In vitro effects of cyclosporin A on the expression of adhesion

molecules on human umbilical vein endothelial cells

Snezana Markovic a,b, Markus Raab a, Heide Daxecker a, Andrea Griesmacher a,Alireza Karimi a, Mathias M. Muller a,*

aInstitute of Laboratory Diagnostics and Ludwig Boltzmann Institute for Cardiothoracic Research, Kaiser Franz Josef Hospital,

Kundratstraße 3, A-1100 Vienna, AustriabInstitute of Medical Biochemistry, Clinical Center of Serbia, Visegradska 26, 11000 Belgrade, Yugoslavia

Received 1 August 2001; accepted 14 August 2001

Abstract

Background: Among the different factors playing crucial roles in endothelial cell activation, cytokines such as interleukin-1

(IL-1), interleukin-6 (IL-6), tumor necrosis factor-a (TNF-a) and interferon-g (IFN-g) have been reported to demonstrate

profound effects on this cell type. It has been shown that the increased release of IFN-a/g and TNF-a causes structural and

functional modulations of the endothelial cell. These cytokines participate in the recruitment and activation of the immune

system. CsA is an immunosuppressive drug that is necessary at high levels in human recipients of vascularised xenografts. This

drug could contribute to a prolonged graft survival by modulation of endothelial cell activation. Methods: The present study

deals with the effects of cyclosporin A on adhesion molecule expression (i.e. ICAM-1, VCAM-1, E-selectin, P-selectin,

PECAM-1 and the L-selectin ligand CD 34) on the surface of cytokine stimulated HUVECs. The in vitro model described

herein mimics the stimulation of endothelial cells by cytokines as seen during inflammatory processes after transplantation.

Therefore, HUVECs were activated either with TNF-a, IL-1b or with a cytokine mixture consisting of those stimulants present

at an elevated level in sera of patients during allograft rejection (i.e. IL-1b, IL-2, IL-4, IL-6, IL-10, TNF-a and IFN-g). Results:The results obtained show that the immunosuppression of CsA is not only achieved by inhibiting lymphocyte proliferation, but

also by decreasing the expression of adhesion molecules on endothelial cells, which are the first target of the cellular rejection

process. Conclusion: Co-incubation of stimulated endothelial cells with a final CsA concentration of 5 mg/ml revealed a

0009-8981/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.

PII: S0009-8981 (01 )00732 -X

Abbreviations: CsA, cyclosporin A; CD, cluster of differentiation; ECGS, endothelial cell growth supplement; E-selectin, endothelial

leucocyte adhesion molecule (ELAM-1, CD 62E); FACS, fluorescence activated cell scanning; FCS, fetal calf serum; FITC, fluorescein

isothiocyanate; HFN, human fibronectin; HUVEC, human umbilical vein endothelial cells; ICAM-1, intercellular adhesion molecule (CD 54);

IFN-g, interferon-g; IL-1b, interleukin-1b; MFI, mean fluorescence intensity; PBS, phosphate buffered saline; PE, R-phycoerythrin; PECAM-1,

platelet endothelial cell adhesion molecule (CD 31); P-selectin, CD 62P; TNF-a, tumor necrosis factor-a; VCAM-1, vascular cell adhesion

molecule (CD 106).* Corresponding author. Tel.: +43-1-60191-3301; fax: +43-1-60191-3309.

E-mail address: mathias.mueller@kfj.magwien.gv.at (M.M. Muller).

www.elsevier.com/locate/clinchim

Clinica Chimica Acta 316 (2002) 25–31

significant down-regulating influence on the surface expression of E-selectin and VCAM-1. D 2002 Elsevier Science B.V. All

rights reserved.

Keywords: Cyclosporin A; Endothelial cells; Adhesion molecules; Fluorescence activated cell scanning

1. Introduction

Previous studies have shown that CsA induces a

concentration-dependent endothelial injury, which is

characterised by vascular thrombosis, cytostatic ef-

fects and alteration of prostaglandin and cytokine syn-

theses. IL-6 mRNA is transiently expressed during

folliculogenesis and the formation of the maternal

decidua in early post-implantation development, sug-

gesting that in a situation where vascular turnover is

high, IL-6 could play an important role in endothelial

cell proliferation [1,2].

Vascular endothelial cells hold a crucial position

in the maintenance of tissue homeostasis, forming the

interface between blood and the surrounding tissue.

Among other pivotal factors playing parts in endo-

thelial cell activation, cytokines such as IL-1b, IL-6,TNF-a and IFN-g have been shown to profoundly

affect this cell type [3]. The vascular endothelium

produces IL-1, IL-6 and IL-8 as a response to sti-

mulation with IL-1, IL-2, IL-6 and IFN-g. It has beenshown that the increased release of IFN-a/g and

TNF-a causes structural and functional modulations

of the endothelial cell. These molecules participate in

the recruitment and activation of the immune system

[4].

CsA is an immunosuppressive drug that selectively

inhibits proliferation and activation of CD 4 + T cells

primarily by inhibition of IL-2 gene transcription

through interference with the T cell receptor signal

transducing pathway. CsA is used by T cells and

bound to the cyctosolic protein cyclophilin, an immu-

nophilin with isomerase/rotamase activity. Cyclophi-

lin activity is inhibited by bound CsA. The CsA–

cyclophilin complex binds to calmodulin and to the

two calcineurin subunits, thus inhibiting the Ca2 + -

activated phosphatase activity of calcineurin. Defi-

cient dephosphorylation of the cytosolic nuclear factor

of T cell activation prevents its translocation into the

nucleus, and therefore a lack of the IL-2 promoter

occurs. As a result, transcription of the IL-2 gene is

suppressed [5]. Because of this CsA is necessary at

high levels in recipients of vascularised xenografts

[6]. Additionally, studies have been performed to

investigate the effects of CsA on vascular endothelial

functions, and both protective [7,8] and adverse [9,10]

effects have been reported.

In this study we stimulated endothelial cells either

with TNF-a or IL-1b or a mixture of those cytokines

(IL-1b, IL-2, IL-4, IL-6, IL-10, TNF-a and IFN-g),which were found at an elevated level in sera of

patients with acute phase graft rejection. Furthermore,

cytokine-treated endothelial cells were co-incubated

with CsA in a final concentration (5 mg/ml), which can

be found at peak levels in serum of patients receiving

immunosuppressive therapy after organ transplanta-

tions. The influence of this drug on the surface

expression of a number of adhesion molecules (i.e.

ICAM-1, VCAM-1, E-selectin, P-selectin, PECAM-1

and the L-selectin ligand CD 34) was determined by

means of FACS.

2. Materials and methods

2.1. Materials

Medium 199 (Hepes-modification), heparin and

CsA were ordered from Sigma-Aldrich, Steinheim,

Germany. Penicillin/streptomycin, L-glutamine, tryp-

sin/EDTA (0.05%/0.02% in Hanks balanced salt sol-

ution) and PBS (without Ca2 + and Mg2 + ) were

purchased from Gibco-Life Technologies, Merelbeke,

Belgium. Cytokines used for stimulation were ordered

from R&D Systems, Abingdon, UK. Fluorescence

labelled (FITC, PE) human monoclonal antibodies di-

rected against PECAM-1, ICAM-1, VCAM-1, E-

selectin, P-selectin and the L-selectin ligand CD 34

were obtained from Serotec, Eching, Germany. Fur-

thermore, mouse IgG1 (Becton Dickinson, San Jose,

CA, USA), ECGS (Upstate Biotechnologies, Waltham,

MA, USA), HFN (Chemicon, Hofheim, Germany),

collagenase type 2 (Worthington Biochemical, Free-

S. Markovic et al. / Clinica Chimica Acta 316 (2002) 25–3126

hold, NJ, USA) and FCS (PromoCell, Heidelberg,

Germany) were used.

2.2. Cell culture

Endothelial cells were isolated from freshly deliv-

ered humans umbilical venous tissue using pre-

warmed collagenase type II as described previously

[11], and were maintained in Medium 199 containing

20% FCS, 10,000 U/ml penicillin, 10,000 mg/ml

streptomycin, 100 mg/l low molecular weight heparin,

3 mmol/l L-glutamine and 30 mg/l ECGS on fibro-

nectin-coated culture flasks [12]. Confluent mono-

layers of single isolates at first passage were used

for all experiments. The cells were cultured in a

humidified incubator set at 37.4 �C and 5% CO2.

2.3. Stimulation of endothelial cells

In this paper the effects of cytokine stimulation

(final concentrations are given in brackets) and cyto-

kine/CsA co-stimulation on the expression of cell

surface molecules were investigated. MFI of unsti-

mulated cells functions as the control group. Endo-

thelial cells were stimulated with TNF-a (10 ng/ml),

IL-1b (10 ng/ml) or with a mixture of different

cytokines, which were found to be elevated in sera

of patients with acute phase graft rejection, i.e. IL-1b(10 ng/ml), IL-2 (20 ng/ml), IL-4 (20 ng/ml), IL-6

(20 ng/ml), IL-10 (20 ng/ml), IFN-g (100 ng/ml),

TNF-a (10 ng/ml) [13–16]. Additionally, endothelial

cells were co-incubated with CsA (10 ml of a 0.5 mg/

ml ethanolic stock solution per milliliter medium,

giving a final concentration of 5 mg/ml). Surface

expression of adhesion molecules (ICAM-1,

VCAM-1, E-selectin, P-selectin, PECAM-1 and the

L-selectin ligand CD 34) was measured after an

incubation period of 16 h.

2.4. FACS analysis

Endothelial cells were detached enzymatically

applying the trypsinisation protocol optimised by

Mutin et al. [17]. Adhesion molecule expression was

investigated via flow-cytometric experiments employ-

ing a FACScan analyzer (Becton Dickinson). Cellular

samples were incubated with an excess of the specific

antibody for 30 min at 4 �C. The following cell sur-

face antigens were determined via FACS analysis: E-

selectin (FITC), CD 34 (PE), P-selectin (FITC),

ICAM-1 (PE), VCAM-1 (FITC) and PECAM-1

(PE). MFI obtained for stimulated cells were com-

pared with the MFI of unstimulated cells serving as

control values. All incubation experiments were per-

formed five times.

2.5. Statistical methods

Data obtained are given as percentage changes of

MFI in cell surface expression versus control and are

expressed as meanF confidence interval (P= 0.95).

3. Results

3.1. E-selectin surface expression

Of all adhesion molecules investigated E-selectin

expression was influenced most significantly. After

stimulation with TNF-a E-selectin expression was

up-regulated to 1127F 27% (for results see Table 1).

Under concomitant stimulation with CsA the E-

selectin level was significantly down-regulated, re-

sulting in 701F154% compared to control values.

IL-1b stimulation slightly increased E-selectin sur-

face expression to 123F 16%. This effect is reversed

under co-incubation with IL-1b/CsA reaching a level

of 77F 12%. The cytokine mixture used for mim-

icking the conditions of acute phase graft rejection

resulted in an increase in E-selectin surface expres-

sion up to 805F 112%. By adding CsA to the

incubation medium, this effect can be diminished to

679F 145% compared to control.

3.2. VCAM-1 surface expression

VCAM-1 surface expression is also statistically

significantly influenced by the different cytokines

used. TNF-a stimulation led to an increase in MFI

to 501F109%. Concomitant incubation of endothe-

lial cells with TNF-a plus CsA significantly reduced

VCAM-1 expression (298F 82%). IL-1b gave a

slight but significant rise in VCAM-1 expression

(126F 11%), which was lowered below control val-

ues when CsAwas included in the incubation medium

(82F 15%). Incubation of cells with the mixture

S. Markovic et al. / Clinica Chimica Acta 316 (2002) 25–31 27

Table 1

Effects of CsA on the cell surface expression of adhesion molecules on cultured HUVECs under the influence of cytokine stimulation

Adhesion molecule Conjugate Cytokine Incubation conditions MeanF confidence

intervala

E-selectin FITC TNF-a TNF-a 1127F 27 *

TNF-a/CsA 701F154 * , * *

CsA 103F 3

IL-1b IL-1b 123F 16 *

IL-1b/CsA 77F 12 * , * *

CsA 101F1

mixture mixture 805F 112 *

mixture/CsA 679F 145 *

CsA 103F 8

VCAM-1 FITC TNF-a TNF-a 501F109 *

TNF-a/CsA 298F 82 * , * *

CsA 98F 27

IL-1b IL-1b 126F 11 *

IL-1b/CsA 82F 15 * , * *

CsA 99F 11

mixture mixture 648F 57 *

mixture/CsA 358F 106 * , * *

CsA 116F 21

ICAM-1 PE TNF-a TNF-a 162F 19 *

TNF-a/CsA 164F 31 *

CsA 115F 12

IL-1b IL-1b 126F 15 *

IL-1b/CsA 132F 18 *

CsA 113F 8

mixture mixture 231F 53 *

mixture/CsA 226F 51 *

CsA 124F 13

P-selectin FITC TNF-a TNF-a 143F 25 *

TNF-a/CsA 139F 22 *

CsA 111F 9

IL-1b IL-1b 106F 6

IL-1b/CsA 109F 10

CsA 100F 8

mixture mixture 265F 64 *

mixture/CsA 256F 130 *

CsA 105F 17

PECAM-1 PE TNF-a TNF-a 106F 14

TNF-a/CsA 96F 18

CsA 95F 12

IL-1b IL-1b 103F 7

IL-1b/CsA 91F18

CsA 86F 3

mixture mixture 94F 25

mixture/CsA 54F 13 * , * *

CsA 101F12

CD 34 PE TNF-a TNF-a 106F 16

TNF-a/CsA 111F 27

CsA 85F 18

IL-1b IL-1b 98F 19

IL-1b/CsA 88F 28

CsA 76F 19

S. Markovic et al. / Clinica Chimica Acta 316 (2002) 25–3128

mentioned above resulted in an uprise in VCAM-1

surface expression to 648F 57%, an effect that can be

significantly reduced by adding CsA to the incubation

medium (358F 106%).

All other cell surface molecules tested were not

influenced in a comparable extent under any of the

incubation conditions described above.

4. Discussion

CsA is a cyclic undecapeptide with highly specific

immunosuppressive effects used in the treatment of

allograft rejection. Previous immunological studies

have revealed that CsA blocked calcium and antigen-

dependent proliferation of T lymphocytes by inhibit-

ing specific nuclear transcription factors that regulate

expression of cytokine genes such as IL-2 [18].

Formation of complexes between CsA and the cyto-

solic protein cyclophilin A, an immunophilin with

isomerase/rotamase activity, blocks the calcium-acti-

vated phosphatase activity of calcineurin, resulting in

the absence of nuclear factor of activated T cells

(NFAT) dephosphorylation, inhibition of its trans-

location into the nucleus and thus leading to the lack

of IL-2 gene expression. CsA is also reported to

inhibit transcription of IL-3, IL-4, granulocyte-mac-

rophage colony-stimulating factor (GM-CSF), TNF-aand IFN-g [19]. However, other data suggest that

CsA inhibits TNF-a production by blocking secre-

tion without depressing either cell-associated TNF

(translation) or TNF mRNA (transcription) [20].

The in vitro model described herein is designed to

mimic the stimulatory conditions observed during in-

flammatory and rejection processes after transplanta-

tion [5]. We studied the effects of CsA on adhesion

molecule expression on the surface of cytokine stimu-

lated HUVECs. Therefore, HUVECs were treated

either with TNF-a, IL-1b or a cytokine mixture

consisting of those stimulants elevated in a situation

of acute phase allograft rejection, i.e. IL-1b, IL-2, IL-4, IL-6, IL-10, TNF-a and IFN-g.

The pro-inflammatory cytokine TNF-a signifi-

cantly increased VCAM-1 and slightly up-regulated

ICAM-1 surface expression accompanied by the

induction of E- and P-selectin. While the cytokine

mixture applied in this work basically evoked the

same observations, stimulation with IL-1b influenced

adhesion molecule expression to a much lesser

extent. Concomitant incubation with TNF-a/CsAled to a slight but significant reduction in VCAM-1

and E-selectin expression compared to the values

obtained under stimulation with this cytokine alone.

Concomitant incubation with the cytokine mixture

revealed the same effects. Treatment of cells with IL-

1b/CsA also decreased VCAM-1 and E-selectin

expression under the level of IL-1b stimulated cells.

While incubating stimulated HUVECs with a me-

dium containing CsA significantly down-regulated

the levels of VCAM-1 and E-selectin expression,

ICAM-1 expression was found to be unaffected un-

der these conditions. Treatment of HUVECs with

either the immunosuppressant alone or with the sol-

vent ethanol alone did not significantly alter the

expression of all surface molecules tested compared

to the values of unstimulated HUVECs.

Endothelial cell activation plays an important role

in inflammation, haemostasis and organ rejection of

Adhesion molecule Conjugate Cytokine Incubation conditions MeanF confidence

intervala

CD 34 PE mixture mixture 86F 8 *

mixture/CsA 123F 43

CsA 77F 9

CsA and the respective stimulant were incubated simultaneously in a humidified incubator set at 37.4 �C and 5% CO2 for 16 h. The following

final concentrations were applied during incubation: TNF-a (10 ng/ml), IL-1b (10 ng/ml) and CsA (5 mg/ml). The cytokine mixture applied

consists of IL-1b, IL-2 (20 ng/ml), IL-4 (20 ng/ml), IL-6 (20 ng/ml), IL-10 (20 ng/ml), TNF-a and IFN-g (100 ng/ml). Data are given as

percentage changes in cell surface molecule expression (MFI) versus respective control value ( = 100%) and are expressed as meanF confidence

interval.a Confidence interval for n= 5; t( P,f ) = 2.78; P= 0.95.

* Significance in comparison to control ( P < 0.05).

** Significance in comparison to respective stimulant ( P< 0.05).

Table 1 (continued)

S. Markovic et al. / Clinica Chimica Acta 316 (2002) 25–31 29

allogeneic and xenogeneic transplantation. These pro-

cesses lead to rapid and transient up-regulation of pro-

inflammatory molecules, such as the adhesion mole-

cule E-selectin, the chemotactic cytokine IL-8 and

other molecules that are of eminent importance for

leucocyte transmigration [21–24]. In another study

serum levels of ICAM-1 were found to be raised in

patients receiving immunosuppressive therapy in

comparison to non-transplanted subjects, while E-

selectin serum levels were higher in non-transplanted

healthy subjects [25].

The intention of our study was to investigate the

specific effects of the immunosuppressive drug CsA

on cytokine-induced HUVEC stimulation. We ob-

served that at high concentrations of CsA, which

correspond to peak levels present in the serum of

organ transplanted patients, VCAM-1 and E-selectin

expression was significantly reduced in cultured

endothelial cells. Another study dealing with cultured

porcine endothelial cells treated with lipopolysac-

charide or TNF-a reported that the immunosuppres-

sive drugs CsA, rapamycin, and glucocorticoids

individually inhibited E-selectin protein induction in

a dose-dependent manner [26]. By means of mes-

senger RNA analysis, it has been demonstrated that

the presence of these drugs during endothelial cell

activation inhibited E-selectin and IL-8 at the gene

expression level. Our results concerning E-selectin

are also in good correlation with the results described

by Kagi et al. [27], who have determined the effect

of CsA on soluble E-selectin levels in mono-thera-

peutic treated patients.

Maksymowicz et al. [28] studied the expression of

some adhesion molecules on thoracic duct lympho-

cytes of rats treated with CsA and also found that

ICAM-1 levels remain unaffected, while L-selectin

expression was significantly decreased under these

test conditions.

The experiments described in our report showed

that the immunosuppression by CsA is also achieved

by decreasing the expression of adhesion molecules

on endothelial cells, which are the first target of the

cellular rejection process. In conclusion, the results of

our experiments clearly demonstrate that incubating

cytokine-stimulated endothelial cells with a final CsA

concentration of 5 mg/ml had a significant down-

regulating influence on the surface expression of E-

selectin and VCAM-1.

Acknowledgements

We appreciate the technical support by Mrs.

Christiane Klein and the kind help of the co-workers

at the Department of Gynecology of Kaiser Franz

Josef Hospital in collecting umbilical cords. Snezana

Markovic is also grateful for receiving a fellowship

of the IFCC professional scientific exchange pro-

gram.

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