Functional α1-adrenergic receptors on leukocytes of patients with polyarticular juvenile rheumatoid arthritis

J oof u Neuroimmunology Journal of Neuroimmunology 71 (1996) 223-226

Short Communication

Functional a I-adrenergic receptors on leukocytes of patients with polyarticuku-juvenile rheumatoid arihritis Cobi J. Heijnen “*, Charlotte Rouppe van der Voort a, Nico Wulffraat a, Janjaap van der Net b, Wietse Kuis a, Annemieke Kavelaars a a Department of Imirrunology,University Hospitalfor Children and Youth ‘Het Wilhelmina Kinderziekenhuis’, PO Box 18009, 3501 CA Utreclrt,The Netherlands b Departmen~of Pediatric Physical Therapy, University Hospitalfor Children and Youth ‘Het Wilhelmina Kinderziekenhuis’, Utrecht, The Netherlands Received 5 December 1995; revised 3 May 1996; accepted 20 June 1996

Abstract During the last decade it has been shownthat the central nervoussystemcan influencethe immunesystem.In healthy individuals, catecholaminescan inhibitthe productionof pro-inflammatorycytokineslike interleukin-6(IL-6)and tumornecrosisfactor a (TNF-cx) via interaction with /32-adrenergicreceptors. In contrast, we show here that catecholaminescan stimulate the production of the interleukin-6(IL-6)in childrenwith the chronicinflammatorydiseasepolyarticularjuvenilerheumatoidarthritis(JRA).The inductionof IL-6 is mediated by triggering of al-adrenergic receptors on peripheral blood leukocytesof the patients with polyarticuIarJRA. Functionala l-adrenergicreceptorsare absenton leukocytesof normaldonorsand on leukocytesof patientswith the oligoarticularform of the disease. Keywords: Juvenile rheumatoid arthritis; Autonomic nervous system; Adrenergic receptors; Interleukin-6

1. Introduction Juvenile rheumatoid arthritis (JRA) is a chronic inflammatory joint disease. The oligoarticular form of the disease is characterized by inflammation of less than four joints, whereas polyarticular JRA is characterized by an inflammation of five or more joints. Moreover, in 50–705%0 of the patients with oligoarticular JRA the disease goes into complete remission, whereas the prognosis for children with the polyarticular form of the disease is worse. In only 20% of the patients with polyarticular JRA the disease goes into complete remission (Gare and Fasth, 1995). The disease JRA is associated with clinical symptoms of altered functioning of the autonomous nervous system such as increased perspiration, increased micturition, sleep disturbances and a decreased pain threshold (Kuis and Heijnen, 1994; Hogeweg et al., 1995). Recently, we showed that children with JRA have an increased urinary level of the noradrenergic metabolize 3-methoxy-4-hydroxy-phenylglym](MHPG) (Kuis et al., 1996). This is indicative for

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an increase in the central noradrenergic outflow which will lead to increased vasoconstriction. Due to the increased vasoconstriction, patients with JRA, have diminished catecholamine and cardiovascular responses to orthostatic stress (Kuis et al., 1996). In general the central autonomic outflow from the pens and medulla to the spinal cord activates sympathetic nerve fibers innervating lymphoid organs (Felten et al., 1987). Activation of sympathetic fibers results in secretion of catecholamines which, under normal conditions, leads to a modulation of the ongoing immune response via binding to either ~2- or ci2-adrenergic receptors (Crary et al., 1983; Arnason, 1993; Bisphoric et al., 1980; Severn et al., 1992; Spengler et al., 1994). Spengler et al., have shown, that in vivo activated peritoneal murine macrophages express cz2-adrenergic receptors. Activation of these receptors leads to an upregulation of lipopolysaccharide (LPS)-induced TNF-a production (Spengler et al., 1994). Culture of human monocytes with adrenaline and LPS leads to inhibition of TNF-a production via ~2-adrenergic receptors (Severe et al., 1992). Peripheral blood leukocytes of healthy individuals do not express CY l-adrenergic receptors (Cassale and Kaliner, 1984). However, we show here that

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peripheral blood mononuclear cells (PBMC) of patients with polyarticular JRA respond to a l-adrenergic activation with an increased production of the pro-inflammatory mediator IL-6.

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2. Patients and methods We studied 8 patients with oligoarticular JRA and 12 patients with polyarticular JRA, defined by the criteria of the American College of Rheumatology (ACR) (Cassidy et al., 1989). Characteristics of the patients are summarized in Table 1. Healthy adult volunteers from our department were taken as controls. PBMC were isolated from heparinized blood by Ficoll Isopaque density gradient centrifugation (Pharmacia, Uppsala, Sweden). Cells (1 X IOG/ml) were cultured in flatbottom 24-well plates in RPMI-1640 (Gibco, Grand Island, NY) supplemented with 10% heat-inactivated fetal calf serum (Gibco) and antibiotics. Cultures were stimulated with phenylephrine (Sigma, St. Louis, MO) in the presence or absence of doxazosin (10-9 M, Pfizer, New York, NY) in the concentrations indicated. Supernatants were harvested after 18 hours of culture, which was the optimal time point for activation of IL-6 production as determined in three patients (8, 18 and 48 hours of culture were tested). The level of IL-6 was determined by ELISA (CLB, Amsterdam, The Netherlands).

3. Results and discussion Basal IL-6 production determined in culture supernatants of PBMC without stimulus did not differ between patients and controls [oligoarticular JRA patients (n= 8), 643 + 382 pg/ml; polyarticular JRA patients (n= 12), 678 + 192 pg/ml; controls (n= 10), 856+ 275 pg/ml; mean + sem, F(2,27) = 0.17, p > 0.8]. To investigate whether the leukocytes of patients can respond to a+adrenergic activation, the effect of the a-adrenergic agonist phenylephrine on the production of IL-6 was tested. Culture of peripheral blood mononuclear cells of polyarticular JRA patients with the a-adrenergic receptor agonist

Table 1 Patient characteristics

Patientgroup

Oligoarticular

Polyarticular

Sexe Age

5 male/3 female 5–15 years (median 9.5) 2

5 mafe/7 female 3-16 years (median 11.5)

0

4 O

6 4

HLA-B27 positive Medication NSAID NSAID and methotrexate

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o Fig. 1. Effect of phenylephrine on IL-6 production by PBMC of patients or controls. PBMC from patients with oligoarticular ( A), polyarticular JRA (.) or controls (0) were cultured with or without phenylephrine (10-9 M) for 18 hours, supematants were harvested and the level of Ii-6 was determined by ELISA. Phenylephrine did not influence 11-6production by cells from oligoarticular JRA (n = 8, two-tailed Wilcoxon paired rank test: ns). In patients with polyarticular JRA, phenylephrine induced an increase in IL-6 production in the patient group (n = 12, two-tailed Wilcoxon paired rank test: p < 0.05). In the controls, phenylephrine induced a decrease in IL-6 production (n = 10, two-tailed Wilcoxon paired rank test: p < 0.02). Result represents the mean of triplicate measurements with sd < 5Y0.

phenylephrine results in an increase in IL-6 production in nine out of 12 patients (Fig. 1). In one patient there was no effect of phenylephrine, whereas two patients responded with a decrease in IL-6 production. In contrast, leukocytes of patients with oligoarticular JRA did not respond to the a-adrenergic receptor agonist with an increase in IL-6 production (Fig. 1). In the non-responding patients we did not observe a shift in the dose response curve for phenylephrine, 0.1–10 nM phenylephrine did not stimulate IL-6 production (data not shown). In control individuals, phenylephrine inhibited IL-6 production in all cases (Fig. 1). The capacity of patients with polyarticular JRA to respond to a-adrenergic activation with increased IL-6 production can not be explained by an effect of NSAID medication since oligoarticular JRA patients on NSAID therapy do not respond to the a-adrenergic agonist. The responding and non-responding patients with polyarticular JRA do not differ with respect to indices of disease activity as determined by Ritchie articular index, joint alignment and motion scale or Fuchs joint swelling index, age or duration of the disease. The stimulator effect of phenylephrine was mediated specifically via an a l-adrenergic receptor, since addition of the specific a l-adrenergic receptor antagonist doxazosin completely abolishes the stimulator effect of phenylephrine on IL-6 production (Fig. 2). The inhibitory effect of phenylephrine in the control cultures was not affected by addition of doxazosin (data not shown). We have preliminary data showing that leukocytes of patients contain mRNA encoding for the a la-adrenergic

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Fig. 2. Effect of doxazosin (10-9 M) on phenyiephrine-induced IL-6 production by PBMC of a representative experiment. PBMC were cultured with phenylephrine alone (solid line) or with phenylephrine plus doxazosin (dotted line). Data represent mean and sd of triplicate measurements of three different patients. Statistically different from control without phenylephrine: * ~ < 0.05; * * p <0.01,

receptor as determined by RT-PCR. These data lead us to propose that catecholamines can influence the disease process by enhancing cytokine production via a la-adrenergic receptor-mediated processes. In chronic inflammatory diseases the autonomic balance is shifted towards increased vasoconstriction, which is an cwadrenergic process (Kuis et al,, 1996; Leden et al., 1983). It is a striking finding that the increased noradrenergic tonus is also reflected by altered a-adrenergic reactivity on the level of the immune system. The mechanism(s) leading to tYl-adrenergic receptor expression on PBMC are not yet clear. The genes encoding a I-adrenergic receptor subtypes contain a cAMP responsive element, so that @-adrenergicagonists that cause an increase in cAMP level can upregulate the expression of mRNA for aladrenergic receptors in e.g. smooth muscle cells (Morris et al., 1991; Kanasaki et al., 1994; Izzo et al., 1990). In addition, it has been shown that hypoxia and reoxygenation of cardiac tissue results in enhanced expression of a l-adrenergic receptors (Kurz et al., 1991). In patients with chronic inflammatory diseases like JRA formation of oxygen radicals frequently occurs (Blake et al., 1989; Fairburn et al., 1993). In line with these investigations, we were able to induce a 1-adrenergic receptors by exposing the leukocytes of normal healthy individuals in vitro to hypoxia and subsequent reoxygenation (man in prep). Collectively, our findings lead us to propose the hypothesis that in children with polyarticular JRA cells of the immune system expresses an aberrant response to catecholamines: leukocytes express a l-adrenergic receptors, triggering of which leads to cytokine production. Cytokines in turn may exacerbate disease. The validity and usefulness of our hypothesis can be tested by administering specific a 1-adrenergic receptor antagonists to patients with polyarticular JRA. In this respect it is remarkable that administration of the a 1-adrenergic receptor antagonist prazosin has been shown to suppress experimental autoimmune

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