OBJECTIVE

Rheumatoid arthritis (RA) is a disease where immune disorders and chronic inflammatory reactions lead to joint distraction. This chronic inflammation, among many other abnormalities, is characterized by an increased number of preactivated neutrophils in the blood and heavy infiltration of these cells into the inflamed joint. 1 Based on the results of earlier studies, it has been proposed that metabolic activity of neutrophils may be influenced by neurotransmitters, including agonists of adrenergic and cholinergic receptors. 2 This conclusion was based on the results of pharmacological studies that suggested the existence of several subtypes of functional muscarinic receptors on human neutrophils. 3 For example, the cholinergic agonist, carbachol, has been shown to increase chemiluminescence of peripheral blood neutrophils. 2 In contrast, the same treatment resulted in a decreased chemiluminescence of neutrophils isolated from synovial fluid of patients with RA. 2 These opposite effects suggested that different subtypes of muscarinic receptors are expressed on neutrophils present in blood and synovial fluid, respectively. Because there are five distinct genes encoding muscarinic receptor subtypes, 4, 5 the aim of this study was to investigate the expression of mRNA encoding these receptor subtypes (ie, m1-m5) in neutrophils isolated from healthy blood donors and patients with RA.

METHODS

Neutrophils were isolated from the peripheral blood of nine healthy donors and from blood and synovial fluid of three patients with RA, using Gradisol G density gradient centrifugation. The final cell preparations contained more than 98% of neutrophils. Total RNA was isolated from neutrophils using TRI zol Reagent (Gibco-BRL) according to the method described by Chomczyński and Sacchi. 6 Prior to reverse transcription (RT), the residual genomic DNA was digested with DNAse I. DNAse-treated RNA (0.5 μg) served as a template for the RT reaction performed using the Gene-Amp RNA PCR kit (Perkin Elmer). PCR was carried out using five sets of sense and antisense primers specific for five muscarinic receptor sequences. The forward primers and an antisense reverse primers used were as published by Steel and Buckley. 7 The RT-PCR products separated via electrophoresis on agarose gels containing ethidium bromide revealed bands of expected size corresponding to m3, m4, and m5 muscarinic receptor subtypes. As the cDNA encoding selected fragments of muscarinic receptors, the identity of each band was further confirmed by DNA sequencing. The intensity of detected bands was evaluated by densitometric scanning and expressed as integrated optical density (IOD). In order to calculate the relative levels of muscarinic receptor expression in each sample, the IODs for mAChR subtypes were normalized based on the IODs of the housekeeping gene, glyceraldehyde phosphate dehydrogenase (GAPDH).

RESULTS

No bands corresponding to the m1 and m2 receptors were detected in any of the studied samples (data not shown). The cDNA corresponding to mRNA encoding m3, m4, and m5 receptor subtypes was present in neutrophils isolated from blood of healthy blood donors or RA patients. The predominant forms of muscarinic receptor subtypes were m3 and m5 (Fig. 1). A similar pattern of expression of muscarinic receptor subtypes was present in neutrophils from both healthy donors and patients with RA. Interestingly, however, neutrophils isolated from synovial fluid of RA patients lacked m4 receptor subtype (Fig. 2).