Clin. exp. Immunol. (1976) 24, 42-48.
The effects of cyclic AMP on leucocyte inhibitory factor (LIF) production and on the inhibition of leucocyte migration RUTH LOMNITZER, A. R. RABSON & H. J. KOORNHOF Department of Microbiology, School of Pathology of University of the Witwatersrand and South African Institute for Medical Research, Johannesburg, South Africa (Received 13 September 1975)
SUMMARY
The effect of drugs known to increase intracellular levels of cyclic AMP were studied in the leucocyte migration inhibition system. It was found that cyclic AMP, dibutyryl cyclic AMP, theophylline, and prostaglandins El and E2 inhibited the production of leucocyte inhibiting factor by PHA pulsed lymphocytes. Inhibition only occurred when the drugs were present during or after the PHA pulse. In addition it was found that these drugs enhanced the migration of polymorphonuclear leucocytes (PMN), in this system. Electrophoretic mobility of PMN cells was not altered by these drugs indicating that the effect is not due to changes in membrane charge. However, granulocyte adhesion was reduced in the presence of these drugs suggesting that adhesion is of primary importance in the migration of polymorphonuclear leucocytes out of capillary tubes. The findings show that cyclic AMP is important in modulating both cell-mediated and inflammatory responses.
INTRODUCTION It has been established that cAMP plays an important role in the biochemical events which initiate lymphocyte activation. It has been shown to effect lymphocyte activation by mitogens (Smith, Steiner & Parker, 1971b), production of MIF (Pick, 1974), cytotoxic factor (Lies & Peter, 1973) and lymphocyte cytotoxicity (Henney & Lichtenstein, 1971). Furthermore, drugs known to increase intracellular levels of cAMP have been shown to produce a doserelated inhibition of both random and directed polymorphonuclear leucocyte (PMN) motility (Tse, Phelps & Urban, 1972) and also reduce adhesion of PMN cells to glass capillary tubes (Bryant & Sutcliff, 1974). Pick (1972) has shown that the migration of guinea-pig macrophages out of capillary tubes is inhibited by cAMP, dibutyryl cAMP and isoproterenol when added in the presence of theophylline, and these agents have been shown to reverse the activity of MIF on macrophage migration (Koopman, Gillis & David, 1973). The leucocyte migration inhibition test has been shown to be a specific measure of cellmediated immunity in man (S0borg & Bendixen, 1967) and relies upon the production of a lymphokine, leucocyte inhibitory factor (LIF) which specifically inhibits human PMN migration (Rocklin, 1974). In view of the effect of cAMP on both PMN movement and lymphocyte activation it was decided to study its effects on the leucocyte migration inhibition system and the adhesion of PMN to nylon fibre columns. Preliminary experiments in our laboratory indicated that the direct inhibition of leucocyte migration by PHA was abolished in the presence of drugs known to increase intracellular Correspondence: Dr Ruth Lomnitzer, S.A.I.M.R., P.O. Box 1038, Johannesburg, South Africa.
42
Effect of cAMP on leucocyte migration
43
levels of cAMP. The work presented here investigates the effect of these drugs on both LIF production and the target PMN cells. The system used employs a modification of the previously described two-stage assay of LIF production (Lomnitzer, Rabson & Koornhof, 1975) whereby lymphocytes are initially pulsed with PHA and the resulting supernatant is then assessed for LIF activity using normal PMN as indicator cells in a capillary migration system. MATERIALS AND METHODS Production ofLIF. Human tonsils obtained at tonsilectomy were washed once in minimal essential medium (MEM) containing 100 u/ml penicillin, 100 pgfml streptomycin and 3 ,ug/ml amphotericin B. After rinsing they were placed in fresh medium, cut into small pieces and the cells dispersed by teasing. The cell suspension was then collected into plastic tubes which were left standing for 3-5 min to allow clumps to sediment. The suspension was then collected and transferred into 50 ml plastic Falcon centrifuge tubes. The cells were centrifuged at 200 g for 7 min, washed twice in MEM and the lymphocyte count was adjusted to 4 x 106/ml viable cells in Hepes buffered MEM containing 10% foetal calf serum (Burroughs Wellcome), 2 mM Lglutamine (Flow Laboratory), 100 u/ml penicillin, 100 pg/ml of streptomycin and 3 ,ug/ml of amphotericin B. The lymphocytes were cultured in plastic tissue culture flasks (Falcon), in the presence and absence of 20
uig/ml PHA (reagent grade Burroughs Wellcome) for 2 hr at 370C in an atmosphere of 5% CO2 in air. After 2 hr the cells were washed three times in MEM, resuspended to the original volume and incubated for a further 72 hr. Cell-free supernatants were collected by centrifugation at 250 g for 10 min. To control for the removal of PHA, samples of similarly treated cells were incubated at 40C for 72 hr and the supernatants assessed for the presence of LIF activity. Preparation of drugs. The drugs to be tested were freshly prepared for each experiment. Adenosine 3:5cyclic monophosphoric acid (cAMP), N602-dibutyryl adenosine 3',5'-cyclic monophosphoric acid sodium salt (DBcAMP) and theophylline were obtained from Sigma Chemical Company (St Louis, Missouri) and were dissolved in MEM and diluted in the culture media to give a final concentration of 10-3 M. Prostaglandins E1 (PGE1) and E2 (PGE2) were kindly supplied by Dr John Pike (Upjohn Company, Kalamazoo, Michigan). Stock solutions were prepared by dissolving 2 mg of each in 0-2 ml of absolute ethanol and made up to 1 mg/ml in MEM. Aliquots were stored at - 20°C and were further diluted in the culture media to give a final concentration of 5 ,ug/ml. The effect of drugs which increase cAMP levels on LIF production. This was tested in three ways: (A) Lymphocytes were incubated with the above drugs for 2 hr after which they were washed twice and pulsed with PHA for 2 hr. The cultures were then treated as previously described. Control cultures were pulsed with the drugs but PHA was not added. (B) Lymphocytes were incubated with the drugs for 2 hr, PHA was then added to the flasks for a further 2 hr and the cells washed three times, resuspended in fresh medium and incubated for 72 hr. Control cultures were treated with the drugs but no PHA was added. (C) Lymphocytes were pulsed with PHA for 2 hr, washed three times and resuspended to their original volume. The drugs were then added to the flasks and the cultures were incubated for 72 hr. Control cultures consisted of non-PHA pulsed lymphocytes which were incubated for the same length of time in the presence of the drugs. The drugs were removed from the cell-free supernatant using a Minicon B 15 (Amicon Corporation,
Lexington, Massachusetts).
LIF assay. Human PMN leucocytes were obtained by separating whole blood diluted 1: 1 in MEM on a
Hypaque-Ficoll gradient. The pellet containing PMN and erythrocytes was treated with 0-83% ammonium chloride at 4°C for 10 min to lyse the erythrocytes. The remaining PMN were centrifuged at 200 g for 5 min and washed three times in MEM. The resultant suspension contained 98-99% PMN. Siliconized
capillary tubes (50 Lambda Yankee disposable micropet Clay Adams) were filled with cell suspension containing 3 x 107 cells/ml. The capillary tubes were then sealed with seal-ease (Clay Adams) and centrifuged for 5 min at 300 g. The tubes were cut at the cell-fluid interface and the capillary stumps were then placed on the floor of plastic disposable migration chamber plates (Sterilin Ltd, Middlesex), and held in place by a drop of silicon grease. The chambers were filled with the supernatants to be tested and sealed with a cover slip. They were then incubated for 18 hr at 37°C in 5% CO2 in air. The migration pattern was projected and traced and the area of migration was measured by planimetry. The indices of migration were calculated as follows: MI = average area of migration in LIF containing supernatants average area of migration in control supernatants In all the experiments the drug-treated controls gave the same migration areas as the non-treated controls.
44
R. Lomnitzer, A. R. Rabson & H. J. Koornhof
The mean ± s.e.m. migrations were determined by averaging the migration from four capillary tubes, and statistical significance was determined by the Student's t-test for paired means. A P value higher than 005 was considered not significant. The effect of the drugs on PMN migration. Aliquots of 1 ml PMN suspensions containing 1P5 x 107 cells were incubated for 2 hr in a 370C water bath in the presence and absence of the drugs. The tubes were shaken every 15 min. At the end of the incubation period the cells were centrifuged and washed twice, packed in capillaries and placed in migration chambers which were then filled with MEM + 10% FCS + 2 mm glutamine. The migration index was calculated as follows: MI area of migration of PMN incubated in the presence of drugs area of migration of PMN incubated in the absence of drugs The effect ofthe drugs on PMN electrophoretic mobility. The electrophoretic mobility of PMN cells treated as above was measured in a Zeiss cytopherometer at 230C. As a positive control PMN cells were treated with the chemotactic factor, C5a known to diminish the negative surface charge of human granulocytes. The cells were suspended in 3 ml of buffered MEM (2-38 g MEM powder, 41-90 g mannitol, 25-68 g sucrose, 1 76 g Hepes and 1-0 ml EDTA (0-1 M) made up to 1 litre with distilled water). The time for each cell to migrate a constant distance along the chamber (three squares on the grid which is 48 pm) was measured in both directions, i.e. before and after reversing the current. Measurements were made in the front stationary plane and at least twenty cells in each group were measured. The current through the cell was 4 0 mA. Adherence assay. The PMN adherence assay was performed according to MacGregor, Spagnalo & Lentnik (1974). Scrubbed nylon fibre (Leuko-pak leucocyte filter, Fenwal Laboratories, Morton Grove, Illinois) was weighed on an analytical balance (Mettler H-20) and 120 mg was packed by means of a wooder applicator stick into a 2 ml disposable plastic syringe (Monoplast, Johannesburg, South Africa). The length of the packed column was adjusted to 15 mm. The packed syringes were placed in suitable holders and warmed to 370C in an incubator. Heparinized whole blood was used for the assay. The drugs to be tested were diluted 1:10 in the blood to give the required concentration, the final total volume being 6 ml. The blood was then incubated in a 370C water bath for 45 min. Control samples consisted of blood to which the appropriate volume of MEM was added prior to incubation. After incubation 1-5-ml aliquots from each specimen were added to the top of three columns and allowed to filter through. 0-2 ml of the effluent or the original blood sample was added to a mixture of 0-6 ml Turk's white cell counting fluid and 0-2 ml of ZAPOglobin (Coulter Electronics, South Africa) and the number of granulocytes counted. The adhesion index was calculated by the following equation: PMN/ml in the effluent sample PMN/ml in the original sample The mean ± s.e.m. PMN/ml were determined by averaging the counts from three columns and statistical significance was calculated by the Student's t-test for paired means. A P value greater than 0 05 was considered not significant.
RESULTS
The effect on LIF production of preincubating lymphocytes with drugs Two types of pre-incubation experiments were performed. In the one, lymphocytes were preincubated with the drugs which were removed before the PHA pulse. In the other, the drugs were not washed off and remained throughout the PHA pulse. As can be seen from Fig. 1, supernatants from lymphocytes pulsed with PHA in the presence of the drugs caused less inhibition than supernatants collected from only PHA pulsed lymphocytes. Those produced in the absence of drugs caused 50% inhibition, whereas supernatants produced in their presence caused only about 70% of normal migration. When the lymphocytes were washed after being treated with the drugs, and then exposed to a PHA pulse, no significant change in the inhibitory activity of the supernatants was detected (Fig. 2). The effect on LIF production of adding drugs after the PHA pulse When lymphocytes were initially pulsed with PHA and then treated with the drugs, the resulting supernatants from which drugs were removed did not cause significant inhibition
45
Effect of cAMP on leucocyte migration 0'7 06 x
.Q_ 'a To TO
0*5 04 0 3
-5;
LUJ
a-
L-
cli
Li
CL
0-2
m
0D 0L
LUI 0D 0l.
0
0
Pvalue*
The effects of cyclic AMP on leucocyte inhibitory factor (LIF) production and on the inhibition of leucocyte migration RUTH LOMNITZER, A. R. RABSON & H. J. KOORNHOF Department of Microbiology, School of Pathology of University of the Witwatersrand and South African Institute for Medical Research, Johannesburg, South Africa (Received 13 September 1975)
SUMMARY
The effect of drugs known to increase intracellular levels of cyclic AMP were studied in the leucocyte migration inhibition system. It was found that cyclic AMP, dibutyryl cyclic AMP, theophylline, and prostaglandins El and E2 inhibited the production of leucocyte inhibiting factor by PHA pulsed lymphocytes. Inhibition only occurred when the drugs were present during or after the PHA pulse. In addition it was found that these drugs enhanced the migration of polymorphonuclear leucocytes (PMN), in this system. Electrophoretic mobility of PMN cells was not altered by these drugs indicating that the effect is not due to changes in membrane charge. However, granulocyte adhesion was reduced in the presence of these drugs suggesting that adhesion is of primary importance in the migration of polymorphonuclear leucocytes out of capillary tubes. The findings show that cyclic AMP is important in modulating both cell-mediated and inflammatory responses.
INTRODUCTION It has been established that cAMP plays an important role in the biochemical events which initiate lymphocyte activation. It has been shown to effect lymphocyte activation by mitogens (Smith, Steiner & Parker, 1971b), production of MIF (Pick, 1974), cytotoxic factor (Lies & Peter, 1973) and lymphocyte cytotoxicity (Henney & Lichtenstein, 1971). Furthermore, drugs known to increase intracellular levels of cAMP have been shown to produce a doserelated inhibition of both random and directed polymorphonuclear leucocyte (PMN) motility (Tse, Phelps & Urban, 1972) and also reduce adhesion of PMN cells to glass capillary tubes (Bryant & Sutcliff, 1974). Pick (1972) has shown that the migration of guinea-pig macrophages out of capillary tubes is inhibited by cAMP, dibutyryl cAMP and isoproterenol when added in the presence of theophylline, and these agents have been shown to reverse the activity of MIF on macrophage migration (Koopman, Gillis & David, 1973). The leucocyte migration inhibition test has been shown to be a specific measure of cellmediated immunity in man (S0borg & Bendixen, 1967) and relies upon the production of a lymphokine, leucocyte inhibitory factor (LIF) which specifically inhibits human PMN migration (Rocklin, 1974). In view of the effect of cAMP on both PMN movement and lymphocyte activation it was decided to study its effects on the leucocyte migration inhibition system and the adhesion of PMN to nylon fibre columns. Preliminary experiments in our laboratory indicated that the direct inhibition of leucocyte migration by PHA was abolished in the presence of drugs known to increase intracellular Correspondence: Dr Ruth Lomnitzer, S.A.I.M.R., P.O. Box 1038, Johannesburg, South Africa.
42
Effect of cAMP on leucocyte migration
43
levels of cAMP. The work presented here investigates the effect of these drugs on both LIF production and the target PMN cells. The system used employs a modification of the previously described two-stage assay of LIF production (Lomnitzer, Rabson & Koornhof, 1975) whereby lymphocytes are initially pulsed with PHA and the resulting supernatant is then assessed for LIF activity using normal PMN as indicator cells in a capillary migration system. MATERIALS AND METHODS Production ofLIF. Human tonsils obtained at tonsilectomy were washed once in minimal essential medium (MEM) containing 100 u/ml penicillin, 100 pgfml streptomycin and 3 ,ug/ml amphotericin B. After rinsing they were placed in fresh medium, cut into small pieces and the cells dispersed by teasing. The cell suspension was then collected into plastic tubes which were left standing for 3-5 min to allow clumps to sediment. The suspension was then collected and transferred into 50 ml plastic Falcon centrifuge tubes. The cells were centrifuged at 200 g for 7 min, washed twice in MEM and the lymphocyte count was adjusted to 4 x 106/ml viable cells in Hepes buffered MEM containing 10% foetal calf serum (Burroughs Wellcome), 2 mM Lglutamine (Flow Laboratory), 100 u/ml penicillin, 100 pg/ml of streptomycin and 3 ,ug/ml of amphotericin B. The lymphocytes were cultured in plastic tissue culture flasks (Falcon), in the presence and absence of 20
uig/ml PHA (reagent grade Burroughs Wellcome) for 2 hr at 370C in an atmosphere of 5% CO2 in air. After 2 hr the cells were washed three times in MEM, resuspended to the original volume and incubated for a further 72 hr. Cell-free supernatants were collected by centrifugation at 250 g for 10 min. To control for the removal of PHA, samples of similarly treated cells were incubated at 40C for 72 hr and the supernatants assessed for the presence of LIF activity. Preparation of drugs. The drugs to be tested were freshly prepared for each experiment. Adenosine 3:5cyclic monophosphoric acid (cAMP), N602-dibutyryl adenosine 3',5'-cyclic monophosphoric acid sodium salt (DBcAMP) and theophylline were obtained from Sigma Chemical Company (St Louis, Missouri) and were dissolved in MEM and diluted in the culture media to give a final concentration of 10-3 M. Prostaglandins E1 (PGE1) and E2 (PGE2) were kindly supplied by Dr John Pike (Upjohn Company, Kalamazoo, Michigan). Stock solutions were prepared by dissolving 2 mg of each in 0-2 ml of absolute ethanol and made up to 1 mg/ml in MEM. Aliquots were stored at - 20°C and were further diluted in the culture media to give a final concentration of 5 ,ug/ml. The effect of drugs which increase cAMP levels on LIF production. This was tested in three ways: (A) Lymphocytes were incubated with the above drugs for 2 hr after which they were washed twice and pulsed with PHA for 2 hr. The cultures were then treated as previously described. Control cultures were pulsed with the drugs but PHA was not added. (B) Lymphocytes were incubated with the drugs for 2 hr, PHA was then added to the flasks for a further 2 hr and the cells washed three times, resuspended in fresh medium and incubated for 72 hr. Control cultures were treated with the drugs but no PHA was added. (C) Lymphocytes were pulsed with PHA for 2 hr, washed three times and resuspended to their original volume. The drugs were then added to the flasks and the cultures were incubated for 72 hr. Control cultures consisted of non-PHA pulsed lymphocytes which were incubated for the same length of time in the presence of the drugs. The drugs were removed from the cell-free supernatant using a Minicon B 15 (Amicon Corporation,
Lexington, Massachusetts).
LIF assay. Human PMN leucocytes were obtained by separating whole blood diluted 1: 1 in MEM on a
Hypaque-Ficoll gradient. The pellet containing PMN and erythrocytes was treated with 0-83% ammonium chloride at 4°C for 10 min to lyse the erythrocytes. The remaining PMN were centrifuged at 200 g for 5 min and washed three times in MEM. The resultant suspension contained 98-99% PMN. Siliconized
capillary tubes (50 Lambda Yankee disposable micropet Clay Adams) were filled with cell suspension containing 3 x 107 cells/ml. The capillary tubes were then sealed with seal-ease (Clay Adams) and centrifuged for 5 min at 300 g. The tubes were cut at the cell-fluid interface and the capillary stumps were then placed on the floor of plastic disposable migration chamber plates (Sterilin Ltd, Middlesex), and held in place by a drop of silicon grease. The chambers were filled with the supernatants to be tested and sealed with a cover slip. They were then incubated for 18 hr at 37°C in 5% CO2 in air. The migration pattern was projected and traced and the area of migration was measured by planimetry. The indices of migration were calculated as follows: MI = average area of migration in LIF containing supernatants average area of migration in control supernatants In all the experiments the drug-treated controls gave the same migration areas as the non-treated controls.
44
R. Lomnitzer, A. R. Rabson & H. J. Koornhof
The mean ± s.e.m. migrations were determined by averaging the migration from four capillary tubes, and statistical significance was determined by the Student's t-test for paired means. A P value higher than 005 was considered not significant. The effect of the drugs on PMN migration. Aliquots of 1 ml PMN suspensions containing 1P5 x 107 cells were incubated for 2 hr in a 370C water bath in the presence and absence of the drugs. The tubes were shaken every 15 min. At the end of the incubation period the cells were centrifuged and washed twice, packed in capillaries and placed in migration chambers which were then filled with MEM + 10% FCS + 2 mm glutamine. The migration index was calculated as follows: MI area of migration of PMN incubated in the presence of drugs area of migration of PMN incubated in the absence of drugs The effect ofthe drugs on PMN electrophoretic mobility. The electrophoretic mobility of PMN cells treated as above was measured in a Zeiss cytopherometer at 230C. As a positive control PMN cells were treated with the chemotactic factor, C5a known to diminish the negative surface charge of human granulocytes. The cells were suspended in 3 ml of buffered MEM (2-38 g MEM powder, 41-90 g mannitol, 25-68 g sucrose, 1 76 g Hepes and 1-0 ml EDTA (0-1 M) made up to 1 litre with distilled water). The time for each cell to migrate a constant distance along the chamber (three squares on the grid which is 48 pm) was measured in both directions, i.e. before and after reversing the current. Measurements were made in the front stationary plane and at least twenty cells in each group were measured. The current through the cell was 4 0 mA. Adherence assay. The PMN adherence assay was performed according to MacGregor, Spagnalo & Lentnik (1974). Scrubbed nylon fibre (Leuko-pak leucocyte filter, Fenwal Laboratories, Morton Grove, Illinois) was weighed on an analytical balance (Mettler H-20) and 120 mg was packed by means of a wooder applicator stick into a 2 ml disposable plastic syringe (Monoplast, Johannesburg, South Africa). The length of the packed column was adjusted to 15 mm. The packed syringes were placed in suitable holders and warmed to 370C in an incubator. Heparinized whole blood was used for the assay. The drugs to be tested were diluted 1:10 in the blood to give the required concentration, the final total volume being 6 ml. The blood was then incubated in a 370C water bath for 45 min. Control samples consisted of blood to which the appropriate volume of MEM was added prior to incubation. After incubation 1-5-ml aliquots from each specimen were added to the top of three columns and allowed to filter through. 0-2 ml of the effluent or the original blood sample was added to a mixture of 0-6 ml Turk's white cell counting fluid and 0-2 ml of ZAPOglobin (Coulter Electronics, South Africa) and the number of granulocytes counted. The adhesion index was calculated by the following equation: PMN/ml in the effluent sample PMN/ml in the original sample The mean ± s.e.m. PMN/ml were determined by averaging the counts from three columns and statistical significance was calculated by the Student's t-test for paired means. A P value greater than 0 05 was considered not significant.
RESULTS
The effect on LIF production of preincubating lymphocytes with drugs Two types of pre-incubation experiments were performed. In the one, lymphocytes were preincubated with the drugs which were removed before the PHA pulse. In the other, the drugs were not washed off and remained throughout the PHA pulse. As can be seen from Fig. 1, supernatants from lymphocytes pulsed with PHA in the presence of the drugs caused less inhibition than supernatants collected from only PHA pulsed lymphocytes. Those produced in the absence of drugs caused 50% inhibition, whereas supernatants produced in their presence caused only about 70% of normal migration. When the lymphocytes were washed after being treated with the drugs, and then exposed to a PHA pulse, no significant change in the inhibitory activity of the supernatants was detected (Fig. 2). The effect on LIF production of adding drugs after the PHA pulse When lymphocytes were initially pulsed with PHA and then treated with the drugs, the resulting supernatants from which drugs were removed did not cause significant inhibition
45
Effect of cAMP on leucocyte migration 0'7 06 x
.Q_ 'a To TO
0*5 04 0 3
-5;
LUJ
a-
L-
cli
Li
CL
0-2
m
0D 0L
LUI 0D 0l.
0
0
Pvalue*
