INTRODUCTION

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Recent work has shown that changes in the peripheral lymphocyte ₂-adrenergic receptor (AR) density correlate with changes in pulmonary AR density (1). However, there has been no systematic examination of the variability of lymphocyte AR density with time in normal individuals or asthmatics, even though peripheral lymphocyte AR densities have been examined in many studies of asthmatics (2, 3). The extent of this variability has obvious implications for the interpretation of the results of these studies.

Numerous medications including ₂-agonists (4), glucocorticoids (5), and mast cell stabilizing agents (6), as well as the severity of asthma (2), are felt to influence AR density on peripheral blood lymphocytes. However, AR density is unequally distributed among lymphocyte subsets, with CD4+ cells having a lower density of ₂-receptors than CD8+ cells (7); thus, changes in AR density could represent change in the lymphocyte subtype composition of the peripheral blood, as has been shown to occur in allergen challenge in asthmatics (8). Therefore we undertook a study to assess the variability of AR density in lymphocytes in normal individuals over a several week time period, and its relationship to lymphocyte subtype composition, specifically CD4+ and CD8+ percentage.

	METHODS

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Ten healthy, nonsmoking, normal subjects (mean age 31.5 ± 3.3 yr; range 28-40 yr) with no history of asthma or pulmonary disease participated in the study after giving written informed consent. Nine of the 10 subjects were males. Each subject was studied on three to five different occasions over a 4- to 12-wk time period. At each visit, peripheral lymphocyte AR density and subtype composition were measured. Subjects fasted overnight and abstained from caffeine for a 12-h period prior to blood withdrawal. Blood was drawn at approximately the same time of day (9:00 A.M.) for each determination. The subject lay supine for 15 min, then 50 ml of blood were withdrawn from an antecubital vein into a heparinized syringe, of which 40 ml were used for determination of AR density and the other 10 ml for flow cytometric analysis (FACScan; Becton Dickenson) to determine CD4+ and CD8+ percentages of total lymphocytes.

AR density on lymphocytes was determined by Brodde's technique (9). Lymphocytes were isolated from 40 ml of blood by density gradient centrifugation with 12 ml Ficoll-Paque and 25 ml of Hank's solution. This was centrifuged at 400 g for 35 min at 4° C. The pellet was agitated with fresh Hank's solution to obtain a homogeneous mixture and an aliquot counted on an automated blood counter (Coulter S+1V; Coulter Instruments). The isolation procedure took 3-4 h and yielded a preparation of 80-90% lymphocytes, confirmed by microscopic identification.

Triplicate samples of 5 × 10⁵ lymphocytes were incubated with eight concentrations of [^I25I] - (-) iodopindolol from 2.5 to 150 pM for 24 h at 4° C. Nonspecific binding was determined using isoproterenol at a high concentration (10 µM) to displace the receptor-bound ligand. The reaction was terminated by adding 10 ml 10 mM Tris HCl, 154 mM NaCl buffer, pH 7.4, and filtration over Whatman CF/C filters (Whatman Inc.). Each filter was then washed with 10 ml of buffer and counted on a gamma counter (Compugamma 1282; LKB Wallac). Specific-binding of [¹²⁵I] - (-) iodopindolol was defined as total minus nonspecific binding. A computer program (Ligand, Biosoft Company) was utilized to determine the maximum binding capacity (Bmax) for each subject.

Data analysis, in terms of coefficient of variation and correlation coefficient, was performed by standard techniques (10).

	RESULTS

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The mean coefficient of variation of repeated estimates of AR in individual samples in our laboratory is 9.8%.

The study results are shown in Table 1. There was a wide variance within and between subjects in peripheral lymphocyte AR density over a 4-12 week time period of the study. The mean (± SD) number of AR's per lymphocyte in the ten individuals was 776 ± 183. The mean individual coefficient of variation was 22.4% and range of receptor density per lymphocyte was 400-1,300 (Table ). The mean K_D value was 2.2 pM ± 0.75 pM. The average CD4+ and CD8+ percentages were 48.7% ± 5.0% and 26.4 ± 6.7%, respectively. The average CD4+ to CD8+ ratio was 2.1 ± 0.6. The mean individual coefficient of variation for CD4+ and CD8+ lymphocyte percentage was 9.5 and 11.1%, respectively. Variations in ₂-adrenergic receptor density significantly correlated (p < 0.001) with the CD4+ and CD8+ composition of the lymphocytes (Figure 1), with a positive correlation with CD8+ cell percentage (correlation coefficient = +0.51) and a negative correlation with both CD4+ percentage (correlation coefficient = 0.58), and CD4+/CD8+ ratio (correlation coefficient = 0.61).

View larger version (11K): [in this window] [in a new window]   Figure 1.   2-Adrenergic receptor density plotted against concentration of CD4+, CD8+, and CD4+/CD8+ ratio.

	DISCUSSION

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The present study demonstrates that there is a relatively large variation in AR density from day to day in normal individuals and that this variation is significantly related to variations in CD4+ and CD8+ percentages of T-lymphocytes. There is only one previously published study (3) evaluating the reproducibility of peripheral lymphocyte AR density: two measurements were performed over a 2-12 wk period in five normal subjects and two asthmatics. No significant variation was noted. We have found significant variation between and within individuals in AR density, very likely due to the greater number of repeated measurements and subjects in the present study.

The group variability of CD4+ and CD8+ lymphocyte percentages has been examined previously and the findings are similar to the present study (11, 12). Maini and coworkers evaluated 286 subjects (195 of whom were females): calculations from their data indicate an average CD4+ and CD8+ composition of 45.6% and 26.3%, respectively, with an average CD4+ to CD8+ ratio of 1.73. These results are very close to the findings of the present study. Individual variability in peripheral CD4+ and CD8+ has been studied previously over three consecutive days (12). Again the findings are very similar to the present study with coefficients of variation of 5.6% for CD4+ and 8.2% for CD8+.

It has previously been demonstrated that there is differential distribution of ₂-adrenergic receptors in lymphocyte subsets, with a significantly higher density on CD8+ cells compared to CD4+ cells (7, 13, 14). Acute changes in CD4+ and CD8+ composition of lymphocytes occur in the airway and peripheral blood in asthma. In sensitized atopic asthmatics 48 h after allergen challenge, a selective increase in CD4+ lymphocytes was demonstrated in the BAL fluid (15). In peripheral blood, 48-72 h after allergen challenge, there is a significant decrease in CD4+ lymphocytes, a small but nonsignificant decrease in CD8+ lymphocytes and an increase in activated lymphocytes (8). CD56+, CD57+, and CD8+ lymphocytes are selectively mobilized after sympathetic stimulation (16). The influx of these ₂-receptor rich cells into the peripheral circulation may account for the increased ₂-receptor density that is observed in unseparated mononuclear leukocytes after sympathetic stimulation.

Changes in lymphocyte composition in peripheral blood or the lung in acute or chronic asthma, or after exposure to asthma medications may have an effect on ₂-adrenergic receptor density, but this relationship has not been studied extensively. Indeed in our study there was a significant positive correlation between CD8+ percentage and receptor density and a significant negative correlation of receptor density and CD4+ percentage as well as the ratio of CD4+ to CD8+ cells. This has not been previously reported. It is possible that previously reported differences in ₂-receptor density on lymphocytes between normal and asthmatic subjects may represent differences in lymphocyte subset composition induced by the acuity and/ or severity of asthma or the administered medications, rather than a true difference in individual lymphocyte receptor density. This possibility is currently under investigation in our laboratory.