Sympathetic Skm Responses Are Decreased and Lymphocyte Beta-Adrenergic Receptors Are Increased in Progressive Multiple Sclerosis Joseph W. Karaszewski, BS, Anthony T. Reder, MD, Ricardo Maselli, MD, Margaret Brown, BS, and Barry G. W. Arnason, MD
Immune abnormalities, including deficient CD8 lymphocyte-mediated suppression, have been implicated in the progression of multiple sclerosis (MS). The peripheral sympathetic branch of the autonomic nervous system innervates the lymphoid organs and affects immune function. Animals with an ablated sympathetic nervous system develop more severe experimental allergic encephalomyelitis than control animals and exhibit an increased density of betaadrenergic receptors on their lymphocytes. Experimental allergic encephalomyelitis shares many features with MS. Accordingly, we investigated the psychogalvanic skin reflex in patients with rapidly progressive MS and found that 13 patients (57%) lacked this sympathetic-mediated response. The density of beta-adrenergic receptors on lymphocyte subsets was increased in progressive MS, most notably on the CD8 suppressodcytotoxic subset. B lymphocytes had the greatest number of receptors with 12.1 ? 1.8 fmol/106cells in control subjects and 18.7 -t 2.6 fmol/106cells in patients with MS. CD8 lymphocytes possessed an intermediate number of receptors with 3.4 0.4 fmol/106 cells in control subjects and 9.1 ? 1.6fmoYl0' cells in patients with MS. CD4 lymphocytes demonstrated the fewest receptors with 1.2 -t 0.1 fmoY106 cells in control subjects and 1.8 -t 0.4 fmol/106cells in patients with MS. No differences in the affinity or function (cyclic adenosine monophosphate levels in response to lop5M ( -)isoproterenol) of the adrenergic receptor were found when patients with progressive MS and control subjects were compared. Autonomic abnormalities in progressive MS and the increased beta-adrenergic receptor density found on CD8 lymphocytes may be related.
*
Karaszewski JW, Reder AT, Maselli R, Brown M, Arnason BGW. Sympathetic skin responses are decreased and lymphocyte beta-adrenergic receptors are increased in progressive multiple sclerosis. Ann Neurol 1990;27:366-372
Multiple sclerosis (MS) is characterized by areas of demyelination and glial scarring. Invading white blood cells are found within plaques and have been implicated in the tissue destruction observed in the disease. The trigger for an immunological reaction within the central nervous system in MS is unknown, but the consequence is a panoply of clinical symptoms that varies from one individual to the next depending on the locations of individual plaques. MS presents most often in a relapsing-remitting manner, but may become persistently progressive. Function of the autonomic nervous system is often compromised in MS. Bladder, bowel, and sexual dysfunction, abnormal cardiovascular responses to the Valsalva maneuver, as well as decreased variations in
heart rate are observed frequently in patients with MS and point to defective autonomic regulation [1-71. The sympathetic nervous system (SNS) innervates the immune system 18, 91. The SNS can be ablated in newborn rodents by chemical axotomy with 6hydroxy-dopamine (60HDA). Adult animals, sympathectomized at birth, have augmented antibody responses to T-cell-independent antigens 181, and exhibit an increase in the severities of experimental allergic encephalomyelitis (EAE) and experimental autoimmune myasthenia gravis [lo, 111. In addition, ablation of the SNS results in an increase in the number of beta-adrenergic receptors present on rodent splenic B and T lymphocytes [9, 12). Beta-adrenergic receptors have also been demon-
From the Department of Neurology and the Brain Research Institute, University of Chicago, Chicago, IL.
Address correspondence to Dr Arnason, Department of Neurology, University of Chicago, 5841 S. Maryland Avenue, BH Box 425, Chicago, IL 60637.
Received Feb 7, 1989, and in revised form Apr 27 and Sep 19. Accepted for publication Sep 30, 1989.
366 Copyright 0 1990 by the American Neurological Association
strated on human lymphocytes 1131. The distribution of beta-adrenergic receptors on T cells in healthy humans is threefold greater on T-suppressor (T,) cells (CD8+, 9.3-) than on cytotoxic T cells (CD8+, 9.3 +). T helper (CD4 +) cells have a very low density of receptors 1141. It seems reasonable to postulate that the influence of the SNS on T, cells should predominate over its influence on other T-cell subsets, owing to the higher density of beta-adrenergic receptors found normally on these cells. CD8 cell-mediated suppressor function is defective in MS 115-201. The derangement in T,-cell function depends on the stage of the disease. T,-cell function decreases during acute attacks and normalizes on remission 120); persistent failure of T,-cell function is observed in progressive disease 115-20). The question can be posed whether deficient SNS input in MS potentiates these immune system abnormalities. The anterior hypothalamus and hippocampus are sites of autonomic integration and send fibers to several areas of the brainstem and spinal cord. Anterior hypothalamic lesions result in peripheral immune alterations 121). MS plaques in limbic areas or, more likely, in those descending pathways that modulate function of the SNS, could lessen sympathetic input to the immune system, compromise T,-cell function, and tilt the balance toward further tissue destruction and a change in the course of MS from relapsing-remitting to progressive. We report physiological evidence for abnormal function of the SNS in patients with progressive MS. In addition, beta-adrenergic receptor density on CD8 lymphocytes is shown to be increased in progressive but not in "stable" MS.
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Methods Subjects All patients had definite MS based on standard criteria {22). Forty-five patients, 19 men (age, 40.1 2 8.6 years) and 26 women (age, 40.7 7.4 years) with progressive MS had lost 1 or more points on the Kurtzke scale in the previous year. The mean duration of the MS was 10.6 ? 1.4 years and average Kurtzke score, 5.1 0.3. All were spastic and none were receiving immunosuppressive therapy. The various experiments were done sequentially and different patients were studied in different ways. Twenty-five patients with stable MS, 11 men (age, 43.0 2 6.4 years) and 14 women (age, 46.3 & 2.1 years), had no evidence of disease activity during the preceding 2 years, and were taking no medications. None had received prior immunosuppressive therapy, and none had received corticotropin or glucocorticoid treatment in the preceding 2 years. Healthy individuals, 36 men (age, 36.3 ? 8.9 years) and 17 women (age, 33.4 2 9.2 years), served as control subjects.
*
*
Sympathetic Skin Responses to Electrical Stimulation Pedal sympathetic skin responses were measured in 23 patients with progressive MS, 14 patients with stable MS, and
24 control subjects in the supine position. Standard electromyograph (EMG) disc electrodes were attached to the sole and dorsum of the right foot. Recordings were obtained from an EMG (TECA TE4; TECA Corp., Pleasantville, NY) with a frequency response from 1.6 Hz to 3.2 kHz. Amplification sensitivity was set between 50 and 500 p,Vl division and sweep speed was 500 msecldivision. Stimulation consisted of square pulses of 200-microsecond duration delivered to the skin. The skin temperature of the patients was maintained above 32°C {23].
Isolation of Peripheral Blood Lymphocytes Peripheral blood was drawn by venipuncture into heparinized 15-ml Vacutainer tubes. Mononuclear cells (MNC) were isolated according to the method of Boyum [24f. B cells were isolated by a single step, rosetting method using Vibrio cbolerae neuraminidase (Calbiochem, La Jolla, CA)treated sheep red blood cells to remove T cells { 171. Monocytes were next depleted by plastic adherence in RPMI 1640 containing 5% fetal bovine serum (FBS) for 1 hour at 37"C, 5% carbon dioxide (COZ). CD4 + and CD8 + cell subsets of T cells were isolated by panning {17, 251. We used negative selection for the isolation of all lymphocyte subsets because antibodies to many cell surface antigens can modulate immune responses and membrane proteins. Purity of the cell subsets, as determined by indirect immunofluorescence using monoclonal antibodies, was greater than 90% at all times, and generally greater than 95%.
Characterization of {'2'1}Cyanopindolol Binding to Intact Lymphocytes Binding of ['251fcyanopindolol ({ '251)CYP) (New England Nuclear, Boston, MA) to lymphocyte subsets was determined by incubating 5 x lo5 cells with {1251)CYPin a total volume of 300 pl of incubation buffer (150 mM sodium chloride, 1 mM ascorbic acid, and 20 mM Tris, p H 7.4, at 30°C) for 90 minutes at 30°C. Nonspecific binding was determined by measuring the amount of ['251)CYP bound to the cells in the presence of 6 p,M dl-propranolol (Sigma Chemical, St. Louis, MO). Specific binding was determined as the difference between ['z51]CYP binding in the absence and in the presence of 6 pM dl-propranolol. Receptor density was determined by incubating the lymphocyte subsets with at least five different concentrations of {'2SI)CYP,ranging from 25 to 200 pM. Association rate constants were determined by incubating T lymphocytes with 25 to 50 PM {'251)CYPfor various times from 0 to 360 minutes. Dissociation rate constants were determined by incubating T lymphocytes with 25 to 50 PM {1251)CYPfor 90 minutes, adding 6 p,M dl-propranolol, and incubating the cells for additional time periods ranging from 95 to 360 minutes. In the displacement binding studies, specific binding was determined by incubating T lymphocytes with 25 to 50 PM {1251)CYPin the presence of different concentrations (10-3-10-8 M) of agonists. The experiments were terminated by dilution with 5 ml of ice-cold incubation buffer followed by vacuum filtration through Whatman GF/C glass fiber filters (Whatman International Ltd, Maidstone, UK). The sample tubes were rinsed with an additional 5 ml of cold buffer and the
Karaszewski et al: Lymphocyte Beta Receptor Density in MS
367
filters washed with an additional 30 ml of ice-cold incubation buffer. The filters were counted in a Beckman Gamma 4000 counter (Beckman Instruments, Inc, Fullerton, CA). Counts per minute were converted to molar equivalents correcting for the specific activity (2,200 Cdmmol) of ['251)CYP and the efficiency of the gamma counter 1261. Analysis of the relationship between specific binding and free ligand concentration at equilibrium was done by linear regression analysis. Rate constants were determined by the program KINETIC (Rodbard and McPherson, Version 1.O). The concentration of agonist displacing 50% ['251)CYP binding and Hill coefficients were determined by log-logit transformation of the displacement data.
Table I . Pedal Symputbetic Skin Response (SSR) in Patients with Progressive MS and Control Subjects
Extraction and Quantitation of Cyclic Adenosine Monophosphate
'p < 0.001 (Mann-Whitney U test).
Number of subjects No. with pedal SSR absent Amplitude of response, when present (FV) SEM Mean Minimum Maximum
*
Control Subjects
with MS
Patients
24
23 13 (57%)"
0
387.5 100 1,000
* 46.5
265.0 50 62 5
?
58.1b
bNot significant (Student's t test).
Cyclic adenosine monophosphate (CAMP)levels were determined by incubating 5 x 10' T cells in the presence of 20 FM isobutylmethykanthine or 20 pM isobutylmethylxanthine and 10 pM ( - )isoproterenol in phosphate-buffered saline solution (pH 7.4) in a total volume of 200 pl for 10 minutes at 37°C. The incubation was terminated by addition of 50 ~1 of 0.5 N hydrochloric acid. Centrifugation at 700 g at 4°C removed precipitated protein, and 200 p1 of the extract was removed and stored at - 70°C. Supernatants were assayed using a Rianen CAMP[125 I) radioimmunoassay kit (Dupont, Specialty Diagnostics, North Billerica, MA).
Results Postganglionic Sympathetic Szldomotor Function The sympathetic skin response or psychogalvanic skin reflex measures a change in skin potential in response to a variety of stimuli and is a sensitive test of postganglionic sympathetic sudomotor function [ 231. We investigated this response seeking evidence for compromised function of the SNS in 23 patients with progressive MS. Pedal sympathetic skin responses to electrical stimulation were present in all control subjects, but were absent in 13 (57%) of the examined patients with progressive MS (Table 1).The mean amplitude of the sympathetic skin responses was not significantly reduced in those patients demonstrating a response. These results point to compromised sympathetic sudomotor function in half the tested patients with progressive MS.
Beta-Adrenergic Receptor Density on Lymphocyte Szlbsets Lymphocyte subsets were isolated from peripheral blood using sheep red blood cell rosetting, plastic adherence, and panning methods. All lymphocyte subsets from both subject groups demonstrated saturable numbers of binding sites, and linear Scatchard plots indicated a single class of binding sites for the beta antagonist ['251]CYP (Fig 1). B cells exhibited the greatest number of receptors per cell, with 7,200 receptors/cell in control subjects and 11,000 receptors/ cell in the patients with progressive MS. C D 4 + T
368 Annals of Neurology Vol 27
Variable
No 4 April 1990
lymphocytes displayed the lowest number of receptors, with 700 receptors/cell in control subjects and 1,100 receptors/cell in patients with progressive MS. CD8 T lymphocytes demonstrated 2,000 receptors/ cell in control subjects versus 5,400 receptors/cell in patients with progressive MS (Table 2). Betaadrenergic receptor density was measured in 5 of the 23 patients whose sudomotor function had been measured. No correlation was noted between the two in this limited sample. When natural killer (NK) cells were removed from the T lymphocytes by panning with Leu l l b antibody, a slight increase in receptor number resulted (data not shown), indicating that NK cells were not the source of the increased receptor number in MS. These results demonstrate that betaadrenergic receptor density is increased on lymphocytes in patients with progressive MS and that the greatest difference is found on the CD8+ subset of lymphocytes.
+
Kinetics and Displacement of {12s1}CYP Binding to T Lymphocytes Association and dissociation rate constants were determined on T lymphocytes from patients with progressive MS and control subjects (Table 3). No difference in the affinity of the receptors for antagonist was detected. This finding contrasts with the apparent E(ds determined from saturation data alone. Representative Scatchard plots (see Fig 1, inset) show an increase in the apparent equilibrium dissociation constant (1/ slope) of CD8 lymphocytes in MS, which reflects the increased density of receptors on these cells in progressive MS. We next addressed the stereospecificity and betareceptor subtype in progressive MS. Binding of agonists to the beta-adrenergic receptor is characterized by different properties from those of antagonists. Only adrenergic receptor agonists are capable of chemically reducing the receptor C27) and distinguishing between the high (agonist dependent) and low (guanosine
14
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[1251]CYP CONCENTRATION (pM)
Fig 1. Specific binding of {12sI)cyanopindolol({12sI}CYP)to B lymphocytes (white box with black dot), CD4 lymphocytes (black diamond), and C D 8 + lymphocytes (black box with white dot) from a representative control subject and a patient with progressive MS as a function of {12sl}CYPconcentration. The insets show Scatchard plots of the binding. Analysis of the relationship between spec;f;cbinding (B) and free (F) ligand concentration at equilibrium was done by linear regression analysis.
+
triphosphate-dependent) affinity states of the receptor 128). Beta-adrenergic agonists expressed stereoselective binding to T lymphocytes in both patients with progressive MS and control subjects (see Table 3). On ' I lymphocytes ' from both groups, ( - )isoproterenol, the physiological ligand, was 10 times more potent than ( + )isoproterenol in inhibiting ['251)CYP-specific binding. Three beta-adrenergic agonists, ( - )isoproterenol, ( - )epinephrine, and ( - )norepinephrine, were examined for their ability to compete for specific ['251)CYP binding sites on intact T lymphocytes from
Table 2. Receptor Density of Lymphocyte Subsets in Patients with Progressive MS and Control Subjects
Control Subjects
*
Subset
Density SEM (frnoVl0' cells)
Mononuclear cells B lymphocytes T lymphocytes CD4 + lymphocytes CD8 + lymphocytes
4.6 0.5 12.1 2 1.8 1.9 0.2 1.2 0.1 3.4 0.4
* * * *
Patients with MS
*
No."
Density SEM (fmoV10' cells)
8 10 8 10 15
8.3 2 1.5 18.7 2 2.6 4.4 2 0.5 1.8 4 0.4 9.1 2 1.6
NO.^
p Value
8 8
< 0.05 < 0.1 < 0.001 < 0.2 < 0.005
9 10 10
"A total of 30 normal individuals were examined: 7 for mononuclear cells alone; 1 for mononuclear, B, CD4 + , and CD8 + cells; 9 for B, CD4 +, and CD8 + lymphocytes; 5 for CD8 + lymphocytes; and 8 additional individuals for T-lymphocyte receptor density. bA total of 22 patients with progressive MS were examined: 3 for mononuclear cells alone; 5 for mononuclear, B, CD4 + , and CD8 + cells; 3 for B, CD4 + , and CD8 + lymphocytes; 2 for CD4 and CD8 lymphocytes; and 9 additional patients for T-lymphocyte receptor density.
+
+
Karaszewski et al: Lymphocyte Beta Receptor Density in MS 369
Table 3. Characteristics of {12sl}Cyanopindolol(CUP) Binding to T Lymphocytes from Control Subjects and Patients with Progressive MS Variable Kinetics of ['251}CYP binding Association rate constant (K, 1 ) Dissociation rate constant (K- 1) Equilibrium dissociation constant (&) Displacement of ['251]CYP binding by agonistsb ( + )Isoproterenol ( - )Isoproterenol (- )Epinephrine ( - )Norepinephrine
Control Valuesa
MS Valuesa
' 1.1 ? 0.2 x 1 0 9 ~ - min-' 0.0034 ? 0.0011 min-' 3.41 r 1.11 x M
1.2 t 0.2 x lo9 M-' min-' 0.0033 ? 0.0006 min-' 3.04 r 0.63 x M
5.0 ? 1.1 3.9 ? 0.7 9.2 c 1.7 2.9 ? 0.6
X X X X
M (0.7845) M (0.5611) M (0.5366) lo-* M (1.0214)
1.2 t 0.3 X lo-* 1.8 t 1.2 x 1.3 t 0.2 ? lo-* 2.2 t 0.5 x lo-*
M (0.7553) M (0.5769) M (0.5471) M (1.0437)
"N = 5. bValues are concentration of agonist capable of displacing 50% ['''I]CYP coefficient.
patients with progressive MS and control subjects. The beta-adrenergic agonists competed for [1251)CYPbinding sites on T cells from control subjects and patients with MS (see Table 3), with an order of potency of ( - )isoproterenol greater than ( - )epinephrine, which was greater than ( - )norepinephrine, indicating that the receptor in patients with MS and control subjects is of the beta-2 type. Hill coefficients derived from the competitive binding curves were all less than unity with the exception of ( - )norepinephrine (see Table 3). Hill coefficients less than one (biphasic curve) may indicate the existence of: (1) receptors of different affinities, or (2) receptors of the same affinity, some of which are compartmentalized in such a way that they fail to interact with hydrophilic ligands.
Basal and ( -)Isoproterenol-Stimulated cAMP in T Lymphocytes Basal and ( - )isoproterenol-stimulated production of CAMP,in the presence of isobutylmethylxanthine, was examined (Fig 2). Basal levels of cAMP were 2.66 -t 0.28 pmoYl0' cells (n = 12) in control subjects and 3.33 -t 0.44 pmoYl0' cells in patients with progressive MS (n = 21; not significant). In response to l o p 5 M (-)isoproterenol, cAMP levels rose to 13.02 +2.37 pmoYlO' cells in control subjects and 15.79 2.10 pmoYlO' cells in the patients with progressive MS (not significant). These findings indicate that both basal and 10- M ( - )isoproterenol-stimulated levels of cAMP are similar in patients with progressive MS and in control subjects.
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CD8 Lymphocyte Receptor Density and Sympathetic Skin Responses in Stable MS and i n Diabetic Neuropathy CD8 lymphocytes were isolated from peripheral blood of patients with stable MS and diabetic patients, and beta-adrenergic receptor density was determined.
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370 Annals of Neurology Vol 27 N o 4 April 1990
binding. In parentheses are p values as determined by the Hill
STIMULATED
BASAL cAMP LEVEL
Fig 2. Basal and lo-' M (-)isoproterenol-stimulated cAMP levels in T (ymphocytes of control subjects and patients with progressive MS. The figure shows results determined from a single lot of the Rianen cAMP kit on the same day. Intra-assay variance was 10%. The data represent 14 controlsubjects and 21 patients with progressive MS. Two control basal values were excluded using Dixon's Gap Test at the 0.005 confidence level. Statistics were computed using Student's t test.
The density of beta-adrenergic receptors on CD8+ lymphocytes from patients with stable MS was 4.9 k 0.9 fmoYl0' cells (n = 13), a value not significantly different from control values. Pedal sympathetic skin responses were determined in 14 additional patients with stable MS. Three (21.4%) of the 14 patients had an absent sympathetic skin response; the mean amplitude of response, when present, was not significantly different from that of control values. CD8 receptor density was also determined in 10 diabetic patients with peripheral neuropathy and evidence of compromised function of the SNS. Receptor density of the diabetic patients was 4.2 0.5 fmoV10' cells, again not statistically significantly different from control values.
*
Discussion The major new finding reported here is that there is a twofold to threefold increase over normal values in the number of beta-adrenergic receptors on CD8+ cells from patients with progressive MS. No such increase is observed in patients with stable MS, indicating that upregulation of beta-adrenergic receptors occurs as the disease evolves. The basis for this upregulation remains uncertain. One possibility is denervation hypersensitivity. The lymphoid organs receive an extensive intraparenchymal SNS innervation and ablation of this innervation in mice is followed by an upregulation of beta-adrenergic receptors on splenic lymphocytes 1121. We show here that function of the SNS is much more frequently compromised in progressive than in stable MS, findings which corroborate and extend earlier work by others 11-71. Compromised function of the SNS may have immunological consequences. EAE is more severe in SNS-ablated rats than in controls {lo], indicating that one normal role of an intact SNS may be to exert a downregulating action on certain immune responses. Might compromised function of the SNS, when it occurs during the course of MS, have as a consequence a resetting of the immune rheostat which switches the disease into a progressive course? Cells of the T-cell subset that mediate suppressor function (CD8+, 9.3-) have, in health, more betaadrenergic receptors than T cells of other subsets, suggesting, perhaps, that the putative downregulating role of the intact SNS on the immune response might be mediated, at least in part, by activation of suppressor cells. A corollary would be that in the absence of SNS innervation, the potential of these cells for activation might be diminished. CD8 + cell-mediated suppressor function is known to be deficient in progressive MS 115-201, a finding consistent with the formulation advanced here. Specific binding of ['251)CYP to lymphocyte subsets both in normal individuals and in patients with progressive MS is saturable, stereospecific, and competitive. The receptors in progressive MS have equilibrium dissociation constants for { '251)CYP equal to those of normal lymphocytes. Competitive binding of agonists reveals that the adrenergic receptor is of the beta-2 subtype. Hill coefficients of competitive binding curves indicate that the beta-2 receptors of patients with progressive MS and control subjects do not have a homogeneous affinity for agonists. We attribute the biphasic nature of the competition binding curves to the presence of high- and low-affinity cell surface receptors, as proposed by other investigators 129-3 11. The beta adrenergic receptors are functional in progressive MS; ( - )isoproterenol induces the production of CAMP. Beta-agonist-induced increases in cAMP
accumulation do not correlate with receptor density, since cells from patients with progressive MS and control subjects show comparable rises. Stimulation of cAMP production has been shown to be maximal at lo-' M ( - )isoproterenol(32). Possibly a difference in cAMP production between cells from patients with progressive MS and control subjects would be seen following stimulation with less than maximal concentrations of ( - )isoproterenol. Alternatively, if the receptors in progressive MS are largely sequestered internally and nonfunctional, then stimulation of cAMP by ( - )isoproterenol need not be expected to parallel their increased density. In addition, the mean age of the patients with progressive MS (41.1 -+ 11.2 years) was higher than that of the normal control subjects (33.7 9.9 years) and, while no correlation exists between age or sex and beta-adrenergic receptor number on lymphocytes, a decreased beta-adrenergic responsiveness with increasing age has been noted 1331. Patients with orthostatic hypotension have been shown to have upregulated beta-adrenergic receptors on their peripheral blood lymphocytes, a finding attributed to denervation hypersensitivity 134, 351. Yet, we find no significant upregulation of beta-adrenergic receptors in diabetic patients with autonomic neuropathy. In experimental diabetic neuropathy, certain autonomic pathways are spared 1361 and it is conceivable that the SNS innervation of lymphoid organs is similarly spared in diabetic neuropathy in man. It is also possible that an upregulation of lymphocyte betaadrenergic receptors cannot simply be attributed to denervation hypersensitivity. MS is an inflammatory disease. Perhaps upregulation of beta-adrenergic receptors on lymphocytes is characteristic of chronic inflammatory diseases in general, and in no way specific to MS. Regardless of specificity, it remains possible that beta-adrenergic agonists could influence the course of MS. The beta-agonist ( - )isoproterenol was recently shown to favorably alter the course of EAE 1101.
*
This study was supported in part by a research grant (MS:RG1130D-19) from the National Multiple Sclerosis Society to D r Arnason, and by a program project grant (1P01-NS-24575-01A1) from the National Institutes of Health. D r Reder is a Harry Weaver Neuroscience Scholar (JF2027-A-1) of the National Multiple Sclerosis Society and is supported by National Institutes of Health-Clinical Investigator Development Award 1 KO8 NS 01068-01. We thank Dr Sol Rajfer for his assistance in the development of this project, as well as many supportive discussions; Dr J Jaspan for providing the diabetic patients; and Ms Madeleine Murphy for her assistance in the preparation of this manuscript. This work was presented in part at the 112th Annual Meeting of the American Neurological Association, San Francisco, CA, Oct 20, 1987.
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35.
36.
cyte suppression and interferon production in multiple sclerosis. Proc Natl Acad Sci USA 1979;76:476-480 Antel JP, Arnason BGW, Medof ME. Suppressor cell function in multiple sclerosis: correlation with clinical disease activity. Ann Neurol 1979;5:338-342 Cross RJ, Markesbury WR, Brooks WH, Roszman TL. Hypothalamic-immune interactions: neuromodulation of natural killer activity by lesioning of the anterior hypothalamus. Immunology 1984;51:399-405 Poser CM, Pary DW, Scheinberg L, et al. New diagnostic criteria for multiple sclerosis: guidelines for research protocols. Ann Neurol 1983;13:227-23 1 Shahani BT, Halperin JJ, Bolu P, Cohen J. Sympathetic skin response: a method of assessing unmyelinated axon dysfunction in peripheral neuropathies. J Neurol Neurosurg Psychiatry 1984;47:536-542 Boyum A. Isolation of mononuclear cells and granulocytes from human blood. Scand J Clin Lab Invest 1968;21(suppl97):77-89 Wysocki LJ, Sat0 VL. Panning for lymphocytes: a method for cell selection. Proc Natl Acad Sci USA 1978;75:2844-2848 Doyle VM, Buhler FR, Burgisser E. Inappropriate correction for radioactive decay in fully iodinated adrenergic radioligands. Eur J Pharmacol 1984;99:353-356 Wong A, Hwang SM, Cheng H , Crooke ST. Structure activity relationships of beta-adrenergic receptor-coupled adenylate cyclase: implications of a redox mechanism for the action of agonists at beta-adrenergic receptors. Mol Pharmacol 1987;31: 368-376 Hoffman BB, Lefiowitz RJ. Radioligand binding studies of adrenergic receptors: new insights into molecular and physiological regulation. Annu Rev Pharmacol Toxic01 1980;20:581-608 Staehelin M, Simons P, Jaeggi K, Wigger N. CGP-12177. A hydrophilic beta-adrenergic receptor radioligand reveals high affinity binding of agonists to intact cells. J Biol Chem 1983;258:3496-3502 Harden TK, Cotton CU, Waldo GL, et al. Catecholamineinduced alteration in sedimentation behavior of membrane bound beta-adrenergic receptors. Science 1980;210:441-443 De Blasi A, Lipartiti M, Motulsky HJ, et al. Agonist-induced redistribution of beta-adrenergic receptors on intact human mononuclear leukocyte redistributed receptors are nonfunctional. J Clin Endocrinol Metab 1985;61:1081-1088 Ebstein RP, Oppenheim G, Stessman J. Alzheimer’s disease: isoproterenol and prostaglandin El-stimulated cyclic AMP accumulation in lymphocytes. Life Sci 1984;34:2239-2243 Halper JP, Mann JJ, Weksler ME, et al. Beta adrenergic receptors and CAMP levels in intact human lymphocytes: effects of age and gender. Life Sci 1984;35:855-863 Bannister R, Boylston AW, Davies IB, et al. Beta-receptor numbers and thermodynamics in denervation hypersensitivity.J Physiol 1981;319:369-377 Hui KKP, Connolly ME. Increased numbers of beta-receptors in orthostatic hypotension due to autonomic dysfunction. N Engl J Med 1981;304:1473-1476 Schmidt RE, Plurad DA, Roth KA. Noradrenergic and peptidergic innervation of rat ileum in chronic experimental diabetes. Ann Neurol 1988;24:473-474
Immune abnormalities, including deficient CD8 lymphocyte-mediated suppression, have been implicated in the progression of multiple sclerosis (MS). The peripheral sympathetic branch of the autonomic nervous system innervates the lymphoid organs and affects immune function. Animals with an ablated sympathetic nervous system develop more severe experimental allergic encephalomyelitis than control animals and exhibit an increased density of betaadrenergic receptors on their lymphocytes. Experimental allergic encephalomyelitis shares many features with MS. Accordingly, we investigated the psychogalvanic skin reflex in patients with rapidly progressive MS and found that 13 patients (57%) lacked this sympathetic-mediated response. The density of beta-adrenergic receptors on lymphocyte subsets was increased in progressive MS, most notably on the CD8 suppressodcytotoxic subset. B lymphocytes had the greatest number of receptors with 12.1 ? 1.8 fmol/106cells in control subjects and 18.7 -t 2.6 fmol/106cells in patients with MS. CD8 lymphocytes possessed an intermediate number of receptors with 3.4 0.4 fmol/106 cells in control subjects and 9.1 ? 1.6fmoYl0' cells in patients with MS. CD4 lymphocytes demonstrated the fewest receptors with 1.2 -t 0.1 fmoY106 cells in control subjects and 1.8 -t 0.4 fmol/106cells in patients with MS. No differences in the affinity or function (cyclic adenosine monophosphate levels in response to lop5M ( -)isoproterenol) of the adrenergic receptor were found when patients with progressive MS and control subjects were compared. Autonomic abnormalities in progressive MS and the increased beta-adrenergic receptor density found on CD8 lymphocytes may be related.
*
Karaszewski JW, Reder AT, Maselli R, Brown M, Arnason BGW. Sympathetic skin responses are decreased and lymphocyte beta-adrenergic receptors are increased in progressive multiple sclerosis. Ann Neurol 1990;27:366-372
Multiple sclerosis (MS) is characterized by areas of demyelination and glial scarring. Invading white blood cells are found within plaques and have been implicated in the tissue destruction observed in the disease. The trigger for an immunological reaction within the central nervous system in MS is unknown, but the consequence is a panoply of clinical symptoms that varies from one individual to the next depending on the locations of individual plaques. MS presents most often in a relapsing-remitting manner, but may become persistently progressive. Function of the autonomic nervous system is often compromised in MS. Bladder, bowel, and sexual dysfunction, abnormal cardiovascular responses to the Valsalva maneuver, as well as decreased variations in
heart rate are observed frequently in patients with MS and point to defective autonomic regulation [1-71. The sympathetic nervous system (SNS) innervates the immune system 18, 91. The SNS can be ablated in newborn rodents by chemical axotomy with 6hydroxy-dopamine (60HDA). Adult animals, sympathectomized at birth, have augmented antibody responses to T-cell-independent antigens 181, and exhibit an increase in the severities of experimental allergic encephalomyelitis (EAE) and experimental autoimmune myasthenia gravis [lo, 111. In addition, ablation of the SNS results in an increase in the number of beta-adrenergic receptors present on rodent splenic B and T lymphocytes [9, 12). Beta-adrenergic receptors have also been demon-
From the Department of Neurology and the Brain Research Institute, University of Chicago, Chicago, IL.
Address correspondence to Dr Arnason, Department of Neurology, University of Chicago, 5841 S. Maryland Avenue, BH Box 425, Chicago, IL 60637.
Received Feb 7, 1989, and in revised form Apr 27 and Sep 19. Accepted for publication Sep 30, 1989.
366 Copyright 0 1990 by the American Neurological Association
strated on human lymphocytes 1131. The distribution of beta-adrenergic receptors on T cells in healthy humans is threefold greater on T-suppressor (T,) cells (CD8+, 9.3-) than on cytotoxic T cells (CD8+, 9.3 +). T helper (CD4 +) cells have a very low density of receptors 1141. It seems reasonable to postulate that the influence of the SNS on T, cells should predominate over its influence on other T-cell subsets, owing to the higher density of beta-adrenergic receptors found normally on these cells. CD8 cell-mediated suppressor function is defective in MS 115-201. The derangement in T,-cell function depends on the stage of the disease. T,-cell function decreases during acute attacks and normalizes on remission 120); persistent failure of T,-cell function is observed in progressive disease 115-20). The question can be posed whether deficient SNS input in MS potentiates these immune system abnormalities. The anterior hypothalamus and hippocampus are sites of autonomic integration and send fibers to several areas of the brainstem and spinal cord. Anterior hypothalamic lesions result in peripheral immune alterations 121). MS plaques in limbic areas or, more likely, in those descending pathways that modulate function of the SNS, could lessen sympathetic input to the immune system, compromise T,-cell function, and tilt the balance toward further tissue destruction and a change in the course of MS from relapsing-remitting to progressive. We report physiological evidence for abnormal function of the SNS in patients with progressive MS. In addition, beta-adrenergic receptor density on CD8 lymphocytes is shown to be increased in progressive but not in "stable" MS.
+
Methods Subjects All patients had definite MS based on standard criteria {22). Forty-five patients, 19 men (age, 40.1 2 8.6 years) and 26 women (age, 40.7 7.4 years) with progressive MS had lost 1 or more points on the Kurtzke scale in the previous year. The mean duration of the MS was 10.6 ? 1.4 years and average Kurtzke score, 5.1 0.3. All were spastic and none were receiving immunosuppressive therapy. The various experiments were done sequentially and different patients were studied in different ways. Twenty-five patients with stable MS, 11 men (age, 43.0 2 6.4 years) and 14 women (age, 46.3 & 2.1 years), had no evidence of disease activity during the preceding 2 years, and were taking no medications. None had received prior immunosuppressive therapy, and none had received corticotropin or glucocorticoid treatment in the preceding 2 years. Healthy individuals, 36 men (age, 36.3 ? 8.9 years) and 17 women (age, 33.4 2 9.2 years), served as control subjects.
*
*
Sympathetic Skin Responses to Electrical Stimulation Pedal sympathetic skin responses were measured in 23 patients with progressive MS, 14 patients with stable MS, and
24 control subjects in the supine position. Standard electromyograph (EMG) disc electrodes were attached to the sole and dorsum of the right foot. Recordings were obtained from an EMG (TECA TE4; TECA Corp., Pleasantville, NY) with a frequency response from 1.6 Hz to 3.2 kHz. Amplification sensitivity was set between 50 and 500 p,Vl division and sweep speed was 500 msecldivision. Stimulation consisted of square pulses of 200-microsecond duration delivered to the skin. The skin temperature of the patients was maintained above 32°C {23].
Isolation of Peripheral Blood Lymphocytes Peripheral blood was drawn by venipuncture into heparinized 15-ml Vacutainer tubes. Mononuclear cells (MNC) were isolated according to the method of Boyum [24f. B cells were isolated by a single step, rosetting method using Vibrio cbolerae neuraminidase (Calbiochem, La Jolla, CA)treated sheep red blood cells to remove T cells { 171. Monocytes were next depleted by plastic adherence in RPMI 1640 containing 5% fetal bovine serum (FBS) for 1 hour at 37"C, 5% carbon dioxide (COZ). CD4 + and CD8 + cell subsets of T cells were isolated by panning {17, 251. We used negative selection for the isolation of all lymphocyte subsets because antibodies to many cell surface antigens can modulate immune responses and membrane proteins. Purity of the cell subsets, as determined by indirect immunofluorescence using monoclonal antibodies, was greater than 90% at all times, and generally greater than 95%.
Characterization of {'2'1}Cyanopindolol Binding to Intact Lymphocytes Binding of ['251fcyanopindolol ({ '251)CYP) (New England Nuclear, Boston, MA) to lymphocyte subsets was determined by incubating 5 x lo5 cells with {1251)CYPin a total volume of 300 pl of incubation buffer (150 mM sodium chloride, 1 mM ascorbic acid, and 20 mM Tris, p H 7.4, at 30°C) for 90 minutes at 30°C. Nonspecific binding was determined by measuring the amount of ['251)CYP bound to the cells in the presence of 6 p,M dl-propranolol (Sigma Chemical, St. Louis, MO). Specific binding was determined as the difference between ['z51]CYP binding in the absence and in the presence of 6 pM dl-propranolol. Receptor density was determined by incubating the lymphocyte subsets with at least five different concentrations of {'2SI)CYP,ranging from 25 to 200 pM. Association rate constants were determined by incubating T lymphocytes with 25 to 50 PM {'251)CYPfor various times from 0 to 360 minutes. Dissociation rate constants were determined by incubating T lymphocytes with 25 to 50 PM {1251)CYPfor 90 minutes, adding 6 p,M dl-propranolol, and incubating the cells for additional time periods ranging from 95 to 360 minutes. In the displacement binding studies, specific binding was determined by incubating T lymphocytes with 25 to 50 PM {1251)CYPin the presence of different concentrations (10-3-10-8 M) of agonists. The experiments were terminated by dilution with 5 ml of ice-cold incubation buffer followed by vacuum filtration through Whatman GF/C glass fiber filters (Whatman International Ltd, Maidstone, UK). The sample tubes were rinsed with an additional 5 ml of cold buffer and the
Karaszewski et al: Lymphocyte Beta Receptor Density in MS
367
filters washed with an additional 30 ml of ice-cold incubation buffer. The filters were counted in a Beckman Gamma 4000 counter (Beckman Instruments, Inc, Fullerton, CA). Counts per minute were converted to molar equivalents correcting for the specific activity (2,200 Cdmmol) of ['251)CYP and the efficiency of the gamma counter 1261. Analysis of the relationship between specific binding and free ligand concentration at equilibrium was done by linear regression analysis. Rate constants were determined by the program KINETIC (Rodbard and McPherson, Version 1.O). The concentration of agonist displacing 50% ['251)CYP binding and Hill coefficients were determined by log-logit transformation of the displacement data.
Table I . Pedal Symputbetic Skin Response (SSR) in Patients with Progressive MS and Control Subjects
Extraction and Quantitation of Cyclic Adenosine Monophosphate
'p < 0.001 (Mann-Whitney U test).
Number of subjects No. with pedal SSR absent Amplitude of response, when present (FV) SEM Mean Minimum Maximum
*
Control Subjects
with MS
Patients
24
23 13 (57%)"
0
387.5 100 1,000
* 46.5
265.0 50 62 5
?
58.1b
bNot significant (Student's t test).
Cyclic adenosine monophosphate (CAMP)levels were determined by incubating 5 x 10' T cells in the presence of 20 FM isobutylmethykanthine or 20 pM isobutylmethylxanthine and 10 pM ( - )isoproterenol in phosphate-buffered saline solution (pH 7.4) in a total volume of 200 pl for 10 minutes at 37°C. The incubation was terminated by addition of 50 ~1 of 0.5 N hydrochloric acid. Centrifugation at 700 g at 4°C removed precipitated protein, and 200 p1 of the extract was removed and stored at - 70°C. Supernatants were assayed using a Rianen CAMP[125 I) radioimmunoassay kit (Dupont, Specialty Diagnostics, North Billerica, MA).
Results Postganglionic Sympathetic Szldomotor Function The sympathetic skin response or psychogalvanic skin reflex measures a change in skin potential in response to a variety of stimuli and is a sensitive test of postganglionic sympathetic sudomotor function [ 231. We investigated this response seeking evidence for compromised function of the SNS in 23 patients with progressive MS. Pedal sympathetic skin responses to electrical stimulation were present in all control subjects, but were absent in 13 (57%) of the examined patients with progressive MS (Table 1).The mean amplitude of the sympathetic skin responses was not significantly reduced in those patients demonstrating a response. These results point to compromised sympathetic sudomotor function in half the tested patients with progressive MS.
Beta-Adrenergic Receptor Density on Lymphocyte Szlbsets Lymphocyte subsets were isolated from peripheral blood using sheep red blood cell rosetting, plastic adherence, and panning methods. All lymphocyte subsets from both subject groups demonstrated saturable numbers of binding sites, and linear Scatchard plots indicated a single class of binding sites for the beta antagonist ['251]CYP (Fig 1). B cells exhibited the greatest number of receptors per cell, with 7,200 receptors/cell in control subjects and 11,000 receptors/ cell in the patients with progressive MS. C D 4 + T
368 Annals of Neurology Vol 27
Variable
No 4 April 1990
lymphocytes displayed the lowest number of receptors, with 700 receptors/cell in control subjects and 1,100 receptors/cell in patients with progressive MS. CD8 T lymphocytes demonstrated 2,000 receptors/ cell in control subjects versus 5,400 receptors/cell in patients with progressive MS (Table 2). Betaadrenergic receptor density was measured in 5 of the 23 patients whose sudomotor function had been measured. No correlation was noted between the two in this limited sample. When natural killer (NK) cells were removed from the T lymphocytes by panning with Leu l l b antibody, a slight increase in receptor number resulted (data not shown), indicating that NK cells were not the source of the increased receptor number in MS. These results demonstrate that betaadrenergic receptor density is increased on lymphocytes in patients with progressive MS and that the greatest difference is found on the CD8+ subset of lymphocytes.
+
Kinetics and Displacement of {12s1}CYP Binding to T Lymphocytes Association and dissociation rate constants were determined on T lymphocytes from patients with progressive MS and control subjects (Table 3). No difference in the affinity of the receptors for antagonist was detected. This finding contrasts with the apparent E(ds determined from saturation data alone. Representative Scatchard plots (see Fig 1, inset) show an increase in the apparent equilibrium dissociation constant (1/ slope) of CD8 lymphocytes in MS, which reflects the increased density of receptors on these cells in progressive MS. We next addressed the stereospecificity and betareceptor subtype in progressive MS. Binding of agonists to the beta-adrenergic receptor is characterized by different properties from those of antagonists. Only adrenergic receptor agonists are capable of chemically reducing the receptor C27) and distinguishing between the high (agonist dependent) and low (guanosine
14
14
13
7 1-2
13
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12 i
11
11
10
10 CDL
9
7
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[1251]CYP CONCENTRATION (pM)
Fig 1. Specific binding of {12sI)cyanopindolol({12sI}CYP)to B lymphocytes (white box with black dot), CD4 lymphocytes (black diamond), and C D 8 + lymphocytes (black box with white dot) from a representative control subject and a patient with progressive MS as a function of {12sl}CYPconcentration. The insets show Scatchard plots of the binding. Analysis of the relationship between spec;f;cbinding (B) and free (F) ligand concentration at equilibrium was done by linear regression analysis.
+
triphosphate-dependent) affinity states of the receptor 128). Beta-adrenergic agonists expressed stereoselective binding to T lymphocytes in both patients with progressive MS and control subjects (see Table 3). On ' I lymphocytes ' from both groups, ( - )isoproterenol, the physiological ligand, was 10 times more potent than ( + )isoproterenol in inhibiting ['251)CYP-specific binding. Three beta-adrenergic agonists, ( - )isoproterenol, ( - )epinephrine, and ( - )norepinephrine, were examined for their ability to compete for specific ['251)CYP binding sites on intact T lymphocytes from
Table 2. Receptor Density of Lymphocyte Subsets in Patients with Progressive MS and Control Subjects
Control Subjects
*
Subset
Density SEM (frnoVl0' cells)
Mononuclear cells B lymphocytes T lymphocytes CD4 + lymphocytes CD8 + lymphocytes
4.6 0.5 12.1 2 1.8 1.9 0.2 1.2 0.1 3.4 0.4
* * * *
Patients with MS
*
No."
Density SEM (fmoV10' cells)
8 10 8 10 15
8.3 2 1.5 18.7 2 2.6 4.4 2 0.5 1.8 4 0.4 9.1 2 1.6
NO.^
p Value
8 8
< 0.05 < 0.1 < 0.001 < 0.2 < 0.005
9 10 10
"A total of 30 normal individuals were examined: 7 for mononuclear cells alone; 1 for mononuclear, B, CD4 + , and CD8 + cells; 9 for B, CD4 +, and CD8 + lymphocytes; 5 for CD8 + lymphocytes; and 8 additional individuals for T-lymphocyte receptor density. bA total of 22 patients with progressive MS were examined: 3 for mononuclear cells alone; 5 for mononuclear, B, CD4 + , and CD8 + cells; 3 for B, CD4 + , and CD8 + lymphocytes; 2 for CD4 and CD8 lymphocytes; and 9 additional patients for T-lymphocyte receptor density.
+
+
Karaszewski et al: Lymphocyte Beta Receptor Density in MS 369
Table 3. Characteristics of {12sl}Cyanopindolol(CUP) Binding to T Lymphocytes from Control Subjects and Patients with Progressive MS Variable Kinetics of ['251}CYP binding Association rate constant (K, 1 ) Dissociation rate constant (K- 1) Equilibrium dissociation constant (&) Displacement of ['251]CYP binding by agonistsb ( + )Isoproterenol ( - )Isoproterenol (- )Epinephrine ( - )Norepinephrine
Control Valuesa
MS Valuesa
' 1.1 ? 0.2 x 1 0 9 ~ - min-' 0.0034 ? 0.0011 min-' 3.41 r 1.11 x M
1.2 t 0.2 x lo9 M-' min-' 0.0033 ? 0.0006 min-' 3.04 r 0.63 x M
5.0 ? 1.1 3.9 ? 0.7 9.2 c 1.7 2.9 ? 0.6
X X X X
M (0.7845) M (0.5611) M (0.5366) lo-* M (1.0214)
1.2 t 0.3 X lo-* 1.8 t 1.2 x 1.3 t 0.2 ? lo-* 2.2 t 0.5 x lo-*
M (0.7553) M (0.5769) M (0.5471) M (1.0437)
"N = 5. bValues are concentration of agonist capable of displacing 50% ['''I]CYP coefficient.
patients with progressive MS and control subjects. The beta-adrenergic agonists competed for [1251)CYPbinding sites on T cells from control subjects and patients with MS (see Table 3), with an order of potency of ( - )isoproterenol greater than ( - )epinephrine, which was greater than ( - )norepinephrine, indicating that the receptor in patients with MS and control subjects is of the beta-2 type. Hill coefficients derived from the competitive binding curves were all less than unity with the exception of ( - )norepinephrine (see Table 3). Hill coefficients less than one (biphasic curve) may indicate the existence of: (1) receptors of different affinities, or (2) receptors of the same affinity, some of which are compartmentalized in such a way that they fail to interact with hydrophilic ligands.
Basal and ( -)Isoproterenol-Stimulated cAMP in T Lymphocytes Basal and ( - )isoproterenol-stimulated production of CAMP,in the presence of isobutylmethylxanthine, was examined (Fig 2). Basal levels of cAMP were 2.66 -t 0.28 pmoYl0' cells (n = 12) in control subjects and 3.33 -t 0.44 pmoYl0' cells in patients with progressive MS (n = 21; not significant). In response to l o p 5 M (-)isoproterenol, cAMP levels rose to 13.02 +2.37 pmoYlO' cells in control subjects and 15.79 2.10 pmoYlO' cells in the patients with progressive MS (not significant). These findings indicate that both basal and 10- M ( - )isoproterenol-stimulated levels of cAMP are similar in patients with progressive MS and in control subjects.
*
CD8 Lymphocyte Receptor Density and Sympathetic Skin Responses in Stable MS and i n Diabetic Neuropathy CD8 lymphocytes were isolated from peripheral blood of patients with stable MS and diabetic patients, and beta-adrenergic receptor density was determined.
+
370 Annals of Neurology Vol 27 N o 4 April 1990
binding. In parentheses are p values as determined by the Hill
STIMULATED
BASAL cAMP LEVEL
Fig 2. Basal and lo-' M (-)isoproterenol-stimulated cAMP levels in T (ymphocytes of control subjects and patients with progressive MS. The figure shows results determined from a single lot of the Rianen cAMP kit on the same day. Intra-assay variance was 10%. The data represent 14 controlsubjects and 21 patients with progressive MS. Two control basal values were excluded using Dixon's Gap Test at the 0.005 confidence level. Statistics were computed using Student's t test.
The density of beta-adrenergic receptors on CD8+ lymphocytes from patients with stable MS was 4.9 k 0.9 fmoYl0' cells (n = 13), a value not significantly different from control values. Pedal sympathetic skin responses were determined in 14 additional patients with stable MS. Three (21.4%) of the 14 patients had an absent sympathetic skin response; the mean amplitude of response, when present, was not significantly different from that of control values. CD8 receptor density was also determined in 10 diabetic patients with peripheral neuropathy and evidence of compromised function of the SNS. Receptor density of the diabetic patients was 4.2 0.5 fmoV10' cells, again not statistically significantly different from control values.
*
Discussion The major new finding reported here is that there is a twofold to threefold increase over normal values in the number of beta-adrenergic receptors on CD8+ cells from patients with progressive MS. No such increase is observed in patients with stable MS, indicating that upregulation of beta-adrenergic receptors occurs as the disease evolves. The basis for this upregulation remains uncertain. One possibility is denervation hypersensitivity. The lymphoid organs receive an extensive intraparenchymal SNS innervation and ablation of this innervation in mice is followed by an upregulation of beta-adrenergic receptors on splenic lymphocytes 1121. We show here that function of the SNS is much more frequently compromised in progressive than in stable MS, findings which corroborate and extend earlier work by others 11-71. Compromised function of the SNS may have immunological consequences. EAE is more severe in SNS-ablated rats than in controls {lo], indicating that one normal role of an intact SNS may be to exert a downregulating action on certain immune responses. Might compromised function of the SNS, when it occurs during the course of MS, have as a consequence a resetting of the immune rheostat which switches the disease into a progressive course? Cells of the T-cell subset that mediate suppressor function (CD8+, 9.3-) have, in health, more betaadrenergic receptors than T cells of other subsets, suggesting, perhaps, that the putative downregulating role of the intact SNS on the immune response might be mediated, at least in part, by activation of suppressor cells. A corollary would be that in the absence of SNS innervation, the potential of these cells for activation might be diminished. CD8 + cell-mediated suppressor function is known to be deficient in progressive MS 115-201, a finding consistent with the formulation advanced here. Specific binding of ['251)CYP to lymphocyte subsets both in normal individuals and in patients with progressive MS is saturable, stereospecific, and competitive. The receptors in progressive MS have equilibrium dissociation constants for { '251)CYP equal to those of normal lymphocytes. Competitive binding of agonists reveals that the adrenergic receptor is of the beta-2 subtype. Hill coefficients of competitive binding curves indicate that the beta-2 receptors of patients with progressive MS and control subjects do not have a homogeneous affinity for agonists. We attribute the biphasic nature of the competition binding curves to the presence of high- and low-affinity cell surface receptors, as proposed by other investigators 129-3 11. The beta adrenergic receptors are functional in progressive MS; ( - )isoproterenol induces the production of CAMP. Beta-agonist-induced increases in cAMP
accumulation do not correlate with receptor density, since cells from patients with progressive MS and control subjects show comparable rises. Stimulation of cAMP production has been shown to be maximal at lo-' M ( - )isoproterenol(32). Possibly a difference in cAMP production between cells from patients with progressive MS and control subjects would be seen following stimulation with less than maximal concentrations of ( - )isoproterenol. Alternatively, if the receptors in progressive MS are largely sequestered internally and nonfunctional, then stimulation of cAMP by ( - )isoproterenol need not be expected to parallel their increased density. In addition, the mean age of the patients with progressive MS (41.1 -+ 11.2 years) was higher than that of the normal control subjects (33.7 9.9 years) and, while no correlation exists between age or sex and beta-adrenergic receptor number on lymphocytes, a decreased beta-adrenergic responsiveness with increasing age has been noted 1331. Patients with orthostatic hypotension have been shown to have upregulated beta-adrenergic receptors on their peripheral blood lymphocytes, a finding attributed to denervation hypersensitivity 134, 351. Yet, we find no significant upregulation of beta-adrenergic receptors in diabetic patients with autonomic neuropathy. In experimental diabetic neuropathy, certain autonomic pathways are spared 1361 and it is conceivable that the SNS innervation of lymphoid organs is similarly spared in diabetic neuropathy in man. It is also possible that an upregulation of lymphocyte betaadrenergic receptors cannot simply be attributed to denervation hypersensitivity. MS is an inflammatory disease. Perhaps upregulation of beta-adrenergic receptors on lymphocytes is characteristic of chronic inflammatory diseases in general, and in no way specific to MS. Regardless of specificity, it remains possible that beta-adrenergic agonists could influence the course of MS. The beta-agonist ( - )isoproterenol was recently shown to favorably alter the course of EAE 1101.
*
This study was supported in part by a research grant (MS:RG1130D-19) from the National Multiple Sclerosis Society to D r Arnason, and by a program project grant (1P01-NS-24575-01A1) from the National Institutes of Health. D r Reder is a Harry Weaver Neuroscience Scholar (JF2027-A-1) of the National Multiple Sclerosis Society and is supported by National Institutes of Health-Clinical Investigator Development Award 1 KO8 NS 01068-01. We thank Dr Sol Rajfer for his assistance in the development of this project, as well as many supportive discussions; Dr J Jaspan for providing the diabetic patients; and Ms Madeleine Murphy for her assistance in the preparation of this manuscript. This work was presented in part at the 112th Annual Meeting of the American Neurological Association, San Francisco, CA, Oct 20, 1987.
Karaszewski et al: Lymphocyte Beta Receptor Density in MS
371
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20.
21.
22.
23.
24. 25. 26.
27.
28.
29.
30.
31.
32.
33.
34.
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