Scand. J. Immunol. 10, 479-486, 1979
Rheumatoid Synovial Lymphocytes Lack Concanavalin-A-activated Suppressor Cell Activity C. CHATTOPADHYAY, H. CHATTOPADHYAY, J. B. NATVIG & O. J. MELLBYE Institute of Immunology and Rheumatology, Rikshospitalet, The National Hospital, Oslo, Norway
Chattopadhyay, C , Chattopadhyay, H., Natvig, J.B. & Mellbye, O.J. Rheumatoid Synovial Lymphocytes Lack Concanavalin-A-activated Suppressor Cell Activity. Scand. J. Immunol. 10, 479-486, 1979. Synovial lymphocytes eluted by enzyme treatment from eleven patients with rheumatoid arthritis (RA) were investigated for the presence of concanavalin A (Con A)-activated suppressor cell activity as compared with that of peripheral blood lymphocytes of twenty normal donors. In addition, two patients with psoriatic arthritis and juvenile rheumatoid arthritis (JRA) were also investigated. Synovial lymphocytes from the eleven RA patients showed a mean augmentation of 28±13.30, and thus clearly lacked suppressor activity, whereas the mean suppression in the lymphocytes from the twenty normal donors was 13 ±14.40. Synovial lymphocytes from one patient with JRA and one with psoriatic arthritis showed a normal suppressor activity. C. Chattopadhyay, Institute of Immunology and Rheumatology, F. Qvamsgt. 1, Oslo 1, Norway.
Rheumatoid arthritis (RA) is a disease of unknown aetiology associated with striking immune abnormalities. The main immunopathological changes are manifested in the rheumatoid joint [19]. The large number of lymphocytes infiltrating the joint has been the subject of many investigations in recent years [2, 5, 15]. Although there is some controversy about the relative proportion of the various lymphoid cell types in the synovial fluid [13], it seems that in the synovial cells eluted by enzyme treatment the T cells are the predominant cell type. These T cells are functionally largely similar to the T cells in normal peripheral blood [3]. It is now well established both in human and in murine systems that the T cells are heterogeneous, possibly with different functions [14]. One such function is to suppress the blastogenic response in the presence of concanavalin A (Con A)-activated cells [17]. Using this method of investigation, abnormalities involving suppressor cells have been demonstrated in various
disease conditions and in old age [1, 10, 11]. However, our knowledge of suppressor cell systems in rheumatoid arthritis and the possible role it plays in this condition is meagre. We have recently reported that the eluted synovial T cells from many RA patients are incapable of suppressing immunoglobulin production at high T-cell to B-cell ratios in contrast to T cells from normal peripheral blood [8], which show strong suppression of Ig synthesis by B cells. The object of the present study was to investigate whether a similar trend also was present in T-cell to T-cell interaction in these patients as measured by Con A-activated suppressor cell system. We have been able to demonstrate that lymphocytes from the synovial tissue of many RA patients indeed lack suppressive activity in this system.
MATERIALS AND METHODS Patients and controls. Eleven patients with clinically
0300-9475/79/1100-0479 $02.00 © 1979 Blackwell Scientific Publications
479
480
C. Chattopadhyay et al.
active RA were investigated. Two other patients were also studied, one with psoriatic arthritis and the other with juvenile rheumatoid arthritis (JRA). Twenty healthy donors served as controls. Isolation of mononuclear cells from synovial tissue. The cells from the synovial tissues were eluted according to the procedure described previously [2]. In brief, the synovial tissue, obtained at operation, was cut into small pieces and treated with deoxyribonuclease (Sigma Dn 100) and collagenase (Sigma Cn 0130). After enzyme treatment for 45 min the cells were passed through a nylon mesh to get rid of contaminating fat. The cells were then centrifuged at 4''C and subsequently incubated overnight in plastic tissue culture flasks with 10% fetal calf serum (FCS) in RPMI-1640 with antibiotics. The next morning the cells were separated on Isopaque/Ficoll (Lymphoprep. Nyegaard A/S, Oslo) according to the method of Boyum [4]. The cells thus separated were washed three times in Hanks' balanced salt solution (BSS) and finally resuspended in RPMI-1640 supplemented with Lglutamine 20 mM, 20% fetal calf serum, penicillin (100 IU/ml), and streptomycin (100 (Xg/ml). The cell concentration was adjusted to 1x10° cells/ml. Separation of peripheral blood mononuclear cells (PBL). Blood was collected in heparinized tubes and the mononuclear cells were separated on Isopaque/Ficoll gradient. The cells were washed three times in Hanks' BSS and resuspended in the culture medium at a concentration of 1 X 10" cells/ml. Suppressor cell assay. Generally 4-8 X 10° cells were incubated with or without Con A for 48 h in a humidified atmosphere enriched with 5% COj. After 48 h of incubation the cells were treated with mitomycin-C for 30 min and then washed three times. These cells are called efl"ector cells. In some experiments the cells were washed twice with 0.3 M solution of a-methyl-Dmannoside to remove residual Con A before the washing. Subsequently the cells were washed three more times and resuspended in culture medium; the cell concentration was then adjusted to 1x10° cells/ml. Responder cells from a normal donor were isolated in the same way. One of us (C. Chattopadhyay) acted
as a donor in most of the experiments. The responder cell concentration was adjusted to 1 x 10° cells/ml. The responder cells were incubated for 72 h in the presence of either Con-A-treated or untreated cells (control cells). The experiments were done in triplicate or in quadruplicate in microculture plates to which 0.05 ml of responder cells, 0.05 ml of suppressor or control cell suspension, 0.020 ml of Con A (150 (xg/ml), and 0.05 ml of culture medium were added. In control culture plates 0.020 ml of culture medium was added to ensure a constant volume. In some experiments normal donor cells were treated with enzyme in the same manner as synovial tissue. To each well 0.5 (xCi of ^H-thymidine (specific activity 2 Ci/mmol; The Radiochemical Centre, Amersham, England) was added 18 h before the cells were harvested. This was done with a semiautomatic harvesting machine (Skatron A/S, Norway) on glass fibre filter papers, and incorporation of 'H-thymidine was measured by liquid scintillation counting. Data were corrected for quenching and expressed as mean dpm (disintegrations per minute), and the degree of suppression was calculated according to the following formula:
(
dpmj Con A Percentage suppression = dpmc Con A
RESULTS Characterization of the suppressor cell assay Tofindan optimal system for testing rheumatoid synovial lymphocytes, preliminary experiments were first done to evaluate the effect of
% Suppression! with effector cells from:
20 10 5
100
where dpm^ Con A is the dpm of the responder cells in the presence of control cells (not stimulated with Con A) and Con A, and dpm^ Con A is the dpm of the responder cells in the presence of Con-A-stimulated cells and Con A. The statistical significance of the suppression was calculated by Student's t test.
TABLE 1. The efl'ect of various concentrations of concanavalin A (Con A) on generation of suppressor cell activity of the effector cells*
Concentration of Con A ((xg/ml)
\ /
Normal donor (PBL)}: -19±20§ -13±21 - 6±10
Synovial cells (donor J.A.) + 107 + 83 + 80
* The responder cells were from the peripheral blood of a normal donor and were stimulated with the mitogenic concentration of Con A (20|xg/ml). t A plus sign preceding the percentage suppression means augmentation; a minus sign means suppression. A'^=no. of donors tested. t Peripheral blood lymphocytes. § ± Standard error of the mean.
Suppressor Cells in RA Synovium
481
TABLE II. Effect of varying the effector cell concentration on the percentage suppression of blastogenic response of the responder cells* % Suppression! with effector cells from: No. of cells added 5x10^ 10x10"
Normal donors
Synovial tissue (donors A.F. and S.W.)
-7.55±6.4t -19.47 ±7.0
-24.5±10.9 -16±1.0
* The responder cells were obtained from the peripheral blood of normal donors. Both the effector and the responder cells were stimulated with the mitogenic concentration of concanavalin A (Con A) (20 (xg/ml). t A minus sign preceding the percentage suppression means suppression. N=no. of donors tested. } ± Standard error of the mean.
various doses of Con A required to generate the maximum suppressor cell activity. A mitogenic (20 [J-g/ml) and a submitogenic dose of Con A (10 (J-g/ml) were equally effective in generating suppressor cell activity (Table 1), although a submitogenic dose of 5 (ig/ml seemed less effective. The optimal mitogenic concentration was determined in pilot experiments and was 20 [Ag/ml of culture. Furthermore, the minimum time required to generate optimal suppressor activity was 24 h of exposure to Con A. We also investigated the influence of the effector cell concentration on the degree of suppression (Table II). The doubling of the effector cell concentration increased the suppressor cell activity. Further increase of the effector cell concentrations was not done, to avoid cell overcrowding and subsequent inhibition in our test system.
Next we tried to ascertain the effect of various doses of the Con A during stimulation of the responder cells. Our data revealed that maximum suppression is brought about at submitogenic concentrations of the Con A, but differences between mitogenic and submitogenic concentrations in suppressor effect were small (Table III). We also investigated the effect of doubling the responder cell concentration. This tended to decrease the suppression seen at a 1:1 ratio of the responder to effector cells (Table IV). Four experiments were performed in which the responders cells were obtained from C.C. and other normal donors. Although the percentage suppression varied, the mean percentage suppression did not differ significantly between the experiments (Table V). On the basis of these studies further experiments were performed with 20 (xg/ml of Con A
TABLE III. Effect of various concentrations of concanavalin A (Con A) added to the responder cells during stimulation* , Suppression! of the effector cells from: Concentration of Con A (fig/ml)
Normal donors
Synovial tissue (donors J.A. and A.A.)
20 10 5
-18.84±6.38t -32.1 ±5.97 -32.18+8.17
+ 57±5O + 17±32 + 12±24
* The responder cells were obtained from the normal donors. The effector cells were stimulated with the mitogenic concentration of Con A (20 [xg/ml). t A plus sign preceding the percentage suppression means augmentation; a minus sign means suppression. A'=no. of donors tested. t ± Standard error of the mean.
482
C. Chattopadhyay et al.
TABLE IV. Effect of increasing the responder cell concentration on suppression of the blastogenic response of normal responder cells*
CONTROLS
PATIENTS
-60 Concentrations of cells added
-50
% Suppression!
(A'=4)t
z-40
5x10* 10x10*
o
ll.75+7.42§ -0.045 + 5.58
•
w
* Both the effector and the responder cells were obtained from the peripheral blood of the normal donors and were stimulated with the mitogenic concentration of concanavalin A (Con A) (20 |xg/ml). ! N=r\o. of donors tested. t A plus sign preceding the percentage suppression means augmentation; a minus sign means suppression. § + Standard error of the mean.
t -10 (/)
0
Suppressor activity of the lymphocytes of the rheumatoid tissue In marked contrast to the mean suppressor effect of 13 ± 4.40% in the twenty normal blood donors, the mean effect of the lymphocytes isolated from rheumatoid synovial tissue in the same test system was an augmented response. Thus, the mean augmentation of lymphocytes from eleven patients tested was 28 ±13.30%.
3*30 •
+60^ +110 ' +120
•
FIG. 1. Concanavalin-A-activated suppressor cell activity in patients and controls. Solid lines represent the means. A minus sign means suppression, and a plus sign means augmentation.
Only three patients out of eleven had positive suppressor cell activity, and the rest of the patients did not have any suppressor cell activity (Fig. 1, Table VII). The mean response was significantly different compared with the peripheral blood lymphocytes of the normal donors (P
Rheumatoid Synovial Lymphocytes Lack Concanavalin-A-activated Suppressor Cell Activity C. CHATTOPADHYAY, H. CHATTOPADHYAY, J. B. NATVIG & O. J. MELLBYE Institute of Immunology and Rheumatology, Rikshospitalet, The National Hospital, Oslo, Norway
Chattopadhyay, C , Chattopadhyay, H., Natvig, J.B. & Mellbye, O.J. Rheumatoid Synovial Lymphocytes Lack Concanavalin-A-activated Suppressor Cell Activity. Scand. J. Immunol. 10, 479-486, 1979. Synovial lymphocytes eluted by enzyme treatment from eleven patients with rheumatoid arthritis (RA) were investigated for the presence of concanavalin A (Con A)-activated suppressor cell activity as compared with that of peripheral blood lymphocytes of twenty normal donors. In addition, two patients with psoriatic arthritis and juvenile rheumatoid arthritis (JRA) were also investigated. Synovial lymphocytes from the eleven RA patients showed a mean augmentation of 28±13.30, and thus clearly lacked suppressor activity, whereas the mean suppression in the lymphocytes from the twenty normal donors was 13 ±14.40. Synovial lymphocytes from one patient with JRA and one with psoriatic arthritis showed a normal suppressor activity. C. Chattopadhyay, Institute of Immunology and Rheumatology, F. Qvamsgt. 1, Oslo 1, Norway.
Rheumatoid arthritis (RA) is a disease of unknown aetiology associated with striking immune abnormalities. The main immunopathological changes are manifested in the rheumatoid joint [19]. The large number of lymphocytes infiltrating the joint has been the subject of many investigations in recent years [2, 5, 15]. Although there is some controversy about the relative proportion of the various lymphoid cell types in the synovial fluid [13], it seems that in the synovial cells eluted by enzyme treatment the T cells are the predominant cell type. These T cells are functionally largely similar to the T cells in normal peripheral blood [3]. It is now well established both in human and in murine systems that the T cells are heterogeneous, possibly with different functions [14]. One such function is to suppress the blastogenic response in the presence of concanavalin A (Con A)-activated cells [17]. Using this method of investigation, abnormalities involving suppressor cells have been demonstrated in various
disease conditions and in old age [1, 10, 11]. However, our knowledge of suppressor cell systems in rheumatoid arthritis and the possible role it plays in this condition is meagre. We have recently reported that the eluted synovial T cells from many RA patients are incapable of suppressing immunoglobulin production at high T-cell to B-cell ratios in contrast to T cells from normal peripheral blood [8], which show strong suppression of Ig synthesis by B cells. The object of the present study was to investigate whether a similar trend also was present in T-cell to T-cell interaction in these patients as measured by Con A-activated suppressor cell system. We have been able to demonstrate that lymphocytes from the synovial tissue of many RA patients indeed lack suppressive activity in this system.
MATERIALS AND METHODS Patients and controls. Eleven patients with clinically
0300-9475/79/1100-0479 $02.00 © 1979 Blackwell Scientific Publications
479
480
C. Chattopadhyay et al.
active RA were investigated. Two other patients were also studied, one with psoriatic arthritis and the other with juvenile rheumatoid arthritis (JRA). Twenty healthy donors served as controls. Isolation of mononuclear cells from synovial tissue. The cells from the synovial tissues were eluted according to the procedure described previously [2]. In brief, the synovial tissue, obtained at operation, was cut into small pieces and treated with deoxyribonuclease (Sigma Dn 100) and collagenase (Sigma Cn 0130). After enzyme treatment for 45 min the cells were passed through a nylon mesh to get rid of contaminating fat. The cells were then centrifuged at 4''C and subsequently incubated overnight in plastic tissue culture flasks with 10% fetal calf serum (FCS) in RPMI-1640 with antibiotics. The next morning the cells were separated on Isopaque/Ficoll (Lymphoprep. Nyegaard A/S, Oslo) according to the method of Boyum [4]. The cells thus separated were washed three times in Hanks' balanced salt solution (BSS) and finally resuspended in RPMI-1640 supplemented with Lglutamine 20 mM, 20% fetal calf serum, penicillin (100 IU/ml), and streptomycin (100 (Xg/ml). The cell concentration was adjusted to 1x10° cells/ml. Separation of peripheral blood mononuclear cells (PBL). Blood was collected in heparinized tubes and the mononuclear cells were separated on Isopaque/Ficoll gradient. The cells were washed three times in Hanks' BSS and resuspended in the culture medium at a concentration of 1 X 10" cells/ml. Suppressor cell assay. Generally 4-8 X 10° cells were incubated with or without Con A for 48 h in a humidified atmosphere enriched with 5% COj. After 48 h of incubation the cells were treated with mitomycin-C for 30 min and then washed three times. These cells are called efl"ector cells. In some experiments the cells were washed twice with 0.3 M solution of a-methyl-Dmannoside to remove residual Con A before the washing. Subsequently the cells were washed three more times and resuspended in culture medium; the cell concentration was then adjusted to 1x10° cells/ml. Responder cells from a normal donor were isolated in the same way. One of us (C. Chattopadhyay) acted
as a donor in most of the experiments. The responder cell concentration was adjusted to 1 x 10° cells/ml. The responder cells were incubated for 72 h in the presence of either Con-A-treated or untreated cells (control cells). The experiments were done in triplicate or in quadruplicate in microculture plates to which 0.05 ml of responder cells, 0.05 ml of suppressor or control cell suspension, 0.020 ml of Con A (150 (xg/ml), and 0.05 ml of culture medium were added. In control culture plates 0.020 ml of culture medium was added to ensure a constant volume. In some experiments normal donor cells were treated with enzyme in the same manner as synovial tissue. To each well 0.5 (xCi of ^H-thymidine (specific activity 2 Ci/mmol; The Radiochemical Centre, Amersham, England) was added 18 h before the cells were harvested. This was done with a semiautomatic harvesting machine (Skatron A/S, Norway) on glass fibre filter papers, and incorporation of 'H-thymidine was measured by liquid scintillation counting. Data were corrected for quenching and expressed as mean dpm (disintegrations per minute), and the degree of suppression was calculated according to the following formula:
(
dpmj Con A Percentage suppression = dpmc Con A
RESULTS Characterization of the suppressor cell assay Tofindan optimal system for testing rheumatoid synovial lymphocytes, preliminary experiments were first done to evaluate the effect of
% Suppression! with effector cells from:
20 10 5
100
where dpm^ Con A is the dpm of the responder cells in the presence of control cells (not stimulated with Con A) and Con A, and dpm^ Con A is the dpm of the responder cells in the presence of Con-A-stimulated cells and Con A. The statistical significance of the suppression was calculated by Student's t test.
TABLE 1. The efl'ect of various concentrations of concanavalin A (Con A) on generation of suppressor cell activity of the effector cells*
Concentration of Con A ((xg/ml)
\ /
Normal donor (PBL)}: -19±20§ -13±21 - 6±10
Synovial cells (donor J.A.) + 107 + 83 + 80
* The responder cells were from the peripheral blood of a normal donor and were stimulated with the mitogenic concentration of Con A (20|xg/ml). t A plus sign preceding the percentage suppression means augmentation; a minus sign means suppression. A'^=no. of donors tested. t Peripheral blood lymphocytes. § ± Standard error of the mean.
Suppressor Cells in RA Synovium
481
TABLE II. Effect of varying the effector cell concentration on the percentage suppression of blastogenic response of the responder cells* % Suppression! with effector cells from: No. of cells added 5x10^ 10x10"
Normal donors
Synovial tissue (donors A.F. and S.W.)
-7.55±6.4t -19.47 ±7.0
-24.5±10.9 -16±1.0
* The responder cells were obtained from the peripheral blood of normal donors. Both the effector and the responder cells were stimulated with the mitogenic concentration of concanavalin A (Con A) (20 (xg/ml). t A minus sign preceding the percentage suppression means suppression. N=no. of donors tested. } ± Standard error of the mean.
various doses of Con A required to generate the maximum suppressor cell activity. A mitogenic (20 [J-g/ml) and a submitogenic dose of Con A (10 (J-g/ml) were equally effective in generating suppressor cell activity (Table 1), although a submitogenic dose of 5 (ig/ml seemed less effective. The optimal mitogenic concentration was determined in pilot experiments and was 20 [Ag/ml of culture. Furthermore, the minimum time required to generate optimal suppressor activity was 24 h of exposure to Con A. We also investigated the influence of the effector cell concentration on the degree of suppression (Table II). The doubling of the effector cell concentration increased the suppressor cell activity. Further increase of the effector cell concentrations was not done, to avoid cell overcrowding and subsequent inhibition in our test system.
Next we tried to ascertain the effect of various doses of the Con A during stimulation of the responder cells. Our data revealed that maximum suppression is brought about at submitogenic concentrations of the Con A, but differences between mitogenic and submitogenic concentrations in suppressor effect were small (Table III). We also investigated the effect of doubling the responder cell concentration. This tended to decrease the suppression seen at a 1:1 ratio of the responder to effector cells (Table IV). Four experiments were performed in which the responders cells were obtained from C.C. and other normal donors. Although the percentage suppression varied, the mean percentage suppression did not differ significantly between the experiments (Table V). On the basis of these studies further experiments were performed with 20 (xg/ml of Con A
TABLE III. Effect of various concentrations of concanavalin A (Con A) added to the responder cells during stimulation* , Suppression! of the effector cells from: Concentration of Con A (fig/ml)
Normal donors
Synovial tissue (donors J.A. and A.A.)
20 10 5
-18.84±6.38t -32.1 ±5.97 -32.18+8.17
+ 57±5O + 17±32 + 12±24
* The responder cells were obtained from the normal donors. The effector cells were stimulated with the mitogenic concentration of Con A (20 [xg/ml). t A plus sign preceding the percentage suppression means augmentation; a minus sign means suppression. A'=no. of donors tested. t ± Standard error of the mean.
482
C. Chattopadhyay et al.
TABLE IV. Effect of increasing the responder cell concentration on suppression of the blastogenic response of normal responder cells*
CONTROLS
PATIENTS
-60 Concentrations of cells added
-50
% Suppression!
(A'=4)t
z-40
5x10* 10x10*
o
ll.75+7.42§ -0.045 + 5.58
•
w
* Both the effector and the responder cells were obtained from the peripheral blood of the normal donors and were stimulated with the mitogenic concentration of concanavalin A (Con A) (20 |xg/ml). ! N=r\o. of donors tested. t A plus sign preceding the percentage suppression means augmentation; a minus sign means suppression. § + Standard error of the mean.
t -10 (/)
0
Suppressor activity of the lymphocytes of the rheumatoid tissue In marked contrast to the mean suppressor effect of 13 ± 4.40% in the twenty normal blood donors, the mean effect of the lymphocytes isolated from rheumatoid synovial tissue in the same test system was an augmented response. Thus, the mean augmentation of lymphocytes from eleven patients tested was 28 ±13.30%.
3*30 •
+60^ +110 ' +120
•
FIG. 1. Concanavalin-A-activated suppressor cell activity in patients and controls. Solid lines represent the means. A minus sign means suppression, and a plus sign means augmentation.
Only three patients out of eleven had positive suppressor cell activity, and the rest of the patients did not have any suppressor cell activity (Fig. 1, Table VII). The mean response was significantly different compared with the peripheral blood lymphocytes of the normal donors (P
