British Journal of Rheumatology 1992;31:663-667
INFLUENCE OF STEROID HORMONES ON PROLIFERATION OF PERIPHERAL BLOOD MONONUCLEAR CELLS IN PATIENTS WITH RHEUMATOID ARTHRITIS BY H. R. VAN DEN BRINK, M. J. G. VAN WIJK AND J. W. J. BIJLSMA Department of Rheumatology, University Hospital Utrecht, The Netherlands
KEY WORDS:
Rheumatoid arthritis, T-Lymphocyte, Oestradiol, Progesterone, Testosterone, Cortisol, PHA, Anti-CD3 mAb.
IN both experimental animals and in man there is a preponderance of autoimmune diseases in females, compared with males. Sex steroids are believed to influence the immune system although the precise mechanism is unknown. It is suggested that gonadal steroids may mediate their effects on the immune system directly on lymphocytes and perhaps also indirectly via the hypothalamus-pituitary-gonadal (HPG) axis. Beside sex steroids, corticosteroids are of major importance in immunoendocrinology. Corticosteroids are known to influence the immune system, even in physiological concentrations. Several reviews on the role of steroid hormones in immunoendocrinology have been published [1-6]. In rheumatoid arthritis (RA) sex steroids are believed to be beneficial [7]. During the menstrual cycle joint symptoms are less when oestradiol and progesterone levels are high [8]. Disease activity during pregnancy is often reduced, possibly due to high levels of steroid hormones [9]. Oral contraceptives and pregnancy may protect against the development of RA [10,11] and some preliminary evidence suggested that oestrogens may modulate disease activity [12]. In animal models oestrogens can suppress collagen-induced arthritis [13]. In man specific oestrogen receptors on T-lymphocytes have been identified [14,15]. Corticosteroids are the most effective agents in suppressing disease activity in patients with RA [16] and may be immunomodulating even at low dose [6,17]. There is no consensus regarding the dosage of corticosteroids needed or mode of administration when used for suppressing disease activity. The circadian rhythm of cortisol secretion might influence immune reactivity and disease activity in patients with RA [18].
Recently it has been shown that the circadian rhythm of cortisol secretion in patients with RA is disturbed depending on disease activity [19]. The aim of this study was to investigate whether peripheral blood mononuclear cells (PBMC) from patients with RA could be influenced by steroid hormones in vitro. The influence of oestradiol, progesterone, testosterone and cortisol on PBMC proliferation was tested in postmenopausal female patients with RA and compared with a control group. PBMC were stimulated with phytohaemagglutinin (PHA) and immobilized anti-CD3 monoclonal antibodies (anti-CD3 mAb). Stimulation with PHA requires accessory cells (monocytes) for optimal stimulation while stimulation with plate-coated immobilized anti-CD3 mAb provides a strong T-lymphocyte activation which is accessory cell-independent and induces stronger proliferation than soluble anti-CD3 mAb [20,21]. PATIENTS AND METHODS Patients and controls Thirty-two postmenopausal female patients with definite or classical RA [22] were studied. Mean age in years ( ± S D ) was 63.1 (±5.9). None of the patients was taking exogenous sex steroids or corticosteroids. Most patients were receiving a non-steroidal anti-inflammatory drug (NSAID) in combination with a disease-modifying drug (hydroxychloroquine, oral or intramuscular gold, D-penicillamine, sulphasalazine or methotrexate). Based on disease activity, measured by Ritchie articular score, number of swollen joints and erythrocyte sedimentation rate (ESR), patients were divided into three groups. Group 1 consisted of nine patients with an inactive or mild disease, with at least two of the following criteria: a Ritchie score less than 10, number of swollen joints less than 5 and an ESR less than 20 mm/h. Group 2 consisted of 12 patients with a moderately active disease who had
Submitted 3 June; revised version accepted 3 September 1991. Correspondence to Dr H. van den Brink, Department of Rheumatology, F02.223, Academisch Ziekenhuis Utrecht, Postbus 85500, 3508 GA Utrecht, The Netherlands.
© 1992 British Society for Rheumatology
0263-7103/92/010661 + 05 $08.00/0 663
Downloaded from http://rheumatology.oxfordjournals.org/ at The University of British Colombia Library on June 28, 2015
SUMMARY Sex steroids are believed to modulate the immune system in rheumatoid arthritis (RA). Since receptors for sex steroids are present on T-lymphocytes, which are thought to play a major role in the pathogenesis of RA, it is suggested that gonadal steroids can mediate their immunomodulating effect directly on T-lymphocytes. Recently a specific method for activating T-lymphocytes with immobilized anti-CD3 monoclonal antibodies was described. We investigated the influence of oestradiol, progesterone, testosterone and cortisol on lymphocytes stimulated by anti-CD3 mAb and PHA of postmenopausal women, comparing female patients with rheumatoid arthritis and age-matched control patients. The results show that oestradiol, progesterone and testosterone do not influence lymphocyte proliferation when stimulated with anti-CD3 mAb or phytohaemagglutinin (PHA). Cortisol, however, can suppress lymphocyte proliferation even at physiological concentrations in both patients with RA and controls. Inhibition of proliferation by cortisol is dose-related and has no significant correlation with RA disease activity. This inhibition differs individually and might explain the often variable response to corticosteroids in vivo.
664
BRITISH JOURNAL OF RHEUMATOLOGY VOL. XXXI NO. 10 TABLE I PROLIFERATIVE RESPONSE OF PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMC) IN /i CONTROL GROUP AND PATIENTS WITH RA
[3H]TdR incorporation (c.p.m., mean±SD) with:
Control PBMC PBMC RA group 1 PBMC RA group 2 PBMC RA group 3
PHA
anti-CD3 mAb
120.442±32.087 (n = ll) 136.868±37.845 (n= 9) 127.011±52.236 (w=12) 58.921±57.702*(« = l l )
87.450±25.555 (n= 9) 67.964±33.782 (n= 8) 72.975±34.255 (« = 12) 48.125±16.805**(/i=9)
at least two of the following criteria: a Ritchie score between 10 and 20, number of swollen joints between 5 and 10 and an ESR between 20 and 50 mm/h. Group 3 were 11 patients with a severe disease activity who had two or more of the following criteria: a Ritchie score of more than 20, at least 10 or more swollen joints and an ESR more than 50 mm/h. The control group consisted of 11 postmenopausal patients who had osteoarthrosis without any signs of inflammation, low back pain or soft tissue rheumatism. Mean age in years (±SD) was 65.5 (±8.4). PBMC proliferation studies were performed in all patients, but it was not possible to study the influence of all the different steroid hormones in all separate patients. Preparation of peripheral blood mononuclear cells Peripheral blood mononuclear cells (PBMC) were isolated from fresh heparinized peripheral blood by density gradient centrifugation on Ficoll-Paque (Pharmacia, Uppsala, Sweden) and washed twice with MEM/TRIS before incubation. Cell cultures All cultures were performed in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% heatinactivated fetal calf serum (FCS), 1% glutamine, lOOU/ml penicillin, 10u.g/ml streptomycin and 25 UM 2-mercaptoethanol. Lymphocytes were cultured at 5 x 104 cells per flat-bottom microtitre plate well (Nuclon, Nunc, Denmark) for 5 days at 37°C in a humidified atmosphere of 5%CO 2 -95% air. Cellular proliferation was measured by the addition of 1 uCi=37 kBq/well [3H]thymidine (Amersham, England) during the last 16 h of incubation. The cells were then collected on filter discs by using a multi-harvester apparatus (Titertek). Radioactivity was determined in a liquid scintillation spectrometer. Proliferation is expressed as the mean counts per minute (c.p.m.) of quadruplicate cultures. Anti-CD3 mAb were purchased from the Central Laboratorium Bloedtransfusiedienst (Amsterdam, The Netherlands). Culture wells were coated overnight with a solution containing anti-CD3 mAb 2 fig/ml in phosphatebuffered saline (PBS). After incubation the wells were washed twice with PBS. PHA was obtained from Wellcome Diagnostics (Dartford, England) and used in a concentration of 20ug/ml. Oestradiol, progesterone, testosterone and cortisol were purchased from Sigma
Chemical Company (St Louis, Missouri, USA). Each steroid was dissolved in absolute ethanol and used in concentrations betwen 1 ng and 10 ug per ml with tenfold dilutions and added at the beginning of the culture. All steroids tested, except for oestradiol, did not decrease cell viability as determined by trypan blue dye exclusion. Only oestradiol in highest concentration (10 ug/ml) decreased cell viability to 60% after 5 days of culture. Absolute ethanol was used as a solvent of the steroids at concentrations not exceeding 1.0% final volume. This concentration had no inhibitory effect on lymphocyte cultures (data not shown). Statistical analysis Differences between group means were determined by the Mann-Whitney U or Wilcoxon test, using Number Cruncher Statistical System (NCSS). RESULTS Proliferation response of PBMC of patients with RA and control PBMC after stimulation with PHA and anti-CD3 mAb Patients were divided into groups according to disease activity. The mean values of the proliferation after stimulation with PHA and anti-CD3 mAb of different groups is shown in Table I. There was no statistically significant difference between the control group and patients with 140-1 120100-
2
80 604O20-
RA2
RA3
FIG. 1.—Peripheral blood mononuclear cell proliferation after stimulation with anti-CD3 mAb. N=control group («=9). RAl=group 1 with no or minimal disease activity (n=8). RA2=group 2 with moderate disease activity (n=12). RA3=group 3 with severe disease activity (n=9). Inserted bars indicate mean proliferation. *P
INFLUENCE OF STEROID HORMONES ON PROLIFERATION OF PERIPHERAL BLOOD MONONUCLEAR CELLS IN PATIENTS WITH RHEUMATOID ARTHRITIS BY H. R. VAN DEN BRINK, M. J. G. VAN WIJK AND J. W. J. BIJLSMA Department of Rheumatology, University Hospital Utrecht, The Netherlands
KEY WORDS:
Rheumatoid arthritis, T-Lymphocyte, Oestradiol, Progesterone, Testosterone, Cortisol, PHA, Anti-CD3 mAb.
IN both experimental animals and in man there is a preponderance of autoimmune diseases in females, compared with males. Sex steroids are believed to influence the immune system although the precise mechanism is unknown. It is suggested that gonadal steroids may mediate their effects on the immune system directly on lymphocytes and perhaps also indirectly via the hypothalamus-pituitary-gonadal (HPG) axis. Beside sex steroids, corticosteroids are of major importance in immunoendocrinology. Corticosteroids are known to influence the immune system, even in physiological concentrations. Several reviews on the role of steroid hormones in immunoendocrinology have been published [1-6]. In rheumatoid arthritis (RA) sex steroids are believed to be beneficial [7]. During the menstrual cycle joint symptoms are less when oestradiol and progesterone levels are high [8]. Disease activity during pregnancy is often reduced, possibly due to high levels of steroid hormones [9]. Oral contraceptives and pregnancy may protect against the development of RA [10,11] and some preliminary evidence suggested that oestrogens may modulate disease activity [12]. In animal models oestrogens can suppress collagen-induced arthritis [13]. In man specific oestrogen receptors on T-lymphocytes have been identified [14,15]. Corticosteroids are the most effective agents in suppressing disease activity in patients with RA [16] and may be immunomodulating even at low dose [6,17]. There is no consensus regarding the dosage of corticosteroids needed or mode of administration when used for suppressing disease activity. The circadian rhythm of cortisol secretion might influence immune reactivity and disease activity in patients with RA [18].
Recently it has been shown that the circadian rhythm of cortisol secretion in patients with RA is disturbed depending on disease activity [19]. The aim of this study was to investigate whether peripheral blood mononuclear cells (PBMC) from patients with RA could be influenced by steroid hormones in vitro. The influence of oestradiol, progesterone, testosterone and cortisol on PBMC proliferation was tested in postmenopausal female patients with RA and compared with a control group. PBMC were stimulated with phytohaemagglutinin (PHA) and immobilized anti-CD3 monoclonal antibodies (anti-CD3 mAb). Stimulation with PHA requires accessory cells (monocytes) for optimal stimulation while stimulation with plate-coated immobilized anti-CD3 mAb provides a strong T-lymphocyte activation which is accessory cell-independent and induces stronger proliferation than soluble anti-CD3 mAb [20,21]. PATIENTS AND METHODS Patients and controls Thirty-two postmenopausal female patients with definite or classical RA [22] were studied. Mean age in years ( ± S D ) was 63.1 (±5.9). None of the patients was taking exogenous sex steroids or corticosteroids. Most patients were receiving a non-steroidal anti-inflammatory drug (NSAID) in combination with a disease-modifying drug (hydroxychloroquine, oral or intramuscular gold, D-penicillamine, sulphasalazine or methotrexate). Based on disease activity, measured by Ritchie articular score, number of swollen joints and erythrocyte sedimentation rate (ESR), patients were divided into three groups. Group 1 consisted of nine patients with an inactive or mild disease, with at least two of the following criteria: a Ritchie score less than 10, number of swollen joints less than 5 and an ESR less than 20 mm/h. Group 2 consisted of 12 patients with a moderately active disease who had
Submitted 3 June; revised version accepted 3 September 1991. Correspondence to Dr H. van den Brink, Department of Rheumatology, F02.223, Academisch Ziekenhuis Utrecht, Postbus 85500, 3508 GA Utrecht, The Netherlands.
© 1992 British Society for Rheumatology
0263-7103/92/010661 + 05 $08.00/0 663
Downloaded from http://rheumatology.oxfordjournals.org/ at The University of British Colombia Library on June 28, 2015
SUMMARY Sex steroids are believed to modulate the immune system in rheumatoid arthritis (RA). Since receptors for sex steroids are present on T-lymphocytes, which are thought to play a major role in the pathogenesis of RA, it is suggested that gonadal steroids can mediate their immunomodulating effect directly on T-lymphocytes. Recently a specific method for activating T-lymphocytes with immobilized anti-CD3 monoclonal antibodies was described. We investigated the influence of oestradiol, progesterone, testosterone and cortisol on lymphocytes stimulated by anti-CD3 mAb and PHA of postmenopausal women, comparing female patients with rheumatoid arthritis and age-matched control patients. The results show that oestradiol, progesterone and testosterone do not influence lymphocyte proliferation when stimulated with anti-CD3 mAb or phytohaemagglutinin (PHA). Cortisol, however, can suppress lymphocyte proliferation even at physiological concentrations in both patients with RA and controls. Inhibition of proliferation by cortisol is dose-related and has no significant correlation with RA disease activity. This inhibition differs individually and might explain the often variable response to corticosteroids in vivo.
664
BRITISH JOURNAL OF RHEUMATOLOGY VOL. XXXI NO. 10 TABLE I PROLIFERATIVE RESPONSE OF PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMC) IN /i CONTROL GROUP AND PATIENTS WITH RA
[3H]TdR incorporation (c.p.m., mean±SD) with:
Control PBMC PBMC RA group 1 PBMC RA group 2 PBMC RA group 3
PHA
anti-CD3 mAb
120.442±32.087 (n = ll) 136.868±37.845 (n= 9) 127.011±52.236 (w=12) 58.921±57.702*(« = l l )
87.450±25.555 (n= 9) 67.964±33.782 (n= 8) 72.975±34.255 (« = 12) 48.125±16.805**(/i=9)
at least two of the following criteria: a Ritchie score between 10 and 20, number of swollen joints between 5 and 10 and an ESR between 20 and 50 mm/h. Group 3 were 11 patients with a severe disease activity who had two or more of the following criteria: a Ritchie score of more than 20, at least 10 or more swollen joints and an ESR more than 50 mm/h. The control group consisted of 11 postmenopausal patients who had osteoarthrosis without any signs of inflammation, low back pain or soft tissue rheumatism. Mean age in years (±SD) was 65.5 (±8.4). PBMC proliferation studies were performed in all patients, but it was not possible to study the influence of all the different steroid hormones in all separate patients. Preparation of peripheral blood mononuclear cells Peripheral blood mononuclear cells (PBMC) were isolated from fresh heparinized peripheral blood by density gradient centrifugation on Ficoll-Paque (Pharmacia, Uppsala, Sweden) and washed twice with MEM/TRIS before incubation. Cell cultures All cultures were performed in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% heatinactivated fetal calf serum (FCS), 1% glutamine, lOOU/ml penicillin, 10u.g/ml streptomycin and 25 UM 2-mercaptoethanol. Lymphocytes were cultured at 5 x 104 cells per flat-bottom microtitre plate well (Nuclon, Nunc, Denmark) for 5 days at 37°C in a humidified atmosphere of 5%CO 2 -95% air. Cellular proliferation was measured by the addition of 1 uCi=37 kBq/well [3H]thymidine (Amersham, England) during the last 16 h of incubation. The cells were then collected on filter discs by using a multi-harvester apparatus (Titertek). Radioactivity was determined in a liquid scintillation spectrometer. Proliferation is expressed as the mean counts per minute (c.p.m.) of quadruplicate cultures. Anti-CD3 mAb were purchased from the Central Laboratorium Bloedtransfusiedienst (Amsterdam, The Netherlands). Culture wells were coated overnight with a solution containing anti-CD3 mAb 2 fig/ml in phosphatebuffered saline (PBS). After incubation the wells were washed twice with PBS. PHA was obtained from Wellcome Diagnostics (Dartford, England) and used in a concentration of 20ug/ml. Oestradiol, progesterone, testosterone and cortisol were purchased from Sigma
Chemical Company (St Louis, Missouri, USA). Each steroid was dissolved in absolute ethanol and used in concentrations betwen 1 ng and 10 ug per ml with tenfold dilutions and added at the beginning of the culture. All steroids tested, except for oestradiol, did not decrease cell viability as determined by trypan blue dye exclusion. Only oestradiol in highest concentration (10 ug/ml) decreased cell viability to 60% after 5 days of culture. Absolute ethanol was used as a solvent of the steroids at concentrations not exceeding 1.0% final volume. This concentration had no inhibitory effect on lymphocyte cultures (data not shown). Statistical analysis Differences between group means were determined by the Mann-Whitney U or Wilcoxon test, using Number Cruncher Statistical System (NCSS). RESULTS Proliferation response of PBMC of patients with RA and control PBMC after stimulation with PHA and anti-CD3 mAb Patients were divided into groups according to disease activity. The mean values of the proliferation after stimulation with PHA and anti-CD3 mAb of different groups is shown in Table I. There was no statistically significant difference between the control group and patients with 140-1 120100-
2
80 604O20-
RA2
RA3
FIG. 1.—Peripheral blood mononuclear cell proliferation after stimulation with anti-CD3 mAb. N=control group («=9). RAl=group 1 with no or minimal disease activity (n=8). RA2=group 2 with moderate disease activity (n=12). RA3=group 3 with severe disease activity (n=9). Inserted bars indicate mean proliferation. *P
