NATURAL KILLER CELL ACTIVITY AND LYMPHOCYTE SUBPOPULATIONS DURING DOBUTAMINE INFUSION IN MAN

British Journal of Anaesthesia 1993; 71: 218-221

NATURAL KILLER CELL ACTIVITY AND LYMPHOCYTE SUBPOPULATIONS DURING DOBUTAMINE INFUSION IN MAN Y. NOMOTO, H. JHONOKOSI AND S. KARASAWA

SUMMARY

KEY WORDS Immune response: natural killer cell activity. dobutamine.

Pharmacology

NK lymphocytes are a subset of lymphocytes exerting spontaneous cytotoxicity against a variety of tumour cells. NK cells have been shown in mice to play a role in primary immune defence against tumour formation and defence against metastatic spread of tumours. Human NK lymphocytes have been identified and shown to be cytotoxic to human tumour target cells in vitro [1]. NK cell activity is enhanced after premedication and during anaesthesia and surgery [2], followed by a reduction for 3-6 days after major surgery [3, 4]. Several studies have shown that administration of adrenaline to human subjects produces an increase in NK cell activity [5]. This change is prevented by simultaneous incubation with the beta-adrenoceptor antagonist, propranolol [6]. It has been suggested that adrenaline pretreatment enhances NK cell activity via lymphocyte beta-adrenoceptors. There have been no reports to date on NK cell activity after administration of dobutamine to human subjects. PATIENTS AND METHODS

With local Ethics Committee approval and informed patient consent, we studied 16 adult patients with

TABLE I. Patient data (mean (range or SD)) Group 1 (dobutamine) No. patients Sex (M/F) Age (yr) Weight (kg) Duration of op. (min)

9 7/2

25 (16-^5) 66(9) 146(48)

Group 2 (isotonic saline) 7 6/1

29 (20-43) 63 (10) 163 (55)

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Sampling time

1 2 3 4 5 6

Before induction of anaesthesia Before infusion of dobutamine or isotonic saline 15 min after beginning of infusion 30 min after beginning of infusion 30 min after stopping infusion 90 min after stopping infusion

YUKIKO NOMOTO,

M.D.; HIDEO JHONOKOSI, M.D. ;

SHUNJI

KARASAWA, M.D. ; Department of Anaesthesiology, Kanto Rosai Hospital, 2035 Kizukisumiyoshi-cho, Nakahara-ku, Kawasakicity, Kanagawa-kcn 211, Japan. Accepted for Publication: February 15, 1993.

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We have studied the effects of dobutamine on natural killer (NK) cell activity and the distribution of lymphocyte subpopu/ations in peripheral blood in 16 patients under general anaesthesia. Patients were allocated to receive either dobutamine (n = 9) or isotonic saline (n = 7) for 30 min. There was a significant decrease in the ratio of 7'-helpery'inducer cells (CD4) to T-suppressorI cytotoxic cells (CD8) in all patients after induction of anaesthesia. Dobutamine infusion induced an increase in NK cell activity, accompanied by a selective increase in circulating NK cells. However, compared with preinfusion values, CD4.CD8 cell ratio did not change during and after dobutamine infusion. In the isotonic saline group, NK cell activity and CD4.CD8 cell ratio remained unchanged. These results suggest that dobutamine induced an increase in NK cell activity and alterations in lymphocyte subpopu/ations. (Br. J. Anaesth. 1 993; 7 1 : 218-221)

lumbar disc herniation undergoing elective partial laminectomy. The patients were otherwise healthy with normal cardiac, renal and hepatic functions; none had endocrine disorders and none was receiving any medications. The patients were allocated randomly to receive either dobutamine (n = 9) or isotonic saline (n = 7) (table I). Premedication and general anaesthesia were identical for all patients: premedication comprised atropine 0.5 mg and hydroxyzine 50 mg i.m. 1 h before operation; anaesthesia was induced with thiopentone 5 mg kg"1 and intubation was performed after administration of vecuronium 0.16 mg kg"1. Anaesthesia was maintained by artificial ventilation with enflurane and 66 % nitrous oxide in oxygen. In all patients, i.v. infusions during surgery were restricted to Ringer lactate solution at a rate of 10 ml kg"1 during the first 1 h and thereafter at 2-4 ml kg" h"1. After steady-state conditions were established, the patients were allocated randomly to two groups to receive a continuous i.v. infusion of either dobutamine 5 |ig kg"1 min"1 or isotonic saline, for 30 min.

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FIG. 1. Individual changes in NK cell activity expressed as % cytotoxicity before, during and after infusion of dobutamine or isotonic saline. Shaded area represents period of infusion. Sample Nos: 1 = Before induction; 2 = before infusion; 3, 4 = 15, 30 min after beginning of infusion; 5, 6 = 30, 90 min after stopping infusion. P < 0.05: fcompared with sample 1; *compared with sample 2.

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RESULTS

There was no difference in age, weight or duration of surgery between the two groups (table I). Individual changes in NK cell activity before, during and after infusion of dobutamine or isotonic saline are shown in figure 1. A significant increase in NK cell cytotoxicity occurred throughout the infusion of dobutamine, and this returned to pre-infusion values 30 min after completing infusion. In the control group, NK cell cytotoxicity did not change significantly during and after the infusion. After induction of anaesthesia, a small but significant increase in NK cell cytotoxicity occurred in both groups. The numbers of circulating leucocytes, lymphocytes and the pan T-cells (CD3) and NK cells (CD 16) are shown in figure 2. In the dobutamine group, absolute leucocytosis and lymphocytosis occurred. The pan T-cells showed no significant change, but the number of NK cells increased during and after the infusion of dobutamine. In the control groups, the number of NK cells did not change significantly. The ratio of CD4 and CD8 cells was significantly reduced after induction of anaesthesia in all patients (fig. 3). However, the ratio did not change during and after infusion of either dobutamine or saline. In both groups, serum cortisol, plasma adrenaline and noradrenaline concentrations did not change significantly throughout the study. In the dobutamine group, mean systolic arterial pressure increased from HOmmHg (range 85-145 mm Hg) to a peak value of 130 mm Hg (range 115-155 mm Hg) (P < 0.05), but there were no significant changes in diastolic arterial pressure. Heart rate also increased, from 73 to 85 beat min"1, in the dobutamine group. In the isotonic saline group, neither arterial pressure nor heart rate changed significantly during the study. DISCUSSION

We have found that dobutamine selectively increased NK cell activity and increased the absolute number of circulating NK cells in vivo.

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Haemorrhage did not exceed 200 ml and blood substitutes or sympathomimetic agents were not required. Arterial blood samples were obtained as shown in table II, for measurement of NK cell activity, distribution of lymphocyte subpopulations, blood leucocyte counts and measurement of serum cortisol, plasma adrenaline and noradrenaline concentrations. Plasma adrenaline and noradrenaline concentrations were measured by high pressure liquid chromatography, serum cortisol by radioimmunoassay. The pan T-cell population (CD3) was measured with Leu4 antibody, the T-helper/inducer cells (CD4) with Leu3a antibody, the T-suppressor/ cytotoxic cells (CD8) with Leu2a antibody and NK cells (CD 16) with Leull antibody (Becton Dickinson Monoclonal Center, Mountain View, CA, U.S.A.) using flow cytometry (Ortho Spectrum III, Ortho Diagnostic Systems Inc.). NK cell activity was measured against K-562 target cells in a chromium-51 release assay. Five x 103 51Cr-labelled target cells of the human erythroleukaemic cell line K562 were mixed with different concentrations of mononuclear cells from patients, for use as effector cells, to obtain effector to target cell ratios 100:1, 50:1, 25:1 and 12:1. Cell suspensions were incubated at 37 °C in humidified air and 5 % carbon dioxide for 4 h. After incubation, the radioactivity of 100 ul of the cell-free supernatant was counted with an automatic well-type gamma counter. Percentage cytotoxicity was computed from the formula:

BRITISH JOURNAL OF ANAESTHESIA

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FIG. 3. Number of T-helper/inducer cells (CD4), T-suppressor/cytotoxic cells (CD8) and CD4:CD8 ratio in peripheral blood (mean, SD) before, during and after infusion of dobutamine (O) or isotonic saline ( # ) . Shaded area represents period of infusion. Sample Nos as in figure 1. fP < 0.05 compared with sample 1.

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400-

DOBUTAMINE AND NK CELL ACTIVITY

by interacting networks of helper/inducer (CD4) and suppressor/cytotoxic (CD8) T cells. The ratio of CD4 cells to CD8 cells, a commonly used indicator of immunoregulatory cell imbalance, was altered after induction of anaesthesia in our study. Although the absolute number of CD8 cells remained unchanged after induction of anaesthesia, the CD4:CD8 ratio declined because the absolute number of CD4 declined, but the CD4: CD8 ratio did not change after administration of dobutamine. Crary and others found a significant decrease in the CD4:CD8 ratio after administration of adrenaline [5]. These results are in contrast with our findings. We suggest that the NK cell regulatory effect of dobutamine may be mediated via betaadrenoceptors, because it is consistent with the findings after administration of adrenaline in other studies. REFERENCES 1. Stevenson GW, Hall SC, Rudnick S, Seleny FL, Stevenson HC. The effect of anesthetic agents on the human immune response. Anesthesxology 1990; 72: 542-552. 2. Tannesen E, Mickley H, Grunnct N. Natural killer cell activity during premedicarion, anaesthesia and surgery. Acta Anaesthesiologtca Scandinavica 1983; 27: 238-241. 3. Tennesen E, Brinklov MM, Christensten NJ, Olesen AS, Madsen T. Natural killer cell activity and lymphocyte function during and after coronary artery bypass grafting in relation to the endocrine stress response. Anesthestology 1987; 67: 526-533. 4. Salo M, Nissila M. Cell-mediated and humoral immune responses to total hip replacement under spinal or general anaesthesia. Acta Anaesthesiologica Scandinavica 1990; 34:

241-248. 5. Crary B, Hauser SL, Borysenko M, Kutz I, Hoban C, Ault KA, Weiner HL, Benson H. Epinephrine-induced changes in the distribution of lymphocyte subsets in peripheral blood of humans. Journal of Immunology 1983; 131: 1178-1181. 6. Hellstrand K, Hermodsson S, Strannegard O. Evidence for a (J-adrenoceptor-mediated regulation of human natural killer cells. Journal of Immunology 1985; 134: 4095-^099. 7. Kendall RA, Targon S. The dual effect of prostaglandin (PGEj) and cthanol on the natural killer cytolytic process: effector activation and NK cell-target cell conjugate lyric inhibition. Journal of Immunology 1980; 126: 2770-2778.

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This increase in NK cell activity was consistent with changes observed in other studies, after administration of adrenaline [5,6]. Tennesen, Mickley and Grunnet observed that, after administration of adrenaline s.c. to healthy individuals, NK cell activity increased significantly within 15-30 min [2]. It has been shown also that the percentage of NK cells in peripheral blood increased after administration of adrenaline [5]. This enhancement of NK cell activity correlated with an increased number of circulating NK cells during operation. Dobutamine is a potent adrenergic agonist. There have been no reports describing NK cell activity after administration of dobutamine to humans. Circulating human lymphocytes possess a large number of beta-adrenoceptors [6], but the biological significance of this is unknown. Hellstrand demonstrated that adrenaline enhanced NK cell activity in vivo. The increased activity was prevented consistently by simultaneous incubation with the non-selective beta-adrenoceptor antagonist, propranolol [6]. Thus it may be assumed that adrenaline pretreatment enhanced NK cell activity via lymphocyte beta-adrenoceptors. Adrenalineinduced beta-adrenoceptor activation is believed to increase intracellular cAMP via the adenylate cyclase complex. Similar results have been obtained with prostaglandin E2, a potent inducer of intracellular cAMP [7], which was shown to enhance NK cell activity in low, physiological concentrations. Although the mechanism of lymphocyte release is not known, our data clearly demonstrate that dobutamine altered the distribution of circulating subsets. The rapid increase in NK cells may be explained by mobilization of mature NK cells from the extravascular space into the circulation or by differentiation of pre-NK cells to CD 16 mature cells [2]. Adrenaline may cause this "mobilization" of lymphocytes directly, by activation of lymphoid adrenergic receptors, or indirectly, by activation of adrenergic receptors in other tissues. The human immune system is regulated, in part,

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