Clinical Science (1598) 94, 537-540 (Printed in Great Britain)
537
A circadian variation exists for soluble levels of
intercellular adhesion molecule=I and EIselectin in
healthy volunteers
C. MAPLE, G. KIRK, M. McLAREN, D. VEALE and 1.1. F. BELCH
University Deportment #Medicine, Ninewells Hospital and Medical School, Dundee, Scotland DDI 9SY. U.K.
1. We have previously shown a circadian variation in
leucocyte activation and endothelial function which may
explain why some inflammatory and vascular diseases
show a circadian variation in disease activity/
occurrence.
2. We have investigated the circadian variation of two
soluble cell adhesion molecules, intercellular adhesion
moleculel and Eselectin, in 10 healthy Volunteers.
Soluble intercellular adhesion moleculel is released
from both activated leucocytes and endothelial cells
while soluble E-selectin is released only from activated
endothelium.
3. Results show a circadian variation exists for both
soluble intercellular adhesion moleculel and Eselectin
(both P < O.OOO1, analysis of variance) with a peak
activity at 12:00 h for both measures and a minimum
activity at 04:00 h for intercellular adhesion moleculel
and 00 :00 h for Eselectin.
4. These results demonstrate the existence of a diurnal
variation in cell adhesion molecules, providing evidence
in support of a diurnal pattern in endothelial and
leucocyte activation. An alteration in this biological
rhythm may help to explain the diurnal variation in
disease activity in certain inflammatory and vascular
disease states. Furthermore, it stresses the importance
of sample time point standardization in clinical studies.
ation in disease activity. Patients with rheumatoid
arthritis have increased symptoms of joint stiffness
and discomfort in the morning, which eases off
throughout the day [8,9].
Cell adhesion molecules (CAMs), which are present
on the surface membrane of many cells including
leucocytes and endothelial cells, are important in the
trafficking of leucocytes from the circulation into
the tissues [101. Intercellular adhesion molecule-1
(ICAM-1) is a member of the immunoglobulin family
of CAMs and is found on endothelial cells and leucocytes. E-selectin is only expressed on cytokineactivated endothelial cells. Recently, soluble forms of
these molecules have been identified in the blood [l 11.
These molecules are released from the bound form
after stimulation by inflammatory mediators and may
be markers for endothelial and leucocyte activation.
They have been identified in healthy volunteers [111,
with elevated levels in certain inflammatory [12,13]
and neoplastic disease states [14].
The aim of this study was to identify any circadian
variation in soluble cell adhesion molecule (SCAM)
levels, which may be related to the diurnal variation in
disease activity or occurrence of certain disorders, in
addition to potentially showing a need for standardization of time points for sampling in clinical studies.
SUBJECTS AND METHODS
Subjects
INTRODUCTION
For many disease states there is a daily cycle of
disease activity. It has been well documented that there
is a peak incidence of cerebrovascular [l] and cardiovascular [2] events in the early hours of the morning.
This peak seems to match an early morning dip in
fibrinolytic activity [3], a rise in blood viscosity [4] and
platelet aggregation [5], all of which predispose to
thrombosis. Previously we have shown a circadian
variation in leucocyte activation [6] and endothelial
function. [7]. This may be important in the pathophysiology of thrombotic events but also in inflammatory disorders, which also show a circadian vari-
This study was performed on normal volunteers
who agreed to spend a 24 h period in Ninewells
Hospital and Medical School. The study was approved
by the local Medical Ethics Committee. Ten male
subjects who gave informed consent were recruited
from laboratory and medical staff. They had a median
age of 24 (range 17-33) years, all were non-smokers
and none had taken any medication in the previous 14
days. During the study, subjects were asked to carry
out their usual daily activities but to refrain from
rigorous activities. They remained ambulant until
00: 10 h; thereafter they were asked to retire to bed
and remain recumbent until 08 : 10 h. During the 24 h
Keywords: circadian rhythm, Eselectin. intercellular adhesion molecule-I.
Abbreviations: CAM, cell adhesion molecule; ICAM-I, intercellular adhesion molecule-I ; SCAM. soluble cell adhesion molecule.
Correspondence: Professor J. J. F. Belch.
538
C.Maple and others
-*-
Table I Time schedule
sE-Selectin
-A-
SICAM-1
Time (h)
12:00 Day I
12: 10
18:00
18: 10
00:00
00:10
04:00
08:00
08: 10
09:00
12:00 Day 2
1st sample
Lunch
2nd sample
Dinner
3rd sample
Retire to bed
4th sample
5th sample
Breakfast, subjects arise from bed
6th sample
7th sample
40 i i 0 0 1600 2000 O O i O
of the study, subjects ate as self-selected diet according
to the schedule shown in Table 1.
Blood sampling and slCAM-I and sE-Selectin assays
Ten millilitres of blood were drawn at each time
point. Each sample was obtained from a different site
within the antecubital fossa without the use of venostasis and using a 19-gauge butterfly needle. The
samples were allowed to coagulate at 37 "C for 1 h
before being centrifuged and stored at -70 "C until
analysis.
Serum levels of SICAM-1 and sE-selectin were
measured using commercially available sandwichlinked immunosorbent assay (ELISA) kits (R&D
Systems, U.K.). In brief, specific antibody conjugated
to horseradish peroxidase was added to murine antihuman sICAM- 1 or sE-selectin antibody-coated
microtitre ELISA plates. Standards and diluted
samples were then added to the plates, which were
covered and incubated for 1.5 h at room temperature.
After washing, substrate (tetramethylbenzidine) was
added to each well, the plate again covered and
incubated. After 30 min, the stop solution (sodium
azide) was added and the absorbance of each well was
determined using a spectrophotometer. The results
were calculated from a standard curve.
Statistical analysis
The results were analysed by repeated measures
analysis of variance looking for any statistically
significant circadian variation in sICAM-I and sEselectin levels allowing for inter-subject differences.
Significance values refer to the P values obtained after
operating multivariate analysis of variance for all time
points and subjects. The null hypothesis was rejected
at P < 0.05. All analyses were performed using the
statistical package INSTAT (GraphPAD Software,
CA, U.S.A.).
RESULTS
The results are given in Figure 1. There is a
statistically significant circadian variation in both sEselectin and SICAM-1 levels ( P < 0.0001). Soluble
04iO 0 8 0 0
1;Oo
Time ( 2 4 hours)
Figure I Diurnal variation in soluble E-selectin and
soluble ICAM-I in 10 male healthy volunteers ( P < 0.ooOl)
(mean and S.E.M.)
ICAM-1 hadamaximumvalueat 12:OO h [238.3(17.1)
ng/ml; mean (S.E.M.)], falling to a minimum at
04 :00 h [203.6 (13.8) ng/ml]. Soluble E-selectin levels
also reached a peak at 12:OO [53.88 (8.8) ng/ml], but
fell to a trough at 0O:OO h [48.14 (7.3) ng/ml]. Serum
levels of sE-selectin and SICAM-1 measured by other
workers [ 151from controlled subjects were 53 17 ng/
ml and 283 f 19 ng/ml respectively. These are very
similar to our own results and show that our assay
methodology is producing expected results.
DISCUSS10 N
Cell adhesion molecules are important in promoting
the inflammatory response and, by allowing the
inflammatory cell access to infected tissues, attenuating infection. These CAMs may also be implicated in
pathological inflammation [16] and may have a role in
many inflammatory diseases, for example rheumatoid
arthritis [12,13]. They may also be linked to the
metastasis of neoplastic cells [14].
There are three families of CAMs. E-selectin is a
member of the selectin family. It is present only on the
surface of endothelial cells activated by inflammatory
mediators such as interleukin- 1 and tumour necrosis
factor-a. Its function is to promote the rolling of the
leucocyte along the surface of the endothelium within
the blood vessels [17]. This then allows tighter bonds to
form between other CAMs such as members of the
integrin and immunoglobulin families. ICAM-1 is one
member of the immunoglobulin family of CAMs and
can be found on the surface membrane of endothelial
cells and leucocytes [lo]. Recently, circulating or
soluble isoforms have been identified in the blood [ 1 11.
Evidence suggests that these molecules may be cleaved
or released from the bound forms [ 181, perhaps by
proteolytic cleavage. The mechanism responsible for
their release, however, is not fully understood nor is
their function. However, it has been shown that sEselection retains its ability to bind to leucocytes,
activates the integrin CDI 1b/CD18 (mac-I) [19] and
also acts as a chemoattractant to leucocytes [20]. These
functions only occur at concentrations greater than
Circadian variation in cell adhesion molecules
would be expected to be found in the circulation.
However, the necessary concentrations may be attained locally at sites of inflammation. Similarly, a
biological function has been shown for SICAM-1. This
soluble molecule has been shown to be a receptor for
the rhinovirus [2 11 and also plasmodium-infected
erythrocytes [22]. It too retains its ability to bind to its
ligand on leucocytes [23]. This may be important in
promoting the de-adhesion of leucocytes from the
endothelium, thus allowing their trans-endothelial
migration.
Soluble E-selectin and sICAM- 1 molecules have
been identified in the blood in healthy subjects with
elevated levels being found in certain disease states
[24]. Soluble E-selectin levels are elevated in patients
with multiple sclerosis [25], vasculitis [26] and in septic
shock [27]. In the latter, levels seemed to correlate
inversely with disease outcome. Since E-selectin is
produced by and expressed solely on activated endothelial cells, the presence of its soluble form in the
circulation may be a good indication of endothelial cell
activation. The low levels of E-selectin identified in
healthy subjects may relate to the small inflammatory
and vascular insults the body is subjected to throughout the day. Soluble ICAM-1 can also be measured in
plasma in healthy subjects. Elevated levels have been
shown to occur in many inflammatory disorders
including systemic sclerosis [28], systemic lupus erythematosus [29] and Wegener’s granulomatosis [30]. It
has also been identified in infections such as malaria
[31] and in patients with malignancies [14]. Levels of
SICAM-1 have been found to correlate with other
markers of inflammatory disease activity, for example
the erythrocyte sedementation rate.
No one has clearly described the expected SCAM
half-life in blood. The SCAMSare released into the
plasma by proteolytic cleavage [24]. In vitro cytokine
stimulation of endothelial cells produces E-selectin
expression after 4 h, which returns to baseline 12 h
later. ICAM-1 expression occurs after 24 h and remains elevated for 96 h [32]. It is interesting to
speculate as to whether all the CAMS such as vascular
cell adhesion molecules (vCAM) exhibit a circadian
variation. We have previously shown such a rhythm to
exist in healthy volunteers for sP-selectin, the platelet
cell adhesion molecule, which totally mirrors the
circadian fluctuation seen in platelet count [33].
In conclusion, we have shown, for the first time, that
a circadian variation in SICAM-1 and sE-selectin levels
exists in healthy volunteers. This further confirms the
circadian variation in endothelial cell and leucocyte
activation which we [6,7,34] and others [8] have
previously shown. It may help to explain why some
symptoms of inflammatory and thrombotic conditions
are maximal in the early morning. It may be of interest
to postulate that this rhythm may be altered in
inflammatory diseases, as we have previously shown in
thrombotic disease [35]. Further studies are underway.
In addition, the findings from this study identify the
need to standardize the time of blood sampling in
future studies where these molecules are to be assessed.
539
ACKNOWLEDGMENTS
C.M. was an Arthritis and Rheumatism Council
clinical research fellow. J.J.F.B. and C.M. gratefully
acknowledge support from the Raynaud’s and Scleroderma Association and the Arthritis and Rheumatism
Council. G.K. held a Pfizer PhD studentship. We
would like to thank Pfizer Ltd for supplying the
ELISA kits for this study.
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