EFFECTS OF THIOPENTONE ON IMMUNOGLOBULIN PRODUCTION IN VITRO

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1 Br. J. Anaesth. (1989), 63, EFFECTS OF THIOPENTONE ON IMMUNOGLOBULIN PRODUCTION IN VITRO M. SALO is known to affect neutrophil and lymphocyte functions in vitro. It decreases spontaneous neutrophil migration and chemotaxis [1, 2], neutrophil adherence [3], phagocytosis [4] and oxidative microbicidal capacity [5], lymphocyte transformation responses [6, 7] and cytotoxicity in a -dependent way [8]. To the author's knowledge, no studies have been published to date on the effects of thiopentone on immunoglobulin production. These effects were studied using pokeweed mitogen (PWM)-activated lymphocytes with thiopentone in s occurring during induction of anaesthesia, and barbiturate treatment for brain protection. Quantification of immunoglobulins produced in a culture is a specific measure of B-lymphocyte function. MATERIALS AND METHODS Blood samples for lymphocyte isolation were obtained from 12 healthy volunteers (seven men and five women, mean age 32 (SD 9) yr, weight 67 () kg and height 1.75 (.11) m) between 7:3 and 8:. Processing of the samples was started immediately. Immunoglobulin production was measured with PWM-activated lymphocytes exposed to thiopentone, 1, 5,, 25,, or ug ml" 1 (, 3.6, 18, 36, 91, 182, 363 or 182 umol litre" 1, respectively) over the entire 7-day culture period and,,, or [ig ml" 1 (, 3.6, 182, 363 or 182 umol litre" 1, respectively) over a preincubation period of only 1, 2 or 3 h. The study was approved by the joint Ethics Committee of the Turku University Medical Faculty and the University Central Hospital. M. SALO, M.D.; Department of Anaesthesiology, University Centra] Hospital, SF-252 Turku, Finland. Accepted for Publication: June 16, SUMMARY is known to depress several granulocyte and lymphocyte functions. This study assessed the effects of thiopentone on, and production by pokeweed mitogen-activated lymphocytes as a measure of B-lymphocyte function. decreased production at ^ 25 fig ml~ 1 (91 fimol litre'') s and that of and at ^ fig ml' 1 (182 fimol litre' 1 ) s over 7 days of culture. Preincubation of lymphocytes for 1 h with thiopentone - fig ml' 1 (-182 fimol litre' 7 ) had no effect on immunoglobulin production, whereas incubation for 2 and 3 h decreased and production with toxic thiopentone s of fig ml' 1. Hydrocortisone-sensitive suppressor cells and concanavalin A-inducible suppressor cells were more sensitive to high s of thiopentone than the other cell populations. These findings may be important in patients receiving barbiturate treatment for cerebral protection. Isolation of lymphocytes The mononuclear cells were separated on Ficoll-Isopaque (Ficoll-Paque, Pharmacia Fine Chemicals, Uppsala, Sweden) by centrifugation at 4 for 4 min at 4 C [9]. The purity of the lymphocyte yield was evaluated, and the viability of the lymphocytes was confirmed as > 95 % by the trypan blue exclusion method. Culture and activation of cells Five x 5 mononuclear cells were cultured in 1 ml of RPMI 164 (Gibco, Grand Island, NY, U.S.A.) supplemented with gentamicin ug ml" 1, % fetal calf serum (FCS, Gibco Biocult, Glasgow) and.2 % sodium bicarbonate, together

2 IMMUNOGLOBULIN PRODUCTION AND THIOPENTONE 717 with thiopentone, 1, 5,, 25,, or ug ml" 1 as final. A pretested FCS lot without mitogenic activity was used throughout the study. The cells were activated with PWM (Gibco) 2.5 ul ml" 1 in the absence and presence of hydrocortisone ~ 5 mol litre" 1 or Con A.75 ug ml" 1 (Pharmacia Fine Chemicals). The cultures were maintained in 12x75 mm round-bottomed tissue culture tubes (Falcon 258, Falcon, Oxnard, CA, U.S.A.) at 37 C in humidified air and 15% carbon dioxide for 7 days []. Lymphocytes were preincubated with thiopentone,,, or SOOugml" 1 in the second part of the study by incubating the cells in RPMI 164 supplemented with gentamicin ug ml" 1, % fetal calf serum and.2% sodium bicarbonate in Falcon 258 tissue culture tubes at 37 C in humidified air and 15% carbon dioxide for 1, 2 or 3 h. After incubation, the cells were centrifuged and washed twice with Hanks' balanced salt solution (HBSS, Orion, Helsinki, Finland). After preincubation, the cells were cultured (as noted above), but without thiopentone and activated only with PWM 2.5 ul ml" 1. The ph of the cultures, measured with the phmeter 28 (Radiometer, Copenhagen, Denmark), was 8.7 (.4) (n = 4) in unstimulated cultures, 8.5 (.4) («= 4) with thiopentone ug ml" 1, 8.4 (.3) (n = 8) with thiopentone ug ml" 1 and 8.9 (.4) («= 8) with thiopentone ug ml" 1. s were measured randomly in the cultures by high performance liquid chromatography with a C 18 column (HPLC). The values obtained were ug ml" 1 without thiopentone, ugml" 1 with thiopentone 5 ug ml" 1, ug ml" 1 with thiopentone 25 ug ml" 1 and ug ml" 1 with thiopentone ug ml" 1. Measurement of immunoglobulins After culture, the supernatants were collected for measurement of immunoglobulin s by a trapping antibody enzyme-linked immunosorbent assay (ELISA) [11]. Wells of microtitre plates (Linbro/Titertek, Flow Laboratories Inc., Hamden, CT, U.S.A.) were coated with rabbit antihuman, or (Dakoimmunoglobulins a/s, Copenhagen, Denmark). Thereafter, supernatants from lymphocyte cultures were added to the wells in appropriate dilutions. Cytophilic immunoglobulins were not removed, as the amount was less than the detection limit of the method ( ng ml" 1 ). Bound, and were detected with peroxidaseconjugated rabbit anti-human, and (Dako-immunoglobulins a/s), respectively. Absorbance at 492 nm was measured with a Titertek Multiskan photometer (Eflab Oy, Helsinki, Finland). The amount of class-specific immunoglobulin was determined from standard curves obtained in samples with known amounts of immunoglobulin (Behringwerke AG, Marburg, West Germany). The detection limit of the assay was ng ml" 1 for, and. All tests were performed in duplicate. Statistics Analysis of variance for repeated measurements and Dunnett's test were used for analysis of the data. Logarithmic transformation was used to normalize the distribution of immunoglobulin values. RESULTS ^ 25 ug ml" 1 decreased and ^ ug ml" 1 decreased and production by PWM-activated lymphocytes over the 7-day exposure (P <.1) (fig. 1). > 25 ug ml" 1 also decreased unstimulated production (P <.5), but and were depressed only at thiopentone s ^ ug ml" 1 (P <.1, <.5, respectively (table I). By contrast, _ I 2 1,1.8 I concn (pgml' 1 ) FIG. 1. Production of immunoglobulins, and in culture medium by PWM-activated lymphocytes with thiopentone, 1, 5,, 25,, or ug ml" 1 (, 3.6, 18, 36, 91, 182, 363 or 182 umol litre" 1, respectively). Log I means and SEM. n = \2. **P<.1 compared with thiopentone ug ml" 1 values.

3 718 BRITISH JOURNAL OF ANAESTHESIA lymphocyte incubation for 1 h with thiopentone - ug ml" 1 before culture had no effect on, or production, but preincubation for 2 and 3 h depressed and production at the highest thiopentone s ( ug ml" 1 ) (P <.1) (table II). However, they had no effect on production. In unstimulated cultures thiopentone \ig ml" 1 decreased production only after preincubation for 3 h (P <.1), and it had no effect on or production (table III) or after 1 or 2 h of preincubation (data not shown). With hydrocortisone " 5 mol litre" 1 or Con A.75 ug ml" 1, production by PWM-activated lymphocytes started to decrease at the same 25-ug ml" 1 thiopentone as it did without the addition of hydrocortisone or Con A. In contrast, and production decreased with hydrocortisone or Con A even with thiopentone 25- ng ml" 1, compared with loougml" 1 without them (data not shown). A change occurred also, at high s of thiopentone, in the ratios of immunoglobulin produced with and without hydrocortisone or Con A. The ratio for with hydrocortisone or without, which was 2. (.27) (mean (SEM)) without thiopentone, decreased to TABLE I. Production of immunoglobulins, and in culture medium by unstimulated lymphocytes with thiopentone, 1, 5,, 25,, or fig ml' 1 (, 3.6, 18, 36, 91, 182, 363 or 182 ftmol litre' 1, respectively). Log l means (SEAT), n = 12. *P <.5; **P <.1 compared with thiopentone ng ml' 1 values (Hgml- 1 ) (.69) (.77) 2.2 (.53) (.68) (.52)* (.72)** 2.45 (.61)** (.69)** 2.29 (.227) 2.14(.226) 1.973(.283) 2.329(.115) (.142) 1.788(.254) 1.2(.279)** 1.5(.283)** 1.945(.269) 1.829(.262) (.265) 1.934(.266) 2.96 (.21) 1.682(.298) 1.465(.317)* 1.43(.39)* TABLE II. Production of immunoglobulins, and in culture medium by PWM-activated lymphocytes after preincubation of lymphocytes with thiopentone,,, or fig ml' 1 (fi, 36, 182, 363 or 182 fimol litre' 1, respectively) for 1, 2 or 3 h. Log means (SEM). n = 8. **P <.1, compared with thiopentone fig ml' 1 values (Ug ml" 1 ) (ng ml" 1-h incubation 2-h incubation 3-h incubation 2.949(.141) 3.149(.6) 3.159(.99) 3.6(.118) 3.65 (.121) 3.156(.133) (.99) 3.237(.123) (.1) (.22)** 2.925(.166) 2.966(.181) 2.948(.178) 2.928(.166) 2.463(.)** 3.13(.89) 3.267(.5) 3.262(.111) 3.239(.128) 3.169(.143) 3.9(.132) 3.218(.1) 3.2(.116) 3.74(.113) (.367)** 3.66(.9) 3.98(.123) 3.112(.125) 3.28(.124) (.92)** (.89) 2.543(.122) (.84) (.329) (.117) 2.622(.115) (.76) 2.779(.8) 2.654(.151) 2.7(.2) (.353) (.362) (.36) (.117) 1.97(.295)

4 IMMUNOGLOBULIN PRODUCTION AND THIOPENTONE 719 TABLE III. Production of immunoglobulins, and in culture medium by unstimulated lymphocytes after preincubation of lymphocytes with thiopentone,,, or ng ml' 1 (, 36, 182, 363 or 182 fimol litre' 1, respectively) for 3 h. Log l means (SEM). n = 8. ** P <.1 compared with thiopentone ng ml' 1 values (Hg ml" 1 ) (ng ml l ) 1.718(.378) 1.896(.314) 1.618(.37) 1.975(.287) 1.136(.375)** 1.43(.445) (.489) 1.229(.481).999 (.495) 1.31 (.423) 2.11 (.3) 1.717(.388) 1.74(.386) 1.76(.392) 1.612(.362) 1.22 (.) with thiopentone ug ml" 1 (P <.1) and to 1.39 (.31) with thiopentone ug ml" 1 (P <.5). The corresponding ratio for decreased from the initial value of 1.63 (.27) without thiopentone to 1.1 (.21) with thiopentone ug ml" 1 (P <.5), whereas the ratio 1.23 (.14) for without thiopentone did not change. In contrast, the ratio for with or without Con A, which was initially.48 (.9), increased to.77 (.7) with thiopentone ug ml" 1 (P <.1), to.92 (.9) with thiopentone loougml- 1 (P<.1) and to 1.15 (.16) with thiopentone SOOugml" 1 (P <.1). The ratios for (.46 (.4) and for (.64 (.)) did not change from baseline values. DISCUSSION depressed humoral immune responses. It decreased immunoglobulin production by polyclonally PWM-activated lymphocytes at s used for induction of anaesthesia (2-8 ug ml" 1 ) [12, 13] and surgical anaesthesia with thiopentone (39-42 ug ml" 1 ) [14], but the effects were observed only after 7 days of exposure. In contrast, exposure for 1 h had no effect on production of immunoglobulin, whereas exposure for 2 and 3 h to toxic s of thiopentone ( ug ml" 1 ) was enough to decrease and production by PWM-activated lymphocytes. However, such s do not occur in clinical practice. Thus at clinical anaesthesia s and exposure times, thiopentone does not depress immunoglobulin production in vitro. In contrast, exposure to thiopentone - ng ml" 1 over several days, which occurs during barbiturate treatment for status epilepticus or cerebral protection [15, 16], is sufficient to decrease PWM-activated immunoglobulin production. In this study hydrocortisone " 5 mol litre" 1 was added to the culture because it inhibits hydrocortisone-sensitive suppressor cells [17]. Similarly, Con A.75 ug ml" 1 was used because it induces Con A-inducible suppressor cells [18]. These two functional suppressor cell populations are used widely in immunological research. The effect of thiopentone on these suppressor cell populations was tested, as alterations in suppressor cell activity have been observed during surgery [19]. With high s of thiopentone, hydrocortisone decreased and Con A increased the ratios of immunoglobulins produced. These findings suggest that hydrocortisone-sensitive and Con A-inducible suppressor cells are more sensitive to increased thiopentone s than are the other lymphocyte subpopulations. This is in accordance with earlier findings of decreased Con A-inducible suppressor cell activity after open-heart surgery [2] or hysterectomy [21]. Although caution should be exercised in extrapolating in vitro observations to clinical practice, the above findings may be clinically important, especially in patients receiving high-dose barbiturate treatment for cerebral protection. The association found between thiopentone treatment and development of bacterial pneumonias in patients with cerebral injury [22] may be a result partly of decreased humoral immune responses, but also of decreased neutrophil [1,4, 23] and T- lymphocyte functions [6] observed at the same s of thiopentone. After major surgery, thiopentone may be a contributory factor to decreased immunoglobulin production by PWMactivated lymphocytes [24] and to decreased

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