40) may play a role in the activation of innate immune mechanisms in the progression and deterioration of CAD.

    Currently it is not well characterized how antiatherosclerotic pharmacotherapy may influence hTLR4 expression patterns and downstream signaling. To the best of our knowledge, besides an effect of statins on hTLR4 expression and signaling (H. Methe, MD, unpublished data, 2005),41 no results have been published with regard to an impact of ACE inhibitors, ;-blockers, aspirin, or clopidogrel on hTLR4 signaling. Importantly, patients in the present study did not show significant differences in antiatherosclerotic pharmacotherapy. The high frequency of hTLR4+/CD14+ monocytes in patients with ACS, despite being on statin therapy, emphasizes even more the potential role of hTLR4 in progression and deterioration of CAD.

    In conclusion, our study demonstrated that patients with UA and AMI exhibit enhanced monocytic expression of hTLR4 and signaling events downstream of hTLR4. In agreement with other reports, circulating HSP60 might be an endogenous ligand for hTLR4 in ACS. Therefore, hTLR4 may provide a link between innate and adaptive immunity, thereby enhancing the cellular immune response to antigens such as HSP60 in patients with ACS.

    Acknowledgments

    This study was supported in part by a grant from the F;rderprogramm für Forschung und Lehre, University of Munich, to H. Methe. We thank Dr Douglas T. Golenbock, University of Massachusetts Medical School, Worcester, for providing us with the CHO cell lines. We are grateful to Vera Tolbert for her expert technical assistance and Dr Mark Vangel, Massachusetts Institute of Technology Clinical Research Center, for his valuable help.

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