Background: β2 Adrenoreceptor expression on peripheral blood mononuclear cells is increased in progressive multiple sclerosis. This increase has been correlated with disease activity in relapsing-remitting multiple sclerosis.
Objective: To determine the β2 adrenoreceptor expression in primary and secondary progressive multiple sclerosis in relation to findings on magnetic resonance imaging (MRI) and clinical disease activity.
Methods: 10 patients with multiple sclerosis were studied (five with primary progressive and five with secondary progressive forms of the disease) over a period of six months. Monthly clinical and MRI assessments of the brain and spinal cord were carried out. β2 Adrenoreceptor expression was assessed monthly using a ligand binding assay with [125I]iodocyanopindolol. Expression of β2 adrenoceptors on peripheral blood mononuclear cells was also assessed in five normal controls over a similar period.
Results: The mean (SEM) value of β2 adrenoreceptor density for the five normal controls was 1346 (183) sites/cell, with affinity Kd of 120 (40) pM. MRI disease activity in primary progressive multiple sclerosis was reported on two occasions and on those occasions the expression of β2 adrenoreceptors was increased in excess of 1900 sites/cell; in the remaining 28 observations β2 adrenoreceptor expression was within the normal range (800 to 1900 sites/cell). In patients with secondary progressive disease, MRI disease activity was observed on 16 occasions. In these patients expression of β2 adrenoreceptors was increased in excess of 2000 sites/cell in all measurements except in one subject who did not show MRI activity throughout the six months period of study. The affinity of the receptors was within the normal range in all cases.
Conclusions: Increased expression of β2 adrenoreceptors was correlated with MRI disease activity in two patients with primary progressive multiple sclerosis. In secondary progressive multiple sclerosis, increased expression of β2 adrenoreceptors tended not to correlate with MRI disease activity. This may reflect a persistent Th1 immune reaction in the secondary progressive form of the disease.
- multiple sclerosis
- β adrenoreceptor expression
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Catecholamines drive a T helper (Th)2 shift at the level of both antigen presenting cells and Th1 cells. Noradrenaline and adrenaline potently inhibit the production of interleukin (IL)-12 while enhancing the production of IL-10 in human whole blood cultures stimulated by lipopolysaccharide ex vivo.1 These effects are mediated by stimulation of β2 adrenoreceptors and they are completely prevented by propranolol, a β adrenergic antagonist. Non-selective β adrenergic and selective β2 adrenergic agonists inhibit the production of IL-12 in vitro and in vivo.2,3 β2 Adrenergic agonists also inhibit the development of Th1 type cells, while promoting Th2 cell differentiation.2
β2 Adrenoreceptors are expressed on Th1 cells but not on Th2 cells.4 This might provide an additional mechanism for a differential effect of catecholamines on Th1/Th2 function. In murine and human systems, β2 adrenergic agonists inhibit γ interferon (IFNγ) production by Th1 cells, but do not affect IL-4 production by Th2 cells.4,5 The differential effects of catecholamines on type Th1/Th2 cytokine production also operate in vivo. Thus increasing sympathetic outflow in mice by selective β2 adrenergic antagonists or the application of β adrenergic agonists results in inhibition of lipopolysaccharide induced tumour necrosis factor α (TNFα) and IL-12.3,6,7 In humans, the administration of the β2 adrenergic agonist salbutamol results in inhibition of IL-12 production ex vivo,2 while acute brain trauma, which is followed by a massive release of catecholamines, triggers secretion of substantial amounts of systemic IL-10.8 Both glucocorticoids and catecholamines—through inhibition and stimulation of type 1 and type 2 cytokine secretion, respectively—cause selective suppression of cellular immunity and a shift towards Th2 mediated humoral immunity. Stress hormones inhibit the effector function of cellular immunity components, notably the activity of natural killer (NK) cells, T cytotoxic (Tc) cells, and activated macrophages. Catecholamines are potent inhibitors of NK cell activity, both directly, acting on β2 adrenoreceptors expressed on these cells, or indirectly, through suppression of the production of IL-12 and INFγ—cytokines essential for NK cell activity.1,9,10
Increased β2 adrenoreceptor density on peripheral blood mononuclear cells (PBMC) has been well documented in multiple sclerosis in several previous studies.11–,13 It has been shown that this increase in β2 adrenoreceptor density is related to high affinity IL-2 receptors on PBMC and to disease activity in the relapsing-remitting form of the disease.14 As β2 adrenoreceptors are expressed on NK cells, monocytes, Th1 cells, and Tc cells, increased expression in multiple sclerosis may reflect activation of Th1 cells and a predominately cellular immune activity. While cellular Th1 immune activity is considered to be the major contributor to disease activity in multiple sclerosis, humoral immune activity may also play an important role, especially in axonal damage. There is increasing evidence that in primary progressive multiple sclerosis axonal loss contributes to disability, while the inflammatory changes may be less pronounced.15–,18 Expression of β2 adrenoreceptors in primary progressive multiple sclerosis would therefore indirectly reflect the state of immunocompetent cells participating in cellular immune reactions.
To compare β2 adrenoreceptor expression in patients with secondary progressive and primary progressive multiple sclerosis we designed a longitudinal six months study during which clinical and magnetic resonance imaging (MRI) findings were compared with β2 adrenoreceptor expression on PBMC.
In our six month follow up study (with monthly assessments) we included 10 patients with multiple sclerosis diagnosed according to the standard criteria of Poser et al.19 Five had primary progressive multiple sclerosis, defined as a clear history of progressive neurological deterioration from the onset of the disease without relapse or remission; and five had secondary progressive disease, defined as clear history of progressive neurological deterioration for at least six months, following an initial relapsing/remitting course.20 All had oligoclonal bands in the cerebrospinal fluid. The age range of the patients with the primary progressive form of the disease was 35 to 49 years (mean 40.5) and of those with the secondary progressive form, 36 to 46 years (mean 38).
All patients were followed up at monthly intervals for six months by DK, who was blinded to the laboratory studies. A full history was taken from each subject, followed by a complete neurological examination. Disability was scored using the expanded disability status scale (EDSS).21 At the monthly assessment the patients were questioned about new or recurring symptoms, were re-examined, and their disability scored.
Clinical criteria were used to define relapse and remission. Relapse was defined as the occurrence of a symptom or symptoms of neurological dysfunction, with or without objective confirmation, lasting more than 24 hours; remission was defined as a definite improvement in signs or symptoms lasting for one month or more.19 None of the patients had received immunosuppressive or immunomodulatory treatment before the study period.
Five normal controls aged 35 to 50 years (mean 39) were studied serially and the expression of β2 adrenoreceptors on PBMC was assessed.
The studies were approved by the ethics committee of the National Hospital for Neurology and Neurosurgery, Queen Square, London. Informed consent was given by all patients and control subjects.
All scans were carried out using a Signa 1.5 T superconducting system (GE Medical Systems, Milwaukee, Wisconsin, USA) at the time of each clinical assessment.
T2 and proton density weighted images of the brain were acquired using a fast variable echo sequence (time of repetition (TR), 3500 ms; time of echo (TEef), 18 and 90 ms; echo train length, 8; image matrix, 192 × 256). The brain was surveyed in 34 × 4 mm contiguous interleaved axial slices. T1 weighted images (TR 570 ms, TE 23 ms) were acquired 5–10 minutes after injection of gadolinium-DTPA, 0.1 mmol/kg.
Sagittal spinal cord MRI
Two sets of 3 mm thick contiguous interleaved sagittal T2 weighted fast spin echo (FSE) images were obtained using a multiarray spinal coil (GE Medical Systems) with a 48 cm field of view, which enabled the entire cord to be visualised on a single image. Sequence parameters were as follows: TR, 2500 ms; TEef, 102 ms; echo train length 16; image matrix 512 × 512; two excitations; phase encoding superior-inferior. T1 weighted sagittal images (TR, 500 ms; TE, 19 ms) were acquired 15–20 minutes after injection of gadolinium-DTPA.
Brain and sagittal spinal cord scans were analysed by a neuroradiologist who was blinded to the clinical details.
β2 Adrenoreceptor assay
Isolation of PBMC
Peripheral blood was drawn by venepuncture into heparinised 10 ml Vacutainer tubes. PBMC were isolated according to the method of Boyum.22 The mononuclear layer was washed twice in phosphate buffered saline. Viability was 95% as assessed by eosin (2% wt/vol) exclusion.
PBMC were washed in phosphate buffered saline and resuspended at a concentration 5 × 106 cells/ml. A ligand binding assay with [125I]iodocyanopindolol (CYP) (Amersham, Aylesbury, Buckinghamshire, UK), specific activity 2000 μCi/mmol, was used as previously described.13 All assays were carried out by YZ, blinded to clinical and MRI information.
Data are presented as mean (SEM) when appropriate. The Wilcoxon rank sum test was used for the analysis of the differences in densities of β adrenoreceptors. The probability of coexistence of clinical or MRI activity with high expression of β adrenoreceptor densities on PBMC was analysed by the χ2 test or Fisher’s exact test. Statistical significance was accepted at the 95% confidence level (p < 0.05).
All data are presented in tables 1⇓ and 2⇓, while the densities of β2 adrenoreceptor densities in patients with primary and secondary progressive multiple sclerosis are presented as histograms in figs 1⇓ and 2⇓, respectively. An increased β2 adrenoreceptor density was considered to be present if the value was above 1900 sites/cell (mean value of the controls + 3 SEM: 1346 + 3 × 183 sites/cell). In secondary progressive multiple sclerosis there was increased expression of β2 adrenoreceptors compared with normal controls. There was no significant difference in the expression of β2 adrenoreceptors in primary progressive multiple sclerosis compared with the controls (fig 3⇓).
MRI spinal cord disease activity with enhancing lesions was recorded in two patients with primary progressive multiple sclerosis. This was correlated with an increased expression of β2 adrenoreceptor density on PBMC.
There was only one patient who had clinical relapses in the secondary progressive group of patients. In this group, MRI activity was observed on 16 occasions, while clinical relapses were reported on five occasions in the same patient. Increased β2 adrenoreceptor densities on PBMC were observed on 24 occasions. The probability of coexistence of increased β2 adrenoreceptor densities on PBMC and clinical or MRI evidence of activity in secondary progressive multiple sclerosis was not significant, though increased expression was observed on all occasions when there was MRI or clinical evidence of activity.
During the study, the disability score (EDSS) increased by 0.5 to 1 point in all except two patients, irrespective of whether they had primary progressive or secondary progressive disease. The two exceptions were patient 3 from the primary progressive group and patient 5 from the secondary progressive group. Neither of these patients showed MRI or clinical evidence of activity, and the β2 adrenoreceptors on their PBMC were within the normal range.
An increase in β2 adrenoreceptors on PBMC is associated with disease activity and sympathetic autonomic dysfunction in multiple sclerosis.11,13 This increase in β2 adrenoreceptors mainly affects CD8+ cells.12 Further studies have indicated that β2 adrenoreceptor expression is linked to disease activity and IL-2 receptor expression in relapsing-remitting multiple sclerosis,14 indicating that the increased density of β2 adrenoceptors is related to immune activation and inflammation. A comparative study of patients with multiple sclerosis and increased β2 adrenoreceptors on PBMC with patients with autonomic failure suggests that in multiple sclerosis the increased density of β2 adrenoreceptors is more likely to be linked to inflammation than to autonomic dysfunction.23
Activated lymphocytes express increased densities of β2 adrenoreceptors.13 As only Th1 cells, NK cells, and activated macrophages4 express β2 adrenoceptors, the increased expression of those receptors in multiple sclerosis may indirectly reflect an increased cellular rather than humoral activity.
Multiple sclerosis is a chronic inflammatory disease of the central nervous system postulated to be a T cell mediated autoimmune disease.24 IFNγ, a cytokine that is the hallmark of Th1 type immune responses, plays an important role in disease pathogenesis, as increased production of IFNγ precedes clinical attacks.25,26 Within the nervous system, the inflammatory process is characterised by increased IFNγ and IL-12 p40 expression.27,28 Alterations in IL-10 mRNA expression in multiple sclerosis have been shown to be dependent on disease activity.29,30
There is evidence from in vitro studies that stimulation of β2 adrenoreceptors results in a shift from a Th1 to a Th2 reaction by influencing the production of TNFα, IL-12, and IL-10.2,3,7,8 The increased expression of β2 adrenoreceptors in relapsing multiple sclerosis may be a recovery mechanism as Th1 cells become vulnerable to stress hormone stimuli, potentially allowing a shift to the Th2 type reaction. The therapeutic potential of using β adrenergic agonists in multiple sclerosis has been highlighted by the studies of Makhlouf et al.31,32
Potential immunological differences between the various forms of multiple sclerosis are of paramount importance as these could determine the response to various immunotherapies and underline different pathogenic mechanisms. Unlike the relapsing-remitting and secondary progressive forms of the disease, primary progressive multiple sclerosis is characterised by deterioration without relapses, and by fewer and smaller gadolinium enhancing lesions.33–,35 This suggests that other mechanisms leading to progressive axonal injury, apart from inflammation, may play a crucial role in primary progressive multiple sclerosis.36,37
In our study a distinct difference in the expression of β2 adrenoreceptors on PBMC was observed between patients with primary progressive and secondary progressive multiple sclerosis. Persistently increased densities of β2 adrenoreceptors were seen in secondary progressive but not in primary progressive disease, despite the observed deterioration in the disability score in both groups. In patients with secondary progressive disease there was no clear correlation between clinical activity, MRI activity, and expression of β2 adrenoreceptor densities, as is seen in the relapsing-remitting form.14 This observation further supports the lack of any clear correlation between clinical, MRI, and immunological variables and disease progression, and may reflect different underlying mechanisms contributing to disability in multiple sclerosis. The lack of correlation between MRI findings and disability progression in multiple sclerosis has been documented in several studies.38,39 Furthermore, attempts to identify reliable immunological markers of disease progression in this disease have so far failed.
Although β2 adrenoreceptor density on PBMC could not be used as a reliable disease activity marker in multiple sclerosis, the distinct differences in receptor expression in patients with the primary progressive and secondary progressive forms of the disease, and the fact that β2 adrenoreceptors are indirectly linked to cellular immune reaction and Th1 type of immune activity, provides support for other observations of immunological differences between promary progressive, relapsing-remitting, and secondary progressive multiple sclerosis.17,40,41
Increased β2 adrenoreceptor expression in patients with secondary progressive as compared with primary progressive multiple sclerosis suggests a persistent increase in cellular immune activity in the former.
This study was supported by the Multiple Sclerosis Society of Great Britain and Northern Ireland.
Competing interests: none declared
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