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Lymphocyte activation status, expression of adhesion molecules and adhesion to human endothelium in rheumatoid arthritis — relationship to disease activity
0
Res. Immunol.
INSTITUT PASTEUR/ELSEVIER
Paris 1994
1994, 145, 101-108
Lymphocyte activation status, expression of adhesion molecules and adhesion to human endothelium in rheumatoid arthritis - relationship to. disease activity A.V. Mertens, L.S. de Clerck, M.M. Moens, C.H. Bridts and W. J. Stevens Department
of Immunology,
Allergology and Rheumatology, WA, Ant werp (Belgium)
University
of Antwerp,
SUMMARY
The synovial tissue of patients with rheumatoid arthritis WA) is characterized by infiltration with inflammatory cells, mainly memory helper cells (CD4+CD29+). An important initiating step in tissue infiltration Is the adhesion of peripheral blood lymphocytes to the vascular endothelium. Therefore, we studied lymphocyte-endothelium adhesion in 40 RA patients and in 19 controls by a sensitive fluorimetric assay, using human umbilical vascular endothelial cells. Furthermore, expression of adhesion molecules VLA (CD291 and LFA-I (CD1 la) on CD4+ and CD8+ T cells was determined. In order to evaluate the activation state of lymphocytes, the soluble interleukin-2 receptor (slL2R) was measured. The relationship to disease activity was evaluated using the Ritchie artitular index. RA patients had a higher percentage of CD4+ cells (p c 0.0051 and a lower percentage of CD8+ cells (p < 0.001) than controls did. The CD4+CD29+/ CD4+CD29- ratio and the CD8+CD29+/CD8+CD29ratio were increased in patients with active RA (p < 0.01 and p c 0.05, respectively) and in patients with inactive disease (p= 0.09 and p < 0.005, respectively) compared with controls. LFA-1 (CD1 la) was present on almost all T lymphocytes and its density did not differ between patients and controls. Serum levels of slL2R were significantly higher in both patient groups compared with controls (p < 0.0005); patients with active disease showed significantly higher levels than patients with inactive disease (p < 0.05). Lymphocyte-endothelium adhesion was not increased in patients, although the expression of the adhesion molecule CD29 on T lymphocytes of RA patients was higher. These results demonstrate that peripheral blood lymphocytes of RA patients are activated, especially in patients with active disease, as demonstrated by elevated slL2R levels and increased numbers of CD29+ cells. Nevertheless, there was no increased lymphocyte adhesion to endothelial cells in RA patients compared with controls, demonstrating that functional adhesion properties are not always correlated. with the mere presence of CD29 or other adhesion molecules on the cell membrane. Key-words: T lymphocyte, Inflammation, Rheumatoid arthritis, Endothelium; Adhesion, Cell activation, IL2, Disease activity.
Submitted September20, 1993,acceptedDecember29, 1993. Corresponding address: Prof. W.J. Stevens, Department of Immunology, Allergology and Rheumatology, University of Antwerp, UIA, Universiteitsplein 1, 2610 Antwerp. Belgium.
A. V. MERTENS
102 INTRODUCTION
Rheumatoid arthritis (R4) is a systemic inflammatory disease associated with chronic joint inflammation. Histopathologic findings consist of hyperplasia of the synovium, generation of new blood vessels and infiltration of mononuclear cells (Kulka et al., 1955). The cellular infiltrates are mainly built up by T lymphocytes, especially memory helper cells (CD4+ CD29+) (Pitzalis et al., 1982; Cush and Lipsky, 1991). For the recruitment of these cells from the peripheral blood, adhesion to and migration through vascular endothelium are important. Adhesion is regulated by adhesion molecules on circulating lymphocytes, and these molecules bind to their ligand on endothelial cells (Springer, 1990). The aim of this study was to investigate adhesion of peripheral blood lymphocytes to human umbilical vascular endothelial cells, by using a sensitive fluorimetric method. Together with functional adhesion, phenotypic analysis of adhesion molecule LFA- 1 (CD 11 a/CD 18) and activation marker/adhesion molecule VLA (CD29) on T lymphocytes was performed. Since the lymphocyte infiltration in synovium is much more pronounced in patients with aggressive disease (Gupta and Talal, 1985), we wanted to correlate adhesion properties with clinical disease activity, assessed by the Ritchie articular index, and with T-cell activation, evaluated by measuring soluble interleukin-2 receptor (sIL2R) in serum (Keystone et al., 1988).
MATERIALS
AND METHODS
Patients Forty-three patients (mean age 58 years, range 24-81) with classical or definite RA according to the ARA-criteria (Ropes et al., 1958) were studied. Activity of the disease was scored with the Ritchie artitular index (Ritchie et al., 1968). A Ritchie score
EC FCS
= endothelial cell. = foetal calf serum.
ET AL. of more than 10 was considered as active RA (16 patients). None of the patients used corticosteroids ; 39 out of 43 used non-steroidal antiinflammatory drugs with or without other antirheumatic medication: oral (2) or IM (4) gold, antimalarials (3), sulphasalazine (1 l), azathioprine (5), methotrexate (11) or penicillamin (1). Nineteen healthy volunteers (mean age 34 years, range 24-48) were included as control group. Cell separation Leukocytes were isolated from IO ml venous blood by discontinuous Percoll gradient centrifugation (60 % and 72.5 % isotonic Percoll). The mononuclear cells were suspended in CO,-independent medium (Gibco Life Technologies, Ghent, Belgium, 041-08045 M) supplemented with 1 % foetal calf serum (J?CS ; Gibco), 100 U/ml penicillin and 100 $/ml streptomycin (Gibco) and were added to culture flasks (Primaria, Falcon, Becton Dickinson, Mountain View, CA, USA) for 30 min, to eliminate the monocytes by adherence. For further experiments, the purity of the lymphocyte population was always 30 % or more (median 94 %, range 80-98 To). Flow cytometric
analysis
The P,-integrin VLA (CD29) and the P,-integrin LFA-1 (CD1 la/CD18) were determined on CD4+ and on CDS+ cells. Fifty ~1 of peripheral blood were incubated with 20 ~1 fluorescein-isothiocyanateand 20 ~1 phycoerythrin-labelled monoclonal antibody (purchased from Immunotech, Marseille, France and from Dako, Glostrup, Denmark) for 15 min at 4°C. After incubation, the remaining red blood cells were lysed with cold NH&l-lysis buffer and the leukocytes were fvted in 1 % paraformaldehyde in phosphate-buffered saline. Flow cytometric analysis was performed on a “Facscan” (Becton Dickinson), standardized with fluorescent beads (Fluoresbrite intensity kit, Polysciences, Warrington, PA, USA), according to Schmid (Schmid et al., 1988). Fluorescence of 10,000 cells was evaluated, and Iymphocytes were gated on forward and site scatter characteristics. The percentage of positive lymphocytes was evaluated, and the mean density for each marker was determined using the mean fluorescence intensity, expressed as mean channel number (Schmid et al., 1988).
HUVEC RA
= =
human umbilical vein endothelial cell. rheumatoid arthritis.
L YMPHOCYTE-ENDOTHELIUM
ADHESION
Adhesion assay Endothelial
IN RHEUMATOID
103
With this method, an adhesion of 6,000 cells or more can be measured.
cell culture Detection
Endothelial cells (EC, i.e. human umbilical vascular endothelial cells, HUVEC) were obtained from umbilical cords by collagenase-dispase (Boehringer, Mannheim, Germany) digestion, using the technique of Jaffe et al. (1973) with minor modifications. The EC were suspended in growth medium Ml99 (Gibco) supplemented with 20 % FCS (Gibco), 100 U/ml penicillin and 100 yg/ml streptomycin (Gibco), 30 yg/ml EC growth supplement (Sigma Chemie, Brussels, Belgium) and 10 U/ml sodium heparin (Sigma). EC were seeded at a density of 3,000/cm2 in culture flasks (Falcon 25 cm2, Becton Dickinson) pretreated with 2 % gelatin (Sigma) in water for 30 min and cultured in a 5 % CO, humidified incubator (Jouan, Saint Herblain, France) at 37°C for 2 to 5 days, until confluence was observed. At confluence, EC were detached using 0.125 % trypsinEDTA in modified Pucks saline A (Gibco). The cells were confirmed to be EC by positive immunofluorescent staining for Factor-VIII-related antigen.
HUVEC
ARTHRITIS
adhesion assays
From the second to the fourth passage, EC were transferred into flat-bottomed, gelatin-coated 96well microtitre plates (Falcon), at a concentration of 35,000 EC per well. After overnight culture, confluent monolayers were obtained and used in the adhesion experiments. EC were stimulated with 100 ~1 recombinant interleukin-1-P (rIL1P; Janssen Biochimica, Beerse, Belgium ; 20 U/ml in growth medium) or were suspended in growth medium as control and incubated for 6 h at 37°C. Afterwards, the EC were washed twice with CO,-independent medium (Gibco, 041-08045 M) supplemented with 1 % FCS (Gibco), 100 U/ml penicillin and 100 d/ml streptomycin (Gibco). Lymphocytes (10’ cells/ml) were labelled with 300 pg/ml 5(6)-carboxyfluorescein-diacetate (CFDA, Sigma) for 15 min at 37°C (Struyf et al., 1990). After labellingd the cells were washed twice and concentrated at 10 cells/ml The remaining red blood cells were lysed with cold NH,Cl-lysis buffer, and after washing, ILl-stimulated and unstimulated EC were incubated with 100,000 lymphocytes per well at 37°C for 30 min. After incubation, total fluorescence (F,) was measured on a “Cytofluor 2300” (Millipore, Bedford, MA, USA) fluorometer. The non-adherent cells were removed by washing four times. The remaining fluorescence (F,) was measured, and the percentage of adhesion was calculated using the following formula: 070adhesion= 100 x (F./F,).
of sJL2R in serum
The quantitative determination of serum sIL2R was done with a sandwich enzyme-linked immunosorbent assay (Eurogenetics, Tessenderlo, Belgium), with two monoclonal antibodies recognizing different epitopes of the IL2R. The serum levels were expressed in U/ml. Statistical
analysis
The Student t test, Mann-Whitney CJ test and Pearson and Spearman rank correlation test were used, where appropriate. .
RESULTS Phenotypical
analysis
(table I)
RA patients had significantly more peripheral blood T helper cells (CD4+) and less T suppressor cells (CD8+) than controls had, resulting in a higher CD4/CD8 ratio. The percentage of memory T helper cells (CD4+CD29+) was significantly higher in patients with active disease and in patients with inactive disease compared with controls. Within the CD4+ subset, the ratio of CD4+CD29+/CD4+CD2gm cells was higher in patients with active disease compared with controls. The percentage of memory T suppressor cells (CD8+CD29+) was lower only in patients with active disease, due to the lower number of CD8+ cells. Indeed, when the ratio of CD8+CD29+/CD8+CD29was made, RA patients showed a significantly higher fraction of CD8+ cells bearing CD29 than controls did CD1 la was present on almost all T cells .of patients (median (range) : 100 (40-100) Vo) and controls (100 (74-100) Vo). Densities of adhesion molecules CD29 and CD1 la, measured on CD4+ and CD8+ subsets, did not differ between the study groups (table II). There was a weak but significant correlation between the density of CD29 on CD4+ cells and the Ritchie articular index (r =0.33, p < 0.05; fig. 1).
104
A. V. MERTENS
ET AL.
Table I. T-lymphocyte subsets (percentage positive cells and ratio (median (range)) in controls and in RA patients with inactive disease (Ritchie score < 10) and RA patients with active disease (Ritchie score > 10).
Vo CD4+ “70 CD4+CD29+ CD4+CD29+/ CD4+CD29% CD8+ % CD8+CD29+ CD8+CD29+/ CD8+CD29CD4+ /CD8 +
Controls (n= 18)
total (n = 41)
RA patients inactive (n = 26)
active (n = 15)
42 (32-61) 20 (12-29) 0.85 (0.41-1.79)
52 (23-79) (***) 27 (11-55) (****) 1.27 (0.38-3.67) (*)
51 (23-77) (*‘) 27 (1 l-42) (***) 1.21 (0.38-3.36)
55 (37-79) (***I 28 (20-55) (****) 1.29 (0.80-3.67) (“)
31 (16-47) 16 (10-32) 1.27 (0.45-5.0)
19 (5-43) (****)
15 (5-38) (****)
;;&?&Jo)
1.5 (0.9-2.9)
2.7 (1.0-15.8) (****)
(***)
11 (2-33) (‘) ::7?,?~7-6.60)
(‘)
2.5 (1 .O-9.6) (***)
3.4 (1.0-15.8) (****)
(*) p < 0.05, (**) p < 0.01, (***) p < 0.005 and (****) p < 0.001 RA versus controls.
sIL2R levels in sera from controls and RA patients The serum sIL2R levels in patients were significantly higher than in controls (median (range): 110 (99-293) U/ml verSu.s 101 (96-130) U/ml, p < 0.0005; fig. 2). Levels in patients with active disease were significantly higher than
levels in patients with inactive disease (115 (104-293) U/ml versus 108 (99-277) U/ml, p c 0.05). A positive correlation was found between level of sIL2R and the CD4+CD29+ /CD4+CD29ratio (r=0.30, p < 0.05) and also between sIL2R level and the CD8+CD29+ /CD8+CD29ratio (r =0.35, p < 0.01). Adhesion of peripheral blood lymphocytes to HUVEC (fig. 3) Adhesion was measured on unstimulated endothelium and on ILl-stimulated endothelium. Percentage of adherent cells was comparable for RA patients and controls in both unstimulated and stimulated conditions. No differences were found between pafients with inactive diseqse and patients with active disease. Adhesion on ILl-stimulated HUVEC was, however, increased in all subjects compared with adhesion on unstimulated endothelium
(p < 0.05 in controls, p < 0.0005 in RA patients with active disease and p c 0.0001 in RA patients with inactive disease). DISCUSSION
T lymphocytes certainly play a major role in the pathogenesis of RA. The cellular infiltrates in the synovial tissue are built up mainly by memory helper cells (CD{+CD29+) (Pitzalis et al., 1988; Cush and Lipsky, 1991). To infiltrate the synovium, peripheral blood lymphocytes first have to adhere to endothelial cells before they can pass through the vascular endothelium and migrate to the site of inflammation. The binding of adhesion molecules on lymphocytes with their ligand on endothelium is an important mechanism in adhesion (Springer, 1990). In this study, we evaluated several aspects of lymphocyte function in paiients with &4 : (1) the adhesion of peripheral blood lymphocytes to endothelial celis, (2) the expression of adhesioq molecules on circulating T lymphocytes, (3) the activation state of lymphocytes and (4) the relationship of all these parameters to disease activity. In comparison with a control population, we found a higher percentage of peripheral blood CD4+ T helper cells and a lower percentage of
L YMPHOCYTE-ENDOTHELIUM
ADHESION
IN RHEUMATOID
ARTHRITIS
105
Table II. Densities of the adhesion molecules (expressed in mean channel number (median (range)) on CD4+ and CDS+ cells in controls and in RA patients with inactive disease (Ritchie score < 10) and RA patients with active disease (Ritchie score > 10). Controls (n= 18)
Density CD29 on CD4+ CD1 la on CD4+ CD29 on CD8 + CDlla on CD8+
cells cells cells cells
404 415 374 436
total (n = 41)
(262-456) (250-471) (265-418) (275-525)
CD29 DENSITY 45oj
391 427 378 478
(281449) (282-486) (287-459) (302-532)
RA patients inactive (n = 26) 389 436 378 496
(281-433) (282-486) (287-432) (302-530)
active (n = 15) 400 424 380 467
(321-449) (354-479) (3 16-459) (394-532)
sIL-ZR(U/ml) 0 0
x OC 0
400 i
0
00 0
0 0
0
0 0
0 0
0
0
I50 v
0 0
+ ;
y=2.47~+368
*: %
(r=0.33)
Fig. 1. CD29 density on CD4+ peripheral blood T lymphocytes of RA patients in relation to clinical arthritis activity as measured by the Ritchie articular index.
Fig. 2. sIL2R in peripheral blood of controls (CTRL, n = 19) and patients with FM (n = 43).
CDS+ T suppressor cells in RA patients, resulting in a higher CD4KD8 ratio. This finding confirms the results of Smith (Smith and Roberts-Thomson, 1990), but is in contrast to others (vaurer et al., 1992). Although there are controv,$esial results in the literature, an increased CD4/CDg ratio mostly due to a decrease in CD!+ cells in patients with gctive disease is reported by many authors (for review see Cush and Lipsky, 1991; Creamer, 1992). In the present study, the significantly higher ratio of CD4+CD29+/CD4+CD29cells in
RA patients with active disease and of the CDS+CD29+/CD8+CD29ratio in both RA groups indicate activation of these cells (Sanders et al., 1988). Although not unexpected for a chrBnjc inflammatory diseasf, changes in the memory phenotype have ngt been found by SO~F other groups (Garcia-Ujcuna et al., 1992 ; Maurer et al., 1992 ; Postigo ef al., 1992) possibly due to smaller numbers and/or selection criteria of patients. It is known that corticosteroids can influence lymphocyte phenotypes (Raziuddin et al., 1990; Frey et al., 1984).
A. V. MERTENS ET AL.
106
Adhesion
(%I A
B _J35.3
_L
2b.5
20-
Fig. 3. Adhesion of T lymphocytes from controls (A), RA patients with inactive disease(Ritchie score< 10)(B), andRA patientswith activedisease (Ritchiescore> 10)(C) on unstimulated(( -)ILl) and on ILl-stimulated (( +)ILl) endothelium.
In order to avoid this bias, all patients using corticosteroids were excluded from the study. In the RA population studied, a significantly higher level of sIL2R was found, especially in patients with active disease, also demonstrating lymphocyte activation, as has been reported (Keystone et al., 1988). Moreover, increased expression of cell surface IL2 receptor has also been described in RA (Maurer et al., 1992). Since RA patients express more CD29 on their peripheral blood T cells, and since mainly CD29+ cells are found in synovial tissue (Cush and Lipsky, 1988; Laffon et al., 1991; Van Dinther-Janssen et al., 1991), we further investigated, in the second part of this study, the role of CD29 as adhesion molecule. Indeed, in addition to its function as activation marker, CD29 is also an important adhesion molecule. Furthermore, another adhesion molecule, i.e. CD1 la, was also determined, since it has been shown that at least two different molecular pathways exist that affect lymphocyte adhesion to cultured endothelial cells: the leukocyte integrin LFA-1 (CDlla/CD18) is responsible for the binding of lymphocytes to unstimulated endothelial cells via its ligand ICAM- or ICAM- (Haskard et al., 1986), whereas the interaction between the leu-
kocyte integrin VLA-4 (CD29/CD49d) with its ligand VCAM-1 on endothelial cells is important for the adhesion of lymphocytes to cytokinestimulated endothelium (Carlos et al., 1990). Thus, in the part of this work dealing with functional aspects, we studied the adhesion of lymphocytes to cultured human endothelial cells in relation to expression of the adhesion molecules CD29 and CD1 1a. Since CD 1la is present on almost alI T lymphocytes, we also determined the density of the adhesion molecules by the use of flow cytometric analysis. In contrast with synovial tissue lymphocytes (Cush et al., 1992), peripheral blood lymphocytes of the RA patient group did not express more CD29 or CDlla than controls did. Nevertheless, a correlation was found between the density of CD29 on CD4+ cells and the Ritchie articular index, suggesting that more aggressive disease is related to activation of peripheral blood lymphocytes. Although there is no direct evidence that the adhesion of lymphocytes to HUVEC in vitro is an equivalent process to the adhesion to microvascular endothelial cells in vivo, the validity of the model is supported by in vitro experiments (Bender et al., 1987; Haskard et al., 1987). In order to mimic inflammation in vitro,
L YMPHOCYTE-ENDOTHELIUM
ADHESION
endothelial cells were stimulated with IL1 (Cotran and Pober, 1988). Our sensitive in vitro model for adhesion, on both unstimulated and ILl-stimulated HUVEC, gave comparable results for controls and patients, although adhesion was clearly increased on stimulated endothelium for all subjects ; thus the presence of more T cells expressing CD29 in RA patients was not reflected in increased adhesion. In conclusion, the results of this study demonstrate that peripheral blood lymphocytes of RA patients are activated, especially in patients with active disease, as indicated by elevated sIL2R levels and increased expression of CD29 on T lymphocytes. Nevertheless, there was no increased lymphocyte adhesion to endothelial cells in RA patients compared with controls, demonstrating that functional adhesion properties are not always correlated with the mere presence of CD29 or of other adhesion molecules on the cell membrane.
Acknowledgements The authors wish to thank Mrs. Chris De Beul and Mrs. lnge Vereecke for expert technical assistance. ELISA kits for determination of slL2R were kindly provided by Dr. Bosmans (Eurogenetics, Belgium). This paper presents research results of the Belgian programme on Interuniversity poles of attraction initiated by the Belgian State, Prime Minister’s Office, Science Policy Programming. The scientific responsibility is assumed by its authors. This study was also supported by Levenslijn Grant no. 7.0044.91 of the Nationaal Fonds voor Wetenschappelijk Onderzoek.
IN RHEUMATOID
ARTHRITIS
107
sion lymphocyte-endothklium dans 40 cas d’AR et chez 19 tCmoins, par un test fluorimetrique sensible, utilisant des cellules endothkliales ombilicales. De plus, I’expression des molicules d’adhesion VLA
(CD29) et LFA-1 (CD1 la) sur les cellules T CD4+ et CD8+ a &e analysee. Pour ttudier 1’Ctat d’activation des lymphocytes, le rkcepteur IL2 soluble (sIL2R) a ttt &al&. La relation avec 1’activitC de la
maladie a et6 analyske g l’aide de I’index de Ritchie. Les malades atteints d’AR ont un pourcentage de CD4+
(p < 0,005) plus ClevC que celui des tCmoins
et un pourcentage de CD8+ plus bas (p < 0,001). Le rapport CD4+CD29+/CD4+CD29et le rapport CDS+CD29+/CD8+CD29sont augment& dans 1’AR active (p < 0,Ol et p < 0,05 respectivement) et dans I’AR non active (p= 0,09 et p < 0,005 respectivement) par rapport aux tCmoins. La molecule LFA-1 (CD1 la) est prCsente sut presque tous les lymphocytes T et la densitC de cette prCsence est iden-
tique chez les malades et les ttmoins.
Les taux ski-
ques d’sIL2R sont significativement plus ClevCs dans les deux groupes de malades par rapport aux ttmoins (p < 0,OOOS); les malades atteints d’AR active ont des taux significativement plus Bevts par rapport aux AR non actives (p < 0,05). L’adhtsion lymphocyteendothClium n’est pas accrue chez les malades, bien que l’expression de la molCcule d’adhCsion CD29 sur les lymphocytes T soit plus importante dans I’AR. Ces rtsultats montrent que les lymphocytes p&iphtriques sont activCs dans I’AR, surtout dans I’AR active, comme l’indiquent les taux eleves d’sIL2R et
le nombre ClevC de cellules CD29+.
Cependant on
n’observe pas d’augmentation de 1’adhCsion lymphocytaire aux cellules endothCliales dans I’AR, par rapport aux tCmoins, ce qui demontre que les proprittCs fonctionnelles d’adhbion ne sont pas toujours en corrClation avec la simple presence de CD29 ou d’autres molCcules d’adhesion sur la membrane cel-
lulaire. Mets-cl&s: Lymphocyte T, Inflammation, Arthrite rhumatdide, Endothelium; AdhCrence, Activation cellulaire, IL2, ActivitC de la maladie.
Activation lymphocytaire, expression de I’adhCsion mol&ulaire et adhesion a I’endotht?lium vasculaire dans I’arthrite rhumatdide, relations avec I’activitC de la maladie Le tissu synovial de malades atteints d’arthrite rhumato’ide (AR) est caracttrisk par un infiltrat de cellules inflammatoires, notamment de cellules (( helper )) CD4+CD29+. Dans I’infiltration cellulaire, une &ape d’initiation capitale est I’adhksion des lymphocytes sanguins pkriphiriques g I’endoth&lium vasculaire. C’est pourquoi nous avons Ctudie I’adhC-
References Bender, J. R., Pardi, R., Karasek, M.A. & Engleman, E.G. (1987), Phenotype and functional characterization of lymphocytes that bind human microvascular endothelial cells in vitro. Evidence for preferential binding of natural killer cells. J. Clin. Invest., 79, 1679-1688. Carlos, T.M., Schwartz, B.R., Kovach, N.L., Yee, E., Rosso, M., Osborn, L., Chi-Rosso, G., Newman, B., Lobb, R. & Harlan, J.M. (1990), Vascular cell adhe-
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A. V. MERTENS
sion molecule-l (VCAM-1) mediates lymphocyte adherence to cytokine-activated cultured human endothelial cells. Blood, 76, 965-970. Cotran, R.S. & Pober, J.S. (1988). Endothelial activation, in “Endothelial cell biology” (pp. 335-347). Plenum Publ. Cor., New York. Creamer, P. (1992), Flow cytometry and rheumatic disease. Brit. J. Rheumatol., 31, 465472. Cush, J.J. & Lipsky, P.E. (1988), Phenotypic analysis of synovial tissue and peripheral blood lymphocytes isolated from patients with rheumatoid arthritis. Arthritis Rheum., 31, 1230-1238. Cush, J.J. & Lipsky, P.E. (1991), Cellular basis for rheumatoid inflammation. Clin. Orthop. Res.. 205, 9-22. Gush, J.J., Pietschmann, P., Oppenheimer-Marks, N. & Lipsky, P.E. (1992), The intrinsic migratory capacity of memory T cells contributes to their accumulation in rheumatoid synovium. Arthritis Rheum., 35, 1434-1444. Frey, B.M., Walker, C., Frey, J.D. & de Week, A.L. (1984), Pharmacokinetics and pharmacodynamics of three different prednisolone prodrugs : effect on circulating lymphocyte subsets and function. J. Immunol., 133, 2479-2487. Garcia-Vicuna, R., Humbria, A., Postigo, A.A., LopezElzaurdia, C., de Landazuri, M.O., Sanchez-Madrid, F. & Laffon, A. (1992), VLA family in rheumatoid arthritis : evidence for in vivo regulated adhesion of synovial fluid T cells to fibronectin through VLA-5 integrin. Clin. exp. Immunol., 88, 435441. Gupta, S. & Talal, N. (1985), Pathogenetic mechanisms, in “Immunology of rheumatic diseases” (pp. 517542). Plenum Publ. Co., New York. Haskard, D.O., Cavender, D., Beatty, P., Springer, T.A. & Ziff, M. (1986), T-lymphocyte adhesion to endothelial cells : mechanisms demonstrated by antiLFA-1 monoclonal antibodies. J. Immunol., 137, 2901-2906. Haskard, D.O., Cavender, D., Fleck, R.M., Sontheimer, R. & Ziff, M. (1987), Human dermal microvascular endothelial cells behave like umbilical vein endothelial cells in T-cell adhesion studies. J. Invest. Dermatol., 88, 340-344. Jaffe, E.A., Nachman, R.L., Becker, C.G. & Minick, C.R. (1973), Culture of human endothelial cells derived from umbilical veins. J. Clin. Invest., 52, 2745-2756. Keystone, E.C., Snow, K.M., Bombardier, C., Chang, C.H., Nelson, D.L. & Rubin. L.A. (1988), Elevated soluble interleukin-2 receptor levels in the sera and synovial fluids of patients with rheumatoid arthritis. Arthritis Rheum., 3 1, 844-849. Kulka, J.P., Backing, D., Ropes, M.W. & Bauer, W. (1955), Early joint lesions of rheumatoid arthritis. Arch. Pathol. Lab. Med., 59, 129-150. Laffon, A., Garcia-Vicuna, R., Humbria, A., Postigo, A.A., Corbi, A.L., de Landazuri, M.O. & SanchezMadrid, F. (1991), Upregulated expression and func-
ET AL. tion of VLA4 fibronectin receptors on human activated T cells in rheumatoid arthritis. J. Clin. Invest., 88, 546-552. Maurer, D., Felzmann, T., Holter, W., Petera, P., Smolen, J. & Knapp, W. (1992), Evidence for the presence of activated CD4 T cells with naive phenotype in the peripheral blood of patients with rheumatoid arthritis. Clin. exp. Immungl., 87, 429-434. Pitzalis, C., Kingsley, G., Haskard, D. & Panayi, Cj. (1988) The preferential accumulation of helperinducer T lymphocytes in inflammatory lesions : evidence for regulation by selective endothelial and homotypic adhesion. Eur. J. Immunol., 18, 1397-1404. Postigo, A.A., Garcia-Vicuna, R., Diaz-Gonzalez, F., Arroyo, A.G., de Landazuri, M.O., Chi-Rosso, G., Lobb, R.R., Laffon, A. & Sanchez-Madrid, F. (1992), Increased binding of synovial T lymphocytes from rheumatoid arthritis to endothelial-leukocyte adhesion molecule-l (ELAM-1) and vascular cell adhesion molecule-l (VCAM-1). J. Clin. Invest., 89, 1445-1452. Raziuddin, S., Nur, M.A. & Al Wabel, A.A. (1990), Increased circulating HLA-DR+ CD4+ T cells in systemic lupus erythematosus : alterations associated with prednisolone therapy. Stand. J. Zmmunol., 31, 139-145. Ritchie, D.M., Boyle, J.A. & McInnes, J.M. (1968), Clinical studies with an articular index for the assessment of joint tenderness in patients with rheumatoid arthritis. Quaterly J. Med., 37, 393-406. Ropes, M.W., Bennett, G.A., Caleb, S., Jacox, R. & Jessar, R.A. (1958), Revision of diagnostic criteria for rheumatoid arthritis. Bull. Rheum. Dis., 9, 175-176. Sanders, M.E., Malegapuru, W., Shaw, M. Jr Shaw, S. (1988), Human naive and memory T cells : reinterpretation of helper-inducer and suppressor-inducer subsets. Zmmunol. Today, 9, 195-199. Schmid, I., Schmid, P. & Giorgi, J.V. (1988), Conversion of logarithmic channel numbers into relative linear fluorescence intensity: Cytometry, 9, 533-538. Smith, M.D. & Roberts-Thomson, P.J. (1990), Lympho: cyte surface marker expression in rheumatic disease ; evidence for prior activation of lymphocytes in vivo. Ann. Rheum. Dis., 49, 81-87. Springer, T.A. (1990), Adhesion receptors of the immune system. Nature (Lond.), 346, 425-434. Struyf, N. J., Snoeck, H. W., Bridts, C.H., de Clerck, L.S. & Stevens, W. J. (1990), Natural killer cell activity in Sjogren’s syndrome and systemic lupus erythematosus : stimulation with interferons and interleukin-2 and correlation with immune complexes. Ann. Rheum. Dis., 49, 690-693. Van Dinther-Janssen, A.C,, Horst, E., Koopman, G:r Newmann, W., Scheper, R.J., Meijer, C.J. & Pals, S.T. (1991). The VLA:UVCAM-1 pathway is involved in lymphocyte adhesion to endothelium in rheumatoid synovium. Jr Zmmunol., 147,4207-4210.
Res. Immunol.
INSTITUT PASTEUR/ELSEVIER
Paris 1994
1994, 145, 101-108
Lymphocyte activation status, expression of adhesion molecules and adhesion to human endothelium in rheumatoid arthritis - relationship to. disease activity A.V. Mertens, L.S. de Clerck, M.M. Moens, C.H. Bridts and W. J. Stevens Department
of Immunology,
Allergology and Rheumatology, WA, Ant werp (Belgium)
University
of Antwerp,
SUMMARY
The synovial tissue of patients with rheumatoid arthritis WA) is characterized by infiltration with inflammatory cells, mainly memory helper cells (CD4+CD29+). An important initiating step in tissue infiltration Is the adhesion of peripheral blood lymphocytes to the vascular endothelium. Therefore, we studied lymphocyte-endothelium adhesion in 40 RA patients and in 19 controls by a sensitive fluorimetric assay, using human umbilical vascular endothelial cells. Furthermore, expression of adhesion molecules VLA (CD291 and LFA-I (CD1 la) on CD4+ and CD8+ T cells was determined. In order to evaluate the activation state of lymphocytes, the soluble interleukin-2 receptor (slL2R) was measured. The relationship to disease activity was evaluated using the Ritchie artitular index. RA patients had a higher percentage of CD4+ cells (p c 0.0051 and a lower percentage of CD8+ cells (p < 0.001) than controls did. The CD4+CD29+/ CD4+CD29- ratio and the CD8+CD29+/CD8+CD29ratio were increased in patients with active RA (p < 0.01 and p c 0.05, respectively) and in patients with inactive disease (p= 0.09 and p < 0.005, respectively) compared with controls. LFA-1 (CD1 la) was present on almost all T lymphocytes and its density did not differ between patients and controls. Serum levels of slL2R were significantly higher in both patient groups compared with controls (p < 0.0005); patients with active disease showed significantly higher levels than patients with inactive disease (p < 0.05). Lymphocyte-endothelium adhesion was not increased in patients, although the expression of the adhesion molecule CD29 on T lymphocytes of RA patients was higher. These results demonstrate that peripheral blood lymphocytes of RA patients are activated, especially in patients with active disease, as demonstrated by elevated slL2R levels and increased numbers of CD29+ cells. Nevertheless, there was no increased lymphocyte adhesion to endothelial cells in RA patients compared with controls, demonstrating that functional adhesion properties are not always correlated. with the mere presence of CD29 or other adhesion molecules on the cell membrane. Key-words: T lymphocyte, Inflammation, Rheumatoid arthritis, Endothelium; Adhesion, Cell activation, IL2, Disease activity.
Submitted September20, 1993,acceptedDecember29, 1993. Corresponding address: Prof. W.J. Stevens, Department of Immunology, Allergology and Rheumatology, University of Antwerp, UIA, Universiteitsplein 1, 2610 Antwerp. Belgium.
A. V. MERTENS
102 INTRODUCTION
Rheumatoid arthritis (R4) is a systemic inflammatory disease associated with chronic joint inflammation. Histopathologic findings consist of hyperplasia of the synovium, generation of new blood vessels and infiltration of mononuclear cells (Kulka et al., 1955). The cellular infiltrates are mainly built up by T lymphocytes, especially memory helper cells (CD4+ CD29+) (Pitzalis et al., 1982; Cush and Lipsky, 1991). For the recruitment of these cells from the peripheral blood, adhesion to and migration through vascular endothelium are important. Adhesion is regulated by adhesion molecules on circulating lymphocytes, and these molecules bind to their ligand on endothelial cells (Springer, 1990). The aim of this study was to investigate adhesion of peripheral blood lymphocytes to human umbilical vascular endothelial cells, by using a sensitive fluorimetric method. Together with functional adhesion, phenotypic analysis of adhesion molecule LFA- 1 (CD 11 a/CD 18) and activation marker/adhesion molecule VLA (CD29) on T lymphocytes was performed. Since the lymphocyte infiltration in synovium is much more pronounced in patients with aggressive disease (Gupta and Talal, 1985), we wanted to correlate adhesion properties with clinical disease activity, assessed by the Ritchie articular index, and with T-cell activation, evaluated by measuring soluble interleukin-2 receptor (sIL2R) in serum (Keystone et al., 1988).
MATERIALS
AND METHODS
Patients Forty-three patients (mean age 58 years, range 24-81) with classical or definite RA according to the ARA-criteria (Ropes et al., 1958) were studied. Activity of the disease was scored with the Ritchie artitular index (Ritchie et al., 1968). A Ritchie score
EC FCS
= endothelial cell. = foetal calf serum.
ET AL. of more than 10 was considered as active RA (16 patients). None of the patients used corticosteroids ; 39 out of 43 used non-steroidal antiinflammatory drugs with or without other antirheumatic medication: oral (2) or IM (4) gold, antimalarials (3), sulphasalazine (1 l), azathioprine (5), methotrexate (11) or penicillamin (1). Nineteen healthy volunteers (mean age 34 years, range 24-48) were included as control group. Cell separation Leukocytes were isolated from IO ml venous blood by discontinuous Percoll gradient centrifugation (60 % and 72.5 % isotonic Percoll). The mononuclear cells were suspended in CO,-independent medium (Gibco Life Technologies, Ghent, Belgium, 041-08045 M) supplemented with 1 % foetal calf serum (J?CS ; Gibco), 100 U/ml penicillin and 100 $/ml streptomycin (Gibco) and were added to culture flasks (Primaria, Falcon, Becton Dickinson, Mountain View, CA, USA) for 30 min, to eliminate the monocytes by adherence. For further experiments, the purity of the lymphocyte population was always 30 % or more (median 94 %, range 80-98 To). Flow cytometric
analysis
The P,-integrin VLA (CD29) and the P,-integrin LFA-1 (CD1 la/CD18) were determined on CD4+ and on CDS+ cells. Fifty ~1 of peripheral blood were incubated with 20 ~1 fluorescein-isothiocyanateand 20 ~1 phycoerythrin-labelled monoclonal antibody (purchased from Immunotech, Marseille, France and from Dako, Glostrup, Denmark) for 15 min at 4°C. After incubation, the remaining red blood cells were lysed with cold NH&l-lysis buffer and the leukocytes were fvted in 1 % paraformaldehyde in phosphate-buffered saline. Flow cytometric analysis was performed on a “Facscan” (Becton Dickinson), standardized with fluorescent beads (Fluoresbrite intensity kit, Polysciences, Warrington, PA, USA), according to Schmid (Schmid et al., 1988). Fluorescence of 10,000 cells was evaluated, and Iymphocytes were gated on forward and site scatter characteristics. The percentage of positive lymphocytes was evaluated, and the mean density for each marker was determined using the mean fluorescence intensity, expressed as mean channel number (Schmid et al., 1988).
HUVEC RA
= =
human umbilical vein endothelial cell. rheumatoid arthritis.
L YMPHOCYTE-ENDOTHELIUM
ADHESION
Adhesion assay Endothelial
IN RHEUMATOID
103
With this method, an adhesion of 6,000 cells or more can be measured.
cell culture Detection
Endothelial cells (EC, i.e. human umbilical vascular endothelial cells, HUVEC) were obtained from umbilical cords by collagenase-dispase (Boehringer, Mannheim, Germany) digestion, using the technique of Jaffe et al. (1973) with minor modifications. The EC were suspended in growth medium Ml99 (Gibco) supplemented with 20 % FCS (Gibco), 100 U/ml penicillin and 100 yg/ml streptomycin (Gibco), 30 yg/ml EC growth supplement (Sigma Chemie, Brussels, Belgium) and 10 U/ml sodium heparin (Sigma). EC were seeded at a density of 3,000/cm2 in culture flasks (Falcon 25 cm2, Becton Dickinson) pretreated with 2 % gelatin (Sigma) in water for 30 min and cultured in a 5 % CO, humidified incubator (Jouan, Saint Herblain, France) at 37°C for 2 to 5 days, until confluence was observed. At confluence, EC were detached using 0.125 % trypsinEDTA in modified Pucks saline A (Gibco). The cells were confirmed to be EC by positive immunofluorescent staining for Factor-VIII-related antigen.
HUVEC
ARTHRITIS
adhesion assays
From the second to the fourth passage, EC were transferred into flat-bottomed, gelatin-coated 96well microtitre plates (Falcon), at a concentration of 35,000 EC per well. After overnight culture, confluent monolayers were obtained and used in the adhesion experiments. EC were stimulated with 100 ~1 recombinant interleukin-1-P (rIL1P; Janssen Biochimica, Beerse, Belgium ; 20 U/ml in growth medium) or were suspended in growth medium as control and incubated for 6 h at 37°C. Afterwards, the EC were washed twice with CO,-independent medium (Gibco, 041-08045 M) supplemented with 1 % FCS (Gibco), 100 U/ml penicillin and 100 d/ml streptomycin (Gibco). Lymphocytes (10’ cells/ml) were labelled with 300 pg/ml 5(6)-carboxyfluorescein-diacetate (CFDA, Sigma) for 15 min at 37°C (Struyf et al., 1990). After labellingd the cells were washed twice and concentrated at 10 cells/ml The remaining red blood cells were lysed with cold NH,Cl-lysis buffer, and after washing, ILl-stimulated and unstimulated EC were incubated with 100,000 lymphocytes per well at 37°C for 30 min. After incubation, total fluorescence (F,) was measured on a “Cytofluor 2300” (Millipore, Bedford, MA, USA) fluorometer. The non-adherent cells were removed by washing four times. The remaining fluorescence (F,) was measured, and the percentage of adhesion was calculated using the following formula: 070adhesion= 100 x (F./F,).
of sJL2R in serum
The quantitative determination of serum sIL2R was done with a sandwich enzyme-linked immunosorbent assay (Eurogenetics, Tessenderlo, Belgium), with two monoclonal antibodies recognizing different epitopes of the IL2R. The serum levels were expressed in U/ml. Statistical
analysis
The Student t test, Mann-Whitney CJ test and Pearson and Spearman rank correlation test were used, where appropriate. .
RESULTS Phenotypical
analysis
(table I)
RA patients had significantly more peripheral blood T helper cells (CD4+) and less T suppressor cells (CD8+) than controls had, resulting in a higher CD4/CD8 ratio. The percentage of memory T helper cells (CD4+CD29+) was significantly higher in patients with active disease and in patients with inactive disease compared with controls. Within the CD4+ subset, the ratio of CD4+CD29+/CD4+CD2gm cells was higher in patients with active disease compared with controls. The percentage of memory T suppressor cells (CD8+CD29+) was lower only in patients with active disease, due to the lower number of CD8+ cells. Indeed, when the ratio of CD8+CD29+/CD8+CD29was made, RA patients showed a significantly higher fraction of CD8+ cells bearing CD29 than controls did CD1 la was present on almost all T cells .of patients (median (range) : 100 (40-100) Vo) and controls (100 (74-100) Vo). Densities of adhesion molecules CD29 and CD1 la, measured on CD4+ and CD8+ subsets, did not differ between the study groups (table II). There was a weak but significant correlation between the density of CD29 on CD4+ cells and the Ritchie articular index (r =0.33, p < 0.05; fig. 1).
104
A. V. MERTENS
ET AL.
Table I. T-lymphocyte subsets (percentage positive cells and ratio (median (range)) in controls and in RA patients with inactive disease (Ritchie score < 10) and RA patients with active disease (Ritchie score > 10).
Vo CD4+ “70 CD4+CD29+ CD4+CD29+/ CD4+CD29% CD8+ % CD8+CD29+ CD8+CD29+/ CD8+CD29CD4+ /CD8 +
Controls (n= 18)
total (n = 41)
RA patients inactive (n = 26)
active (n = 15)
42 (32-61) 20 (12-29) 0.85 (0.41-1.79)
52 (23-79) (***) 27 (11-55) (****) 1.27 (0.38-3.67) (*)
51 (23-77) (*‘) 27 (1 l-42) (***) 1.21 (0.38-3.36)
55 (37-79) (***I 28 (20-55) (****) 1.29 (0.80-3.67) (“)
31 (16-47) 16 (10-32) 1.27 (0.45-5.0)
19 (5-43) (****)
15 (5-38) (****)
;;&?&Jo)
1.5 (0.9-2.9)
2.7 (1.0-15.8) (****)
(***)
11 (2-33) (‘) ::7?,?~7-6.60)
(‘)
2.5 (1 .O-9.6) (***)
3.4 (1.0-15.8) (****)
(*) p < 0.05, (**) p < 0.01, (***) p < 0.005 and (****) p < 0.001 RA versus controls.
sIL2R levels in sera from controls and RA patients The serum sIL2R levels in patients were significantly higher than in controls (median (range): 110 (99-293) U/ml verSu.s 101 (96-130) U/ml, p < 0.0005; fig. 2). Levels in patients with active disease were significantly higher than
levels in patients with inactive disease (115 (104-293) U/ml versus 108 (99-277) U/ml, p c 0.05). A positive correlation was found between level of sIL2R and the CD4+CD29+ /CD4+CD29ratio (r=0.30, p < 0.05) and also between sIL2R level and the CD8+CD29+ /CD8+CD29ratio (r =0.35, p < 0.01). Adhesion of peripheral blood lymphocytes to HUVEC (fig. 3) Adhesion was measured on unstimulated endothelium and on ILl-stimulated endothelium. Percentage of adherent cells was comparable for RA patients and controls in both unstimulated and stimulated conditions. No differences were found between pafients with inactive diseqse and patients with active disease. Adhesion on ILl-stimulated HUVEC was, however, increased in all subjects compared with adhesion on unstimulated endothelium
(p < 0.05 in controls, p < 0.0005 in RA patients with active disease and p c 0.0001 in RA patients with inactive disease). DISCUSSION
T lymphocytes certainly play a major role in the pathogenesis of RA. The cellular infiltrates in the synovial tissue are built up mainly by memory helper cells (CD{+CD29+) (Pitzalis et al., 1988; Cush and Lipsky, 1991). To infiltrate the synovium, peripheral blood lymphocytes first have to adhere to endothelial cells before they can pass through the vascular endothelium and migrate to the site of inflammation. The binding of adhesion molecules on lymphocytes with their ligand on endothelium is an important mechanism in adhesion (Springer, 1990). In this study, we evaluated several aspects of lymphocyte function in paiients with &4 : (1) the adhesion of peripheral blood lymphocytes to endothelial celis, (2) the expression of adhesioq molecules on circulating T lymphocytes, (3) the activation state of lymphocytes and (4) the relationship of all these parameters to disease activity. In comparison with a control population, we found a higher percentage of peripheral blood CD4+ T helper cells and a lower percentage of
L YMPHOCYTE-ENDOTHELIUM
ADHESION
IN RHEUMATOID
ARTHRITIS
105
Table II. Densities of the adhesion molecules (expressed in mean channel number (median (range)) on CD4+ and CDS+ cells in controls and in RA patients with inactive disease (Ritchie score < 10) and RA patients with active disease (Ritchie score > 10). Controls (n= 18)
Density CD29 on CD4+ CD1 la on CD4+ CD29 on CD8 + CDlla on CD8+
cells cells cells cells
404 415 374 436
total (n = 41)
(262-456) (250-471) (265-418) (275-525)
CD29 DENSITY 45oj
391 427 378 478
(281449) (282-486) (287-459) (302-532)
RA patients inactive (n = 26) 389 436 378 496
(281-433) (282-486) (287-432) (302-530)
active (n = 15) 400 424 380 467
(321-449) (354-479) (3 16-459) (394-532)
sIL-ZR(U/ml) 0 0
x OC 0
400 i
0
00 0
0 0
0
0 0
0 0
0
0
I50 v
0 0
+ ;
y=2.47~+368
*: %
(r=0.33)
Fig. 1. CD29 density on CD4+ peripheral blood T lymphocytes of RA patients in relation to clinical arthritis activity as measured by the Ritchie articular index.
Fig. 2. sIL2R in peripheral blood of controls (CTRL, n = 19) and patients with FM (n = 43).
CDS+ T suppressor cells in RA patients, resulting in a higher CD4KD8 ratio. This finding confirms the results of Smith (Smith and Roberts-Thomson, 1990), but is in contrast to others (vaurer et al., 1992). Although there are controv,$esial results in the literature, an increased CD4/CDg ratio mostly due to a decrease in CD!+ cells in patients with gctive disease is reported by many authors (for review see Cush and Lipsky, 1991; Creamer, 1992). In the present study, the significantly higher ratio of CD4+CD29+/CD4+CD29cells in
RA patients with active disease and of the CDS+CD29+/CD8+CD29ratio in both RA groups indicate activation of these cells (Sanders et al., 1988). Although not unexpected for a chrBnjc inflammatory diseasf, changes in the memory phenotype have ngt been found by SO~F other groups (Garcia-Ujcuna et al., 1992 ; Maurer et al., 1992 ; Postigo ef al., 1992) possibly due to smaller numbers and/or selection criteria of patients. It is known that corticosteroids can influence lymphocyte phenotypes (Raziuddin et al., 1990; Frey et al., 1984).
A. V. MERTENS ET AL.
106
Adhesion
(%I A
B _J35.3
_L
2b.5
20-
Fig. 3. Adhesion of T lymphocytes from controls (A), RA patients with inactive disease(Ritchie score< 10)(B), andRA patientswith activedisease (Ritchiescore> 10)(C) on unstimulated(( -)ILl) and on ILl-stimulated (( +)ILl) endothelium.
In order to avoid this bias, all patients using corticosteroids were excluded from the study. In the RA population studied, a significantly higher level of sIL2R was found, especially in patients with active disease, also demonstrating lymphocyte activation, as has been reported (Keystone et al., 1988). Moreover, increased expression of cell surface IL2 receptor has also been described in RA (Maurer et al., 1992). Since RA patients express more CD29 on their peripheral blood T cells, and since mainly CD29+ cells are found in synovial tissue (Cush and Lipsky, 1988; Laffon et al., 1991; Van Dinther-Janssen et al., 1991), we further investigated, in the second part of this study, the role of CD29 as adhesion molecule. Indeed, in addition to its function as activation marker, CD29 is also an important adhesion molecule. Furthermore, another adhesion molecule, i.e. CD1 la, was also determined, since it has been shown that at least two different molecular pathways exist that affect lymphocyte adhesion to cultured endothelial cells: the leukocyte integrin LFA-1 (CDlla/CD18) is responsible for the binding of lymphocytes to unstimulated endothelial cells via its ligand ICAM- or ICAM- (Haskard et al., 1986), whereas the interaction between the leu-
kocyte integrin VLA-4 (CD29/CD49d) with its ligand VCAM-1 on endothelial cells is important for the adhesion of lymphocytes to cytokinestimulated endothelium (Carlos et al., 1990). Thus, in the part of this work dealing with functional aspects, we studied the adhesion of lymphocytes to cultured human endothelial cells in relation to expression of the adhesion molecules CD29 and CD1 1a. Since CD 1la is present on almost alI T lymphocytes, we also determined the density of the adhesion molecules by the use of flow cytometric analysis. In contrast with synovial tissue lymphocytes (Cush et al., 1992), peripheral blood lymphocytes of the RA patient group did not express more CD29 or CDlla than controls did. Nevertheless, a correlation was found between the density of CD29 on CD4+ cells and the Ritchie articular index, suggesting that more aggressive disease is related to activation of peripheral blood lymphocytes. Although there is no direct evidence that the adhesion of lymphocytes to HUVEC in vitro is an equivalent process to the adhesion to microvascular endothelial cells in vivo, the validity of the model is supported by in vitro experiments (Bender et al., 1987; Haskard et al., 1987). In order to mimic inflammation in vitro,
L YMPHOCYTE-ENDOTHELIUM
ADHESION
endothelial cells were stimulated with IL1 (Cotran and Pober, 1988). Our sensitive in vitro model for adhesion, on both unstimulated and ILl-stimulated HUVEC, gave comparable results for controls and patients, although adhesion was clearly increased on stimulated endothelium for all subjects ; thus the presence of more T cells expressing CD29 in RA patients was not reflected in increased adhesion. In conclusion, the results of this study demonstrate that peripheral blood lymphocytes of RA patients are activated, especially in patients with active disease, as indicated by elevated sIL2R levels and increased expression of CD29 on T lymphocytes. Nevertheless, there was no increased lymphocyte adhesion to endothelial cells in RA patients compared with controls, demonstrating that functional adhesion properties are not always correlated with the mere presence of CD29 or of other adhesion molecules on the cell membrane.
Acknowledgements The authors wish to thank Mrs. Chris De Beul and Mrs. lnge Vereecke for expert technical assistance. ELISA kits for determination of slL2R were kindly provided by Dr. Bosmans (Eurogenetics, Belgium). This paper presents research results of the Belgian programme on Interuniversity poles of attraction initiated by the Belgian State, Prime Minister’s Office, Science Policy Programming. The scientific responsibility is assumed by its authors. This study was also supported by Levenslijn Grant no. 7.0044.91 of the Nationaal Fonds voor Wetenschappelijk Onderzoek.
IN RHEUMATOID
ARTHRITIS
107
sion lymphocyte-endothklium dans 40 cas d’AR et chez 19 tCmoins, par un test fluorimetrique sensible, utilisant des cellules endothkliales ombilicales. De plus, I’expression des molicules d’adhesion VLA
(CD29) et LFA-1 (CD1 la) sur les cellules T CD4+ et CD8+ a &e analysee. Pour ttudier 1’Ctat d’activation des lymphocytes, le rkcepteur IL2 soluble (sIL2R) a ttt &al&. La relation avec 1’activitC de la
maladie a et6 analyske g l’aide de I’index de Ritchie. Les malades atteints d’AR ont un pourcentage de CD4+
(p < 0,005) plus ClevC que celui des tCmoins
et un pourcentage de CD8+ plus bas (p < 0,001). Le rapport CD4+CD29+/CD4+CD29et le rapport CDS+CD29+/CD8+CD29sont augment& dans 1’AR active (p < 0,Ol et p < 0,05 respectivement) et dans I’AR non active (p= 0,09 et p < 0,005 respectivement) par rapport aux tCmoins. La molecule LFA-1 (CD1 la) est prCsente sut presque tous les lymphocytes T et la densitC de cette prCsence est iden-
tique chez les malades et les ttmoins.
Les taux ski-
ques d’sIL2R sont significativement plus ClevCs dans les deux groupes de malades par rapport aux ttmoins (p < 0,OOOS); les malades atteints d’AR active ont des taux significativement plus Bevts par rapport aux AR non actives (p < 0,05). L’adhtsion lymphocyteendothClium n’est pas accrue chez les malades, bien que l’expression de la molCcule d’adhCsion CD29 sur les lymphocytes T soit plus importante dans I’AR. Ces rtsultats montrent que les lymphocytes p&iphtriques sont activCs dans I’AR, surtout dans I’AR active, comme l’indiquent les taux eleves d’sIL2R et
le nombre ClevC de cellules CD29+.
Cependant on
n’observe pas d’augmentation de 1’adhCsion lymphocytaire aux cellules endothCliales dans I’AR, par rapport aux tCmoins, ce qui demontre que les proprittCs fonctionnelles d’adhbion ne sont pas toujours en corrClation avec la simple presence de CD29 ou d’autres molCcules d’adhesion sur la membrane cel-
lulaire. Mets-cl&s: Lymphocyte T, Inflammation, Arthrite rhumatdide, Endothelium; AdhCrence, Activation cellulaire, IL2, ActivitC de la maladie.
Activation lymphocytaire, expression de I’adhCsion mol&ulaire et adhesion a I’endotht?lium vasculaire dans I’arthrite rhumatdide, relations avec I’activitC de la maladie Le tissu synovial de malades atteints d’arthrite rhumato’ide (AR) est caracttrisk par un infiltrat de cellules inflammatoires, notamment de cellules (( helper )) CD4+CD29+. Dans I’infiltration cellulaire, une &ape d’initiation capitale est I’adhksion des lymphocytes sanguins pkriphiriques g I’endoth&lium vasculaire. C’est pourquoi nous avons Ctudie I’adhC-
References Bender, J. R., Pardi, R., Karasek, M.A. & Engleman, E.G. (1987), Phenotype and functional characterization of lymphocytes that bind human microvascular endothelial cells in vitro. Evidence for preferential binding of natural killer cells. J. Clin. Invest., 79, 1679-1688. Carlos, T.M., Schwartz, B.R., Kovach, N.L., Yee, E., Rosso, M., Osborn, L., Chi-Rosso, G., Newman, B., Lobb, R. & Harlan, J.M. (1990), Vascular cell adhe-
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sion molecule-l (VCAM-1) mediates lymphocyte adherence to cytokine-activated cultured human endothelial cells. Blood, 76, 965-970. Cotran, R.S. & Pober, J.S. (1988). Endothelial activation, in “Endothelial cell biology” (pp. 335-347). Plenum Publ. Cor., New York. Creamer, P. (1992), Flow cytometry and rheumatic disease. Brit. J. Rheumatol., 31, 465472. Cush, J.J. & Lipsky, P.E. (1988), Phenotypic analysis of synovial tissue and peripheral blood lymphocytes isolated from patients with rheumatoid arthritis. Arthritis Rheum., 31, 1230-1238. Cush, J.J. & Lipsky, P.E. (1991), Cellular basis for rheumatoid inflammation. Clin. Orthop. Res.. 205, 9-22. Gush, J.J., Pietschmann, P., Oppenheimer-Marks, N. & Lipsky, P.E. (1992), The intrinsic migratory capacity of memory T cells contributes to their accumulation in rheumatoid synovium. Arthritis Rheum., 35, 1434-1444. Frey, B.M., Walker, C., Frey, J.D. & de Week, A.L. (1984), Pharmacokinetics and pharmacodynamics of three different prednisolone prodrugs : effect on circulating lymphocyte subsets and function. J. Immunol., 133, 2479-2487. Garcia-Vicuna, R., Humbria, A., Postigo, A.A., LopezElzaurdia, C., de Landazuri, M.O., Sanchez-Madrid, F. & Laffon, A. (1992), VLA family in rheumatoid arthritis : evidence for in vivo regulated adhesion of synovial fluid T cells to fibronectin through VLA-5 integrin. Clin. exp. Immunol., 88, 435441. Gupta, S. & Talal, N. (1985), Pathogenetic mechanisms, in “Immunology of rheumatic diseases” (pp. 517542). Plenum Publ. Co., New York. Haskard, D.O., Cavender, D., Beatty, P., Springer, T.A. & Ziff, M. (1986), T-lymphocyte adhesion to endothelial cells : mechanisms demonstrated by antiLFA-1 monoclonal antibodies. J. Immunol., 137, 2901-2906. Haskard, D.O., Cavender, D., Fleck, R.M., Sontheimer, R. & Ziff, M. (1987), Human dermal microvascular endothelial cells behave like umbilical vein endothelial cells in T-cell adhesion studies. J. Invest. Dermatol., 88, 340-344. Jaffe, E.A., Nachman, R.L., Becker, C.G. & Minick, C.R. (1973), Culture of human endothelial cells derived from umbilical veins. J. Clin. Invest., 52, 2745-2756. Keystone, E.C., Snow, K.M., Bombardier, C., Chang, C.H., Nelson, D.L. & Rubin. L.A. (1988), Elevated soluble interleukin-2 receptor levels in the sera and synovial fluids of patients with rheumatoid arthritis. Arthritis Rheum., 3 1, 844-849. Kulka, J.P., Backing, D., Ropes, M.W. & Bauer, W. (1955), Early joint lesions of rheumatoid arthritis. Arch. Pathol. Lab. Med., 59, 129-150. Laffon, A., Garcia-Vicuna, R., Humbria, A., Postigo, A.A., Corbi, A.L., de Landazuri, M.O. & SanchezMadrid, F. (1991), Upregulated expression and func-
ET AL. tion of VLA4 fibronectin receptors on human activated T cells in rheumatoid arthritis. J. Clin. Invest., 88, 546-552. Maurer, D., Felzmann, T., Holter, W., Petera, P., Smolen, J. & Knapp, W. (1992), Evidence for the presence of activated CD4 T cells with naive phenotype in the peripheral blood of patients with rheumatoid arthritis. Clin. exp. Immungl., 87, 429-434. Pitzalis, C., Kingsley, G., Haskard, D. & Panayi, Cj. (1988) The preferential accumulation of helperinducer T lymphocytes in inflammatory lesions : evidence for regulation by selective endothelial and homotypic adhesion. Eur. J. Immunol., 18, 1397-1404. Postigo, A.A., Garcia-Vicuna, R., Diaz-Gonzalez, F., Arroyo, A.G., de Landazuri, M.O., Chi-Rosso, G., Lobb, R.R., Laffon, A. & Sanchez-Madrid, F. (1992), Increased binding of synovial T lymphocytes from rheumatoid arthritis to endothelial-leukocyte adhesion molecule-l (ELAM-1) and vascular cell adhesion molecule-l (VCAM-1). J. Clin. Invest., 89, 1445-1452. Raziuddin, S., Nur, M.A. & Al Wabel, A.A. (1990), Increased circulating HLA-DR+ CD4+ T cells in systemic lupus erythematosus : alterations associated with prednisolone therapy. Stand. J. Zmmunol., 31, 139-145. Ritchie, D.M., Boyle, J.A. & McInnes, J.M. (1968), Clinical studies with an articular index for the assessment of joint tenderness in patients with rheumatoid arthritis. Quaterly J. Med., 37, 393-406. Ropes, M.W., Bennett, G.A., Caleb, S., Jacox, R. & Jessar, R.A. (1958), Revision of diagnostic criteria for rheumatoid arthritis. Bull. Rheum. Dis., 9, 175-176. Sanders, M.E., Malegapuru, W., Shaw, M. Jr Shaw, S. (1988), Human naive and memory T cells : reinterpretation of helper-inducer and suppressor-inducer subsets. Zmmunol. Today, 9, 195-199. Schmid, I., Schmid, P. & Giorgi, J.V. (1988), Conversion of logarithmic channel numbers into relative linear fluorescence intensity: Cytometry, 9, 533-538. Smith, M.D. & Roberts-Thomson, P.J. (1990), Lympho: cyte surface marker expression in rheumatic disease ; evidence for prior activation of lymphocytes in vivo. Ann. Rheum. Dis., 49, 81-87. Springer, T.A. (1990), Adhesion receptors of the immune system. Nature (Lond.), 346, 425-434. Struyf, N. J., Snoeck, H. W., Bridts, C.H., de Clerck, L.S. & Stevens, W. J. (1990), Natural killer cell activity in Sjogren’s syndrome and systemic lupus erythematosus : stimulation with interferons and interleukin-2 and correlation with immune complexes. Ann. Rheum. Dis., 49, 690-693. Van Dinther-Janssen, A.C,, Horst, E., Koopman, G:r Newmann, W., Scheper, R.J., Meijer, C.J. & Pals, S.T. (1991). The VLA:UVCAM-1 pathway is involved in lymphocyte adhesion to endothelium in rheumatoid synovium. Jr Zmmunol., 147,4207-4210.