Matthias Wahle

Failure of catecholamines to shift T-cell cytokine responses toward a Th2 profile in patients with rheumatoid arthritis

To further understand the role of neuro-immunological interactions in the pathogenesis of rheumatoid arthritis (RA), we studied the influence of sympathetic neurotransmitters on cytokine production of T cells in patients with RA. T cells were isolated from peripheral blood of RA patients or healthy donors (HDs), and stimulated via CD3 and CD28. Co-incubation was carried out with epinephrine or norepinephrine in concentrations ranging from 10-5 M to 10-11 M. Interferon (IFN)-γ, tumour necrosis factor (TNF)-α, interleukin (IL)-4, and IL-10 were determined in the culture supernatant with enzyme-linked immunosorbent assay. In addition, IFN-γ and IL-10 were evaluated with intracellular cytokine staining. Furthermore, basal and agonist-induced cAMP levels and catecholamine-induced apoptosis of T cells were measured. Catecholamines inhibited the synthesis of IFN-γ, TNF-α, and IL-10 at a concentration of 10-5 M. In addition, IFN-γ release was suppressed by 10-7 M epinephrine. Lower catecholamine concentrations exerted no significant effect. A reduced IL-4 production upon co-incubation with 10-5 M epinephrine was observed in RA patients only. The inhibitory effect of catecholamines on IFN-γ production was lower in RA patients as compared with HDs. In RA patients, a catecholamine-induced shift toward a Th2 (type 2) polarised cytokine profile was abrogated. Evaluation of intracellular cytokines revealed that CD8-positive T cells were accountable for the impaired catecholaminergic control of IFN-γ production. The highly significant negative correlation between age and catecholamine effects in HDs was not found in RA patients. Basal and stimulated cAMP levels in T-cell subsets and catecholamine-induced apoptosis did not differ between RA patients and HDs. RA patients demonstrate an impaired inhibitory effect of catecholamines on IFN-γ production together with a failure to induce a shift of T-cell cytokine responses toward a Th2-like profile. Such an unfavorable situation is a perpetuating factor for inflammation.

Reduced catecholarnine response of lymphocytes from patients with rheumatoid arthritis

Catecholamines modulate lymphocyte function via stimulation of beta2-adrenergic receptors (beta2R). Previous investigations revealed a decreased density of beta2R on peripheral blood mononuclear cells (PBMC) in patients with chronic rheumatic diseases. Aim of the present study was to determine the impact of this decrease on catecholamine response of PBMC from patients with rheumatoid arthritis (RA) in vitro. PBMC from 17 patients with RA and 12 healthy blood donors (HD) were investigated. Beta2R were determined by a radioligand binding assay. The effects of epinephrine (E) and norepinephrine (NE) on PBMC proliferation were studied using cells activated with pokeweed mitogen (PWM) and monoclonal anti-CD3-antibodies (OKT3), respectively. In parallel, alpha1- or beta-receptor antagonist were added to the culture to determine the specificity of the catecholaminergic effects. The results showed that depending on the stimulus and the catecholamine concentration employed E and NE exert inhibitory (OKT3) or stimulatory signals (PWM) on lymphocyte proliferation. Inhibitory effects could be abolished by adding beta-antagonist, while stimulatory signals were diminished after addition of alpha1- of beta-antagonist. Patients with RA showed a significantly reduced density of beta2R compared to HD paralleled by a significantly reduced influence of catecholamines on lymphocyte function. The study demonstrates the intricate relationship between PBMC reactivity and catecholamine effects that are mediated via alpha1- and beta-adrenergic receptors. In this respect the reduced catecholamine response of PBMC from RA patients may contribute to the pathogenic process of RA.

Immunopathogenesis of Rheumatic Diseases in the Context of Neuroendocrine Interactions

Abstract: Growing evidence supports the hypothesis that alterations of the stress response and interactions between the neuroendocrine and immune systems contribute to the pathogenesis of rheumatic diseases such as rheumatoid arthritis (RA). In particular, the hypothalamus‐pituitary‐adrenal (HPA) axis and the autonomic nervous system (ANS) are of special interest. Polymorphisms of the corticotropin‐releasing hormone (CRH)‐regulating region have been described recently. These polymorphisms are differentially distributed in RA patients and healthy subjects of various ethnic origin, thus supporting the hypothesis that they represent a new genetic marker for RA susceptibility. The decreased expression of β2‐adrenergic receptors (β2‐R) on lymphatic cells in rheumatic diseases like RA, together with an impaired influence of catecholamines on immune function in these patients, further underlines the concept of a dysfunction of the ANS in rheumatic diseases. Results from work in this field will provide more insight into the pathogenesis of RA and help to establish novel therapies for this chronic rheumatic disease.

Disease activity related catecholamine response of lymphocytes from patients with rheumatoid arthritis.

ABSTRACT: In patients with chronic rheumatic diseases, a decreased density of β2‐adrenergic receptors (β2R) on peripheral blood mononuclear cells (PBMC) could be demonstrated negatively correlating with various disease activity parameters. The aim of the present study was to determine the impact of this decrease on catecholamine response of PBMC from patients with rheumatoid arthritis (RA) in vitro. PBMC from 17 patients with RA and 6 healthy blood donors (HD) were investigated. The effects of epinephrine (E) and norepinephrine (NE) on PBMC proliferation were studied using cells activated with phytohemagglutinin (PHA) and monoclonal anti‐CD3‐antibodies (OKT3), respectively. The results revealed that lymphocytes of patients with RA showed a significantly reduced influence of catecholamines on PBMC function. In RA patients with high disease activity only, a shift to α1‐adrenergic‐mediated catecholamine effects upon PBMC reactivity could be observed. The study demonstrates the intricate relationship between PBMC reactivity and catecholamine effects that is mediated via α‐ and β‐adrenergic receptors due to disease activity. In this respect the altered catecholamine response of PBMC from patients with RA may contribute to the pathogenic process of RA.

Decreased catecholamine-induced cell death in B lymphocytes from patients with rheumatoid arthritis.

Abstract: The expression of β2‐adrenergic receptors (β2‐R) on B lymphocytes and agonist‐induced cAMP production is reduced in patients with rheumatoid arthritis (RA). To further study functional consequences of the diminished β2‐R density on B lymphocytes in RA patients, agonist‐induced cell death was evaluated and compared to healthy controls. B lymphocytes from patients with RA and healthy controls were activated with anti‐IgM‐antibody. Coincubation was carried out with isoprenaline (iso, 0.001‐10 μM). Apoptotic and necrotic cells were determined using Annexin‐V and propidium‐iodide staining. β2‐R‐induced cell death in B cells from healthy volunteers was stimulated after 24 h (medium, 21.2 ± 1.6%; iso, 34.6 ± 4.4%; increase 59.3 ± 10.1%). However, in RA patients the increase in cell death following β2‐R stimulation (21.8 ± 8.9%) was significantly impaired (p= 0.02). Our data demonstrate that catecholamine‐induced cell death after stimulation of β2‐R on B lymphocytes is decreased in RA patients, possibly contributing to the pathogenesis of the disease.

Sequence variants of the CRH 5'-flanking region : Effects on DNA-protein interactions studied by EMSA in PC12 cells

Abstract:  Recently, studies in adult rheumatoid arthritis patients have shown an association with four single‐nucleotide polymorphisms (SNPs) in the 3.7‐kb regulatory region of human corticotropin‐releasing hormone (hCRH) gene located at positions −3531, −3371, −2353, and −684 bp. Three of these novel polymorphisms are in absolute linkage disequilibrium, resulting in three combined alleles, named A1B1, A2B1, and A2B2. To study whether the described polymorphic nucleotide sequences in the 5′ region of the hCRH gene interfere with binding of nuclear proteins, an electric mobility shift assay (EMSA) was performed. At position −2353 bp, a specific DNA protein complex was detected for the wild‐type sequence only, possibly interfering with a binding site for the activating transcription factor 6 (ATF6). In contrast, no difference could be detected for the other SNPs. However, at position −684, a quantitative difference in protein binding due to cAMP incubation could be observed. To further investigate whether these SNPs in the CRH promoter are associated with an altered regulation of the CRH gene, we performed a luciferase reporter gene assay with transiently transfected rat pheochromocytoma cells PC12. Incubation with 8‐Br‐cAMP alone or in combination with cytokines enhanced significantly the promoter activity in PC12 cells. The promoter haplotypes studied exhibited a differential capacity to modulate CRH gene expression. In all our experiments, haplotype A1B1 showed the most pronounced influence on promoter activity. Taken together, our results demonstrate a differential binding capacity of nuclear proteins of the promoter polymorphisms resulting in a different gene regulation. Most probably the SNP at position −2,353 plays a major role in mediating these differences.