OBJECTIVE To investigate further the influence of the autonomic nervous system on chronic rheumatic diseases.
METHODS The density and affinity of β2 adrenergic receptors (β2R) on CD19+ lymphocytes in patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and systemic sclerosis (SSc), as well as intracellular cAMP levels in patients with RA and SLE, were determined. Human peripheral blood mononuclear cells were separated from venous blood of patients and healthy controls by Ficoll-Hypaque density centrifugation. CD19+ lymphocytes were purified by magnetic cell sorting, and β2R were determined by a radioligand binding assay with [125I]iodocyanopindolol. Intracellular cAMP levels and β2R agonist induced cell death were measured by a radioimmunoassay and flow cytometry using annexin-V binding, respectively. Systemic disease activity of the patients was evaluated using multifactorial scoring systems.
RESULTS The density of β2R on peripheral CD19+ lymphocytes was significantly decreased in patients with RA, SLE, and SSc compared with healthy controls. In patients with RA and SSc β2R density was negatively correlated with systemic disease activity. Furthermore, although basal intracellular cAMP levels were raised in patients with RA and SLE, the increase of cAMP upon stimulation of β2R was significantly reduced in these patients compared with control subjects. Preliminary data suggest that β2R agonist induced cell death is diminished in patients with RA exhibiting decreased β2R densities.
CONCLUSIONS The results of this study show a reduction of β2R densities on B lymphocytes mirrored by an impaired intracellular cAMP generation in patients with chronic rheumatic diseases, indicating a decreased influence of the autonomic nervous system on B cells in these conditions.
- β adrenergic receptor
- cyclic AMP
- lymphocyte subsets
- rheumatic diseases
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Although the cause of inflammatory autoimmune diseases like rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and systemic sclerosis (SSc) is unknown, it is well accepted that cellular and humoral immune responses underlie the distinct pathology of these diseases. A hallmark of chronic rheumatic diseases is the formation of autoantibodies—for example, rheumatoid factor in RA, antibodies against double stranded DNA (dsDNA) and Smith antigen (Sm) in SLE, and against topoisomerase-II (Scl-70) in SSc. Additionally, immune complexes and hypergammaglobulinaemia are common features, indicating a B cell hyperactivity.1
A growing body of evidence points towards a modulation of immune responses in vitro as well as in vivo and the inflammatory activity of autoimmune diseases by the autonomous nervous system. It has been shown that lymphocytes and noradrenergic varicosities form synapse-like conjunctions in lymphoid organs2 ,3 and that β2 adrenergic receptors (β2R) are expressed on various lymphocyte subpopulations.4 ,5
Previous studies of our group showed that in patients with chronic rheumatic diseases the density of β2R is decreased on peripheral blood mononuclear cells (PBMC). Further investigations showed that β2R are modulated differentially on lymphocyte subsets because β2R densities were reduced on CD8+ lymphocytes but not on CD4+ cells.6 ,7 However, little is known about β2R on B lymphocytes in these disease entities.
Therefore we investigated the characteristics of β2R on peripheral blood B lymphocytes (CD19+ mononuclear cells) in patients with RA, SLE, and SSc, together with the systemic disease activity, and determined the coupling of β2R to the intracellular signal transduction cascade.
PATIENTS AND CONTROL SUBJECTS
Patients with RA (n=24), SLE (n=13), and SSc (n=6) according to classical diagnostic criteria8-10 and a group of healthy blood donors (n=16) were included in the study. We excluded patients in whom other factors were supposed to influence β2R (that is, infectious and atopic diseases, hyperthyroidism/hypothyroidism, hypertonia, treatment with sympathomimetic/sympatholytic agents, cancer).11 Patients were examined by taking a history, physical examination, and laboratory findings (erythrocyte sedimentation rate (ESR), C reactive protein (CRP), haemoglobin, packed cell volume, leucocytes, lymphocytes, platelets, autoantibodies, creatinine). Inflammatory disease activity in RA, SLE, and SSc was determined by different multifactorial scoring systems. For patients with RA we used a modified total disease activity score according to Farr et al, (TAI)12 which includes morning stiffness, the Ritchie articular index,13ESR, CRP, and haemoglobin. For patients with SLE the SLE activity measure (SLAM) was used.14 For the patients with SSc a new scoring system adapted from the SLAM was developed and called the SSc score (SSS, table 1). Table 2 summarises the clinical characteristics of patients and control subjects. Treatment with non-steroidal antirheumatic drugs or steroids up to 7.5 mg prednisolone equivalent a day were allowed in all patient groups (RA 15/24 patients, range 2–7.5 mg prednisolone equivalent a day, SLE 9/13, range 2.5–7.5 mg, SSc 5/6, range 2–7.5 mg). No patient received disease modifying antirheumatic drugs.
Cell death induced by isoprenaline was determined in four patients with RA and five healthy controls.
PURIFICATION OF CD19+ LYMPHOCYTES
PBMC were separated from heparinised venous blood by Ficoll-Hypaque (Seromed, Berlin, Germany) density gradient centrifugation.15 Cells were then washed three times in phosphate buffered saline supplemented with 0.5% bovine serum albumin (PBS/BSA) and adjusted to a cell concentration of 1×108/ml. CD19+ lymphocytes were purified by magnetic cell sorting.16 To separate CD19+ cells, PBMC of patients or control subjects were incubated with a monoclonal antihuman CD19 antibody coupled to supramagnetic microparticles for 20 minutes in a refrigerator (Miltenyi Biotec, Bergisch-Gladbach, Germany). Cells were then washed in cold PBS/BSA and CD19+ cells were separated by filtration through a magnetised steel wool column (MACS cell separator, MACS column A2 and LS, Miltenyi Biotec). The efficacy of the magnetic separation was evaluated by flow cytometry using a FACScan (Becton Dickinson, Heidelberg, Germany). The purity of the isolated CD19+ lymphocytes averaged 95% in all experiments.
DETERMINATION OF β2R
The number of β2R on purified CD19+ cells was determined as previously described.17 In brief, aliquots of 0.6×106 CD19+ cells were incubated for 60 minutes at 37°C with six different concentrations of [125I]iodocyanopindolol (ICYP, DuPont, Boston, MA, USA), ranging from 12.5 to 200 pmol/l ICYP. Cells were then harvested immediately on Whatman GF/B filters (Whatman Inc, Clifton, NJ, USA). Filters were washed to remove unbound radioactivity, and radioactivity on wet filters was determined in a multicrystal gammacounter (LB 2103, Berthold, Wildbad, Germany). Unspecific binding of ICYP was measured in parallel by incubating the CD19+ cells in the presence of high concentrations of propranolol (1 μmol/l), a competitive antagonist of ICYP. Specific binding of ICYP was determined by subtracting unspecific binding from the total binding capacity. The maximum number of ICYP binding sites, representing the number of β2R, and dissociation constant (KD) for ICYP of β2R were calculated according to the method of Scatchard.18
DETERMINATION OF BASAL AND STIMULATED INTRACELLULAR CAMP
The concentration of basal and stimulated levels of cAMP in CD19+ lymphocytes was determined in patients with RA (n=5), SLE (n=4), and controls (n=8) by a radioimmunoassay. Aliquots of 1×106 CD19+ cells in PBS/BSA containing 50 μM theophylline and 10 μM ascorbic acid were either incubated for 15 minutes at 37°C to determine basal cAMP levels or incubated with 10 μM (−)-isoprenaline for 15 minutes to determine stimulated cAMP levels.19 ,20 After boiling and centrifugation, cAMP in the supernatant was measured by a [125I]cAMP radioimmunoassay (Dianova-Imunotech, Hamburg, Germany) according to the guidelines of the manufacturer. The increase of cAMP upon β2R stimulation with isoprenaline was calculated by subtraction of basal from stimulated levels.
INDUCTION AND DETERMINATION OF CELL DEATH IN ACTIVATED CD19+ LYMPHOCYTES
Cell death induced by stimulation of β2R in activated CD19+ lymphocytes was determined in patients with RA (n=4) and control subjects (n=5). Aliquots of 2×105 CD19+ cells in serum free RPMI medium supplemented with penicillin/streptomycin,l-glutamine, and 2% TCH defined serum supplement (ICN, Eschwege, Germany) were activated with 1 μg/ml plate bound anti-IgM polyclonal antibody (BioSource, Camarillo, CA, USA) for 16 hours in the presence or absence of 10 μM isoprenaline. After washing in binding buffer (150 mM NaCl, 2 mM CaCl2, 10 mM HEPES) the number of apoptotic and necrotic cells was evaluated using annexin-V-FITC (Bender Med Systems, Vienna, Austria) and propidium iodide staining. At least 5000 events were counted on a FACScan (Becton Dickinson) using the Cell Quest software (Becton Dickinson). Spontaneous and β2R agonist induced cell death (apoptotic and necrotic cells) was determined with a histogram and two dimensional dot plots.
Values in tables and figures are given as means and standard errors of the mean (SEM) if not otherwise indicated. Correlations between β2R characteristics and disease activity scores were calculated by the Pearson product moment correlation. A comparison of independent single variables between the groups was calculated by one way analysis of variance (ANOVA) followed by Tukey's procedure. When normality test failed, Kruskal-Wallis one way ANOVA on ranks was used. A comparison of isoprenaline induced cell death in patients with RA and healthy controls was calculated by a pairedt test. p Values less than 0.05 were considered to be significant.
β2R DENSITY AND DISSOCIATION CONSTANTS
The number of β2R on CD19+ cells was significantly decreased in patients with chronic rheumatic diseases compared with control subjects (p<0.001). The mean (SEM) number of β2R was 1041 (44) binding sites (bs)/cell in patients with RA, 1278 (103) bs/cell in patients with SLE, and 1035 (83) bs/cell in patients with SSc, respectively. On CD19+ cells derived from healthy donors 2060 (106) bs/cell were detected (fig1). No significant difference was found between the β2R density in the various patient groups.
The KD values of β2R for ICYP were reduced in the patient groups (RA 5.89 (0.53) pmol/l; SLE 6.36 (0.92) pmol/l; SSc 5.9 (0.76) pmol/l) compared with the control group (9.86 (1.36) pmol/l). However, when the Kruskal-Wallis test was used the difference between patient groups and control subjects did not reach significance (p=0.113).
Stratifying patients with RA and SLE according to the use of corticosteroid drugs did not disclose a difference in β2R characteristics between the respective groups: patients with RA receiving corticosteroid treatment 1036 (62.6) bs/cell (KD 5.9 (0.7) pmol/l; n=15) v patients with RA without corticosteroid drugs 1048 (56.2) bs/cell (KD 5.8 (0.8) pmol/l; n=9); and patients with SLE receiving corticosteroid drugs 1287 (140) bs/cell (KD 6.4 (1.2) pmol/l; n=9) v patients with SLE without corticosteroids 1273 (144) bs/cell (KD 6.3 (1.5) pmol/l; n=4).
CORRELATION OF β2R STATUS WITH DISEASE ACTIVITY IN RA, SLE, AND SSC
In patients with RA and SSc, β2R density on CD19+ lymphocytes showed a significant negative correlation with disease activity. In patients with RA a negative correlation between the β2R density and the TAI (r=−0.76, p<0.0001; fig 2), and in the SSc group a negative correlation with SSS (r=−0.90, p=0.01) was found. In the SLE group β2R density on CD19+ lymphocytes was negatively correlated with SLAM, but the correlation was not significant (r=−0.52, p=0.07). This was because one patient had high disease activity as well as high β2R density (fig 2). When this patient was omitted a significant negative correlation between β2R density and disease activity (r=−0.79, p=0.002) was found. No significant correlation was found between the KD values of the various patient groups and the disease activity (p>0.05).
BASAL AND STIMULATED INTRACELLULAR CAMP LEVELS IN RA, SLE, AND CONTROL SUBJECTS
Intracellular cAMP levels were investigated in patients with RA (n=5) and SLE (n=4) in comparison with healthy controls (n=8). Basal cAMP levels were significantly raised in patients with SLE (3.88 (0.54) pmol/106 cells) compared with control subjects (1.77 (0.17) pmol/106 cells, p<0.001). In patients with RA basal cAMP levels were slightly raised (2.17 (0.19) pmol/106 cells), with no significant difference from healthy controls.
The relative increase of cAMP above basal levels after isoprenaline stimulation was markedly decreased in RA (3.51 (1.86)%) and SLE (1.69 (0.51)%) in comparison with the control group (9.43 (1.30)%, p<0.05, fig 3). No significant correlation was found between cAMP levels and β2R characteristics or disease activity scores, respectively.
β2R AGONIST INDUCED CELL DEATH
Cell death induced by the incubation of activated CD19+ lymphocytes with 10 μM isoprenaline was investigated in patients with RA (n=4) in comparison with healthy controls (n=5). The proportion of dead cells (defined as apoptotic and necrotic cells based upon staining with annexin-V and propidium iodide, respectively) among the CD19+ cells activated with plate bound polyclonal anti-IgM antibody for 16 hours was raised in patients with RA (30.0 (3.6)%, table 3) compared with control subjects (18.9 (3.7)%). Stimulation of β2R with 10 μM isoprenaline slightly increased the number of dead cells in patients with RA (36.1 (6.7)%, relative change 18.1 (9.7)%, NS). In contrast, the number of dead cells significantly increased upon activation of β2R with isoprenaline in the control subjects (32.9 (7.5)%; p<0.05, relative change 79.7 (20.5)%; p<0.05, table3).
The results of our study demonstrate a profound modulation of β2R on B lymphocytes (CD19+ lymphocytes) in chronic rheumatic diseases. The density of β2R on B lymphocytes was decreased in patients with RA, SLE, and SSc compared with healthy control subjects, and a negative correlation between the β2R density on B cells and systemic disease activity scores was found in all patient groups studied except the SLE group. In addition, inducible intracellular cAMP levels in response to stimulation of β2R were reduced in patients with chronic rheumatic diseases, thus demonstrating the functional significance of β2R modulation.
Previous studies showed that β2R density was decreased on PBMC in patients with chronic rheumatic diseases.7 ,17 ,21Recently, it was shown that in patients with RA, β2R were differentially regulated in lymphocyte subpopulations because β2R on CD8+ lymphocytes were down regulated in contrast with unchanged β2R numbers on CD4+ lymphocytes.7 When the results of these studies are taken together the picture emerges that in chronic rheumatic diseases the expression of β2R on T and B lymphocytes is a complex modulated process rather than simply down regulation.
Various mechanisms contributing to the observed differential modulation of β2R expression are possible. A long term increase of catecholamines decreases β2R density through the stimulation of the β adrenoceptor kinase.22 ,23 However, the differential regulation of β2R on CD4+ and CD8+ lymphocyte subsets in patients with RA argues against a simple down regulation of β2R due to an increase of systemic catecholamine concentrations,7 as can be seen in patients with pheochromocytoma.24 Moreover, a correlation with plasma catecholamine levels could not be shown either for systemic disease activity or for β2R density on PBMC in patients with RA.6
The affinity of β2R in human neutrophils and the transcription of β2R mRNA in human lung cells have been shown to be increased by corticosteroids.25 In RA and SLE a decreased cortisol plasma concentration due to alterations in cortisol metabolism in SLE26 ,27 and a disturbed adrenal secretion of cortisol in RA28 have been proposed. Therefore, decreased cortisol concentrations are a possible mechanism for the down regulation of β2R in rheumatic diseases. However, low dose corticosteroids did not influence β2R characteristics in previous investigations, nor in the study presented here. Thus, at least in patients with chronic rheumatic diseases, low dose corticosteroids do not influence β2R characteristics of immune cells.17 ,29
Other factors, such as a differential regulation of high and low affinity β2R and a defect in intrinsic β2R regulation, may also contribute to the disturbed β2R expression.30 ,31Because it has been shown in this study and in previous studies that there was a striking negative correlation between β2R density and the disease activity,6 ,7 it is tempting to speculate that the density of β2R on PBMC is influenced by the inflammatory process. In particular, cytokines and eicosanoids like prostaglandin E2are known to modulate β2R density in vitro.32 ,33Furthermore, investigations of our group showed that interleukin 2 may be a key player in modulating β2R expression differentially in CD4+ and CD8+ cells.34
The generation of cAMP after stimulation of β2R with isoprenaline was impaired in B lymphocytes of patients with RA and SLE. As has been shown previously, the increase of stimulated cAMP rather than basal levels is crucial for the intracellular signal transduction.35 It seems unlikely that changes in intracellular cAMP levels were induced by preparation of CD19 positive cells, as engagement of CD19 does not interfere with cAMP production in B cells.36
As a functional consequence of β2R stimulation, cell death induced by isoprenaline was determined in CD19+ cells activated by B cell receptors. An increase of intracellular cAMP influences B cell function at different stages and has been shown to induce apoptotic cell death in resting B cells as well as in B cells activated by the B cell receptor.37-41 As our preliminary data show, the isoprenaline induced cell death of B cells is diminished in patients with RA, underlining the functional consequence of decreased β2R densities on immune cells of patients with RA.
Hence, our results indicate that the influence of the sympathetic nervous system on B lymphocytes is impaired in chronic rheumatic diseases. Together with interleukin 1 the increase of intracellular cAMP is an essential signal for the primary immune response and the differentiation of B cell precursors into IgM secreting cells through inactivation of protein kinase C.42-44 In contrast, T helper cell function and the secondary immune response are inhibited by activation of β2R and an increase of intracellular cAMP.42-46 An inverse effect on T cell function could be seen after propranolol treatment and sympathectomy.47 ,48Therefore, an impaired cAMP response may have a role in the B cell dysregulation in chronic rheumatic diseases.
In conclusion, our results show that the density of β2R and the agonist induced cAMP production are impaired in B lymphocytes of patients with chronic rheumatic diseases. However, it is not clear whether this phenomenon occurs in response to the inflammatory process or precedes exacerbations of chronic rheumatic diseases. In every case, changes in the regulation of β2R density and coupling to the intracellular signalling machine may be an accelerating factor for B cell dysregulation and predispose to the development of rheumatic diseases.