British Journal of Rheumatology 1996;35:424~429
MONOCYTE ACTIVITY IN BEHCET'S DISEASE
§. §AHlN (OZGUN), R. LAWRENCE, H. DtRESKENELt, V. HAMURYUDAN,* H. YAZICI*
and T. AKOGLU
Department of Immunology, Marmara Medical School and *Department of Rheumatology, Cerrahpasa Medical
School, Istanbul, Turkey
SUMMARY
Monocytes obtained from patients with Behcet's disease (BD) were examined for differentiation markers (expression of CD14
and antigens reacting with monoclonal antibodies 25F9 and G16/1) and for expression of selected adhesion molecules. There
was significantly raised expression of the CD 14 molecule, and increased staining with 25F9 and G16/1 antibodies in monocytes
obtained from patients with BD. A monocyte activation marker, soluble CD 14, was also found to be raised in patients' serum
compared with normal serum (8.1 ± 9.2 us 1.4 ± 0.7 /ig/ml). Furthermore, BD patients' monocyte culture supernatants caused
significantly increased adhesion of normal neutrophils to endothelial cell monolayers in vitro. All these findings show that BD
patients' monocytes are active in vivo and produce a number of pro-inflammatory cytokines which may play a role in the chronic
inflammation found in these patients.
KEY WORDS:
Monocyte, Behcet's disease, Adhesion molecules.
BEH£ET'S disease (BD) is a chronic systemic vasculitis
involving mainly the eyes, joints and mucocutaneous
surfaces. The aetiology of the disease is not known,
but there is some evidence that may indicate a possible
relationship with some bacterial or viral agents, such
as atypical species of streptococci or herpes simplex
virus type I [1-3]. Although streptococci are the
primarily implicated bacterial agent in the aetiology
of BD, antibodies against Gram-negative bacterial
lipopolysaccharides (LPS) have also been found in
patients' sera [4]. The significance of this finding
remains unclear.
Skin pathergy positivity is a characteristic feature of
this disease and histopathology of the pathergy
reaction involves a predominance of neutrophils in
various stages of activation [5]. We have previously
demonstrated an increased adhesion of neutrophils
obtained from patients with BD to endothelial cell
monolayers in vitro [6]. The expression of the adhesion
molecules CD1 la and CD 18 was found to be increased
on the surface of neutrophils from patients with BD.
We also observed instability of the adhesion molecule,
LECAM, in patients' neutrophils in in vitro conditions
[7]. Furthermore, when normal neutrophils were
incubated with patient sera, expression of CD1 la and
CD 18 increased on the cell surface in addition to the
increment of adhesion of neutrophils to endothelial
cells [6], suggesting a possible role in adhesion for
soluble factors present in patients' sera [6]. Indeed,
various pro-inflammatory cytokines, such as IL-1,
IL-6, IL-8 and TNF-a, have been reported to be
elevated in the sera of BD patients [6, 8, 9].
Monocytes are the principal source for some of these
cytokines and may play a part in the pathogenesis of
chronic inflammation in this disorder. This study was
designed to investigate the activity of monocytes in
patients with BD. Expression of adhesion molecules
(CDlla, CDllb and CD 18) and the role of monocyte
supernatants in neutrophil-endothelial cell adhesion
were also investigated.
MATERIALS AND METHODS
Patients
The overall study group consisted of 90 patients with
BD attending the Behcet's disease Multi-disciplinary
Clinic at the Cerrahpasa Medical Faculty, Istanbul.
However, in different experiments different number of
patients and controls were used. All patients fulfilled
the International Study Group Criteria for the
diagnosis of BD [10]. Fifty-five patients had at least one
of the major symptoms of the disease at the time of
study and since there is no laboratory test for the
assessment of disease activity, this was regarded as a
criterion for activity. Fifty normal human sera from
healthy laboratory staff and medical students were also
used as controls. Patients and controls were not age or
sex matched.
Flow cytometric analysis
Ten millilitres of venous blood taken from patients
and controls were placed in tubes containing 0.01%
sodium azide and 0.015% EDTA, and the tubes
immediately cooled in ice-cold water [11]. Next, 0.1 ml
of whole blood was incubated with 20 /il of monoclonal
antibodies 25F9 and G16/1 (Immunotech, France), and
5/xl of anti-CD lla, CD 18 (Dako, Denmark) and
CD 14 (Serotec, UK). Samples were then washed at
4°C with phosphate-buffered saline (PBS) containing
7 mM sodium azide and 0.1% bovine serum albumin
(BSA) (PBS/BSA) and further incubated with 50 /tl
1/1000 diluted fluorescein isothiocyanate (FITC)conjugated goat anti-mouse antibody (Serotec, UK)
for 30min. Following incubation, samples were
Submitted 23 August 1995; reviled version accepted 7 December
1995.
Correspondence to: T. Akofclu, Marmara Tip Fakultesi Hastancsi,
AJtunizade, Istanbul, Turkey.
© 1996 British Society for Rheumatology
424
42S
SAHlN ET AL.: MONOCYTE ACnVITY IN BEHCETS DISEASE
Forward scatter
FIG. 1.—Whole blood samples were used in monoclonal antibody
stainings and, following lysis of erythrocytes by lysing solution,
monocytes were gated in flow cytometric analysis as shown and per
cent staining and/or mean fluorescence intensities were determined.
washed with cold PBS/BSA buffer and erythrocytes
were lysed with erythrocyte lysing solution (Becton
Dickinson). Samples were analysed by FACScan
(Becton Dickinson) with monocyte gating as seen in
Fig. 1, and 7500 cells/sample were counted.
Background staining was determined by staining
of mononuclear cells with FITC-conjugated goat
anti-mouse antibody alone, and this figure was
subtracted from the figure obtained from the samples.
The mean fluorescence intensities for CD 14, CD lla,
CD lib and CD 18 were presented, but since only a
proportion of normal monocytes are positive, the
percentage of the stained cells was presented for 25F9
and G16/1.
Monocyte culture supernatants
These were obtained as described by Mege et al. [8].
Mononuclear cells were separated by Ficoll/Hypaque
(Nycomed/Oslo) from heparinized venous blood taken
from the patients and controls, and suspended in
RPMI medium supplemented with 10% fetal calf
serum (FCS) to obtain a final concentration of 2 x 10*
10'
Patients
Control
415.2 ±63.6
344.8 ± 7 9
(n - 30)
P = 0.0004
25F9
(%)
13.2 ±15.6
FIG. 2.—Representative patients' expression of 25F9 (a) and G16/1
(b) antigens on normal monocytes (dotted area) and monocytes
obtained from patients with BD (dark area). As seen, a significantly
increased number of stained cells were found in patients compared
with normal controls.
cells/ml. The cell suspension was distributed to 24 well
plates, 1 ml to each well, and then incubated for 1 h at
37°C in 7% CO3. After incubation, wells were washed
extensively with RPMI. Cells not adhering to wells
were aspirated and removed. Adhered cells were
incubated for 48 h at 37°C, either in medium alone for
the assessment of in vivo stimulation or in the presence
TABLE II
Effect of monocyte culture supernatant on the adhesion of normal
neutrophils to endothdial cell monolayers in vitro
3.4 ± 3
(i - 29)
P = 0.001
Adhesion ratio (%)
G16/1
15 ± 16.2
1.4 ± 1.4
P - 0.00001
103
Log Fluorescence Intensity
TABLE I
Expression of selected differentiation and activation molecules on
monocytes
CD14
(mean
fluorescence
intensity)
102
Patient
monocytes
• Control
monocytes
Unstimulated
monocyte
culture
supernatant
PMA-stnnulated
monocyte
culture
supernatant
LPS-*timulatMi
monocyte
culture
supernatant
53.4 ± 17.9
55.6 ± 19.8
46.5 ± 1 1 . 3
(n-9)
40.4 ± 13.5
d-7)
P-0.15
(n-9)
41.9 ± 13.8
(i-8)
38.4 ± 9.7
(« = 8)
P - 0.058
(i-8)
P-024
426
BRITISH JOURNAL OF RHEUMATOLOGY VOL. 35 NO. 5
TABLE HI
Expression of adhesion molecules on monocytes (mean fluorescence
intensity)
Patients
Controls
CDlla
CDllb
329.3 ± 57.5
(n-30)
327.2 ± 68
(n - 30)
289.5 ± 47.3
(n-30)
258.9 ± 55.2
(n = 30)
CD18
269.4 ± 79
264 ± 78.1
(n-30)
of 1 /ig/ml LPS (from Escherichia coli 055:B5; Sigma,
USA) or 5 ng/ml phorbol myristate acetate (PMA;
Sigma, USA) for the assessment of monocyte
reactivity, and supernatants were collected and stored
at -40°C.
Endothelial cell culture
Human umbilical vein endothelial cells were
obtained by collagenese digestion (0.1% collagenase
type 1; Sigma Chemical, USA) of umbilical cords as
previously described by Jaffe et al. [12]. Cells were
cultured in RPMI 1640 supplemented with 20% FCS,
50 /ig/ml endothelial cell growth factor (Sigma, USA),
5 IU/ml heparin, 5 /ig/ml gentamycin and 20 /ig/ml
amphotericin B. Cells in the third passage were
transferred to 96-well plates (2 x 104 cells/well)
pre-coated with 0.1 % gelatin. The adhesion assay was
performed after 24 h when cells formed confluent
monolayers at the bottom of wells.
Adhesion assay
This was performed mainly by the method of
Vercellotti et al. [13], but a slightly longer incubation
(45 min) was used. In summary, human peripheral
blood polymorphonucleocytes from two healthy
individuals were isolated in one step from the whole
blood after centrifugation through lymphoprep (Nycomed, Norway) and pooled as described by Ferrante
and Thong [14]. Hypotonic saline lysis was used to
remove residual red blood cells (RBCs). Cells were
observed to be >95% neutrophils using phase-contrast
microscopy. Fifty microlitres of 2 x 106/ml neutrophil
suspension and 50 /il of monocyte culture supernatants
were added onto endothelial cell monolayers in 96-well
plates and then incubated for 45 min at 37°C in 7%
COj. Non-adhered neutrophils were then aspirated and
adhered neutrophils were quantitated by a colorimetric
assay for myeloperoxidase enzyme activity as described
[15]. Briefly, 75/xl of 0.1 M sodium acetate solution
(pH 4.2) containing 1 % (w/v) cetitrimethylammonium
bromide (Sigma) and 0.2 mM tetramethylbenzidine as
peroxidase substrate were added to each well. After
2 min, 75 /il of 0.7 mM HjO2 were added and further
TABLE rv
Soluble CD14 levels in the serum obtained from patients and normal
controls O'g/ml)
8.1 ± 9.2
Patients (n = 50)
1.4 ±0.7
Controls (n - 10)
P •= 0.0001
incubated for 2 min. The reaction was terminated by
the addition of 50 /il of 4 N acetic acid containing
10 mM sodium azide. The optical density of wells was
determined with a multiscan plate reader at 600 nm
(Metertech 960). Total activity was defined as the mean
of the optical density of five wells containing 50 /il of
neutrophil suspension (2 x 10* cells/ml). Background
was measured from wells containing only endothelial
cells. The adhesion ratio was then calculated using the
following formula:
Adhesion ratio (AR) = {(OD of sample containing
monocyte supernatant with non-adherent cells
removed — background)/[Total activity (OD of wells
containing total cells) - background]} x 100
Measurement of soluble CD 14
Soluble CD 14 levels in the sera were determined
using a commercial ELISA kit (IBL Germany) and the
manufacturer's instructions were followed without
modification.
Statistical analysis
Results are given as the mean ± S.D. The MannWhitney (/-test was used to compare differences
between groups.
RESULTS
CD 14 molecule on the monocytes and antigens
reacting with monoclonal antibodies 25F9 and G16/1
were significantly raised in patients with BD compared
with healthy controls (Table I). As seen in Table I, the
mean fluorescence intensity of CD14 was 415.2 ± 63.6
in patients and 344.8 ± 79 in normal controls
(P = 0.0004). Similarly, 25F9 and G16/1 staining were
elevated in patients (13.2 ± 15.6% and 15 ± 16.2%,
respectively) as compared with normal controls
(3.4 ± 3 %
and
1.4 ±1.4%,
.P = 0.001
and
P = 0.00001) (Table I, Fig. 2).
The effect of monocyte culture supernatant on
neutrophil adhesion to endothelial cells is presented in
Table II. Unstimulated PMA- and LPS-stimulated
monocyte culture supernatants from patients with BD
caused increased adhesion of neutrophils to endothelial
cell monolayers as compared with normal monocyte
supernatants, but differences between patient and
control groups did not reach statistical significance
(Table H).
The expression of various adhesion molecules on
monocytes obtained from patients and controls is given
in Table III. As shown, the expressions of CD1 la and
CD 18 molecules were not different between patients
and controls. CDllb expression, however, was found
to be significantly increased in patients with BD as
compared with healthy controls (289.5 ± 47.3 vs
258.9 ± 55.2, P = 0.04).
Levels of the soluble CD 14 molecule were also found
to be significantly higher in patients' sera as compared
SAHlN ET AL:. MONOCYTE ACTIVITY IN BEHCET'S DISEASE
427
VIRAL / MICRO8IAL ANTIGENS
Heat shock proteins, LPS ?
( PPD, Strep., HSV-1 )
STIMULATION OF
ENDOTHEUAL CELLS
I
MEDIATORS
(IL-fl, PAF, etc.)
STIMULATION OF
MONONUCLEAR CELLS
X
/
IMMUNOGENET1CALLY
SUSCEPTIBLE SUBJECTS
(HLA-B51 of others)
MEDIATORS
(TNF, IL-1,IL-6,etc.)
AUGMENTED NEUTROPHIL REACTION
VASCULITIS WITH NEUTROPHIL INFILTRATION
FIG. 3.—Proposed mechanism for immunopathogenesis of Behcet's disease. Some infectious agents may trigger mononudear cells as well as
endothelium through heat shock peptides in genetically susceptible, HLA-B51-positive individuals. At the final step, different mediators released
from activated mononuclear cells and endothelium may in turn activate neutrophils and monocytes, resulting in severe inflammation
to control serum samples (8.1 ± 9.2 w l . 4 ± 0.7 fig/ml,
P = 0.0001) (Table IV).
There were no significant differences between active
and inactive cases in terms of cell surface adhesion
molecule expression or differentiation and activation
markers (CD 14, 25F9 and G16/1), as well as the effect
of monocyte culture supernatants on adhesion of PMN
to VEC. Soluble CD 14 levels were also similar in both
groups.
DISCUSSION
We have previously shown that BD patients' serum
causes increased adhesion of normal neutrophils to
endothelial cell monolayers in vitro [6]. In addition,
Behcet's neutrophils express increased adhesion molecules, LFA-1, (CD 11 a/CD 18) on their surfaces and
also show other signs of activation [6].
Neutrophil activation occurs mainly via soluble
mediators. It has been shown that TNF-a, IL-1, IL-6
and IL-8 are elevated in the sera of patients with BD
[6, 9, 16, 17]. Although many immunologically active
cells and also neutrophils may produce these cytokines
following stimulation, one of the major sources of
TNF-a, IL-1, IL-6 and IL-8 are monocytes. Therefore,
the presence of activated monocytes in patients with
BD is not surprising. Indeed, Warabi el al. [18] have
shown increased chemotactic activity of peripheral
blood monocytes in Behcet's patients.
In this study, the monocyte activation marker
soluble CD 14 was found to be elevated in BD patients'
serum, and also activation/differentiation markers such
as expression of CD 14 and surface antigens which react
with monoclonal antibodies 25F9 and G16/1 were
found to be present in increased numbers on Behcet's
monocytes.
A surface receptor for the monoclonal antibody
25F9 has been shown to be present on tissue
macrophages and some cell lines, but not on normal
peripheral blood monocytes [19]. This monoclonal
antibody detects a novel differentiation antigen on
mature human tissue macrophages. However, cultivation of peripheral blood monocytes for 2-3 days leads
to expression of the 25F9 specific antigen on the
surface. Similarly, antigen reacting with the monoclonal antibody G16/1 is found on cells present in the
red pulp of the spleen, some macrophages in
granulomas, foam cells and epitheloid cells, but not on
normal peripheral blood monocytes [20].
CD 14 is a 55 kDa glycoprotein expressed on the
surface of mature monocytes and macrophages, and is
an important molecule involved in the immune
response. It has been reported to be a receptor for the
complex of LPS and LPS binding protein [21]. The
binding of LPS to CD 14 triggers a series of monocytic
immune responses, including the synthesis and release
of TNF-a, IL-6 and IL-8 [22]. CD14 is also involved
in monocyte-mediated T-cell activation and monocyte
adhesion to endothelial cells [23, 24]. Soluble CD14 can
also bind to LPS and this complex can function as a
co-ligand with LPS and activates endothelial cells [22].
As a further step, we have also studied monocyte
activities functionally and have observed that PMNendothelial cell adhesion slightly increases when
supernatants obtained from BD patients' monocytes
428
BRITISH JOURNAL OF RHEUMATOLOGY VOL. 35 NO. 5
are present in the test system (Table II). Increased
expression of the C D l l b molecule on monocytes
obtained from patients with BD is a further sign of
increased monocyte activation. Indeed, it is well known
that expression of CD11/CD18 molecules on monocytes is closely related to CD14-mediated monocyte
activation [25].
All these findings indicate the presence of functionally active monocytes and also monocytes in the stage
of transition to macrophages in Behcet's patients.
At present, we do not know the cause of monocyte
or neutrophil activation in this disease. Recently, it has
been shown that some bacterial agents such as atypical
streptococci may be implicated in the aetiology of this
disease [3]. It has also been shown that patients with
BD sometimes present with anti-LPS antibodies in
their sera, and this may be a sign of Gram-negative
bacterial infection [4]. Interaction of CD14 with LPS
and endothelial cells [20] suggests a possible mechanism
by which a bacterial antigen might trigger BD.
We previously proposed a working hypothesis for
the aetiopathogenesis of BD [26] (Fig. 3). Some
infectious agents may trigger mononuclear cells [27],
as well as endothelium [28], through heat shock
pep tides in genetically susceptible, HLA-B51-positive
individuals [29,30]. At the final step, different
mediators released from activated mononuclear cells
and endothelium may in turn activate neutrophils and
monocytes, resulting in severe inflammation. Other
factors involved and some steps of this hypothesis,
however, remain to be elucidated in future studies.
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