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Effects of histamine-sensitizing factor and cortisol on lymphocyte adenyl cyclase responses
Effects of histamine-sensitizing factor and cortisol on lymphocyte adenyl cyclase responses Tee-Ping
Lee, Ph.D. Madison, Wis.
Levels qf cyclic adenosime monophosphate (CAMP) in lymphocytes are regulated by P-adrenergic agonists and PGE,. The effect of these agonists is potentiated by cortisol. Incubation of lymphocytes with histamine-sensitizing factor (HSF) leads to loss of response to epinephrine and PGE,. The response can be partially restored by cortisol. Incubation of lymphocytes with /3-adrenergic antagonists such as propranolol leads to the loss of /3-adrenergic agents but not PGE,. The inhibition by propranolol is not reversed by cortisol. These results suggest that the action of HSF and Bordetella pertussisvaccine is not mediated through the inhibition qf padrenergic receptors alone
Vaccination with Bordetella pet-tusk increases sensitivity to anaphylaxis in several species and also increases antibody production, especially homocytotropic (IgE) antibody. l--j In addition, vaccinated mice develop a diminished hyperglycemic response to epinephrine.6 These abnormalities can also be induced by treating animals with propranolol. For this reason, P-adrenergic blockade has been proposed as the mechanism of vaccine action.2 We (Lee and Reed7) found a decreased responsiveness of the adenyl cyclase system in the skin of vaccinated mice. Similar abnormalities also have been detected in lung and liver.x It is generally believed that a protein, histamine-sensitizing factor (HSF), is responsible for these abnormalities induced by pertussis vaccine.g-ll Pertussis vaccine also produces a striking lymphocytosis due to redistribution of lymphocytes from lymphoid organs into the intravascular po01.‘~ Parker and Morsel3 have obtained a protein fraction, lymphocytosis-promoting factor (LPF), from pertussis organisms that produces lymphocytosis and also inhibits elevation of cyclic adenosine monophosphate (CAMP) in lymphocytes on stimulation by isoproterenol or PGE,. LPF has been found to be closely From the Department of Medicine, University of Wisconsin. Supported by National Institutes of Health Grants 2P15 AI 10404 and 2 ROI AI 08106. Received for publication March 19, 1976. Accepted for publication Aug. 27, 1976. Reprint requests to: Tee-Ping Lee, Ph.D., 504 N. Walnut St., Room 305, Madison, Wis. 53705.
associated with HSF. There is evidence suggesting that HSF and LPF are one and the same entity.“, l4 But others15 have suggested that HSF and LPF are different molecules. The present experiments were undertaken to explore the actions of HSF on human lymphocytes in vitro and to compare the effect of HSF with that of the @-adrenergic blocking drug propranolol. MATERIALS Materials HSF*
AND METHODS
was a gift
of Dr. John
Munoz
of the Rocky
Moun-
tain Laboratory, Hamilton, Mont. Prostaglandin El was generously provided by Dr. John Pike of the Upjohn Co., Kalamazoo, Mich. Cortisol, were purchased from Sigma
DL-epinephrine, Chemical Co.,
and Ficoll St. Louis, MO.
Medium 199 was purchasedfrom Gibco, Grand Island, N. Y. Hypaque was purchased from Winthrop Laboratories, New York, N. Y. used to prepare HSF is as follows: Bordetellu perin liquid medium by Eli Lilly and Company were collected by centrifugation, dialyzed against water, extracted with acetone at room temperature, and air-dried. The acetone-treated cells (ATC) were extracted overnight with 1 M NaCl in 0.05 M sodium pyrophosphate at pH 8.5 and centrifuged at 29,000 X g for 2 hr. The clear supernatant fluid (BPE) was dialyzed against water and stored at - 15” C or lyophilized. BPE was passed through a hydroxylapatite column equilibrated in 0.01 M phosphate buffer at pH 6.8. The unabsorbed material was called P-l. Fractions were eluted from the column by increasing the molarity of the buffer to 0. I (p-2 fraction, inactive), 0.2 (p-3, most active fraction), and 0.65 (p-4, contains some activity). The pH of the eluting buffers was 6.8; p-3 is not a pure substance; p-3 is designated as HSF in this report.
*The
procedure
tussis cells grown
Vol.
59, No.
1, pp. 79-82
80
Lee
J. ALLERGY CLIN. IMMUNOL. JANUARY 1977
Preparation
I 0
16’
10-s
10-5
CONCENTRATION
I o-9
OF DRUG(M)
FIG. 1. Effects of isoproterenol phocytes.
and PGE, on CAMP in lym-
CAMP (pmolesllO8
HSF
added h)
(4 0 (B) 2.0 (C) 2.0 (D) 2.0
cells)
0
0* 0
after
15
agonist
added
Epinephrine (1O-5 M)
I.351 1.73 1.80 1.25
60
4.80 5.03 4.95 1.20
PGE, (W5 M)
8.85 9.23 9.75 3.70
*Lymphocytes were incubated with (D) or without (A, B, C) HSF at the start of the preincubation. HSF was added to C 15 min before the termination of preincubation. It was added to B at the termination of preincubation. tAverage of duplicate samples. The differences between duplicate samples are less than 10%.
TABLE II. Effect of different concentrations of HSF on response of lymphocytes to epinephrine and PGE, CAMP
HSF* added kg)
0
0 0.2 0.5 2.0
I .20t I .28 I .28 1.20
(pmoles/106 after agonist
lymphocytes) added
Epinephrine (1O-5 M)
6.75 2.93 2.25 1.88
PGE, (1O-5 M)
13.95 5.25 4.43 3.15
*The cells were preincubated with HSF (0 to 2.0 pg) for 60 min. tAverage of duplicate samples. The differences between duplicate
samples are less than 10%.
of lymphocytes
tion. Ethanol containing HC1 (0.02 N) was added to packed cells and centrifuged again. Ethanol containing CAMP was
TABLE I. Effect of duration of preincubation with use on CAMP response of lymphocytes to epinephrine and PGE, Duration of preincubation (min)
and incubation
Blood was drawn from people with no history of asthma or allergic diseases. Heparin (100 U/ml) was used as an anticoagulant. The procedure used for isolating lymphocytes has been described by Boyum.lfi In summary, the heparinized blood was mixed with three volumes of saline solution and layered over a Ficoll-Hypaque solution (d = 1.077 to 1.076) in polycarbonate tubes. The tubes were then centrifuged at 4.50 x g for 40 min. After aspirating the plasma layer, the layer of lymphocyte was removed and washed with medium 199. Lymphocytes (>97YG) were suspended in medium 199 at a concentration of 2 x IO6 cells/ml. Except as otherwise indicated, 1 x 10fi cells/ml were incubated with agonists for 10 min at 37” C in 5% CO2 and air. After incubation the cells were harvested by centrifuga-
removed and dried under nitrogen. The content of CAMP was assayed by a saturation assay method.17
RESULTS The levels of CAMP in lymphocytes, like those in other mammalian tissues, are stimulated by /3-adrenergic agents and PGE,. On the other hand, phenylepinephrine, PGFz, and glucagon have very little effect on the levels of CAMP in lymphocytes (unpublished observations.) PGEl was more effective than epinephrine in elevating the levels of CAMP in lymphocytes (Fig. 1). Preincubation of lymphocytes with HSF does not change the basal level of CAMP but does lead to a loss of responsiveness to both epinephrine and PGEl (Table I). The inhibition was dose-dependent (Table II). Even the lowest dose (0.2 pg/107 cells/ml) inhibited both adrenergic and prostaglandin responses. The largest dose tested (2.0 pg/107 cells/ml) almost abolished the responses. Under conditions where the /3-adrenergic effect was completely blocked by HSF, cortisol restored the response to the agonists (Table III). Propranolol is more specific, for it does not affect the PGE,-responsive elevation of CAMP. The effect of propranolol does not depend on preincubation as does HSF. Finally, cortisol did not overcome the P-blockade produced by propranolol (Table IV). DISCUSSION HSF or B. perrussis vaccination is known to induce homocytotropic antibody production.4s 5 These abnormalities can also be induced by treating the animal with /3-adrenergic blocking agent such as propranolol or butoxamine.4 But even at a sublethal dosage, /3-adrenergic blocking agents did not approach the efficacy of pertussis vaccine as an adjuvant. Epineph-
Histamine-sensitizing
VOLUME59 NUMBER1
III. Effect of cortisol on responses phocytes to agonists and HSF
TABLE
cells) HSF*
(2.0 ualml)
Cortisol (10m4 MI
+ +
+ +
0 1.58-t 2.85 1.55 1.58
CAMP (pmolesll06 after agonists Epinephrine (1O-5 MI 3.45 15.30 1.28 3.23
of lym-
TABLE
IV.
lymphocyte
Effect of propranolol CAMP response CAMP cytes)
added PGE, (1O-5 Ml
Propranolol (1O-5 MI + +
8.70 22.20 2.63 8.85
factor and cortisol
Cortisol (W4 M) -
0 I .50” 3.00 I .70 3.30
+ +
and cortisol
(pmoles/106 after agonist
on
lymphoadded
Epinephrine (1O-5 M) 3.60 23.70 1.90 2.85
PGE, (1O-5 M) 12.30 43.20 13.55 38.85
*The cells were preincubated with or without HSF for 60 min. tAverage of duplicate samples. The differences between duplicate samples are less than 10%.
*Average of duplicate samples. samples are less than 10%.
rine treatment, on the other hand, decreases the homocytotropic antibody leveL4 Inhibition of the adenyl cyclase response in lymphocytes may be related to the lymphocytosis induced by pertussis vaccine. It is noteworthy that epinephrine and cortisol induce a lymphopenia and have the opposite effect on lymphocyte distribution from pertussis. The action of /?-adrenergic agents is generally considered to be mediated through the activation of the adenyl cyclase system. Propranolol specifically inhibits the /3-adrenergic adenyl cyclase without any effect on PGE,. HSF, on the other hand, is not specific for adrenergic receptors since it also blocks PGE, stimulation of adenyl cyclase. The broader specificity of HSF compared to propranolol in blocking stimulation of adenyl cyclase may be important in producing the greater physiologic abnormalities in vivo if one assumes that CAMP is playing a role in modulating anaphylaxis and antibody production. It is interesing to note that cortisol in vitro is able to restore normal responsiveness after preincubation with HSF. In addition to lymphocytes, intracellular CAMP of tissue slices prepared from normal mouse lung is increased synergistically by P-adrenergic agents and cortisol (unpublished data). This reversal of unresponsiveness to agonists by cortisol might contribute to the mechanism of its beneficial effect in asthma and its reversal of pertussis-increased sensitivity to anaphylactic shock in mice. Considering that various physiologic responses are mediated by CAMP, the ability of corticosteroids to modulate the intracellular level of CAMP may be one of the means by which corticosteroids regulate physiologic responses in target tissues. However, corticosteroids undoubtedly have many actions in addition to facilitating agonist stimulation of adenyl cyclase. For example, at a concentration (lO-‘j M) where cortisol alone has no apparent effect on lymphocyte CAMP
levels, cortisol is far more potent than epinephrine or PGEl in inhibiting lymphocyte H3-thymidine uptake following mitogenic stimulation. ‘a In conclusion, the present study has confirmed that some macromolecular compound(s) in B. perhmis block the hormonal stimulation of adenyl cyclase and has provided additional evidence that the blockade can be reversed by cortisol. Therefore it can be assumed that these inhibitory effects may be responsible for the abnormal physiologic function induced by HSF and that some of the effects of cortisol may be due to the synergistic effect of cortisol and hormones on adenyl cyclase. The author wishes to thank discussions and encouragement and for reading the manuscript.
The differences
81
between
duplicate
Dr. Charles E. Reed for valuable during the course of these studies
REFERENCES I,
Munoz, J., and Bergman, R. K.: Histamine-sensitizing factors from microbial agents, with special reference to Bordetclla perrussis,
Bacreriol.
Rev.
32: 103,
1968.
2. Szentivanyi, A.: The beta adrenergic theory of the atropic abnormality in bronchial asthma, J. ALLERGY 42:203, 1968. 3. Stronk, M. G., and Pittman, M.: The influence of pertussis vaccine on histamine sensitivity of rabbits and guinea pigs and on the blood sugar in rabbits and mice, J. Infect. Dis. 96:152. 1955. 4. Reed. C. E., Benner, M., Lackey. S. D., Enta, T.. Makino. S., and Carr, R. M.: On the mechanism of the adjuvant effect ofBordetel/u perfwrsis vaccine, J. ALLERGY CLIN. IMMUNOL. 49:174. 1972. 5. Munoz, J.: Comparison of Borderella perhrssis cells and Freud’s adjuvant with respect to their antibody inducing and anaphylactogenic properties, J. lmmunol. 90: 132. 1963. 6. Szentivanyi, A., Fishel, C. W., and Talmage, D.: Adrenaline mediation of histamine and serotin hyperglycemia in normal mice and the absence of adrenaline-induced hyperglycemia in pertussis-sensitized mice, J. Infect. Dis. 113536, 1963. 7. Lee, T. P., Busse, W. W., and Reed, C. E.: Epidermal adenyl cyclase of human and mouse. A study of the atopical state, J. ALLERGY CLIN. IMM~NOL. 53:283, 1974.
82
J. ALLERGY CLIN. IMMUNOL. JANUARY 1977
Lee
8. Lee, T. P., and Reed, C. E.: Adenyl cyclase system in pertussis vaccinated mice-an animal model for atopic disease, Adv. Cyclic Nucleotide Res. 5:815, 197.5. 9. Lehrer, S. B., Tan, E. M., and Vaughan, J. H.: Extraction and partial purification of the histamine-sensitizing factor of Borderellaperh~sis, J. Immunol. 113:18, 1974. 10. Sate, Y., and Arai, H.: Leucocytosis-promoting factor of Bordetella pertunis. I. Purification and characterization, Infect. Immunology 6:899, 1972. 11. Sato, Y., Arai, H., and Suzuki, K.: Leucocytosis-promoting factor of Bordetella pertussis. II. Biological properties. Infect. Immunology 7:992, 1973. 12. Morse, S. I., and Riester, S. K.: Studies on the leukocytosis and lymphocytosis induced by Bordetella perhwis. II. The effect of pertussis vaccine on the thoracic duct lymph and lymphocytes of mice, J. Exp. Med. 125:619, 1967. 13. Parker, C. W., and Morse, S. I.: The effect of Bordetella pet-funk on lymphocyte cyclic AMP metabolism, J. Exp. Med. 137:1078, 1973. 14. Lehrer, S. B., Vaughan, J. H., and Tan, E. M.: Immunologic
15.
16.
17.
18.
and biochemical properties of the histamine-sensitizing factor from Bordetella pertunis, J. Immunol. 114 (1 Part):34, 1975. Okuyama, S., Aronson, R. B., Chanana, A. D., Chronkie, E. P., Rai, K. R., and Schiffer, L. M.: Attempts at isolation of lymphocytosis reducing factor from supernatant fluids of Bordetella pertussis cultures, Proc. Sot. Exp. Biol. Med. 133:723, 1970. Boyum, A.: Isolation of mononuclear cells and granulocytes from human blood. Isolation of mononuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g, Stand. J. Clin. Lab. Invest. 21:77, 1968. Brown, B. L., Albano, J. D. M., Edins, R. P., Sgherzi, A. M., and Tampion, W.: A simple and sensitive saturation assay method for the measurement of adenosine 3’: 5’-cyclic monophosphate, Biochem. J. 121:561, 1971. Mendelsohn, J., Multer, M. M., and Booner, R. F.: Enhanced effects of prostaglandin El and dibutryl cyclic AMP upon human lymphocytes in the presence of cortisol, J. Clin. Invest. 52:2129, 1973.
Lee, Ph.D. Madison, Wis.
Levels qf cyclic adenosime monophosphate (CAMP) in lymphocytes are regulated by P-adrenergic agonists and PGE,. The effect of these agonists is potentiated by cortisol. Incubation of lymphocytes with histamine-sensitizing factor (HSF) leads to loss of response to epinephrine and PGE,. The response can be partially restored by cortisol. Incubation of lymphocytes with /3-adrenergic antagonists such as propranolol leads to the loss of /3-adrenergic agents but not PGE,. The inhibition by propranolol is not reversed by cortisol. These results suggest that the action of HSF and Bordetella pertussisvaccine is not mediated through the inhibition qf padrenergic receptors alone
Vaccination with Bordetella pet-tusk increases sensitivity to anaphylaxis in several species and also increases antibody production, especially homocytotropic (IgE) antibody. l--j In addition, vaccinated mice develop a diminished hyperglycemic response to epinephrine.6 These abnormalities can also be induced by treating animals with propranolol. For this reason, P-adrenergic blockade has been proposed as the mechanism of vaccine action.2 We (Lee and Reed7) found a decreased responsiveness of the adenyl cyclase system in the skin of vaccinated mice. Similar abnormalities also have been detected in lung and liver.x It is generally believed that a protein, histamine-sensitizing factor (HSF), is responsible for these abnormalities induced by pertussis vaccine.g-ll Pertussis vaccine also produces a striking lymphocytosis due to redistribution of lymphocytes from lymphoid organs into the intravascular po01.‘~ Parker and Morsel3 have obtained a protein fraction, lymphocytosis-promoting factor (LPF), from pertussis organisms that produces lymphocytosis and also inhibits elevation of cyclic adenosine monophosphate (CAMP) in lymphocytes on stimulation by isoproterenol or PGE,. LPF has been found to be closely From the Department of Medicine, University of Wisconsin. Supported by National Institutes of Health Grants 2P15 AI 10404 and 2 ROI AI 08106. Received for publication March 19, 1976. Accepted for publication Aug. 27, 1976. Reprint requests to: Tee-Ping Lee, Ph.D., 504 N. Walnut St., Room 305, Madison, Wis. 53705.
associated with HSF. There is evidence suggesting that HSF and LPF are one and the same entity.“, l4 But others15 have suggested that HSF and LPF are different molecules. The present experiments were undertaken to explore the actions of HSF on human lymphocytes in vitro and to compare the effect of HSF with that of the @-adrenergic blocking drug propranolol. MATERIALS Materials HSF*
AND METHODS
was a gift
of Dr. John
Munoz
of the Rocky
Moun-
tain Laboratory, Hamilton, Mont. Prostaglandin El was generously provided by Dr. John Pike of the Upjohn Co., Kalamazoo, Mich. Cortisol, were purchased from Sigma
DL-epinephrine, Chemical Co.,
and Ficoll St. Louis, MO.
Medium 199 was purchasedfrom Gibco, Grand Island, N. Y. Hypaque was purchased from Winthrop Laboratories, New York, N. Y. used to prepare HSF is as follows: Bordetellu perin liquid medium by Eli Lilly and Company were collected by centrifugation, dialyzed against water, extracted with acetone at room temperature, and air-dried. The acetone-treated cells (ATC) were extracted overnight with 1 M NaCl in 0.05 M sodium pyrophosphate at pH 8.5 and centrifuged at 29,000 X g for 2 hr. The clear supernatant fluid (BPE) was dialyzed against water and stored at - 15” C or lyophilized. BPE was passed through a hydroxylapatite column equilibrated in 0.01 M phosphate buffer at pH 6.8. The unabsorbed material was called P-l. Fractions were eluted from the column by increasing the molarity of the buffer to 0. I (p-2 fraction, inactive), 0.2 (p-3, most active fraction), and 0.65 (p-4, contains some activity). The pH of the eluting buffers was 6.8; p-3 is not a pure substance; p-3 is designated as HSF in this report.
*The
procedure
tussis cells grown
Vol.
59, No.
1, pp. 79-82
80
Lee
J. ALLERGY CLIN. IMMUNOL. JANUARY 1977
Preparation
I 0
16’
10-s
10-5
CONCENTRATION
I o-9
OF DRUG(M)
FIG. 1. Effects of isoproterenol phocytes.
and PGE, on CAMP in lym-
CAMP (pmolesllO8
HSF
added h)
(4 0 (B) 2.0 (C) 2.0 (D) 2.0
cells)
0
0* 0
after
15
agonist
added
Epinephrine (1O-5 M)
I.351 1.73 1.80 1.25
60
4.80 5.03 4.95 1.20
PGE, (W5 M)
8.85 9.23 9.75 3.70
*Lymphocytes were incubated with (D) or without (A, B, C) HSF at the start of the preincubation. HSF was added to C 15 min before the termination of preincubation. It was added to B at the termination of preincubation. tAverage of duplicate samples. The differences between duplicate samples are less than 10%.
TABLE II. Effect of different concentrations of HSF on response of lymphocytes to epinephrine and PGE, CAMP
HSF* added kg)
0
0 0.2 0.5 2.0
I .20t I .28 I .28 1.20
(pmoles/106 after agonist
lymphocytes) added
Epinephrine (1O-5 M)
6.75 2.93 2.25 1.88
PGE, (1O-5 M)
13.95 5.25 4.43 3.15
*The cells were preincubated with HSF (0 to 2.0 pg) for 60 min. tAverage of duplicate samples. The differences between duplicate
samples are less than 10%.
of lymphocytes
tion. Ethanol containing HC1 (0.02 N) was added to packed cells and centrifuged again. Ethanol containing CAMP was
TABLE I. Effect of duration of preincubation with use on CAMP response of lymphocytes to epinephrine and PGE, Duration of preincubation (min)
and incubation
Blood was drawn from people with no history of asthma or allergic diseases. Heparin (100 U/ml) was used as an anticoagulant. The procedure used for isolating lymphocytes has been described by Boyum.lfi In summary, the heparinized blood was mixed with three volumes of saline solution and layered over a Ficoll-Hypaque solution (d = 1.077 to 1.076) in polycarbonate tubes. The tubes were then centrifuged at 4.50 x g for 40 min. After aspirating the plasma layer, the layer of lymphocyte was removed and washed with medium 199. Lymphocytes (>97YG) were suspended in medium 199 at a concentration of 2 x IO6 cells/ml. Except as otherwise indicated, 1 x 10fi cells/ml were incubated with agonists for 10 min at 37” C in 5% CO2 and air. After incubation the cells were harvested by centrifuga-
removed and dried under nitrogen. The content of CAMP was assayed by a saturation assay method.17
RESULTS The levels of CAMP in lymphocytes, like those in other mammalian tissues, are stimulated by /3-adrenergic agents and PGE,. On the other hand, phenylepinephrine, PGFz, and glucagon have very little effect on the levels of CAMP in lymphocytes (unpublished observations.) PGEl was more effective than epinephrine in elevating the levels of CAMP in lymphocytes (Fig. 1). Preincubation of lymphocytes with HSF does not change the basal level of CAMP but does lead to a loss of responsiveness to both epinephrine and PGEl (Table I). The inhibition was dose-dependent (Table II). Even the lowest dose (0.2 pg/107 cells/ml) inhibited both adrenergic and prostaglandin responses. The largest dose tested (2.0 pg/107 cells/ml) almost abolished the responses. Under conditions where the /3-adrenergic effect was completely blocked by HSF, cortisol restored the response to the agonists (Table III). Propranolol is more specific, for it does not affect the PGE,-responsive elevation of CAMP. The effect of propranolol does not depend on preincubation as does HSF. Finally, cortisol did not overcome the P-blockade produced by propranolol (Table IV). DISCUSSION HSF or B. perrussis vaccination is known to induce homocytotropic antibody production.4s 5 These abnormalities can also be induced by treating the animal with /3-adrenergic blocking agent such as propranolol or butoxamine.4 But even at a sublethal dosage, /3-adrenergic blocking agents did not approach the efficacy of pertussis vaccine as an adjuvant. Epineph-
Histamine-sensitizing
VOLUME59 NUMBER1
III. Effect of cortisol on responses phocytes to agonists and HSF
TABLE
cells) HSF*
(2.0 ualml)
Cortisol (10m4 MI
+ +
+ +
0 1.58-t 2.85 1.55 1.58
CAMP (pmolesll06 after agonists Epinephrine (1O-5 MI 3.45 15.30 1.28 3.23
of lym-
TABLE
IV.
lymphocyte
Effect of propranolol CAMP response CAMP cytes)
added PGE, (1O-5 Ml
Propranolol (1O-5 MI + +
8.70 22.20 2.63 8.85
factor and cortisol
Cortisol (W4 M) -
0 I .50” 3.00 I .70 3.30
+ +
and cortisol
(pmoles/106 after agonist
on
lymphoadded
Epinephrine (1O-5 M) 3.60 23.70 1.90 2.85
PGE, (1O-5 M) 12.30 43.20 13.55 38.85
*The cells were preincubated with or without HSF for 60 min. tAverage of duplicate samples. The differences between duplicate samples are less than 10%.
*Average of duplicate samples. samples are less than 10%.
rine treatment, on the other hand, decreases the homocytotropic antibody leveL4 Inhibition of the adenyl cyclase response in lymphocytes may be related to the lymphocytosis induced by pertussis vaccine. It is noteworthy that epinephrine and cortisol induce a lymphopenia and have the opposite effect on lymphocyte distribution from pertussis. The action of /?-adrenergic agents is generally considered to be mediated through the activation of the adenyl cyclase system. Propranolol specifically inhibits the /3-adrenergic adenyl cyclase without any effect on PGE,. HSF, on the other hand, is not specific for adrenergic receptors since it also blocks PGE, stimulation of adenyl cyclase. The broader specificity of HSF compared to propranolol in blocking stimulation of adenyl cyclase may be important in producing the greater physiologic abnormalities in vivo if one assumes that CAMP is playing a role in modulating anaphylaxis and antibody production. It is interesing to note that cortisol in vitro is able to restore normal responsiveness after preincubation with HSF. In addition to lymphocytes, intracellular CAMP of tissue slices prepared from normal mouse lung is increased synergistically by P-adrenergic agents and cortisol (unpublished data). This reversal of unresponsiveness to agonists by cortisol might contribute to the mechanism of its beneficial effect in asthma and its reversal of pertussis-increased sensitivity to anaphylactic shock in mice. Considering that various physiologic responses are mediated by CAMP, the ability of corticosteroids to modulate the intracellular level of CAMP may be one of the means by which corticosteroids regulate physiologic responses in target tissues. However, corticosteroids undoubtedly have many actions in addition to facilitating agonist stimulation of adenyl cyclase. For example, at a concentration (lO-‘j M) where cortisol alone has no apparent effect on lymphocyte CAMP
levels, cortisol is far more potent than epinephrine or PGEl in inhibiting lymphocyte H3-thymidine uptake following mitogenic stimulation. ‘a In conclusion, the present study has confirmed that some macromolecular compound(s) in B. perhmis block the hormonal stimulation of adenyl cyclase and has provided additional evidence that the blockade can be reversed by cortisol. Therefore it can be assumed that these inhibitory effects may be responsible for the abnormal physiologic function induced by HSF and that some of the effects of cortisol may be due to the synergistic effect of cortisol and hormones on adenyl cyclase. The author wishes to thank discussions and encouragement and for reading the manuscript.
The differences
81
between
duplicate
Dr. Charles E. Reed for valuable during the course of these studies
REFERENCES I,
Munoz, J., and Bergman, R. K.: Histamine-sensitizing factors from microbial agents, with special reference to Bordetclla perrussis,
Bacreriol.
Rev.
32: 103,
1968.
2. Szentivanyi, A.: The beta adrenergic theory of the atropic abnormality in bronchial asthma, J. ALLERGY 42:203, 1968. 3. Stronk, M. G., and Pittman, M.: The influence of pertussis vaccine on histamine sensitivity of rabbits and guinea pigs and on the blood sugar in rabbits and mice, J. Infect. Dis. 96:152. 1955. 4. Reed. C. E., Benner, M., Lackey. S. D., Enta, T.. Makino. S., and Carr, R. M.: On the mechanism of the adjuvant effect ofBordetel/u perfwrsis vaccine, J. ALLERGY CLIN. IMMUNOL. 49:174. 1972. 5. Munoz, J.: Comparison of Borderella perhrssis cells and Freud’s adjuvant with respect to their antibody inducing and anaphylactogenic properties, J. lmmunol. 90: 132. 1963. 6. Szentivanyi, A., Fishel, C. W., and Talmage, D.: Adrenaline mediation of histamine and serotin hyperglycemia in normal mice and the absence of adrenaline-induced hyperglycemia in pertussis-sensitized mice, J. Infect. Dis. 113536, 1963. 7. Lee, T. P., Busse, W. W., and Reed, C. E.: Epidermal adenyl cyclase of human and mouse. A study of the atopical state, J. ALLERGY CLIN. IMM~NOL. 53:283, 1974.
82
J. ALLERGY CLIN. IMMUNOL. JANUARY 1977
Lee
8. Lee, T. P., and Reed, C. E.: Adenyl cyclase system in pertussis vaccinated mice-an animal model for atopic disease, Adv. Cyclic Nucleotide Res. 5:815, 197.5. 9. Lehrer, S. B., Tan, E. M., and Vaughan, J. H.: Extraction and partial purification of the histamine-sensitizing factor of Borderellaperh~sis, J. Immunol. 113:18, 1974. 10. Sate, Y., and Arai, H.: Leucocytosis-promoting factor of Bordetella pertunis. I. Purification and characterization, Infect. Immunology 6:899, 1972. 11. Sato, Y., Arai, H., and Suzuki, K.: Leucocytosis-promoting factor of Bordetella pertussis. II. Biological properties. Infect. Immunology 7:992, 1973. 12. Morse, S. I., and Riester, S. K.: Studies on the leukocytosis and lymphocytosis induced by Bordetella perhwis. II. The effect of pertussis vaccine on the thoracic duct lymph and lymphocytes of mice, J. Exp. Med. 125:619, 1967. 13. Parker, C. W., and Morse, S. I.: The effect of Bordetella pet-funk on lymphocyte cyclic AMP metabolism, J. Exp. Med. 137:1078, 1973. 14. Lehrer, S. B., Vaughan, J. H., and Tan, E. M.: Immunologic
15.
16.
17.
18.
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