Vol. 14, No. 4 Printed in U.S.A.
INFECTION AND IMMUNITY, OCt. 1976, p. 948-950 Copyright © 1976 American Society for Microbiology
Inhibition of Human Neutrophil Chemotaxis In Vitro by Phenothiazines and Related Compounds BENGT BJORKSTIgN' AND PAUL G. QUIE* Department of Pediatrics, University of Minnesota School of Medicine, Minneapolis, Minnesota 55455 Received for publication 1 June 1976
Chlorpromazine (CPZ) and three other phenothiazines and the structurally related antidepressant drugs imipramine and amitriptyline were found to depress human. neutrophil chemotactic responsiveness. A 7 x 10-6 M solution of CPZ inhibited chemotaxis, whereas concentrations of the other tested drugs 10 to 1,000 times greater than this were needed to inhibit chemotaxis. This effect of CPZ could not, however, be demonstrated when testing neutrophils from patients treated with the drug. The inhibition of chemotaxis was reversible when CPZ-incubated neutrophils were washed before testing for chemotactic responsiveness. CPZ affects neutrophil function as well as other aspects of immune response.
Phenothiazine derivates are commonly used as tranquilizers; however, they show a wide variety of biological activity affecting the function of many organs in man (1). These compounds act in a reversible fashion on membranes of mammalian cells and inhibit a variety of cell surface phenomena (12). Chlorpromazine (CPZ) has been shown to inhibit several lymphocyte responses, including the proliferative response and generation of cytotoxic lymphocytes in mixed lymphocyte cultures (2). Ruutu in 1972 showed that a number of phenothiazines inhibited neutrophil phagocytosis (9). Diminished resistance to infection has been reported in patients on high doses of CPZ (7, 13). Since chemotactic responsiveness is an important function of neutrophils in host resistance, the effect of phenothiazines and the struc-
turally closely related antidepressant drugs imipramine and amitriptyline on neutrophil chemotaxis was studied. MATERIALS AND METHODS Leukocytes from healthy young adults and from four patients treated with phenothiazine drugs were separated by allowing heparinized venous blood to sediment spontaneously for 45 min in plastic syringes. The leukocytes were separated into plastic tubes (17 by 100 mm) (Falcon, Oxnard, Calif.). The leukocytes were separated from platelets by centrifugation at 400 x g for 5 min and were suspended in Hanks balanced salt solution (Hanks BSS) at a concentration of 4 x 106 to 8 x 106 polymorphonuclear leukocytes per ml. Leukocytes from one donor were used for all assays done on the same day. Present address: Department of Pediatrics, University of UmeA, UmeA, Sweden. 948
The following phenothiazine derivatives were studied: CPZ and prochlorperazine (Smith, Klein and French Laboratories, Philadelphia, Pa.), perphenazine (Schering Corp., Kenilworth, N.J.), amitriptyline hydrochloride (Merck Sharp & Dohme, West Point, Pa.), thioridazine (Sandoz, Inc., East Hanover, N.J.), and imipramine (Ciba-Geigy, Summit, N.Y.). The drugs were diluted in Hanks BSS with 0.3 mM HEPES buffer (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), pH 7.3. Experimental procedures. Equal volumes of leukocyte suspension and drug solutions at various concentrations or Hanks BSS alone were incubated for 30 min at 37°C. After incubation, a volume of 0.4 ml of leukocyte suspensions was placed in the upper compartment of Boyden chambers on the surface of a 5-,ug cellulose acetate filter (Millipore Corp.). The lower compartment was filled with chemotactic attractant, a culture supernatant from an overnight culture of Escherichia coli (4). The chambers were incubated for 60 min at 37°C, and the filters were fixed and stained as previously described (4). The distance of migration of neutrophils from the top of the filter was measured by determining the leading front of neutrophils in the filter using a magnification of x 300 (4). The effect of drugs on chemotaxis is expressed as a percentage of the distance of migration of neutrophils preincubated with drugs compared with distance of migration by neutrophils not incubated with drugs.
RESULTS There was inhibition of neutrophil chemotaxis by all of the compounds tested, although there was a marked difference in the concentrations necessary for this effect (Fig. 1). CPZ was markedly inhibitory (63%) at a concentration of 5 ,.ug/ml (1.4 x 10-5 M). The concentration of other phenothiazines necessary to inhibit neu-
VOL. 14, 1976
PHENOTHIAZINE EFFECT ON CHEMOTAXIS
949
DISCUSSION 2
(
Ruutu reported that CPZ and related
{\l;4Q 3 se \w 6r
com-
pounds depressed leukocyte phagocytosis and bacterial killing, and he considered this to be an effect of CPZ on the cell membranes, since washing the leukocytes completely reversed the
z .-
inhibition (9, 10). Furthermore, the drug did not interfere with opsonization (9, 10). The con-
0
centrations of CPZ that depressed phagocytosis 0l
were
._
pressed chemotaxis. At
0)
0
T.
E
t
higher
than the concentrations that de-
10-5 M, phagocytosis
a
concentration of 2
x
90% of the normal; ,ug/ml), phagocytosis was was
10- M (17.8 80% of the normal (9).
with 5 x 0
Recently Ferguson et al. (2) reported that CPZ inhibits erative
o0
2
5
1O 510
20 20
o 50
1( )O 100
Drrug Concentration
(jig/ml) FIG. 1. Effect of phenothiazines on leukocyte chemotactic responsiveeness. The effect is expressed as a percentage (mean arnd range) of the chemotaxis of leukocytes not incubatted with any drugs. 1 = chlorpromazine; 2 = prochiIorperazine; 3 = perphenazine; 4 = thioridazine; 5 = camitriptyline; 6 = imipramine.
trophil chemotaxis was 25 ,ug/ml (6 x 10- to 8 10-5 M) or higher , and with imipramine and amitriptyline a con centration of 50 ug/ml (1.5 x 10-4 M) or higher was required for an inhibitory effect. No gr(DSS morphological changes were observed in iricubated cells examined by light microscope, a nd more than 99% of the neutrophils incubat;ed with 12.5 ,ug of CPZ per ml excluded trypan blue. Inhibition of che motaxis by phenothiazines was reversible sincce normal chemotaxis was observed when leukKocytes that had been incubated for 30 min wit;h 12.5 ,g of CPZ per ml (3.5 10-5 M) were washed in Hanks BSS and resuspended in plasma. To investigate a possible correlation between these in vitro results and an in vivo effect of the compounds, we evaluated the chemotaxis of leukocytes from four adults, two men and two women, who were being treated with phenothiazine derivatives. The patients were receiving 400 mg of CPZ twice, 300 mg three times, and 600 mg four times daily or 100 mg of thioridazine three times daily, respectively. Blood was drawn in the morning before the drugs were given and again 90 to 120 min after the first dose of the drugs. As shown in Table 1, chemotaxis of their leukocytes was not depressed by chlorpromazine or thioridazine at therapeutic dosages. x
allogenic stimulation of the prolif-
response
in
mixed
and inhibits the mixed
lymphocyte
lymphocyte
culture
culture
gen-
eration of cytotoxic lymphocytes. At a CPZ conug/ml, there was a 50% inhibi[3Hlthymidine uptake in mixed lymphocyte culture. Chemotaxis was inhibited by concentrations as low as 2.5 ,ug/ml and thus appears to be more
centration of 1.8 tion of
sensitive to the effect of CPZ than phagocytosis. Indeed, Ruutu and Collan demonstrated that at 2 10'; M inhibited pseudopod formation by the leukocytes, although phagocytosis
CPZ
x
remained almost normal (11). This difference in concentration of CPZ necessary to effect chemo-
taxis and phagocytosis might be
a
useful labo-
ratory tool to study neutrophil phagocytosis
un-
der conditions where there is little chemotaxis. The chemotaxis-depressing effect varied between different phenothiazine compounds; prochlorperazine was only slightly inhibitory at 25 ,ug/ml. Amitriptyline and imipramine structurally resemble the phenothiazines and are used in similar clinical conditions; however, these drugs were inhibitory in higher concentrations than CPZ. Ten micrograms of amitriptyline
x
TABLE 1. Chemotaxis of leukocytes from four patients before and 90 to 120 min after oral medication with chlorpromazine (CPZ) or thioridazine (THI) Chemotaxis
Patient Patient
of CPZ or Daily dose THI
(Sm)a Before
1 2 3 4
CPZ (2,400 mg) CPZ (800 mg) CPZ (1,200 mg) THI (300 mg)
90 103 89 79 93 92
After 79 88 105 100
Control Control a Expressed as the distance traveled by the leading front.
950
INFECT. IMMUN.
BJORKSTlN AND QUIE
per milliliter (3.2 x 10-5 M) did not depress chemotaxis, and 50 ,g of imipramine per ml (1.6 x 10-4 M) only slightly depressed chemotaxis (Fig. 1). The concentration of CPZ necessary to demonstrate inhibition of chemotaxis was 2 to 10 times higher than peak plasma levels found in patients receiving the drugs (5, 6). However, the drug is rapidly metabolized, and serum concentrations of the drug and pharmacologically active metabolites of it range from 4 to 22 ,ug/ml in patients receiving the drug orally (6). Measurement of serum concentrations of CPZ are difficult and may not correspond with clinical effectiveness of the drug in patients on longterm treatment (8). CPZ is accumulated in certain tissues, particularly the lungs, where concentrations of up to 80 ,ug/g of wet tissue are found (3). The in vitro effect of CPZ on neutrophil function may well have a clinical counterpart in vivo and may bear relevance to the suggested association between pulmonary infection and CPZ treatment (7, 13). LITERATURE CITED 1. Byck, R. 1975. Drugs and the treatment of psychiatric disorders, p. 152-200. In L. S. Goodman and A. Gilman (ed.), The pharmacological basis of therapeutics. Macmillan Publishing Co., New York. 2. Ferguson, R. M., J. R. Schmidtke, and R. L. Simmons. 1976. Differential effects of chlorpromazine on the in vitro generation and effector function of cytotoxic lymphocytes. J. Exp. Med. 143:232-237. 3. Forrest, I. S., A. G. Bolt, and M. T. Serra. 1968. Distribution of chlorpromazine metabolites in selected or-
4.
5.
6. 7.
8.
9.
10. 11.
12. 13. 14.
gans of psychiatric patients chronically dosed up to the time of death. Biochem. Pharmacol. 17:2061-2070. Hill, H. R., R. D. Estenson, P. G. Quie, N. A. Hogan, and N. D. Goldberg. 1975. Modulation of human neutrophil chemotactic responses by cyclic 3'5' guanosine monophosphate and cyclic 3'5' adenosine monophosphate. Metabolism 24:447-456. Hollister, L. E., S. H. Curry, J. E. Derr, and S. L. Kanter. 1970. Studies of delayed-action medication. V. Plasma levels and urinary excretion of four different dosage forms of chlorpromazine. Clin. Pharmacol. Ther. 11:49-59. Huang, C. L., and B. H. Ruskin. 1964. Determination of serum chlorpromazine metabolites in psychotic patients. J. Nerv. Ment. Dis. 139:381-386. Kline, N. S., J. Barsa, and E. Goline. 1956. Management of the side effects of reserpine and combined reserpine-chlorpromazine treatment. Dis. Nerv. Syst. 17:352-358. Mackay, A. V. P., A. F. Healey, and J. Baker. 1974. The relationship of plasma chlorpromazine to its 7hydroxy and sulphoxide metabolites in a large population of chronic schizophrenics. Br. J. Clin. Pharmacol. 1:425-430. Ruutu, T. 1972. Effect of phenothiazines and related compounds on phagocytosis and bacterial killing by human neutrophilic leukocytes. Ann. Med. Exp. Biol. Fenn. 50:24-36. Ruutu, T. 1972. Factors affecting the inhibition of phagocytosis by chlorpromazine. Ann. Med. Exp. Biol. Fenn. 50:37-41. Ruutu, T., and Y. Collan. 1972. Short-term effect of chlorpromazine on human leukocytes and erythrocytes. An electron microscopic study. Ann. Med. Exp. Biol. Fenn. 50:42-48. Seeman, P. 1972. The membrane effects of anesthetics and tranquilizers. Pharmacol. Rev. 24:583-655. Wardell, D. W. 1957. Untoward reactions to tranquilizing drugs. Am. J. Psychiatry 113:745. Wilkinson, P. C. 1974. Chemotaxis and inflammation, p. 168-172. Churchill Livingstone, Edinburgh.
INFECTION AND IMMUNITY, OCt. 1976, p. 948-950 Copyright © 1976 American Society for Microbiology
Inhibition of Human Neutrophil Chemotaxis In Vitro by Phenothiazines and Related Compounds BENGT BJORKSTIgN' AND PAUL G. QUIE* Department of Pediatrics, University of Minnesota School of Medicine, Minneapolis, Minnesota 55455 Received for publication 1 June 1976
Chlorpromazine (CPZ) and three other phenothiazines and the structurally related antidepressant drugs imipramine and amitriptyline were found to depress human. neutrophil chemotactic responsiveness. A 7 x 10-6 M solution of CPZ inhibited chemotaxis, whereas concentrations of the other tested drugs 10 to 1,000 times greater than this were needed to inhibit chemotaxis. This effect of CPZ could not, however, be demonstrated when testing neutrophils from patients treated with the drug. The inhibition of chemotaxis was reversible when CPZ-incubated neutrophils were washed before testing for chemotactic responsiveness. CPZ affects neutrophil function as well as other aspects of immune response.
Phenothiazine derivates are commonly used as tranquilizers; however, they show a wide variety of biological activity affecting the function of many organs in man (1). These compounds act in a reversible fashion on membranes of mammalian cells and inhibit a variety of cell surface phenomena (12). Chlorpromazine (CPZ) has been shown to inhibit several lymphocyte responses, including the proliferative response and generation of cytotoxic lymphocytes in mixed lymphocyte cultures (2). Ruutu in 1972 showed that a number of phenothiazines inhibited neutrophil phagocytosis (9). Diminished resistance to infection has been reported in patients on high doses of CPZ (7, 13). Since chemotactic responsiveness is an important function of neutrophils in host resistance, the effect of phenothiazines and the struc-
turally closely related antidepressant drugs imipramine and amitriptyline on neutrophil chemotaxis was studied. MATERIALS AND METHODS Leukocytes from healthy young adults and from four patients treated with phenothiazine drugs were separated by allowing heparinized venous blood to sediment spontaneously for 45 min in plastic syringes. The leukocytes were separated into plastic tubes (17 by 100 mm) (Falcon, Oxnard, Calif.). The leukocytes were separated from platelets by centrifugation at 400 x g for 5 min and were suspended in Hanks balanced salt solution (Hanks BSS) at a concentration of 4 x 106 to 8 x 106 polymorphonuclear leukocytes per ml. Leukocytes from one donor were used for all assays done on the same day. Present address: Department of Pediatrics, University of UmeA, UmeA, Sweden. 948
The following phenothiazine derivatives were studied: CPZ and prochlorperazine (Smith, Klein and French Laboratories, Philadelphia, Pa.), perphenazine (Schering Corp., Kenilworth, N.J.), amitriptyline hydrochloride (Merck Sharp & Dohme, West Point, Pa.), thioridazine (Sandoz, Inc., East Hanover, N.J.), and imipramine (Ciba-Geigy, Summit, N.Y.). The drugs were diluted in Hanks BSS with 0.3 mM HEPES buffer (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), pH 7.3. Experimental procedures. Equal volumes of leukocyte suspension and drug solutions at various concentrations or Hanks BSS alone were incubated for 30 min at 37°C. After incubation, a volume of 0.4 ml of leukocyte suspensions was placed in the upper compartment of Boyden chambers on the surface of a 5-,ug cellulose acetate filter (Millipore Corp.). The lower compartment was filled with chemotactic attractant, a culture supernatant from an overnight culture of Escherichia coli (4). The chambers were incubated for 60 min at 37°C, and the filters were fixed and stained as previously described (4). The distance of migration of neutrophils from the top of the filter was measured by determining the leading front of neutrophils in the filter using a magnification of x 300 (4). The effect of drugs on chemotaxis is expressed as a percentage of the distance of migration of neutrophils preincubated with drugs compared with distance of migration by neutrophils not incubated with drugs.
RESULTS There was inhibition of neutrophil chemotaxis by all of the compounds tested, although there was a marked difference in the concentrations necessary for this effect (Fig. 1). CPZ was markedly inhibitory (63%) at a concentration of 5 ,.ug/ml (1.4 x 10-5 M). The concentration of other phenothiazines necessary to inhibit neu-
VOL. 14, 1976
PHENOTHIAZINE EFFECT ON CHEMOTAXIS
949
DISCUSSION 2
(
Ruutu reported that CPZ and related
{\l;4Q 3 se \w 6r
com-
pounds depressed leukocyte phagocytosis and bacterial killing, and he considered this to be an effect of CPZ on the cell membranes, since washing the leukocytes completely reversed the
z .-
inhibition (9, 10). Furthermore, the drug did not interfere with opsonization (9, 10). The con-
0
centrations of CPZ that depressed phagocytosis 0l
were
._
pressed chemotaxis. At
0)
0
T.
E
t
higher
than the concentrations that de-
10-5 M, phagocytosis
a
concentration of 2
x
90% of the normal; ,ug/ml), phagocytosis was was
10- M (17.8 80% of the normal (9).
with 5 x 0
Recently Ferguson et al. (2) reported that CPZ inhibits erative
o0
2
5
1O 510
20 20
o 50
1( )O 100
Drrug Concentration
(jig/ml) FIG. 1. Effect of phenothiazines on leukocyte chemotactic responsiveeness. The effect is expressed as a percentage (mean arnd range) of the chemotaxis of leukocytes not incubatted with any drugs. 1 = chlorpromazine; 2 = prochiIorperazine; 3 = perphenazine; 4 = thioridazine; 5 = camitriptyline; 6 = imipramine.
trophil chemotaxis was 25 ,ug/ml (6 x 10- to 8 10-5 M) or higher , and with imipramine and amitriptyline a con centration of 50 ug/ml (1.5 x 10-4 M) or higher was required for an inhibitory effect. No gr(DSS morphological changes were observed in iricubated cells examined by light microscope, a nd more than 99% of the neutrophils incubat;ed with 12.5 ,ug of CPZ per ml excluded trypan blue. Inhibition of che motaxis by phenothiazines was reversible sincce normal chemotaxis was observed when leukKocytes that had been incubated for 30 min wit;h 12.5 ,g of CPZ per ml (3.5 10-5 M) were washed in Hanks BSS and resuspended in plasma. To investigate a possible correlation between these in vitro results and an in vivo effect of the compounds, we evaluated the chemotaxis of leukocytes from four adults, two men and two women, who were being treated with phenothiazine derivatives. The patients were receiving 400 mg of CPZ twice, 300 mg three times, and 600 mg four times daily or 100 mg of thioridazine three times daily, respectively. Blood was drawn in the morning before the drugs were given and again 90 to 120 min after the first dose of the drugs. As shown in Table 1, chemotaxis of their leukocytes was not depressed by chlorpromazine or thioridazine at therapeutic dosages. x
allogenic stimulation of the prolif-
response
in
mixed
and inhibits the mixed
lymphocyte
lymphocyte
culture
culture
gen-
eration of cytotoxic lymphocytes. At a CPZ conug/ml, there was a 50% inhibi[3Hlthymidine uptake in mixed lymphocyte culture. Chemotaxis was inhibited by concentrations as low as 2.5 ,ug/ml and thus appears to be more
centration of 1.8 tion of
sensitive to the effect of CPZ than phagocytosis. Indeed, Ruutu and Collan demonstrated that at 2 10'; M inhibited pseudopod formation by the leukocytes, although phagocytosis
CPZ
x
remained almost normal (11). This difference in concentration of CPZ necessary to effect chemo-
taxis and phagocytosis might be
a
useful labo-
ratory tool to study neutrophil phagocytosis
un-
der conditions where there is little chemotaxis. The chemotaxis-depressing effect varied between different phenothiazine compounds; prochlorperazine was only slightly inhibitory at 25 ,ug/ml. Amitriptyline and imipramine structurally resemble the phenothiazines and are used in similar clinical conditions; however, these drugs were inhibitory in higher concentrations than CPZ. Ten micrograms of amitriptyline
x
TABLE 1. Chemotaxis of leukocytes from four patients before and 90 to 120 min after oral medication with chlorpromazine (CPZ) or thioridazine (THI) Chemotaxis
Patient Patient
of CPZ or Daily dose THI
(Sm)a Before
1 2 3 4
CPZ (2,400 mg) CPZ (800 mg) CPZ (1,200 mg) THI (300 mg)
90 103 89 79 93 92
After 79 88 105 100
Control Control a Expressed as the distance traveled by the leading front.
950
INFECT. IMMUN.
BJORKSTlN AND QUIE
per milliliter (3.2 x 10-5 M) did not depress chemotaxis, and 50 ,g of imipramine per ml (1.6 x 10-4 M) only slightly depressed chemotaxis (Fig. 1). The concentration of CPZ necessary to demonstrate inhibition of chemotaxis was 2 to 10 times higher than peak plasma levels found in patients receiving the drugs (5, 6). However, the drug is rapidly metabolized, and serum concentrations of the drug and pharmacologically active metabolites of it range from 4 to 22 ,ug/ml in patients receiving the drug orally (6). Measurement of serum concentrations of CPZ are difficult and may not correspond with clinical effectiveness of the drug in patients on longterm treatment (8). CPZ is accumulated in certain tissues, particularly the lungs, where concentrations of up to 80 ,ug/g of wet tissue are found (3). The in vitro effect of CPZ on neutrophil function may well have a clinical counterpart in vivo and may bear relevance to the suggested association between pulmonary infection and CPZ treatment (7, 13). LITERATURE CITED 1. Byck, R. 1975. Drugs and the treatment of psychiatric disorders, p. 152-200. In L. S. Goodman and A. Gilman (ed.), The pharmacological basis of therapeutics. Macmillan Publishing Co., New York. 2. Ferguson, R. M., J. R. Schmidtke, and R. L. Simmons. 1976. Differential effects of chlorpromazine on the in vitro generation and effector function of cytotoxic lymphocytes. J. Exp. Med. 143:232-237. 3. Forrest, I. S., A. G. Bolt, and M. T. Serra. 1968. Distribution of chlorpromazine metabolites in selected or-
4.
5.
6. 7.
8.
9.
10. 11.
12. 13. 14.
gans of psychiatric patients chronically dosed up to the time of death. Biochem. Pharmacol. 17:2061-2070. Hill, H. R., R. D. Estenson, P. G. Quie, N. A. Hogan, and N. D. Goldberg. 1975. Modulation of human neutrophil chemotactic responses by cyclic 3'5' guanosine monophosphate and cyclic 3'5' adenosine monophosphate. Metabolism 24:447-456. Hollister, L. E., S. H. Curry, J. E. Derr, and S. L. Kanter. 1970. Studies of delayed-action medication. V. Plasma levels and urinary excretion of four different dosage forms of chlorpromazine. Clin. Pharmacol. Ther. 11:49-59. Huang, C. L., and B. H. Ruskin. 1964. Determination of serum chlorpromazine metabolites in psychotic patients. J. Nerv. Ment. Dis. 139:381-386. Kline, N. S., J. Barsa, and E. Goline. 1956. Management of the side effects of reserpine and combined reserpine-chlorpromazine treatment. Dis. Nerv. Syst. 17:352-358. Mackay, A. V. P., A. F. Healey, and J. Baker. 1974. The relationship of plasma chlorpromazine to its 7hydroxy and sulphoxide metabolites in a large population of chronic schizophrenics. Br. J. Clin. Pharmacol. 1:425-430. Ruutu, T. 1972. Effect of phenothiazines and related compounds on phagocytosis and bacterial killing by human neutrophilic leukocytes. Ann. Med. Exp. Biol. Fenn. 50:24-36. Ruutu, T. 1972. Factors affecting the inhibition of phagocytosis by chlorpromazine. Ann. Med. Exp. Biol. Fenn. 50:37-41. Ruutu, T., and Y. Collan. 1972. Short-term effect of chlorpromazine on human leukocytes and erythrocytes. An electron microscopic study. Ann. Med. Exp. Biol. Fenn. 50:42-48. Seeman, P. 1972. The membrane effects of anesthetics and tranquilizers. Pharmacol. Rev. 24:583-655. Wardell, D. W. 1957. Untoward reactions to tranquilizing drugs. Am. J. Psychiatry 113:745. Wilkinson, P. C. 1974. Chemotaxis and inflammation, p. 168-172. Churchill Livingstone, Edinburgh.
