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Circulation. 1997;96:4232-4238

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(Circulation. 1997;96:4232-4238.)
© 1997 American Heart Association, Inc.

Articles

Cyclosporin A Inhibits Monocyte Tissue Factor Activation in Cardiac Transplant Recipients

Hans Hölschermann, MD; Oliver Kohl, MD; Ulrich Maus, PhD; Frank Dürfeld, MD; Angelika Bierhaus, PhD; Peter P. Nawroth, MD; Jürgen Lohmeyer, MD; Harald Tillmanns, MD; ; Werner Haberbosch, MD

From the Department of Internal Medicine, Division of Cardiology (H.H., O.K., F.D., H.T., W.H.), and the Department of Internal Medicine, Division of Pneumology (U.M., J.L.), Justus-Liebig-University, Giessen, and the Department of Internal Medicine, Ruprecht-Karls-University Heidelberg (A.B., P.P.N), Germany.

Correspondence to Dr Hans Hölschermann, Department of Internal Medicine, Division of Cardiology, University of Giessen, Klinikstr 36, D-35392 Giessen, Germany. E-mail hans.f.hoelschermann{at}innere.med.uni-giessen.de


*    Abstract
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 *Abstract
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 down arrowMethods
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Background Fibrin deposition and thrombosis have been implicated in both allograft rejection and vasculopathy after cardiac transplantation. Because monocytes play a pivotal role in the pathophysiology of intravascular coagulation activation through their ability to synthesize tissue factor (TF), we asked (1) whether monocyte TF activation occurs in cardiac transplant recipients and (2) whether monocyte TF expression is affected by treatment with cyclosporin A (CsA).

Methods and Results We measured levels of TF activity in peripheral blood mononuclear cells and highly purified monocytes/macrophages from 10 consecutive cardiac transplant recipients and 10 healthy control subjects. TF activity generated by both unstimulated and endotoxin-stimulated cells was significantly higher in transplant recipients than in control subjects (P<.05). Increased monocyte TF expression in transplant recipients was shown to be adversely affected by treatment with CsA: TF induction was markedly reduced by CsA serum concentrations reaching peak CsA drug levels. Inhibition of TF induction in the presence of high CsA blood concentrations was also observed when stimulation of cells was performed with interferon-{gamma} or interleukin-1ß. As shown by reverse transcription–polymerase chain reaction and electrophoretic mobility shift assay, respectively, treatment with CsA leads to decreased TF mRNA expression and reduced activation of the NF-{kappa}B transcription factor, which is known to contribute to the induction of the TF promotor in human monocytes.

Conclusions This study demonstrates that TF activation, occurring in mononuclear cells of cardiac transplant recipients, is inhibited by treatment with CsA. Inhibition of monocyte TF induction by CsA may contribute to its successful use in cardiac transplant medicine and might be useful in managing further settings of vascular pathology also known to involve TF expression and NF-{kappa}B activation.


Key Words: vasculature • transplantation • blood cells • coagulation


*    Introduction
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 *Introduction
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 down arrowDiscussion
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Tissue factor is a cell membrane–bound glycoprotein that plays a key role in the activation of the coagulation cascade.1 When exposed to blood, TF binds to factor VII, and the resulting complex activates factors IX and X, leading to thrombin and fibrin generation.2 Under pathological conditions, monocytes are known to express TF in response to inflammatory mediators such as cytokines, immune complexes, and bacterial toxins.3–5 Aberrant TF expression has been associated with a variety of clinical disorders, such as inflammatory diseases,6 septic shock,7 cancer,8 and cardiovascular disease.9,10 Moreover, monocyte TF expression has been shown to represent a link between defense and coagulation systems in transplant immunology.11,12 In light of these findings, the intravascular initiation of the coagulation cascade by induction of TF on endothelium-adherent monocytes may be responsible for thrombus formation and fibrin deposits observed in cardiac allograft rejection and transplant vasculopathy.13,14

Cardiac allograft vasculopathy is an as yet untreatable obliterative vasculopathy that has emerged as a major limiting factor in long-term survival of heart transplant recipients.15 High-dose regimens of CsA, which is the agent of choice of contemporary immunosuppressive therapy in transplant medicine,16 have been associated with a reduction of both experimental17,18 and human19,20 cardiac allograft vasculopathy. Although the principal mechanism of action of CsA is thought to be an inhibition of IL-2 production by T helper cells,21 additional effects of CsA on various vascular cells have been described in the past few years.22–24 Recently, we found that exposure of cultured monocytes to CsA inhibits LPS induction of monocyte TF expression independently of the presence of regulatory T lymphocytes, an effect that could be ascribed to an inhibition of the transcriptional activation of the monocyte TF gene in vitro.24 The present clinical study was conducted to evaluate whether treatment of transplant recipients with CsA also prevents monocyte TF activation after cardiac transplantation in vivo.


*    Methods
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 up arrowIntroduction
 *Methods
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Patients
Ten consecutive heart transplant recipients (9 men and 1 woman, 54±6 years old [range, 44 to 66 years], 36±25 months [range, 3 to 74 months] after heart transplantation) and 10 healthy volunteers (8 men and 2 women, 31±3 years old [range, 27 to 37 years] drawn from the hospital staff) were included in the study.

All patients were on oral immunosuppressive therapy with CsA (Sandimmune) starting 3 to 4 days after transplantation. Mean CsA dosage was 3.4±0.3 mg · kg-1 · d-1 at the time of the study. In the first week after transplantation, all patients had received azathioprine (dosage adjusted according to white blood cell counts), antithymocyte globulin (5 mg · kg-1 · d-1) for 3 days, and prednisone (1 mg · kg-1 · d-1 orally, tapering after 1 week). All patients received aspirin (100 mg/d) as long-term therapy. Preoperative diagnosis was dilated cardiomyopathy in 6 patients and coronary artery disease in 4. Blood sampling was performed at least 40 days after any invasive procedure or alteration in treatment. No patient or control subject had a history of immunological or inflammatory disease, graft rejection, infection, or cancer in at least 3 months preceding blood sampling. The patients' baseline characteristics are summarized in Table 1Down.


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  		Table 1. Baseline Patient Characteristics

Collection of Blood Samples and Measurement of Blood CsA Levels
Blood samples (40 mL) were aspirated into evacuated heparinized tubes for isolation of mononuclear cells and into tubes containing EDTA for determination of whole-blood CsA concentrations. Blood samples were drawn (1) in the morning before daily CsA administration and (2) 4 hours after daily oral CsA administration. Concentrations of CsA in whole blood were assayed with a commercially available fluorescence polarization immunoassay (Abbot) according to the recommendations of the manufacturer.

Isolation of Cells
PBMCs were isolated by Ficoll-Hypaque density gradient centrifugation.25 Differential cell counts of Pappenheim-stained cytocentrifuge preparations revealed >98% mononuclear cells with 17% to 26% (mean, 21%) monocytes as evidenced by FACS analysis as well as nonspecific esterase staining of cytocentrifuge preparations. Cells were suspended in a defined serum-free culture medium (MSFM; Gibco BRL) and plated in 30-mm 6-well culture plates (Greiner). Final cell suspensions contained 1x106 cells/mL monocytes.

One third of PBMCs were further fractionated by prolonged adherence to plasma-coated tissue culture flasks. Nonadherent cells (lymphocyte-enriched cell preparation, subsequently referred to as lymphocytes) were monitored for homogeneity (<3% monocytes by Pappenheim staining and nonspecific-esterase staining). Adherent cells, subsequently referred to as monocytes/macrophages, were cultured at 1x106 cells/mL MSFM in 30-mm 6-well culture plates (Greiner). The final cultures contained >98% monocytes/macrophages, as evidenced by Pappenheim staining and FACS analysis as well as nonspecific-esterase staining of cytocentrifuge preparations. Cell viability of all cell preparations was always >95% as determined by ethidium bromide staining of cell aliquots and subsequent FACS analysis.

Measurement of TF Activity
Cells were lysed by three freeze-thaw cycles before assays for TF activity. PCA was measured with a one-stage clotting assay as described previously.24 Clotting times were converted to milliunits of PCA by reference to a standard curve established by serial dilutions of a standard rabbit brain thromboplastin preparation (Sigma). Values are given as mU/106 cells. Medium or medium with reagents alone did not show any PCA. Procoagulant activity was characterized as TF in all cases by use of factor VII–deficient plasma and inhibition of clot formation with a neutralizing monospecific antibody directed against human TF (American Diagnostica Inc).

RNA Isolation and RT-PCR
For isolation of total cellular RNA, the acid guanidinium thiocyanate–phenol-chloroform method as described by Chomczynski and Sacchi26 was used. Preparation of complementary DNA and subsequent PCR were performed as previously described.27 Sequences of intron-spanning TF-specific primers were sense, 5'-GCCGCCAACTGGTAGACATG-3' and antisense, 5'-TAGCCAGGATGATGACAAGG-3' and for the housekeeping gene GAPDH, sense, 5'-CGTCTTCACCACCATGGAGA-3' and antisense, 5'-CGGCCATCACGCCACAGTTT-3', respectively. Negative controls were performed routinely by PCR run without cDNA to exclude false-positive amplification products. The specificity of the obtained TF PCR products was verified by subjecting the related PCR product to automated DNA sequencing (model 373A, Applied Biosystems) and comparing the resultant cDNA sequence with the published human TF cDNA sequence.28

Electrophoretic Mobility Shift Assay
Nuclear extracts were prepared as described previously.24 Oligonucleotides of the NF-{kappa}B consensus (5'-AGTTGAGGGGACTTTCCCAGGC-3') were labeled to a specific activity >5x107 cpm/µg DNA. NF-{kappa}B binding was performed in 10 mmol/L HEPES, (pH 7.5), 0.5 mmol/L EDTA, 70 mmol/L KCl, 2 mmol/L DTT, 2% glycerol, 0.025% NP-40, 4% Ficoll, 0.1 mol/L PMSF, 1 mg/mL BSA (DNAase free), and 0.1 µg/µL poly dI/dC in a total volume of 20 µL. Nuclear extracts (10 µg) were incubated for 20 minutes at room temperature in the presence of 1 ng labeled oligonucleotide ({approx}50 000 cpm, Cerenkow radiation). Protein-DNA complexes were separated from the free DNA probe by electrophoresis and autoradiographed as described previously in detail.29 Specificity of binding was ascertained by competition with a 160-fold molar excess of cold NF-{kappa}B consensus oligonucleotides, and characterization was performed with monospecific antibodies directed against NF-{kappa}B family members (obtained from Santa Cruz Inc: anti-p50 sc-114X, anti-p65 sc-109X, anti–c-rel sc-70X, anti-relB sc-226X, and anti-p52 sc-298X).

Experimental Protocol
PBMCs and monocytes/macrophages were prepared from blood samples drawn from cardiac transplant recipients before and 4 hours after daily CsA administration, respectively, as well as from healthy control subjects. CsA blood levels were measured in both samples obtained from the transplant recipients. PBMCs were fractionated into fractions 1 to 3: Fraction 1 was assayed for procoagulant activity immediately after separation. Fraction 2 was analyzed after a 3-hour incubation period for the analysis of TF mRNA expression and a 6-hour incubation period for determination of TF activity, respectively, in the presence or absence of LPS (10 µg/mL; endotoxin 055:B5, Sigma). Fraction 3 was further separated into highly purified monocytes/macrophages and lymphocytes. The monocyte/macrophage preparations were incubated with or without LPS for 6 hours before measurement of procoagulant activity. In selected experiments, cells were stimulated with rIFN-{gamma} (Dr Rentschler; 100 U/mL) and rIL-1ß (Biomol; 100 U/mL), respectively, instead of LPS. NF-{kappa}B binding activity was studied in nuclear extracts prepared from PBMCs collected from transplant recipients before and after CsA administration. In some experiments, PBMCs isolated from transplant recipients before daily CsA administration were fractionated as described, and lymphocytes were treated with 2 µg/mL CsA (concentration comparable to peak CsA blood levels of transplant recipients in vivo) for 4 hours. After washing, pretreated lymphocytes were pooled with the monocytes/macrophages obtained from the same blood sample and assayed for TF activity after 6 hours of incubation with or without LPS.

Statistical Analysis
The statistical analyses were performed with SPSS for Windows. The data were described by median and range. The Wilcoxon signed-rank test for paired data was used to compare levels of TF activity before and after CsA administration. Differences between control and transplant recipient groups were tested by Student's t test for unpaired observations. Differences were assumed to be statistically significant at values of P<.05.


*    Results
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 up arrowMethods
 *Results
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TF Activity
Mononuclear cells obtained from cardiac transplant recipients demonstrated markedly increased (P<.05) TF generation after incubation with or without LPS compared with healthy control subjects (Fig 1Down). The TF activity generated by unstimulated PBMCs obtained from cardiac transplant recipients (median, 62 mU/106 cells; range, 29 to 275 mU/106 cells) was significantly higher than control values (median, 17 mU/106 cells; range, 2 to 27 mU/106 cells) in each transplant recipient investigated (Fig 1Down, left). In addition, cells from cardiac transplant recipients expressed significantly (P<.05) increased procoagulant activity (median, 1925 mU/106 cells; range, 600 to 3950 mU/106 cells) in response to LPS compared with control (median, 700 mU/106 cells; range, 230 to 2250 mU/106 cells; Fig 1Down, right). Similar data were obtained when normalized by protein concentration instead of cell numbers. The increase in TF activity was not related to increased monocyte counts in cardiac transplant recipients. There was no significant difference between transplant recipients and healthy control subjects in the number of peripheral blood mononuclear cells. Moreover, monocyte content was the same in mononuclear cell preparations from transplant recipients as in normal subjects. In contrast to the increased TF expression in plated mononuclear cells, freshly isolated PBMCs from both healthy control subjects and transplant recipients (before and after CsA administration) expressed only scanty amounts of procoagulant activity, without significant differences between the two groups (data not shown).



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  		Figure 1. Increased TF activity in PBMCs of heart transplant recipients. Each dot represents TF activity generated by mononuclear cell cultures derived from control subjects or heart transplant recipients (HTx). TF activity was assayed either in unstimulated cultures (left) or cultures stimulated with LPS (right). Median TF activity is indicated for each group. P<.05 vs control.

We next isolated mononuclear cells from cardiac transplant recipients before and after daily CsA administration. Measurement of the corresponding CsA plasma concentration in each blood sample revealed an increase of CsA blood levels from 233 ng/mL (median; range, 161 to 308 ng/mL) in the sample before daily CsA administration to 691 ng/mL (median; range, 397 to 900 ng/mL) in samples after daily CsA administration (data are given in Table 2Down). The degree of TF activity generated by mononuclear cells was inversely related to the level of CsA blood concentrations: TF activity was reduced from 1925 mU/106 cells (median; range, 600 to 3950 mU/106 cells) in stimulated PBMCs separated from transplant recipients during baseline CsA blood levels (before CsA administration) to 660 mU/106 cells (median; range, 190 to 1800 mU/106 cells) in PBMCs drawn from transplant recipients in the presence of peak CsA blood concentrations (after CsA administration). As shown in Fig 2ADown and Table 2Down, monocyte TF induction was reduced after CsA application in all transplant recipients investigated. Likewise, a similar inverse relationship between CsA blood concentrations and TF inducibility was observed when highly purified monocytes/macrophages were analyzed instead of whole mononuclear cells (Fig 2BDown). The inverse correlation between CsA plasma concentration and monocyte TF induction was reproducible when patients were reanalyzed several times, and the level of TF expression in response to LPS remained essentially constant in individual transplant recipients. Reduced TF generation in monocytes/macrophages obtained during high CsA blood concentrations was observed not only in response to LPS but also when stimulation of cells was performed with rIFN-{gamma} or rIL-1ß as activator instead of LPS (data not shown). As demonstrated in Table 3Down, ex vivo treatment of lymphocytes with CsA at concentrations comparable to peak CsA levels in cardiac transplant recipients (2 µg/mL) before LPS stimulation did not affect TF inducibility of whole mononuclear cells (protocol as described in "Methods").


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  		Table 2. TF Activity of Mononuclear Cells Isolated From Transplant Recipients in Presence of Baseline and Peak CsA Drug Levels



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  		Figure 2. Inhibition of monocyte TF activity in heart transplant recipients after CsA administration. Plots show TF activity generated by stimulated mononuclear cells (A) and purified monocytes/macrophages (B) obtained from same transplant recipients before and after CsA administration. Lines indicate median TF activity.


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  		Table 3. Effect of Ex Vivo Treatment of Lymphocytes With High Concentrations of CsA on TF Inducibility in Mononuclear Cells Isolated From Transplant Recipients During Low Baseline CsA Concentrations

TF mRNA Expression of Mononuclear Cells
To determine whether reduced TF generation in the presence of high CsA drug levels is related to reduced TF mRNA expression in mononuclear cells, TF mRNA expression was analyzed in mononuclear cells separated from transplant recipients before and after CsA administration. Cells obtained from transplant recipients in the presence of low baseline CsA blood levels (sample before CsA administration) exhibited moderate TF mRNA expression in the absence of LPS and a strong TF mRNA expression when challenged with LPS (Fig 3Down). In contrast, no or only weak upregulation of the TF gene transcription was observed in unstimulated and stimulated cells collected after CsA administration.



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  		Figure 3. Inhibition of TF mRNA expression by mononuclear cells from heart transplant recipients after CsA administration. TF mRNA expression was assessed by RT-PCR in stimulated and unstimulated mononuclear cells obtained from transplant recipients before and after CsA administration. Representative photograph of PCR-amplified products of TF and GAPDH mRNA. Lane 1, 123-bp DNA ladder; lane 2, basal TF mRNA expression (ex vivo analysis) in cells obtained before CsA administration; lane 3, TF mRNA expression (after 6 hours' incubation) in unstimulated cells obtained before CsA administration; lane 4, TF mRNA expression in stimulated cells obtained before CsA administration; lane 5, basal TF mRNA expression (ex vivo analysis) in cells obtained after CsA administration; lane 6, TF mRNA expression (after 6 hours' incubation) in unstimulated cells obtained after CsA administration; lane 7, TF mRNA expression in stimulated cells obtained after CsA administration.

Binding Activity of NF-{kappa}B
The observed decrease in TF mRNA expression in mononuclear cells obtained during high CsA blood levels led us to investigate the activation of the transcription factor NF-{kappa}B, known to be required for TF gene transcription in monocytes. EMSAs using NF-{kappa}B consensus oligonucleotides were performed with nuclear extracts of mononuclear cells separated before and after CsA administration. In cells obtained from transplant recipients during low baseline CsA blood levels (before CsA administration), strong NF-{kappa}B binding activity was detected (Fig 4Down), whereas cells separated from blood in the presence of high CsA concentrations exhibited decisively reduced NF-{kappa}B binding activity. Specificity of the binding reaction was shown by the competition with unlabeled consensus oligonucleotides.



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  		Figure 4. NF-{kappa}B activation in peripheral blood mononuclear cells obtained from heart transplant recipients before and after CsA administration. Nuclear extracts of mononuclear cells were subjected to EMSA with radiolabeled oligonucleotides containing the NF-{kappa}B consensus sequence. Nuclear extracts were prepared from blood mononuclear cells freshly isolated from transplant recipients before (lanes 1 through 3) and after (lanes 4 through 6) CsA administration. Protein-DNA complexes were electrophoresed through 5% polyacrylamide gels. Specificity of binding was ascertained by competition with 160-fold molar excess of cold consensus oligonucleotides included in the binding reaction (lane 7). Analysis was performed in triplicate.


*    Discussion
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 *Discussion
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The primary finding of the present study is that the level of monocyte TF activation, which is shown to be increased in heart transplant recipients, varies with the blood level of CsA in vivo.

We demonstrated that mononuclear cells from transplant recipients generate increased TF activity compared with healthy control subjects. The finding that the increased TF production in cells obtained from transplant recipients was observed with or without ex vivo stimulation suggests that cells from cardiac transplant recipients have been preactivated in vivo before separation. This concept is supported by the observation that mononuclear cells from cardiac transplant recipients showed marked activation of the transcription factor NF-{kappa}B, known to be required for TF gene transcription. Our data indicating an increased monocyte TF activity after cardiac transplantation are in line with previous studies demonstrating elevated levels of procoagulant activity in peripheral blood monocytes of kidney transplant recipients.11 TF activation in transplant recipients may be triggered by various agents, such as inflammatory cytokines, mitogens, and/or occupancy of cell adhesion molecules, that have been implicated in the allogeneic immune response after transplantation and that are known to induce TF expression in monocytes.30

TF generation by activated blood monocytes may represent the link between immune response and coagulation system in the development of cardiac transplant vasculopathy. The initial steps in the formation of allograft vasculopathy involve the adherence of circulating monocytes to the vascular endothelium,31,32 followed by local fibrin deposition along the intima of the affected vessels.14 The finding of the present study that monocyte TF activation occurring in heart transplant recipients leads to enhanced TF expression by surface-adherent monocytes after cardiac transplantation supports the pathophysiological concept that aberrant TF expression by monocytes adhering to the activated coronary endothelium may be responsible for the in vivo activation of the coagulation protease cascade and intravascular fibrin formation observed in cardiac allografts.13,33 This hypothesis seems more conceivable the more that {alpha}4ß1 monocyte integrin binding to endothelial cells through vascular cell adhesion molecule-1, an adhesion molecule known to participate in vascular leukocyte adhesion and emigration in transplant vasculopathy,34,35 directly induces TF mRNA accumulation and TF expression in monocytes.36,37

Effective prevention and treatment of transplant vasculopathy are still unsolved clinical problems. Although when CsA was introduced in 1980, it was originally hoped that it would prevent cardiac transplant vasculopathy by providing more effective immune suppression, clinical data concerning the effect of CsA on the incidence of graft vascular disease remain conflicting.19,38,39 More recent studies, however, emphasize the findings that the dosage of CsA may be critical to the development of transplant vasculopathy: whereas low-dose CsA treatment was associated with a reduction of coronary diameter and coronary flow reserve compared with that of conventional CsA doses,20,40 CsA seems to decrease the incidence of cardiac allograft vasculopathy when applied in high-dose regimens.18,41,42 Because the prevalence of transplant vasculopathy has not changed despite the reduction of acute rejection episodes under conventional-dose CsA regimens, the suppression of transplant vasculopathy by high-dose therapy might be related to additional, nonimmunologic effects of CsA. Recently, we demonstrated that exposure of cultured monocytes/macrophages to CsA at concentrations comparable to high CsA blood levels directly inhibits monocyte TF induction in vitro.24 The present clinical study was conducted to evaluate whether the inhibitory effect of CsA on monocyte TF generation observed in vitro is of relevance to TF expression in monocytes of CsA-treated transplant recipients in vivo.

In this study, therefore, TF generation was assayed in peripheral blood mononuclear cells and highly purified monocytes/macrophages, which were separated from cardiac transplant recipients during low baseline CsA blood levels (before daily CsA administration) and in the presence of high peak CsA blood concentrations (4 hours after oral administration). Measurements of the corresponding CsA blood concentrations revealed an average 2.6-fold elevation of CsA level when determined 4 hours after administration of CsA. These data are consistent with previous pharmacokinetic studies demonstrating peak (transient high) to trough (low steady) CsA concentration differences of a factor of 5 or more 1 to 4 hours (mean, 3.8 hours) after oral administration.43 Both mononuclear cells and purified monocytes/macrophages collected from the blood of transplant recipients containing high CsA blood concentrations (after CsA administration) showed decreased TF generation compared with cells collected at low CsA blood levels before CsA administration. Inhibition of monocyte TF generation in the presence of high CsA levels was also observed when cells were exposed to IFN-{gamma} or IL-1ß, cytokines known to be actively involved in the allogeneic reactions after cardiac transplantation.44

Decreased TF formation by mononuclear cells in the presence of high CsA plasma concentrations was shown to be accompanied by a reduced TF gene transcription after CsA administration. The finding of reduced TF formation as well as reduced TF mRNA expression in monocytes after CsA administration suggests that the transcriptional activation of the monocyte TF gene is inhibited in the presence of high CsA blood concentrations in vivo. Indeed, the marked activation of the NF-{kappa}B transcription factor, which is known to play a major role in the regulation of the TF gene,45 was prevented in the presence of high CsA blood concentrations. CsA recently was shown to abolish the inducible phosphorylation and degradation of the cytoplasmic inhibitor protein I-{kappa}B,46 thereby suggesting the mechanism by which CsA interferes with the signaling process leading to NF-{kappa}B activation.

The cellular site of the inhibitory action of CsA, however, is not completely known. Because lymphocytes reportedly facilitate47 or may even be required48 for maximal TF induction in monocytes, the question arises as to whether the observed inhibition of monocyte TF activation in cardiac transplant recipients is based on a direct effect of CsA on monocytes or might be related to cytokine or other molecular imbalances induced by CsA interfering with stimulatory lymphocytes, as was suggested by previous in vitro studies.49,50 Indeed, because CsA inhibits T cell activation and secretion of lymphokines,51 it seems conceivable that inhibition of lymphocyte-monocyte collaboration by CsA may at least in part account for the observed suppression of monocyte TF induction. However, the results of the present clinical study strongly suggest that the inhibitory effect of CsA was directly on monocytes. Thus, inhibition of TF induction was not seen in monocytes separated from transplant recipients in the presence of CsA blood levels within the therapeutic range known to fully inhibit T cell activation.51 Moreover, the finding that reduced inducibility of monocyte TF expression is observed exclusively at high peak CsA levels in vivo is in line not only with our previous data showing similar CsA concentrations to directly inhibit monocyte TF activation in vitro24 but also with previous studies demonstrating that high CsA concentrations are required to exert direct inhibitory effects on NF-{kappa}B binding and function.52 Further support for the concept of direct effects is derived from our experiments showing TF inhibition to similar extents when highly purified monocytes/macrophages are studied instead of whole mononuclear cells, whereas no inhibition was observed in PBMC preparations in which lymphocytes had been separately pretreated ex vivo with CsA concentrations that were comparable to high in vivo CsA drug levels. Taken together, these findings propose a direct interaction of CsA with monocytes, leading to reduced NF-{kappa}B activation and subsequent inhibition of TF expression. With respect to the in vivo situation, however, at present the possibility that CsA also acts through inhibition of lymphocytes cannot be entirely excluded.

Our study revealed a high interindividual variation in monocyte TF inducibility, but it shows that the levels of monocyte TF expression remain essentially constant in individual patients. Further prospective clinical studies after TF generation and clinical outcome of cardiac transplant recipients may determine whether monocyte TF expression serves as a marker of risk for the appearance of graft vasculopathy and/or transplant organ failure.

In conclusion, we have shown that monocyte TF activation occurs after cardiac transplantation. The enhanced TF expression by adherent monocytes observed in cardiac transplant recipients might lead to thrombin formation and fibrin deposition in the affected vessels of transplant vasculopathy. It seems conceivable that the reported inhibition of monocyte TF activation by CsA contributes to its successful use in transplant medicine. Further studies are required (1) to evaluate whether our observations furnish further rationale for high-dose CsA regimens after heart transplantation and (2) to elucidate whether CsA might be useful in managing other pathophysiological settings of cardiovascular disease also known to involve TF expression and NF-{kappa}B activation.


*    Selected Abbreviations and Acronyms
 
CsA = cyclosporin A
EMSA = electrophoretic mobility shift assay
FACS = fluorescence-activated cell sorting
IFN-{gamma} = interferon-{gamma}
IL-1ß = interleukin-1ß
LPS = lipopolysaccharide
MSFM = monocyte serum-free medium
NF = nuclear factor
PBMC = peripheral blood mononuclear cell
PCA = procoagulant activity
rIFN-{gamma} = recombinant human interferon-{gamma}
rIL-1ß = recombinant human interleukin-1ß
RT-PCR = reverse transcription–polymerase chain reaction
TF = tissue factor

Received July 3, 1997; revision received September 4, 1997; accepted September 11, 1997.


*    References
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 up arrowIntroduction
 up arrowMethods
 up arrowResults
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 *References
 

  1. Nemerson Y. Tissue factor and hemostasis. Blood. 1988;71:71–78.[Abstract/Free Full Text]
  2. Carson SD, Brozna JP. The role of tissue factor in the production of thrombin. Blood Coagul Fibrinolysis. 1993;4:281–292.[Medline] [Order article via Infotrieve]
  3. Carlsen E, Flatmark A, Prydz H. Cytokine-induced procoagulant activity in monocytes and endothelial cells: further enhancement by cyclosporine. Transplantation. 1988;46:575–580.[Medline] [Order article via Infotrieve]
  4. Drake TA, Ruf W, Morrissey JH, Edgington TS. Functional tissue factor activity is entirely cell surface expressed on lipopolysaccharide-stimulated human blood monocytes and a constitutively tissue factor-producing cell line. J Cell Biol. 1989;109:389–395.[Abstract/Free Full Text]
  5. Gregory SA, Kornbluth RS, Helin H, Remold HG, Edgington TS. Monocyte pro-coagulant inducing factor: a lymphokine involved in the T-cell instructed monocyte pro-coagulant response to antigen. J Immunol. 1986;141:3231–3239.
  6. Osterud B, Flaestad T. Increased tissue thromboplastin activity in monocytes of patients with meningococcal infection: related to an unfavorable prognosis. Thromb Haemost. 1983;49:5–7.[Medline] [Order article via Infotrieve]
  7. Taylor FBJ, Chang A, Ruf W, Morrissey JH, Hinshaw L, Catlett R, Blick K, Edgington TS. Lethal E. coli septic shock is prevented by blocking tissue factor with monoclonal antibody. Circ Shock. 1991;33:127–134.[Medline] [Order article via Infotrieve]
  8. Rao LVM. Tissue factor as a tumor procoagulant. Cancer Metastasis Rev. 1992;11:249–266.[Medline] [Order article via Infotrieve]
  9. Jude B, Agraou B, McFadden EP, Susen S, Bauters C, Lepelley P, Vanhaesbroucke C, Devos P, Cosson A, Asseman P. Evidence for time-dependent activation of monocytes in the systemic circulation in unstable angina but not in acute myocardial infarction or in stable angina. Circulation. 1994;90:1662–1668.[Abstract/Free Full Text]
  10. Marmur JD, Thiruvikraman SV, Fyfe BS, Guha A, Sharma SK, Ambrose JA, Fallon JT, Nemerson Y, Taubman MB. Identification of active tissue factor in human coronary atheroma. Circulation. 1996;94:1226–1232.[Abstract/Free Full Text]
  11. Halloran P, Aprile M, Haddad G, Robinette M. The significance of elevated procoagulant activity in the monocytes of renal transplant recipients. Transplant Proc. 1982;14:669–672.[Medline] [Order article via Infotrieve]
  12. Hattler BG, Rocklin RE, Ward PA, Rickles FR. Functional features of lymphocytes recovered from a human renal allograft. Cell Immunol. 1973;9:289–296.[Medline] [Order article via Infotrieve]
  13. Labarrere CA, Pitts D, Halbrook H, Faulk WP. Tissue plasminogen activator, plasminogen activator inhibitor-1, and fibrin as indexes of clinical course in cardiac allograft recipients: an immunocytochemical study. Circulation. 1994;89:1599–1608.[Abstract/Free Full Text]
  14. Faulk WP, Labarrere CA. Vascular immunopathology and atheroma development in human allografted organs. Arch Pathol Lab Med. 1992;116:1337–1344.[Medline] [Order article via Infotrieve]
  15. Schroeder JS, Gao SZ, Hunt SA, Stinson EB. Accelerated graft coronary artery disease: diagnosis and prevention. J Heart Lung Transplant. 1992;11:S258–S265.[Medline] [Order article via Infotrieve]
  16. Kahan BD. Cyclosporine. N Engl J Med. 1989;321:1725–1738.[Medline] [Order article via Infotrieve]
  17. Handa N, Hatanaka M, Baumgartner WA, Reitz B, Sandford G, Esa AH, Herskowitz A. Late cyclosporine treatment ameliorates established coronary graft disease in rat allografts. Transplantation. 1993;56:535–540.[Medline] [Order article via Infotrieve]
  18. Anderson HO, Madsen G, Nordestgaard BG, Kjeldsen K, Stender S. Cyclosporine suppresses experimental transplant atherosclerosis. Transplant Proc. 1994;26:1738.[Medline] [Order article via Infotrieve]
  19. Hess ML, Hastillo A, Thompson JA, Sansonetti DJ, Szentpetery S, Barnhart G, Lower RR. Lipid mediators in organ transplantation: does cyclosporine accelerate coronary atherosclerosis? Transplant Proc. 1987;19:71–73.
  20. Vassalli G, Kaski JC, Tousoulis D, Kiowski W, Turina M, Follath F, Gallino A. Low-dose cyclosporine treatment fails to prevent coronary luminal narrowing after heart transplantation. J Heart Lung Transplant. 1996;15:612–619.[Medline] [Order article via Infotrieve]
  21. Wong RL, Winslow CM, Cooper KD. The mechanism of action of cyclosporin A in the treatment of psoriasis. Immunol Today. 1993;14:69–74.[Medline] [Order article via Infotrieve]
  22. Ferns G, Reidy M, Ross R. Vascular effects of cyclosporine A in vivo and in vitro. Am J Pathol. 1990;137:403–413.[Abstract]
  23. Jonasson L, Holm J, Hansson GK. Cyclosporin A inhibits smooth muscle proliferation in the vascular response to injury. Proc Natl Acad Sci U S A. 1988;85:2303–2306.[Abstract/Free Full Text]
  24. Hölschermann H, Dürfeld F, Maus U, Bierhaus A, Heidinger K, Lohmeyer J, Nawroth PP, Tillmanns H, Haberbosch W. Cyclosporine A inhibits tissue factor expression in monocytes/macrophages. Blood. 1996;88:3837–3845.[Abstract/Free Full Text]
  25. Boyum A. Isolation of mononuclear cells and granulocytes from human blood: isolation of mononuclear cells by one centrifugation and of granulocytes by combining centrifugation and sedimentation at 1g. Scand J Clin Lab Invest. 1963;21(suppl 97):77–89.
  26. Chomczynski P, Sacchi N. Single step method of RNA-isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156–159.[Medline] [Order article via Infotrieve]
  27. Maus U, Rosseau S, Seeger W, Lohmeyer J. Separation of human alveolar macrophages by flow cytometry. Am J Physiol. 1997;272:L566–L571.[Abstract/Free Full Text]
  28. Scarpati EM, Wen D, Broze G, Miletich JP, Flandermayer RR, Siegel NR, Sadler E. Human tissue factor: cDNA-sequence and chromosome localization of the gene. Biochemistry. 1987;26:5234–5238.[Medline] [Order article via Infotrieve]
  29. Bierhaus A, Zhang Y, Deng Y, Mackman N, Quehenberger P, Haase M, Luther T, Müller M, Böhrer H, Greten J, Eike M, Bauerle P, Waldherr R, Kisiel W, Ziegler R, Stern DM, Nawroth PP. Mechanism of the TNF{alpha} mediated induction of endothelial tissue factor. J Biol Chem. 1995;270:26419–26432.[Abstract/Free Full Text]
  30. Camerer E, Kolsto A, Prydz H. Cell biology of tissue factor, the principal initiator of blood coagulation. Thromb Res. 1996;81:1–41.[Medline] [Order article via Infotrieve]
  31. Adams DH, Wyner LR, Steinbeck MJ, Karnovsky MJ. Inhibition of graft arteriosclerosis by modulation of the inflammatory response. Transplant Proc. 1993;25:2092–2094.[Medline] [Order article via Infotrieve]
  32. Kuwahara M, Jacobsson J, Kuwahara M, Kagan E, Ramwell PW, Foegh ML. Coronary artery ultrastructural changes in cardiac transplant atherosclerosis in the rabbit. Transplantation. 1991;52:759–765.[Medline] [Order article via Infotrieve]
  33. Lo SK, Cheung A, Zheng Q, Silverstein RL. Induction of tissue factor on monocytes by adhesion to endothelial cells. J Immunol. 1995;154:4768–4777.[Abstract]
  34. Libby P, Salomon RN, Payne DD, Schoen FJ, Pober JS. Functions of vascular wall cells related to the development of transplantation-associated arteriosclerosis. Transplant Proc. 1989;21:3677–3684.[Medline] [Order article via Infotrieve]
  35. Hosenpud JD, Shipley GD, Wagner CR. Cardiac allograft vasculopathy: current concepts, recent developments, and future directions. J Heart Lung Transplant. 1992;11:9–23.[Medline] [Order article via Infotrieve]
  36. Fan S-T, Edgington T-S. Coupling of the adhesive receptor CD11b/CD18 to functional enhancement of effector macrophage tissue factor response. J Clin Invest. 1991;87:50–57.
  37. Fan S-T, Mackman N, Cui M-Z, Edgington TS. Integrin regulation of an inflammatory effector gene: direct induction of the tissue factor promotor by engagement of ß1 or {alpha}4 integrin chains. J Immunol. 1995;154:3266–3274.[Abstract]
  38. Gao SZ, Schroeder JS, Alderman EL, Hunt SA, Valantine HA, Wiederhold V, Stinson EB. Prevalence of accelerated coronary artery disease in heart transplant survivors: comparison of cyclosporine and azathioprine regimens. Circulation. 1989;80(suppl III):III-100-III-105.
  39. Narrod J, Kormos R, Armitage J, Hardesty R, Ladowski J, Griffith B. Acute rejection and coronary artery disease in long-term survivors of heart transplantation. J Heart Transplant. 1989;8:418–420.[Medline] [Order article via Infotrieve]
  40. Treasure CB, Vita JA, Ganz P, Ryan TJJ, Schoen FJ, Vekshtein VI, Yeung AC, Mudge GH, Alexander RW, Selwyn AP. Loss of coronary microvascular response to acetylcholine in cardiac transplant recipients. Circulation. 1992;86:1156–1164.[Abstract/Free Full Text]
  41. Lim TT, Botas J, Ross H, Liang DH, Theodore J, Hunt S, Oesterle SN, Yeung AC. Are heart-lung transplant recipients protected from developing transplant coronary artery disease. Circulation. 1996;94:1573–1577.[Abstract/Free Full Text]
  42. Koskinen PK, Lemström KB, Hägry PK. How cyclosporine modifies histological and molecular events in the vascular wall during chronic rejection of rat cardiac allografts. Am J Pathol. 1995;146:972–980.[Abstract]
  43. Mueller EA, Kovarik JM, van Bree JB, Lison AE, Kutz K. Pharmacokinetics and tolerability of a microemulsion formulation of cyclosporine in renal allograft recipients: a concentration-controlled comparison with the commercial formulation. Transplantation. 1994;57:1178–1182.[Medline] [Order article via Infotrieve]
  44. Schwager I, Jungi TW. Effect of human recombinant cytokines on the induction of macrophage procoagulant activity. Blood. 1994;83:152–160.[Abstract/Free Full Text]
  45. Oeth P, Parry GCN, Mackman N. Regulation of the tissue factor gene in human monocytic cells. Arterioscler Thromb Vasc Biol. 1997;17:365–374.[Abstract/Free Full Text]
  46. Alkalay I, Yaron A, Hatzubai A, Jung S, Avraham A, Gerlitz O, Pashut-Lavon I, Ben-Neriah Y. In vivo stimulation of I{kappa}B phosphorylation is not sufficient to activate NF-{kappa}B. Mol Cell Biol. 1995;15:1294–1301.[Abstract]
  47. Edwards RL, Rickles FR. The role of human T cells and T cell products for monocyte tissue factor generation. J Immunol. 1980;125:606–609.[Abstract]
  48. Levy GA, Schwartz BS, Edgington TS. The kinetics and metabolic requirements for direct lymphocyte induction of human procoagulant monokines by bacterial lipopolysaccharides. J Immunol. 1981;127:357–363.[Medline] [Order article via Infotrieve]
  49. Helin HJ, Edgington TS. Cyclosporin A regulates monocyte/macrophage effector functions by affecting instructor T cells: inhibition of monocyte procoagulant response to allogeneic stimulation. J Immunol. 1984;132:1074–1076.[Medline] [Order article via Infotrieve]
  50. Ferreira OC Jr, Barcinski MA. Autologous induction of the human T-cell-dependent monocyte procoagulant activity: a possible link between immunoregulatory phenomena and blood coagulation. Cell Immunol. 1986;101:259–265.[Medline] [Order article via Infotrieve]
  51. Di Padova FE. Pharmacology of cyclosporine (Sandimmune). Pharmacol Rev. 1989;41:373–405.[Free Full Text]
  52. Emmel EA, Verweij CL, Durand DB, Higgins KM, Lacy E, Crabtree GR. Cyclosporin A specifically inhibits function of nuclear proteins involved in T cell activation. Science. 1989;246:1617–1619.[Abstract/Free Full Text]



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