Mechanism of immunosuppressive effect of alprazolam: Alprazolam suppresses T-cell proliferation by selectively inhibiting the production of IL2 but not acquisition of IL2 receptor

Int. ,L Immunopharmac., Vol. 14, No. 2, pp. 227-237, 1992. Printed in Great Britain.

0192-0561/92 $5.00 + .00 Pergamon Press plc. International Society for lmmunopharmacology.

M E C H A N I S M OF I M M U N O S U P P R E S S I V E EFFECT OF A L P R A Z O L A M : A L P R A Z O L A M SUPPRESSES T-CELL PROLIFERATION BY SELECTIVELY INHIBITING THE P R O D U C T I O N OF IL2 BUT NOT ACQUISITION OF IL2 RECEPTOR M . - P . CHANG,** STEVEN C. CASTLE* a n d DEAN C. NORMAN* *Geriatric Research, Education and Clinical Center (GRECC), VA Medical Center West Los Angeles, Los Angeles, CA 90073; and *Department of Medicine and Jonsson Comprehensive Cancer Center, UCLA, Los Angeles, CA 90024, U.S.A.

(Received 4 February 1991 and in final form 28 June 1991)

Abstrae! - - The purpose of this study was to elucidate the mechanism of action of alprazolam on concanavalin A (Con A)-induced murine T-cell proliferation. Splenic cells of BALB/c mice were first cultured with an optimum dose of Con A in the presence or absence of varying doses of alprazolam to assess effects of alprazolam on T-cell proliferation, interleukin 2 (IL2) production and IL2 receptor (IL2R) expression. Then, Con A-induced T-blast cells from BALB/c mice were cultured with an excess dose of human recombinant IL2 (rlL2) or crude rat IL2 supernate in the presence or absence of alprazolam to assess the effects of alprazolam on the interaction of IL2 and IL2R. The results of these studies clearly demonstrated that alprazolam can inhibit the T-cell proliferation in response to Con A but not to IL2. Alprazolam also reduced the production of IL2 by splenic T-cells, but did not alter the expression of IL2R on Con A-induced T-blast cells. Furthermore, the results also showed that (a) alprazolam did not inhibit the proliferative response of splenic T-cells to a combination of phorbol 12-myristate-13-acetate (PMA) and ionomycin, and (b) the addition of exogenous IL2 reversed the inhibitory effect of alprazolam on T-cell proliferation. Finally, the addition of alprazolam produced a time-dependent inhibiting effect on T-cell proliferation. However, this inhibitory effect of alprazolam was abolished when the drug was added to the cultures of competent cells that fully expressed IL2R. Taken together, these results suggest that alprazolam inhibits murine T-cell proliferation by affecting the mitogenic receptor-mediated events (initiation) rather than the IL2R-mediated events (progression) of ligand-activated T-cells through the cell cycle.

T h e benzodiazepines (BZD) are one of the most c o m m o n l y prescribed drugs which are prescribed to patients for their antianxiolytic, a n t i c o n v u l s a n t a n d h y p n o t i c properties (Skolnick & Paul, 1982). These pleiotropic effects of B Z D , such as c l o n a z e p a m a n d R0 15-1788, are m e d i a t e d via highaffinity central-type receptors t h a t are exclusively localized in the central nervous system (CNS) (Mohler & O k a d a , 1977; B r a e s t r u p & Squires, 1977; Skolnick & Paul, 1981). A n o t h e r class o f BZD, such as 4 - c h l o r o d i a z e p a m (R0 5-4864) can interact with the so-called " p e r i p h e r a l - t y p e r e c e p t o r " which has been f o u n d b r o a d l y in m a n y n o n - n e u r o n a l tissues including kidney, heart, platelets, mast cells, lymphocytes as well as several cultured cell lines (Wang, M o r g a n & Spector, 1984; M a r t i n , 1984).

Evidence has been s h o w n t h a t binding of this class o f B Z D to the peripheral B Z D receptor o n i m m u n e cells results in inhibition o f cell proliferation ( W a n g et al., 1984), induction o f chemotaxis (Ruff, Pert, Weber, W a h l , W a h l & Paul, 1985), e n h a n c e m e n t of a n t i b o d y response to sheep red b l o o d cells (SRBC) (Zavala, H a u m o n t & L e n f a n t , 1984) a n d m o d u l a t i o n o f n o r m a l activity o f m a c r o p h a g e s (Zavala et al., 1984). Recently a t h i r d type o f BZD, such as d i a z e p a m (Valium), has been identified to be a mixed-type B Z D receptor ligand for its ability to b i n d with relatively high affinity to b o t h the central peripheral receptors (Clarke & Ryan, 1980). It has been s h o w n t h a t d i a z e p a m can induce chemotaxis of h u m a n m o n o c y t e s ( R u f f et al., 1985). However, diazepam, as a potent i m m u n o s u p p r e s s a n t , inhibits

+Author to whom correspondence should be addressed. 227

228

M.-P. CHAY~et al.

primary antibody response to SRBC (Descotes, Tedone & Evreux, 1982), delayed hypersensitivity (Descotes, Laschi, Tachon, Tedone & Evreux, 1983), and lymphocyte proliferation (Pawlikowski, Lyson, Kunert-Rodek & Stepien, 1988) in normal mice. Alprazolam is a relatively new benzodiazepine and has a pharmacologic profile similar to that of other benzodiazepines (Dawson, Jue & Brogden, 1984). However, unlike other benzodiazepines, alprazolam also has antidepressant activity and is a more potent anticonvulsant, antiaggressive agent and muscle relaxant than diazepam (Dawson et al., 1984). It was demonstrated that alprazolam can displace clonazepan, known to be a central BZD receptor ligand, but not R0 5-4864 (a peripheral-type BZD receptor ligand) on rat brain membrane. Recently evidence has also shown that administration of a low dose of alprazolam (0.2 mg/kg) can enhance natural killer cell activity, mixed lymphocyte reaction, and mitogen-induced lymphocyte proliferation (Fride, Skolnick & Arora, 1990). However, the enhancing effects of alprazolam were not significantly noted 24 h after administration. Furthermore, higher doses of alprazolam ( 5 - 1 0 mg/kg) did not affect those immune functions measured. Alprazolam is also known to be a potent antagonist of plateletactivating factor (PAF), which can induce a dosedependent inhibition of lymphocyte mitogenesis. Recently, evidence has also shown that both PAF and alprazolam decrease the levels of tumor necrosis factor-a (TNF-a) in the plasma of mice treated with endotoxin (Kim et al., 1990). Previously, we demonstrated that alprazolam, like diazepam, can suppress immune functions of T- and B-cells and macrophages in a dose-dependent fashion (Chang, Castle & Norman, 1991). Although there is clear evidence that alprazolam inhibits the proliferative response of T-cells to concanavalin A (Con A) and production of interleukin 2 (IL2) by splenic T-cells (Chang et al., 1991), the mode of action by which alprazolam impairs cell proliferation has not been established. Therefore, we made an attempt to study mechanisms of the antiproliferative effect of alprazolam at the cellular level. Accordingly, a murine model was used to dissect the mode of action of alprazolam on T-cell proliferation by analyzing the in vitro effect of alprazolam on (i) Con A-induced T-cell proliferation, (ii) production of IL2 by splenic T-cells, (iii) expression of IL2 receptor on Con A-induced T-blast cells, (iv) effect of exogenous IL2 on the proliferative responses of splenic cells to Con A and alprazolam, (v) PMA/ionomycin-induced T-cell proliferation, (vi) interaction of IL2 and IL2R,

(vii) generation of Con A-induced T-blast cells, and (viii) the time-dependent effect of alprazolam on Con A-induced T-cell proliferation. The results of this study demonstrated that alprazolam decreased the proliferative activity of T-cells and the production of IL2 by splenic T-cells. However, alprazolam did not inhibit (a) IL2 receptor expression, (b) the proliferative responses of T-cells to a combination of PMA/ionomycin or IL2, and (c) generation of Con A-induced T-blast cells. Furthermore, alprazolam inhibited T-cell proliferation when it was added for the first 6 h of culture with Con A. This inhibitory effect was not seen when alprazolam was added to the cultures after 24 h of Con A stimulation. Finally, in the continual presence of alprazolam, the addition of exogenous IL2 reversed the inhibitory effect, suggesting that the suppressive effect of the alprazolam on T-cell proliferation is due to inhibition of IL2 production by Con A-stimulated splenic T-cells.

EXPERIMENTAL PROCEDURES

Mice. 2 - 4 month old male BALB/c mice, purchased from Charles River (Wilmington, MA), were used for this study. Cell suspensions. Splenic cell suspensions were prepared as described elsewhere (Chang, Tanaka, Stosic-Grujicic, Yamamoto, Perkins, Strehler & Makinodan, 1982b). In brief, cells were dispersed by teasing individual spleens with a 22-gauge needle in cold RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (Sterile Systems, Logan, UT), 2 mM L-glutamine, 20 mM HEPES, and 50 ~g gentamicin sulfate per ml. Mitogenic response. As described elsewhere (Chang et al., 1991), briefly, a total of 2.5 x 105 spleen cells were delivered in triplicate to flat-bottom microplates (Falcon Microtest II) in a volume of 0.1 ml of complete RPMI 1640 medium. A stock solution of alprazolam (1 mg/ml in double distilled water, equivalent to 3.8 x 103 M) was prepared and stored at - 20°C until use. Varying doses (5 - 50 ng) of alprazolam (provided by Up john Co., Kalamazoo, MI) were added in a volume of 50/A with a volume of 50/A of an optimal dose of Con A (0.5 ~g/culture) (Calbiochem, San Diego, CA) or a combination of phorbol 12-myristate-13-acetate (PMA, 0.5 ng/culture) and ionomycin (100 ng/ culture, Sigma, St. Louis, MO). The optimal dose of

Immunosuppressive Effect of Alprazolam a combination of PMA (0.5 ng/ml) and ionomycin (100ng/ml) was previously established in our laboratory (unpublished data). The cultures were incubated at 37°C in a 5070C O 2 atmosphere for 66 h, and then labeled with 0.1 taCi of 3H-thymidine CH-TdR, spec. act. 6.7 Ci/mM; NEN, DuPont, Boston, MA) in 10 ~1 RPMI 1640 medium for 6 h. The cells from individual culture wells were harvested on glass-filter strips with a MASH 1I harvester and processed for radioactivity in a Beckman scintillation counter (model LS-250). IL2 production and assay. As described elsewhere (Chang, Makinodan, Peterson & Strehler, 1982a), 5 x 10 6 splenic cells/ml were stimulated with 5 tag/ml Con A in complete RPMI 1640 medium containing 5 × 10 -5 M 2-mercaptoethanol (2-ME) and varying doses of alprazolam (5 - 50 ng) at 37°C in a 5070 CO2 humidified incubator for 24 h. The supernatants were harvested and stored at - 7 0 ° C until use. Supernatants were assayed by the method of Gillis et al. (Gillis, Ferm, Ou & Smith, 1978) as described in our previous study (Chang et al., 1982a), using a IL2-dependent cloned mouse cell line, CTLL-2, as indicator cells and crude rat IL2 as the reference standard. In this study, proliferation of CTLL-2 cells was monitored by [3H]-thymidine incorporation during the final 4 - 6 h of a 24-h culture period in the presence of serial two-fold dilutions of a standard IL2 preparation and the experimental supernates. One unit activity of IL2 was defined as the reciprocal dilution required to give a half maximum [3Hjthymidine incorporation of 1 x 104 cells after 24 h of culture. T-blast cell preparations. The method of Andersson et aL (Andersson, Gr6nvik, Larsson & Coutinho, 1979) with a minor modification described elsewhere (Chang, Norman & Makinodan, 1990) was used. Briefly, 2 x 106/ml whole spleen cells were cultured in complete RPMI 1640 medium containing 2/~g/ml Con A. Two to three days later, the cells were harvested, washed in Hank's balanced salt solution containing 10 mg/ml a-methylmannose, centrifuged on a F i c o l l - H y p a q u e (Pharmacia) gradient for 20 rain at 1500 rev/min at room temperature, and the blast T-cells collected for various proliferation experiments in response to recombinant interleukin IL2 (10 units/culture) or crude rat IL2 supernate (1 : 16 dilution). T-blast cells were also generated in the presence of alprazolam for determination of surface marker by flow cytometric scan and proliferation assay. It was established in this laboratory that IL2R is optimally expressed 48 - 72 h after stimulation with Con A (unpublished data). Only viable blast cells were used for the

229

fluorescence flow cytometric analysis and the proliferation studies. IL2-dependent T-blast cell proliferation. Con Astimulated T-blast cells, 5 x 104, were cultured at 37°C in the presence of 10 units of human recombinant IL2 (rlL2) or 1 : 16 dilution of crude rate IL2 supernate for 18 h. The cultures were then pulsed with 0.4 ~Ci of 3H-TdR and 6 h later the cells were harvested and processed for radioactivity counting. The amount of IL2 used in this study is an optimal dose, based on our previous finding (Norman, Chang, Wong, Branch, Castle & Taylor, t990), for IL2-dependent proliferative response of T-blast cells. Flow cytometric analysis o f IL2 receptor (antiTac) positive T-cell markers. As described elsewhere (Norman et al., 1990), Con A-induced T-blast cells at 106/ml were incubated with saturating concentrations o f rat anti-mouse IL2 receptor mAb (clone AMT13, purchased from Boehringer Mannheim Biochemical Co.) for 30 min at 4°C, then subsequently incubated with an excess of FITCconjugated polyclonal rabbit anti-rat IgG for 1 h at 37°C. The mAB AMTI3 can recognize an antigenic determinant of the murine IL2R molecule with tool. wt about 5 0 - 6 0 K (Osawa & Diamantstein, 1984). The fluorescein-labeled cells were then analyzed on an EPICS C flow cytometer (Coulter Electronic, Hileah, FL) equipped with an argon laser, at the UCLA-Jonsson Comprehensive Cancer Center. Statistical analysis. The data are presented as mean _+ S.D. Student's t-test was employed to evaluate significance of differences between the different groups.

RESULTS

Effect o f alprazolam on the proliferative responses o f T-cells to Con A or a combination o f PMA and ionomycin. We first studied the effect of alprazolam on Con A-induced splenic cell proliferation. As reported previously (Chang et al., 1991), the addition of alprazolam at the beginning of cultures stimulated with Con A resulted in a marked suppression (Fig. I(A); P<0.01). The decreased proliferative response of T-cells was not due to toxic effects of alprazolam in the cultures because there was no significant difference in the viability of recovered cells cultured for 3 days in the presence or absence of 50 ng alprazolam (i.e. 57 _ 4.5% vs 63 ___3.6°70, n = 5, P>0.05), as measured by the trypan blue exclusion assay. In contrast, alprazolam had little or

M.-P. CHANGet al.

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no effect on the proliferative response of splenic cells to a combination of PMA and ionomycin stimulation (Fig. I(B), P>0.1). Effect o f alprazolam on Con A-induced IL2 production and IL2 receptor expression. It is known that T-cell proliferation is dependent on (a) production of IL2, (b) expression of IL2R, and (c) interaction between IL2 and IL2R. To determine whether alprazolam-mediated inhibition of T-cell proliferation is due to a lack of IL2R expression and/or a reduced IL2 production, a monoclonal antibody to the mouse IL2R (rat anti-mouse IL2R mAb, clone AMT13) was used to directly evaluate the effect of alprazolam on the expression of this T-cell activation antigen. In addition, we also assessed the effect on the production of IL2 by splenic T-cells. The results (Table 1) confirmed our previous findings that alprazolam suppresses IL2 production by T-cells in a dose-dependent fashion

(Chang et al., 1991), with up to 46O7o inhibition at a concentration of 200 ng/106 cells/ml. However, it had no significant effect on IL2R expression (Fig. 2 and Table 1). Effect o f exogenous IL2 on the proliferative responses o f splenic T-cells to Con A and alprazolam. Because inhibition of IL2 production can result in impaired T-cell proliferation, we would like to determine whether IL2 production is the primary process affected by alprazolam. Therefore, in the continual presence of alprazolam, IL2 was added at various times to the Con A-stimulated cell cultures. The results showed that the addition of exogenous IL2 at the beginning or at 24 h of culture reversed the drug effect. However, exogenous IL2 did not appreciably alter the effect of alprazolam on T-cell proliferation when IL2 was added 48 h after cells cultured in the presence of Con A and alprazolam (Table 2). Effect o f alprazolam on the interaction o f lL2 and IL2 receptor. To determine whether alprazolam inhibits T-cell proliferation by interfering with the interaction between IL2 and IL2R, the ability of Con A-induced splenic T-blast cells, which already fully express IL2R, to proliferate in response to IL2 stimulation in the presence or absence of alprazolam was examined. The results (Fig. 3) showed that alprazolam did not inhibit the proliferative capacities of the T-blast cells to IL2. To further confirm that alprazolam does not interfere with the I L 2 - I L 2 R interaction, cloned CTLL-2 cells, an IL2-dependent cell line, were used to assess their ability to proliferate to IL2 in the presence of alprazolam. The results (Table 3) showed no significant inhibition of proliferation of cells derived from cultures incubated with IL2 and alprazolam (P>0.1). Effect o f alprazolam treatment on generation o f Con A-induced T-blast cells and their responsiveness to IL2. Con A-stimulated cells were cultured in the presence or absence of various doses of alprazolam to assess the effect of alprazolam treatment on the numbers of blast T-cells generated and their responsiveness to IL2. The results (Table 4) showed that alprazolam slightly decreased the number of blast cells generated. However, there was no significant difference in the ability of blast cells, obtained from cultures which were incubated in the presence or absence of alprazolam, to proliferate in response to an excess of IL2 (Table 5). The results again reflect the finding that alprazolam does not inhibit expression of IL2R on Con A-induced T-blast cells. Time-dependent effects o f alprazolam on Con Ainduced T-cell proliferation. To further establish

Immunosuppressive Effect of Alprazolam

231

Table 1. Effect of alprazolam on IL2 production* and IL2 receptor expressiont Alprazolam (ng/106 cells/ml)

IL2 production (units/ml)

% IL2R ÷ cells (fluorescence intensity)

Inhibition (%) 0

9.6 ___0.2

0

25 50 100

8.4 ___0.3 7.3 _+0.4 6.1 ___0.7

12.5 24 36.5

200

5.2 ___0.8

46

Inhibition (%) 91.7 _+ 1.1 (671 _+9.3) --88.2 _+ 3.7 (671 _+ 16.2) 84.5 _ 2.7 (666 _+9.7)

0

3.9* 7.9*

*Splenic cells (5 × 106/ml) were stimulated with Con A (5/ag/ml) in the presence or absence of alprazolam for 24 h. Supernatants were collected and assayed on the IL2-dependent CTLL-2 cell line as described in the Experimental Procedures section. The results (mean +__S.D.) of five BALB/c mice are shown. *3-day Con A blast T-cells were collected and the number of IL2R (Tac) antigen-positive cells was determined by indirect immunofluorescence. The results of two separate experiments (n = 6) shown are obtained from the fluorescence histograms of a flow cytometric analysis. *The inhibition was not significant (P>0.05). whether the antiproliferative effect of alprazolam is on the early events of T-cell activation, we added alprazolam (50 ng/culture) at different time intervals (0, 2, 4, 6, 34, 48 and 66 h) after Con A-stimulation of splenic cell cultures. The proliferative activities of 3-day cultures were then determined by 3H-thymidine incorporation. The results (Table 6) showed that the maximum inhibition of T-cell proliferation occurred when alprazolam was added at zero time, and that the inhibitory effect gradually decreased as the drug was added 2, 4, 6, and 24 h after Con A stimulation. However, the inhibiting effect of the addition of alprazolam was abrogated when the drug was added 24 h later to the cultures stimulated with Con A. Furthermore, the degree of inhibition was even greater when cells were preexposed to alprazolam for 2 h, followed by washing, then were stimulated with Con A than the inhibition seen in cultures in which alprazolam was added at zero time and present throughout the culture period.

DISCUSSION It is well known that alprazolam mediates its principal pharmacological actions via BZD receptors in the CNS (Mohler & Okada, 1977; Braestrup & Squires, 1977; Skolnick & Paul, 1981). Recently both stimulatory (Fride et al., 1990) and inhibitory effects of alprazolam (Chang et aL, 1991; Kim et al., 1990) on various immune functions have been

reported. However, most of the studies have been of phenomenological nature and did not examine the mechanisms underlying the specific action of alprazolam. Thus, this study attempted to elucidate the mechanism of antiproliferative effect of alprazolam on T-cells. The proliferation of T-cells in vitro involves an initial activation by mitogens or antigens, followed by a cascade of biochemical events and requires the interaction of several cell types (Williams, Ransil, Shapiro & Strom, 1985; Davis & Lipsky, 1986). A mitogenic stimulation initiates the entry of resting Tcell from the Go into the G~ phase of the cell cycle and induces the production of IL2 and the expression of IL2 receptor (IL2R). The interaction of the IL2R with IL2 then induces further cell cycle progression from G, into the S phase (Cantrell & Smith, 1984). Several possible events (sites) of T-cell proliferation, which could be affected by alprazolam, include antigenic (or mitogenic) binding, early biochemical and molecular events of T-cell activation, IL2 production, IL2R expression and an interaction of I L 2 - I L 2 R , and post IL2R events leading to DNA synthesis and cell division. There are six lines of evidence presented in this study supporting our view that alprazolam inhibits murine T-cell proliferation by selectively affecting the initiation (mitogenic receptor-mediated events) but not the progression (IL2 receptor-mediated events) of ligand-mediated T-cell through the cell cycle. First, the observations noted here, confirming

232

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Fig. 2. Influence of alprazolam on IL2 receptor (Tac antigen) expression on Con A-induced T-blast cells. Splenic cells (1 × 106 cells/ml) were cultured for 2 - 3 days with Con A (1 ~g/ml) in the presence or absence of alprazolam (100 or 200 ng/ml). 1 × 106 viable blast cells [shown in Figs 2(B), (C) and (D)] were stained with anti-mouse IL2R (p55) mAb (clone AMT13), followed by indirect fluorescent staining with FITC-conjugated polyclonal rabbit anti-rat lgG. The viable blast cells [shown in Fig. 2(A)] were stained with non-specific lgM, then followed by FITC-anti-rat IgG. Vial blast cells/ sample, 5 × 104, were analyzed on a flow cytometric scan. Histograms represent FITC fluorescence reflecting the IL2R (p55) subunit expression. The mean of FITC fluorescence intensity (F.I.) is given in the right side of each histogram and was calculated by the EPICS scan program. The results are representative of two differentexperiments (n = 6).

o u r earlier r e p o r t ( C h a n g et al., 1991), indicate that alprazolam can inhibit

clearly T-cell

p r o l i f e r a t i o n (Fig. 1) a n d IL2 p r o d u c t i o n (Table 1). T h e d i m i n i s h e d p r o l i f e r a t i v e r e s p o n s e o f splenic

Immunosuppressive Effect of Alprazolam

233

Table 2. Addition of exogenous IL2 reverses the effect of alprazolam on Con A-induced splenic T-cell proliferation* Time at which IL2 was added A. Addition of rlL2 No IL2 Day 0,1,2 Day 0 Day t Day 2 B. Addition of rat IL2 No IL2 Day 0,1,2 Day 0 Day 1 Day 2

Con A stimulation

Con A + alprazolam stimulation

% Activity*

55,653 111,171 111,244 112,223 66,202

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28,210 107,661 103,117 103,067 34,173

± 3495 ± 7883 ± 5903 _+ 9849 ± 433

50.7 92.7 96.8 91.8 51.6

57,248 112,617 103,567 126,930 58,645

+ 6604 ± 4924 ± 2261 ± 1830 4- 635

31,770 109,815 99,561 119,650 34,246

± 3787 ± 2440 ± 10552 _+ 8627 ± 2408

55.5 97.5 96.1 94.3 58.3

*2.5 × 10~splenic cells/culture were incubated under the conditions with Con A (0.5 gg/culture), Con A + alprazolam (50 ng/culture), Con A + rlL2 (10 units/culture), or 20 #1 of crude rat IL2 (1:16 dilution), or Con A ÷ IL2 + alprazolam for 72 h including a final 6 h of 3H-TdR incorporation. *Percent activity was calculated on the basis of counts/min obtained from cultures containing alprazolam divided by counts/min obtained from cultures containing no alprazolam x 100%. Sample size per group, five BALB/c mice. Table 3. Alprazolam does not inhibit the responsiveness of CTLL-2 cells (IL2-dependent cell line) to IL2*

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(1:16 dilution) 41,559 41,019 39,280 40,524 40,222

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ALPRAZOLAM (ng/culture) Fig. 3. Influence of alprazolam on the interaction of IL2 and IL2 receptors. Con A-induced blast T-cells per culture, 5 x 104; sample size per group, five BALB/c mice; rIL2 dose, 10 units/culture. The proliferative responses of 24 h cultures were determined with a final 6 h of 3H-TdR incorporation. Horizontal bars, mean of proliferative responses. Vertical bars, one S.D. T-cells is not due to a significant decrease in the n u m b e r o f viable splenic cells resulting f r o m toxic effects o f alprazolam (Chang et al., 1990). Second, the expression o f IL2R (Tac antigen) o n the Con A-activated T-blast cells was not (or only slightly) affected (Fig. 2 and Table 1), although the proliferative responses o f splenic cells to C o n A and p r o d u c t i o n o f IL2 are remarkably diminished by alprazolam. However, these expressed IL2R could be o f low affinity and biologically inactive when alprazolam is continuously present f r o m the

*CTLL-2 cells (1 × 104/culture) were incubated with IL2 for 20 h and then pulsed with 0.4/JCi 3H-TdR for an additional 4 h. The results of three experiments (mean _+ S.D.) are shown. Background without IL2 varied between 300 and 1100 counts/min.

beginning o f culture. On the other h a n d , once cells (such as blast cells) become c o m p e t e n t to proliferate in the presence o f growth factor (or factors), the drug is no longer able to affect the proliferation o f these cells. This may explain why alprazolam can impair C o n A-induced but not IL2-induced T-cell proliferation. Third, alprazolam does not interfere with the interaction o f I L 2 - IL2R (Fig. 3 and Tables 3 and 4). The progression o f cells t h r o u g h the cell cycle requires a p r o p e r interaction between I L 2 - I L 2 R . Decreased T-cell proliferation could also have resulted f r o m a lack o f interaction between I L 2 - I L 2 R , even if sufficient IL2 is available and

M.-P. CHANGet al.

234

Table 4. Effect of alprazolam treatment on the generation of Con A-induced blast T-cells* Total number of viable blast cells/flask (ng/10 6 cells/ml) ( X 10 6) Alprazolam 0 50 100 200

61.2 _+4.3 ~ 56.5 _+ 3.9~ 53.5 _+ 5.2t 51.8 + 4.7 f

% Recovery 100 92.3 87.4 84.6

*3-day cultures of Con A-induced viable blast cells were collected, washed and then counted with trypan blue dye solution (0.1%). Cells obtained from the culture containing no alprazolam were arbitrarily assigned to be 100% recovery. Sample size per group, five BALB/c mice. +The difference was not significant (P>0.05). IL2R fully expressed. Therefore, the effect of alprazolam on the interaction of I L 2 - I L 2 R was studied by incubating Con A-induced blast cells (or CTLL-2 cells), that already fully expressed IL2R, in the presence of an excess of IL2. As expected, alprazolam showed no appreciable inhibition of the proliferative response of blast cells (or CTLL-2) to IL2. Fourth, alprazolam did not significantly inhibit the generation of Con A-induced T-blast cells (Table 3) and was not able to inhibit the responsiveness of Con A-induced blast cells which were generated in the presence of alprazolam to IL2 (Table 4). These results (Tables 1 - 5 and Figs I(A), 2 and 3) taken together indicate that the alprazolam-mediated suppression of T-cell proliferation could be in part due to reduced IL2 production, but not to a defective expression of IL2R, nor to interference with the I L 2 - 1 L 2 R interaction. To further confirm this hypothesis, we assessed effects of exogenous IL2 on the proliferative response of splenic cells to Con A and alprazolam. The fact that addition of exogenous IL2 reversed the impaired T-cell proliferation seen in those cultures containing alprazolam strongly indicates that IL2 production is likely to be the site of the target affected most by alprazolam. Fifth, alprazolam does not inhibit P M A / ionomycin-induced cell proliferation [Fig. I(B)]. The prerequisite for T-cell proliferation is a triggering of the interaction of antigen (or mitogen) and its receptors with the T-cell antigen complex (Meurer, Acuto, Hussey, Hodgdon, Fitzgerald, Schlossman & Reinherz, 1983; Yang, Chouaib & DuPont, 1986; Weiss, Imboden, Hardy, Manger, Terhorst & Stobo, 1986; Meruer, Hussey, Cantrell, Hodgdon, Schlossman, Smith & Reinherz, 1984), followed by activation of protein kinase C (Isakor, Mally, Scholz & Altman, 1987; Nishizuka, 1984) and an increase in

intracellular calcium concentration (Tsien, Pozzan & Rink, 1982; Hesketh, Smith, Moore, Taylor & Metcalfe, 1984; Gelfand, Cheung, Grinstein & Mills, 1986). These events trigger or regulate certain genes, eventually leading to DNA synthesis and cell division. P MA can act as an analog of diacylglycerol to activate protein kinase C (PKC) translocation (Alemseged, Albert, Goldstein & Schmitt-Verhulst, 1985), and ionomycin can substitute for the effect of inositol triphosphate, which causes release of Ca 2 from intracellular stores (Nishizuka, 1984). It is known that neither PMA nor ionomycin alone is mitogenic for murine splenic cells. However, a combination of these two agents can synergistically induce murine splenic cells to proliferate, by-passing the requirement for an antigen- or mitogen-induced signal at the onset of T-cell activation (Alemseged et al., 1985). In order to demonstrate that alprazolam is likely to affect T-cell proliferation at the level of mitogen receptor interaction rather than IL2R interaction, we examined the proliferative response of splenic cells to Con A, or a combination of ionomycin and PMA, in the presence of alprazolam. If the proliferative response of T-cells stimulated by a combination of PMA and ionomycin were suppressed by alprazolam to a similar degree as that of T-cells stimulated by Con A, it would suggest that alprazolam-mediated suppression of T-cell proliferation may be at the level of IL2R-mediated events. However, if the proliferative response of T-cells triggered by a combination of PMA and ionomycin is n o t affected by alprazolam, it strongly suggests that suppression of T-cell proliferation by alprazolam may be at the level of mitogenic receptormediated events. Our results showed that alprazolam does n o t inhibit the proliferative response of splenic cells to an optimal concentration of PMA and ionomycin. Thus, alprazolam is likely to affect T-cell proliferation at the level of mitogenic receptormediated processes. Lastly, we observed that preincubation of alprazolam with cells for a short period of time (2 h), followed by washing, caused a greater inhibition in the proliferative responses of splenic cells to Con A stimulation than did addition of alprazolam at the beginning of cultures. These results indicate that preexposure of cells to alprazolam may cause cell membrane damage and prevent changes in [Ca-~ ]i and/or membrane potential. Evidence supporting this notion is that (a) changes in [Ca ~ ]i are an obligatory event for lectin-induced IL2 production but not necessary for IL2R expression (Mills, Cheung, Grinstein & Gelfand, 1985a,b) and (b) the prevention of changes in [Ca2]i is associated with

lmmunosuppressive Effect of Alprazolam

235

Table 5. Effect of alprazolam treatment on the responsiveness of Con A-induced blast T-cells to IL2* 3H-TdR incorporation (counts/min, X _+ S.D.)

Alprazolom treatment (ng/106 cells/ml)

80,085 73,375 73,303 74,795

0

50 100 200

°70 Inhibition+

_+ 4243 _+ 7902 +_ 8346 + 2294

0

8.4 8.5 6.6

*Splenic cells (2 x 106/ml) were stimulated with Con A (2 tag/ml) in the presence or absence of alprazolam for 2 - 3 days. The T-blast cells were collected, washed and adjusted to 5 x 105 cells/ml. Blast T-cells (5 x 104/culture) were incubated with crude rat IL2 for 18 h and then pulsed with ~H-TdR for 6 h. Sample size, four BALB/c mice. +The inhibition is not significant (P>0.05). Table 6. Time-dependent effects of the addition of alprazolam on Con A-induced T-cell proliferation* Initial incubation (0 h) Con A Con A Con A Con A Con A Con A Con A Con A Pretreatment of alprazolam for 2 h, then Con A was added

Time at which alprazolam (50 ng) was added -0h 2h 4h 6h 24 h 48 h 66 h -2 h

3H-TdR incorporation (counts/min)( _+ S.D.) 64,213 26,709 43,690 48,160 53,186 57,920 61,278 60,634 15,283

_+ 1633 _+ 1627 _+ 3430 _+ 2751 _+ 6776 __.2735 4- 2370 _+ 6298 _+ 1373

°70 Inhibitiont 0 58.4 32 25 17.2 9.2* 4.5* 5.6* 76.2

*Splenic cells (2.5 x 105/culture) were incubated under the conditions with addition of alprazolam at different times after or before cells were stimulated with Con A (0.5/ag/culture). The proliferative responses of 72 h cultures stimulated with Con A only were assigned as 100% activity. +Percent inhibition of cell proliferation is calculated on the basis of ratio of counts/min of cells from experimental cultures/counts/min of cells from the controls (Con A stimulation) × 100. *The inhibition was not significant (P>0.05). cyclosporin A - m e d i a t e d suppression of lectininduced IL2 p r o d u c t i o n a n d T-cell proliferation (Gelfand, C h e u n g & Mills, 1987). Platelet-activating factor ( P A F ) is a p o t e n t s t i m u l a t o r o f acute i n f l a m m a t o r y processes as well as a p o t e n t m o d u l a t o r o f the i m m u n e response (Braquet & Rola-Pleszczynski, 1987). It is also k n o w n t h a t P A F can induce the synthesis o f p r o s t a g l a n d i n s a n d decrease p l a s m a T N F - a level in the e n d o t o x i n treated mice (Kim et al., 1990). As a potent P F A a n t a g o n i s t , a l p r a z o l a m is also able to decrease the level of T N F - a in the p l a s m a o f mice treated with lipopolysaccharide (LPS). H o w e v e r , the suppressive effect o f a l p r a z o l a m can be reversed by pretreating the mice with i n d o m e t h a c i n , suggesting t h a t the effect was d e p e n d e n t in part o n p r o s t a g l a n d i n synthesis (Kim et al., 1990). It is k n o w n t h a t T N F - a

levels in the plasma o f e n d o t o x i n - t r e a t e d patients a n d those suffering f r o m e n d o t o x i n shock are high. T N F - a appears to be responsible, at least in part, for the clinical lesion associated with septic shock. T h u s , if a l p r a z o l a m is able to decrease p l a s m a T N F - a a n d block the deleterious effects o f P A F , a l p r a z o l a m m a y be useful in the clinical m a n a g e m e n t of e n d o t o x i n shock. The i n f o r m a t i o n presented here m a y also facilitate the design of a therapeutic a n d / o r preventive t r e a t m e n t for persons with anxiety disorders, which m a y increase susceptibility to the initial infection by pathogenic bacteria a n d viruses. In s u m m a r y , the findings of this study clearly demonstrated that alprazolam, as an i m m u n o s u p p r e s s a n t at high doses, can inhibit the mitogenic response of T-cells by decreasing IL2 p r o d u c t i o n but not by affecting IL2R expression,

236

M.-P. CHANG et al.

nor by interfering with the IL2 - IL2R interaction in c o m p e t e n t cells that have fully expressed IL2R. F u r t h e r m o r e , addition o f exogenous IL2 is able to reverse the inhibitory effect o f alprazolam on Con Ainduced T-cell proliferation. The results also show that the drug does not affect the P M A / i o n o m y c i n induced cell proliferation. Finally, the results d e m o n s t r a t e d that alprazolam has a t i m e - d e p e n d e n t inhibitory effect on Con A-induced T-cell proliferation. These results taken together suggest that alprazolam inhibits murine T-cell proliferation by selectively affecting the initiation (mitogenic receptor-mediated events) rather than the progression (IL2R-mediated events) o f lectinactivated T-cells t h r o u g h the cell cycle.

Further studies to determine whether alprazolam alters expression o f T-cell receptor (TCR) gene a n d / o r certain specific genes prior to expression o f IL2 and IL2R genes during the c o m p e t e n t phase o f cell cycle are needed. Such studies should provide a better insight into the molecular basis o f alprazolam effects on the i m m u n e system.

Acknowledgements - - We are very grateful to Drs T. Makinodan and T. Hahn for their constructive and helpful suggestions and critical review of the manuscript and to J. Sproul for his preparation of the manuscript. This work was supported in part by VA Medical Research Funds and by a grant from the Upjohn Company.

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