0192-0561/92 $5.00 + .00 Pergamon Press Ltd. ©1992 International Society for lmmunopharmacology.
Int. J. Immunopharmac., Vol. 14, No. 6, pp. 1125-1131, 1992.
Printed in Great Britain.
C O C A I N E - I N D U C E D S U P P R E S S I O N OF I N T E R F E R O N - G A M M A S E C R E T I O N IN L E U K O C Y T E S F R O M Y O U N G A N D OLD C 5 7 B L / 6 MICE B E R N H A R D W A T Z L , * G U A N J I E C H E N , * P H I L I P SCUDERI, t SERGEI P I R O Z H K O V * and R O N A L D R . W A T S O N *
*Department of Family and Community Medicine and tArizona Cancer Center, University of Arizona Health Sciences Center, Tucson, AZ 85724, U.S.A. (Received 27 March 1991 and in final form 4 December 1991)
Abstract -- The effects of cocaine (0.1 - 100 Mg/ml) were studied on interferon-gamma (1FN) secretion in
Con A-stimulated splenocytes from young (4 months) and old (18 months) C57BL/6 mice. IFN secretion was significantly suppressed by cocaine in a concentration-dependent manner. Suppression was observed in splenocytes from both young and old mice. Splenocytes from young mice released higher amounts of IFN (23.40 _+ 1.47 ng/ml) than those from old mice (6.10 _+0.35 ng/ml) after 24 h in culture. The concentration of Con A used to stimulate IFN secretion significantly affected the suppressive effect of cocaine. Pretreatment of splenocytes with cocaine followed by culture in the absence of cocaine did not affect IFN secretion in old mice, while splenocytes from young mice showed a sustained depression in IFN release. This suggests that cocaine suppresses IFN release in vitro and the susceptibility to this inhibitory effect may be age related.
Interferon-gamma (IFN) is a cytokine secreted by T-lymphocytes (Perussia, Mangoni, Engers & Trinchieri, 1980). IFN has multiple distinct biological activities including anti-viral activity (Trinchieri & Perussia, 1985), the capacity to modulate membrane MHC class I and II surface antigen expression (Wong, Clark-Lewis, McKimmBreschkin, Harris & Schrasher, 1983; Skoskiewicz, Colvin, Schneeberger & Russel, 1985), and the ability to enhance macrophage and natural killer cell activity (Ijzermans & Marquet, 1989; Friedman, 1990). The complex integration of IFN in immunoregulation suggests that any impairment of the IFN production and secretion may have severe consequences for the overall immune response. Drugs of abuse, such as cocaine, are known to modulate in vivo the immune response of humans and experimental animals. However, little is known about the mechanism of this immunomodulation. In our review (Watzl & Watson, 1990), we suggested that in vivo cocaine exerts most of its immunomodulating effects via its impact on the neuroendoc r i n e - i m m u n e network. In addition to the in vivo effects, a recent study demonstrated that cocaine can block neutrophil triggering and activation in vitro (Haines, Reibman, Callegan, Abramson, Philips &
Weissmann, 1990). Further, cocaine augmented in vitro the replication of HIV-1 in PHA-activated peripheral blood mononuclear cell (PBMC) via a mechanism that appears to involve transforming growth factor-beta (Peterson, Gekker, Chap, Schut, Molitor & Balfour, 1991). Plasma levels of cocaine in humans using this drug are in the range of 0.1 - 1 Mg/ml (Van Dyke, 1981). Because of the short half-life of cocaine (1 h), these concentrations last only for 1 - 4 h in the blood after a single administration (Chou, Ambre & Ruo, 1986). However, in cocaine-related deaths, plasma cocaine concentrations of up to 20.9 Mg/ml were found (Mittleman & Wetli, 1984). If cocaine is incubated with human plasma, it is rapidly hydrolyzed by plasma esterases with the formation of ecgonine methyl ester, one of cocaine's major metabolites (Stewart, Imaba, Lucassen & Kalow, 1979). The other major metabolite, benzoyl ecgonine, is spontaneously produced when cocaine is kept at 37°C in saline buffered to pH 7.4. After 24 h, 42.3°7o of the cocaine was converted to benzoyl ecgonine (Stewart et al., 1979). To be able to maintain physiologically achievable cocaine levels in vitro over a 72 h time period, we decided to start the cell cultures with low and high cocaine concentrations
1125
1126
B. WATZL et al.
(O.l-lO0/~g/ml). Since cocaine abuse is not restricted to a particular age group, we decided to compare the effects of the drug on both young and old mice. A sensitive sandwich enzyme linked immunosorbent assay (ELISA) system was developed which detects biologically active murine IFN in leukocyte culture supernatants. EXPERIMENTAL PROCEDURES
Materials C o c a i n e - H C l was obtained from the National Institute on Drug Abuse, Rockville, MD. Concanavalin A (Con A) was purchased from Sigma (St. Louis, MO). Recombinant murine IFN, recombinant murine interleukin-1 (IL-1) and tumor necrosis factor (TNF) were purchased from Genzyme, Inc. (Boston, MA) and recombinant murine interleukin-2 (IL-2) was purchased from Collaborative Research, Inc. (Bedford, MA). A rat monoclonal antibody specific for murine IFN was purchased from Lee Biomolecular Research, Inc. (San Diego, CA). Rabbit anti-murine IFN was produced in New Zealand white rabbits by immunizing the animals with 0.1 mg recombinant murine IFN (Genentech) suspended in 200/A of Freund's complete adjuvant, followed by a similar boost after 21 days. Ten days after the second injection of recombinant murine IFN the animal was bled by the lateral ear vein. The anti-serum was compared with preimmune serum from the same animal and assayed for specificity by solid phase immunoassay. The anti-murine IFN exhibited no cross-reactivity with recombinant routine TNF and murine IL-1. Horseradish peroxidase conjugated goat anti-rabbit IgG preadsorbed to human Ig was purchased from American Qualex, Inc. (La Mirada, CA). The peroxidase substrate 2,2'-azino-bis-3ethylbenzthiazoline-6-sulfonic acid (ABTS) was purchased from Sigma Chemical Co. (St. Louis, MO). Methods ELISA. An ELISA system for murine IFN was created by coating the wells of 96-well lmmulon II plates (Dynatech, Inc., McLean, VA) with 0.4 pg/ well of rat anti-mouse IFN monoclonal antibody. The monoclonal antibody was suspended in 50 pl of carbonate coating buffer consisting of 1.59 g NazCO3, 2.93 g NaHCO3 and 0.2 g sodium azide per liter (pH 9.6) and added to the wells. The plates were subsequently incubated at 4°C and after 12 h the coating buffer and residual unbound antibody were
removed from the wells. The plates were then washed twice with 0.01 M phosphate-buffered saline containing 0.05% Tween-20. Remaining proteinbinding sites were blocked with 100 ~1 of 2°70 BSA (bovine serum albumin) in PBS (1 h at 37°C). BSA/ PBS was discarded and 50/A of culture supernatants to be assayed for IFN were added to each well. The plates were incubated for 1 h at room temperature. The plates were washed and 50/A of rabbit antimurine IFN were added to each well and incubated for 1 h at room temperature, followed by a similar wash procedure. The final antibody added was 50 ~1 of a 1 : 5000 dilution (in PBS-Tween) of horseradish peroxidase conjugated goat anti-rabbit IgG which had been preadsorbed to human Ig. After 1 h incubation at room temperature it was washed three times with PBS/Tween and once with PBS. One hundred microliters of ABTS substrate solution were then added. The ABTS substrate solution was made by adding 0.274mg ABTS, 10.5 g citric acid monohydrate to 500 ml of deionized distilled HzO and adjusting the pH to 4.2. Optical density changes prooduced on the ELISA plates were determined by a Titertek Multiscan (Flow Laboratories, McLean, VA) fixed at 405 nm within 30 min of substrate addition. The quantity of IFN contained in culture supernatants was determined by comparison with a set of standards made by adding recombinant murine IFN to complete RPMI-1640. Animals. C57BL/6 female mice, aged 4 months (young) and 18 months (old), were kept in a ventilated animal room with a 12 h l i g h t - d a r k cycle. Regular food and water were supplied ad fibitum. Cell culture o f murine splenocytes. Splenocytes were cultured in 96-well flat-bottom plates (Falcon 3072, Lincoln Park, N J) with RPMI-1640 containing 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 U/ml penicillin and 100 ~g/ml streptomycin, 100/A of splenocytes suspensions (107 cells/ml) were added to the wells. IFN secretion was induced by culturing splenocytes for various lengths of time at 37°C and 5% COz with 2/~g/ml Con A. Splenocytes were also cultured with various concentrations of cocaine (0.1 - 100/~g/ml). After incubation culture plates were centrifuged (180 g for 10 min), and the supernatants were removed for detection of IFN. Determination o f cocaine levels after incubation in RPMI-1640 culture media. C o c a i n e - H C I , 100 or 10/ag/ml, was incubated in RPMI-1640 media 72 h at 37°C. After 0, 8, 24, 48 and 48 h 2.0 ml of the sample were withdrawn and the concentration of cocaine was determined. Some samples containing cocaine were incubated 24 h in the presence of
1127
Cocaine-induced Suppression Table 1. Stability of cocaine during 72 h incubation in RPMI-1640 culture media Time (h)
Percent of the initial concentration
0 8 24 48 72
100 43.2 26.8 3.4 2.8
- 7.3* +-. 3.7* ___0.6* +- 0.2*
Cocaine was added to RPMI-1640 media at concentrations 10 or 100 ~g/ml and incubated 72 h at 37°C as described in Experimental Procedures. Since at various time points similar portions of cocaine were decomposed at both initial concentrations data for 10 and 100 gg/ml were combined. Data are mean _+ S.E. of at least three determinations for each concentration. *Significantly different from beginning of incubation (P
Int. J. Immunopharmac., Vol. 14, No. 6, pp. 1125-1131, 1992.
Printed in Great Britain.
C O C A I N E - I N D U C E D S U P P R E S S I O N OF I N T E R F E R O N - G A M M A S E C R E T I O N IN L E U K O C Y T E S F R O M Y O U N G A N D OLD C 5 7 B L / 6 MICE B E R N H A R D W A T Z L , * G U A N J I E C H E N , * P H I L I P SCUDERI, t SERGEI P I R O Z H K O V * and R O N A L D R . W A T S O N *
*Department of Family and Community Medicine and tArizona Cancer Center, University of Arizona Health Sciences Center, Tucson, AZ 85724, U.S.A. (Received 27 March 1991 and in final form 4 December 1991)
Abstract -- The effects of cocaine (0.1 - 100 Mg/ml) were studied on interferon-gamma (1FN) secretion in
Con A-stimulated splenocytes from young (4 months) and old (18 months) C57BL/6 mice. IFN secretion was significantly suppressed by cocaine in a concentration-dependent manner. Suppression was observed in splenocytes from both young and old mice. Splenocytes from young mice released higher amounts of IFN (23.40 _+ 1.47 ng/ml) than those from old mice (6.10 _+0.35 ng/ml) after 24 h in culture. The concentration of Con A used to stimulate IFN secretion significantly affected the suppressive effect of cocaine. Pretreatment of splenocytes with cocaine followed by culture in the absence of cocaine did not affect IFN secretion in old mice, while splenocytes from young mice showed a sustained depression in IFN release. This suggests that cocaine suppresses IFN release in vitro and the susceptibility to this inhibitory effect may be age related.
Interferon-gamma (IFN) is a cytokine secreted by T-lymphocytes (Perussia, Mangoni, Engers & Trinchieri, 1980). IFN has multiple distinct biological activities including anti-viral activity (Trinchieri & Perussia, 1985), the capacity to modulate membrane MHC class I and II surface antigen expression (Wong, Clark-Lewis, McKimmBreschkin, Harris & Schrasher, 1983; Skoskiewicz, Colvin, Schneeberger & Russel, 1985), and the ability to enhance macrophage and natural killer cell activity (Ijzermans & Marquet, 1989; Friedman, 1990). The complex integration of IFN in immunoregulation suggests that any impairment of the IFN production and secretion may have severe consequences for the overall immune response. Drugs of abuse, such as cocaine, are known to modulate in vivo the immune response of humans and experimental animals. However, little is known about the mechanism of this immunomodulation. In our review (Watzl & Watson, 1990), we suggested that in vivo cocaine exerts most of its immunomodulating effects via its impact on the neuroendoc r i n e - i m m u n e network. In addition to the in vivo effects, a recent study demonstrated that cocaine can block neutrophil triggering and activation in vitro (Haines, Reibman, Callegan, Abramson, Philips &
Weissmann, 1990). Further, cocaine augmented in vitro the replication of HIV-1 in PHA-activated peripheral blood mononuclear cell (PBMC) via a mechanism that appears to involve transforming growth factor-beta (Peterson, Gekker, Chap, Schut, Molitor & Balfour, 1991). Plasma levels of cocaine in humans using this drug are in the range of 0.1 - 1 Mg/ml (Van Dyke, 1981). Because of the short half-life of cocaine (1 h), these concentrations last only for 1 - 4 h in the blood after a single administration (Chou, Ambre & Ruo, 1986). However, in cocaine-related deaths, plasma cocaine concentrations of up to 20.9 Mg/ml were found (Mittleman & Wetli, 1984). If cocaine is incubated with human plasma, it is rapidly hydrolyzed by plasma esterases with the formation of ecgonine methyl ester, one of cocaine's major metabolites (Stewart, Imaba, Lucassen & Kalow, 1979). The other major metabolite, benzoyl ecgonine, is spontaneously produced when cocaine is kept at 37°C in saline buffered to pH 7.4. After 24 h, 42.3°7o of the cocaine was converted to benzoyl ecgonine (Stewart et al., 1979). To be able to maintain physiologically achievable cocaine levels in vitro over a 72 h time period, we decided to start the cell cultures with low and high cocaine concentrations
1125
1126
B. WATZL et al.
(O.l-lO0/~g/ml). Since cocaine abuse is not restricted to a particular age group, we decided to compare the effects of the drug on both young and old mice. A sensitive sandwich enzyme linked immunosorbent assay (ELISA) system was developed which detects biologically active murine IFN in leukocyte culture supernatants. EXPERIMENTAL PROCEDURES
Materials C o c a i n e - H C l was obtained from the National Institute on Drug Abuse, Rockville, MD. Concanavalin A (Con A) was purchased from Sigma (St. Louis, MO). Recombinant murine IFN, recombinant murine interleukin-1 (IL-1) and tumor necrosis factor (TNF) were purchased from Genzyme, Inc. (Boston, MA) and recombinant murine interleukin-2 (IL-2) was purchased from Collaborative Research, Inc. (Bedford, MA). A rat monoclonal antibody specific for murine IFN was purchased from Lee Biomolecular Research, Inc. (San Diego, CA). Rabbit anti-murine IFN was produced in New Zealand white rabbits by immunizing the animals with 0.1 mg recombinant murine IFN (Genentech) suspended in 200/A of Freund's complete adjuvant, followed by a similar boost after 21 days. Ten days after the second injection of recombinant murine IFN the animal was bled by the lateral ear vein. The anti-serum was compared with preimmune serum from the same animal and assayed for specificity by solid phase immunoassay. The anti-murine IFN exhibited no cross-reactivity with recombinant routine TNF and murine IL-1. Horseradish peroxidase conjugated goat anti-rabbit IgG preadsorbed to human Ig was purchased from American Qualex, Inc. (La Mirada, CA). The peroxidase substrate 2,2'-azino-bis-3ethylbenzthiazoline-6-sulfonic acid (ABTS) was purchased from Sigma Chemical Co. (St. Louis, MO). Methods ELISA. An ELISA system for murine IFN was created by coating the wells of 96-well lmmulon II plates (Dynatech, Inc., McLean, VA) with 0.4 pg/ well of rat anti-mouse IFN monoclonal antibody. The monoclonal antibody was suspended in 50 pl of carbonate coating buffer consisting of 1.59 g NazCO3, 2.93 g NaHCO3 and 0.2 g sodium azide per liter (pH 9.6) and added to the wells. The plates were subsequently incubated at 4°C and after 12 h the coating buffer and residual unbound antibody were
removed from the wells. The plates were then washed twice with 0.01 M phosphate-buffered saline containing 0.05% Tween-20. Remaining proteinbinding sites were blocked with 100 ~1 of 2°70 BSA (bovine serum albumin) in PBS (1 h at 37°C). BSA/ PBS was discarded and 50/A of culture supernatants to be assayed for IFN were added to each well. The plates were incubated for 1 h at room temperature. The plates were washed and 50/A of rabbit antimurine IFN were added to each well and incubated for 1 h at room temperature, followed by a similar wash procedure. The final antibody added was 50 ~1 of a 1 : 5000 dilution (in PBS-Tween) of horseradish peroxidase conjugated goat anti-rabbit IgG which had been preadsorbed to human Ig. After 1 h incubation at room temperature it was washed three times with PBS/Tween and once with PBS. One hundred microliters of ABTS substrate solution were then added. The ABTS substrate solution was made by adding 0.274mg ABTS, 10.5 g citric acid monohydrate to 500 ml of deionized distilled HzO and adjusting the pH to 4.2. Optical density changes prooduced on the ELISA plates were determined by a Titertek Multiscan (Flow Laboratories, McLean, VA) fixed at 405 nm within 30 min of substrate addition. The quantity of IFN contained in culture supernatants was determined by comparison with a set of standards made by adding recombinant murine IFN to complete RPMI-1640. Animals. C57BL/6 female mice, aged 4 months (young) and 18 months (old), were kept in a ventilated animal room with a 12 h l i g h t - d a r k cycle. Regular food and water were supplied ad fibitum. Cell culture o f murine splenocytes. Splenocytes were cultured in 96-well flat-bottom plates (Falcon 3072, Lincoln Park, N J) with RPMI-1640 containing 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 U/ml penicillin and 100 ~g/ml streptomycin, 100/A of splenocytes suspensions (107 cells/ml) were added to the wells. IFN secretion was induced by culturing splenocytes for various lengths of time at 37°C and 5% COz with 2/~g/ml Con A. Splenocytes were also cultured with various concentrations of cocaine (0.1 - 100/~g/ml). After incubation culture plates were centrifuged (180 g for 10 min), and the supernatants were removed for detection of IFN. Determination o f cocaine levels after incubation in RPMI-1640 culture media. C o c a i n e - H C I , 100 or 10/ag/ml, was incubated in RPMI-1640 media 72 h at 37°C. After 0, 8, 24, 48 and 48 h 2.0 ml of the sample were withdrawn and the concentration of cocaine was determined. Some samples containing cocaine were incubated 24 h in the presence of
1127
Cocaine-induced Suppression Table 1. Stability of cocaine during 72 h incubation in RPMI-1640 culture media Time (h)
Percent of the initial concentration
0 8 24 48 72
100 43.2 26.8 3.4 2.8
- 7.3* +-. 3.7* ___0.6* +- 0.2*
Cocaine was added to RPMI-1640 media at concentrations 10 or 100 ~g/ml and incubated 72 h at 37°C as described in Experimental Procedures. Since at various time points similar portions of cocaine were decomposed at both initial concentrations data for 10 and 100 gg/ml were combined. Data are mean _+ S.E. of at least three determinations for each concentration. *Significantly different from beginning of incubation (P
