ARTICLE Most
of the connective tissue diseases predominate in women [1]. One of the cardinal
features of connective tissue diseases is vasculitis or vasculopathy involving
small blood vessels. The exact mechanisms by which sex hormones modulate
disease activity are incompletely understood [2, 3]. Injury or dysfunction
of the dermal microvascular endothelial cells with activated expression
of certain cell adhesion molecules (CAMs) was suggested to be the primary
pathogenic event [4, 5]. Sex steroids were shown to affect the endothelial
expression of CAMs and possess immunoregulatory properties [6]. However,
most observations of the in vitro effect of sex steroids on cytokine-stimulated
human endothelial cells were made on cells derived from human umbilical
veins (HUVEC) [7-10]. It has become increasingly recognized that not all
types of endothelial cells are alike [11]. This study was aimed to observe
the effect of dexamethasone (DEX), testosterone (T), 5alpha-dihydrotestosterone
(DHT) and 17beta-estradiol (E2) on the in vitro expression CAMs such
as ICAM-1, VCAM-1 and E-selectin in cytokine-stimulated human microvascular
endothelial cells, with a parallel study on HUVEC.
Material and methods
A transformed human dermal microvascular endothelial cell line, HMEC-1
(generously offered by Dr. F.C. Candal, CDC, USA) and primary culture
of HUVEC were used. Both types of cells, HMEC-1 and HUVEC, were initially
grown in phenol red-free culture medium (EGM, Clonetics, MD, USA) containing
2% fetal bovine serum (FBS). Before administration of cytokines or/and
hormones to the cell cultures, the cells were starved first by switching
the medium from the 2% FBS containing medium (2%) to serum-free medium
for 24 hrs (starvation).
The dynamic of cytokines' effect on the expression of CAMs by HMEC-1
up to 24 hrs was examined first. IL-1beta and TNF-alpha (R&D, MN,
USA), at a final concentration of 50 U/ml each, were used to stimulate
the HMEC-1 cells cultured in 96-well plate (2 x 104 cells/well).
The expression of ICAM-1, VCAM-1 and E-selectin on the cellular surface
was measured by ELISA in triplicate (Dynatech, PA, USA) using specific
monoclonal antibodies (all from Serotec, NC, USA).
In the second step, the individual steroid hormones (T, DHT, DEX and
E2), each at 10- 6 M, were applied to the cell cultures
to see if they could influence the endothelial basal constitutive expression
of CAMs. Then, steroid hormones and IL-1beta/TNF-alpha at the aforementioned
concentration were simultaneously added to the culture medium to examine
the expression of CAMs. After incubation for 2, 4, 6, 12, 18, 24 hrs,
respectively, the supernatants were removed. No cytokines and hormones
were added in controls. The OD ratios, derived by using the control OD
value as baseline, were used to compare the modulating effect among 4
different hormones. A parallel study was done on HUVEC.
To test the presence of androgen receptor (AR) and estrogen receptor
(ER) on endothelial cells, the cell lines were cultured and starved as
before, and T and E2 (each at 10- 6 M) were then supplemented
for 24 hrs to boost the expression of AR and ER, respectively. With testis
and ovary tissues as positive controls, the expression of AR and ER was
studied by immunocytochemical staining using specific monoclonal antibodies
(Dako, Glostrup, Denmark), respectively.
As for statistical analysis, values of ELISA studies represented the
mean ± SE of separate determination in six different wells from three
different experiments. Student's t test was used for comparison
of the means. Differences of p < 0.05 were considered significant.
Results
The basal constitutive expression of ICAM-1, E-selectin and VCAM-1 was
very low in both HMEC-1 and HUVEC (Figs.
1 and 2). The 4 steroid hormones, when given in the absence
of pro-inflammatory cytokines (IL-1beta/TNF-alpha), showed no regulatory
effect on the expression of CAMs (data not shown).
In the cytokine-stimulated HUVEC, maximal expression of E-selectin in
vitro occurred 4 hrs after activation and declined thereafter (Fig.
2b); VCAM-1 appeared at slightly later times than E-selectin, reaching
maximal level at 6-12 hrs (Fig.
2c); the expression of ICAM-1 was strongly upregulated with the maximal
effect observed at 18-24 hrs (Figs.
1 and 2a). The time course of expression of these CAMs in our
experiment was comparable to that in previous reports [12, 13]. DEX exhibited
significantly strong inhibition on cytokine-stimulated expression of E-selectin
at 4 hrs (Fig. 2b), VCAM-1
at 6 hrs (Fig. 2c) and
ICAM-1 at 18 hrs (Fig. 2a).
E2 showed no modulating effect on cytokine-stimulated CAMs expression.
Immunostaining of the HUVEC revealed a very weak reaction of ER and no
AR expression on the cells.
In the cytokine-stimulated HMEC-1, the expression of VCAM-1 and E-selectin
was still undetectable (data not shown), while the expression of ICAM-1
was enhanced (Fig. 1).
When the endothelial cells were treated with steroid hormones, DEX exerted
the most significant inhibition on the cytokine-stimulated expression
of ICAM-1 (Fig. 1), with
maximal effect at 24 hrs of treatment. E2 had no regulatory functions.
Androgens exhibited a small, yet statistically significant inhibitory
effect; with DHT stronger than T. Expression of ER and AR in HMEC-1 was
negative by immunostaining.
Discussion
Variable kinetics of expression of E-selectin, VCAM-1 and ICAM-1 have
been shown in different experimental models of inflammation [14]. Our
results of the IL-1beta/TNF-alpha-induced expression of ICAM-1 in HMEC-1
were comparable to those in a previous study where IL-1alpha, IL-1beta,
and TNF-alpha markedly increased the expression of ICAM-1 in a time- and
dose-dependent manner in primary culture of human dermal microvascular
endothelial cells [15]. Assessment of the effect of steroid hormones on
cytokine-induced expression of CAMs is often confounded by multiple variables
including the timing of administration of cytokines and hormones (simultaneously
at the beginning or sequentially), incomplete delineation of critical
culture conditions and endothelial expression of hormone receptors [7-9,
16].
In our study, the absence of a regulatory effect by E2 in both cell
lines might be due to the scarcity or absence of ER expression caused
by the insufficient duration of E2 pretreatment for only 24 hrs. Reports
concerning expression of the ER in HUVEC are controversial [9, 17, 18].
In one study [9], HUVEC pretreated with high-dose E2 (1,000 ng/ml, about
0.5 x 10- 6 M) for 3 and 24 hrs was shown to be ER-negative,
and the induction of ER expression was first observed after E2 pretreatment
for 48 hrs. Moreover, in these ER-positive HUVECs, E2 was shown to strongly
inhibit IL-1 mediated membrane E-selectin, VCAM-1 and ICAM-1 induction
and the induction was abrogated by addition of one E2 antagonist [9].
The effect of androgens in inflammatory processes
has been less well investigated. In the present study, androgens displayed
statistically significant but lower levels of suppression on cytokine-induced
ICAM-1 only in HMEC-1, compared to the potent inhibitory effect of DEX
in both cell lines (Fig. 1).
DHT appeared to exhibit slightly stronger inhibition than T (Fig.
1). Although the expression of type 15alpha-reductase has been demonstrated
in HMEC-1 [19], the significance of the in situ conversion of T
to DHT by 5alpha-reductase in endothelial cells is unclear. On the other
hand, in contrast to the in vivo demonstration of AR expression
in endothelial cells of cutaneous small vessels [20], AR could not be
detected in either endothelial cell lines in the present study, even after
pretreatment with 10- 6 M T for 24 hrs. If the absence
of AR in our study were not due to methodological limitations, the observed
effects of androgens in cultured endothelial cells might bypass the classic
genomic AR-mediated pathway [21].
DEX has been shown to inhibit cytokine-induced ICAM-1 up-regulation
on several human endothelial cell lines [15, 22]. The action of DEX was
found to be mediated through ligation of corticosteroid receptors [16].
Our study further demonstrated a potent inhibitory effect of DEX on cytokine-induced
expression of VCAM-1 and E-selectin. Taken together, the clinically superior
anti-inflammatory effect of glucocorticoids might be attributable in part
to their suppression on endothelial expression of CAMs, which is crucial
for leukocyte trafficking and migration.
In sum, our study provided some pharmacological evidence for the superior
therapeutic efficacy of potent corticosteroids in systemic vasculitis
or inflammatory dermatoses. Although androgens failed to exhibit a convincing
suppressive effect on the cytokine-activated HMEC-1 in this study, it
remains to be determined if long-term priming of endothelial cells by
androgens could play a protective role in males from connective tissue
diseases
CONCLUSION
Acknowledgements
This study was supported by the grant from National Science Committee
Taiwan (Grant No. 89-2314-B-006-092). We would like to thank Dr. F.C.
Candal (CDC, USA) for providing us the HMEC-1 cell line. We are grateful
to Prof. L.-Y. Chen (Department of Physiology, College of Medicine, National
Cheng Kung University, Tainan, Taiwan) for her valuable advice on the
administration of sex steroids to our culture system. We would also like
to thank Prof. Ch. Zouboulis (Department of Dermatology, University Medical
Center Benjamin-Franklin, The Free University of Berlin, Berlin, Germany)
for his precious suggestion on the preparation of this manuscript.
Article accepted on 10/6/02
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