European Journal of Pharmacology, 37 (1976) 397--399 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
397
Short communication INHIBITION BY 5-HYDROXYTRYPTAMINE OF ANAPHYLACTIC HISTAMINE RELEASE FROM BOVINE GRANULOCYTES MARTIN C. HOLROYDE and PETER EYRE
Pharmacology Laboratory, Department of Biomedical Sciences, University of Guelph, Guelph, Ontario, Canada Received 25 March 1976, accepted 21 April 1976
M.C. HOLROYDE and P. EYRE, Inhibition by 5-hydroxytryptamine of anaphylactic histamine release from bovine granulocytes, European J. Pharmacol. 37 (1976) 397--399. A suspension of granulocytes from calves sensitised to horse plasma released histamine when exposed to the antigen. Histamine release was strongly inhibited by 5-hydroxytryptamine in a dose-related fashion. 5-HT is known to be released from bovine lungs in vitro, arid may regulate the release of histamine in vivo during anaphylaxis. Anaphylaxis
Granulocyte
Histamine
5-Hydroxytryptamine
1. Introduction The release of histamine from tissue mast cells during anaphylaxis has been described in all species so far studied. Histamine has also been shown to be released from basophil leucocytes of man (Lichtenstein and Osler, 1964), guinea pig (Greaves and Burdis, 1968) and rabbit (Greaves and Mongar, 1968). Several pharmacological agents have been shown to modulate the release process in either one or both cell types, including catecholamines, methylxanthines, E prostaglandins, carbamylcholine and histamine itself {Bourne et al., 1974; Ishizaka et al., 1971; Kaliner et al., 1972). All these agents appear to operate through the cyclic nucleotide system {Bourne et al., 1974). We have recently shown that granulocytes from calves sensitised to horse plasma release histamine when exposed to the antigen (Holroyde and Eyre, 1975), a process which is modulated by catecholamines in an unusual manner: fl-adrenoceptor stimulation results in potentiation, rather than inhibition, of histamine release, whereas a-adrenoceptor stimula-
tion results in inhibition (Holroyde and Eyre, 1976). We now present evidence that anaphylactic histamine release from bovine granulocytes is strongly inhibited by 5-hydroxytryptamine (5-HT), a substance known to be released from sensitised bovine lung upon exposure to antigen (Eyre, 1972).
2. Materials and methods As previously described (Holroyde and Eyre, 1975), a pure granulocyte fraction was isolated from the venous blood of 4 calves previously sensitised to horse plasma. Aliquots of this fraction (18 X 106 leukocytes) were incubated at 37°C for 30 min in a tris buffer together with sufficient horse plasma to release 40--60% of the available histamine. 5-HT (10-s--10 -3 M) was added to the incubate 2 min before the antigen. After incubation, the released histamine was extracted and analysed fluorometrically (Holroyde and Eyre, 1976). High concentrations of 5-HT produced a yellow discolouration of the final extract, and a slight increase in background
398
M.C. HOLROYDE, P. EYRE
fluorescence• Suitable controls allowed corrections to be made for this fluorescence where necessary.
3. Results and discussion Histamine release from the granulocytes was significantly inhibited by 5-HT in a doserelated manner over the whole concentration range tested (fig. 1). 10 -3 M 5-HT completely abolished histamine release in each calf. 5-HT is present in bovine tissues and is related both in vitro following exposure to specific antigen (Eyre, 1972), and in vivo following treatment with c o m p o u n d 48/80 (Lewis and Eyre, 1972). Its presence in bovine granulocytes has not been demonstrated, partly due to the lack of an analytical technique of sufficient sensitivity. Hence it is not known whether bovine granulocytes release 5-HT in addition to histamine when exposed to specif. ic antigen. The present results suggest that such a situation could possibly provide the basis for a feedback regulation of the histamine release process in bovine granulocytes. The resting plasma concentration of 5-HT in cattle is in the region of 10 -7 M (Lewis and Eyre, 1972), which is approximately the same
o
100,
'
as in dog and human (Garattini and Valzelli, 1965). The concentration following anaphylaxis is unknown, but presumably could approximate the normal serum concentration, due to release from platelets. In many species, including human, this concentration approaches 10 -s M (Garattini and Valzelli, 1965). Under these conditions, histamine release from bovine granulocytes would be inhibited by 50%. Indeed, the local concentration of 5-HT at the site of release could conceivably be even higher. We believe this is the first report of any response to 5-HT in an immunological system such as this. It is particularly important in view of the proven release of 5-HT from sensitised bovine lung, and its probable contribution to the development of the varied symptoms of systemic anaphylaxis in this species (Eyre et al., 1973). It is somewhat surprising that no similar study has been reported in the rat or mouse: species in which the involvement of 5-HT in inflammation and anaphylaxis is well established (Spector and Willoughby, 1964; Church et al., 1972; Stotland and Share, 1974). As 5-HT is reported to increase cyclic AMP levels in rat mast cells (Gripenberg et al., 1974}, and histamine release from many tissues is inhibited by cyclic AMP (Bourne et al., 1974), it is quite possible that 5-HT may modulate histamine release from rat and mouse tissues.
80,
Acknowledgements -~ z
60
M.C. Holroyde is the holder of an Ontario Graduate Scholarship. This investigation was supported by the Ontario Ministry of Agriculture and Food and by Grant No. A5937 of the National Research Council of Canada.
Lo 20
o-
,;-8
,0-,
(P)l
,0-5
5-HT
,;-,
,0:3
Fig. 1. Inhibition by 5-hydroxytryptamine of anaphylactic release of histamine from bovine granulocytes. Results are mean ± S.E.M., n = 4. All points are significantly different from zero (p < 0.05).
References Bourne, H.R., L.M. Lichtenstein, K.L. Melmon, C.S. Henney, Y. Weinstein and G.M. Shearer, 1974, Modulation of inflammation and immunity by cyclic AMP, Science 184, 19. Church, M.K., H.O.J. Collier and G.W.L. James,
INHIBITION OF HISTAMINE RELEASE 1972, The inhibition by dexamethasone and diso° dium cromoglycate of anaphylactic bronchoconstriction in the rat, Brit. J. Pharmacol. 46, 56. Eyre, P., 1972, Release of 5-hydroxytryptamine from sensitized calf lung by antigen and by compound 48/80, Arch. Intern. Pharmacodyn. Therap. 199, 245. Eyre, P., A.J. Lewis and P.W. Wells, 1973, Acute systemic anaphylaxis in the calf, Brit. J. Pharmacol. 47,504. Garattini, S. and L. Valzelli, 1965, Serotonin (Elsevier, Amsterdam). Greaves, M.W. and B.D. Burdis, 1968, Anaphylactic histamine release from guinea pig basophil leucocytes, Intern. Arch. Allergy Appl. Immunol. 34, 313. Greaves, M.W. and J.L. Mongar, 1968, The histamine content of rabbit leucocytes and its release during in vitro anaphylaxis, Immunology 15, 733. Gripenberg, J., M. Harkonen and S.-E. Jansson, 1974, Stimulation of adenosine 3,5,-monophosphate formation in mast cells by 5-hydroxytryptamine and guanethidine, Acta Physiol. Scand. 9 0 , 6 4 8 . Holroyde, M.C. and P. Eyre, 1975, Immunologic release of histamine from bovine polymorphonuclear l e u k o c y t e s , Amer. J. Vet. Res. 36, 1801. Holroyde, M.C. and P. Eyre, 1976, Immunological re-
399 lease of histamine from bovine leukocytes: unusual adrenergic modulation, Immunology (in press). Ishizaka, T., K. Ishizaka, R.P. Orange and K.F. Austen, 1971, Pharmacologic inhibition of the antigen-induced release of histamine and slow-reacting substance of anaphylaxis (SRS-A) from monkey lung tissues mediated by human IgE, J. Immunol. 106, 1267. Kaliner, M., R.P. Orange and K.F. Austen, 1972, Immunological release of histamine and slow reacting substance of anaphylaxis from human lung. IV. Enhancement by cholinergic and ~-adrenergic stimulation, J. Exptl. Med. 136,556. Lichtenstein, L.M. and A.G. Osler, 1964, Studies on the mechanisms of hypersensitivity phenomena. IX. Histamine release from human leukocytes by ragweed pollen antigen, J. Exptl. Med. 120, 507. Lewis, A.J. and P. Eyre, 1972, Some cardiorespiratory effects of histamine, 5-hydroxytryptamine, and compound 48/80 in the calf, Can. J. Physiol. Pharmacol. 50, 545. Spector, W.G. and D.A. Willoughby, 1964, Vasoactive amines in acute inflammation, Ann. N.Y. Acad. Sci. 116,839. Stotland, L.M. and N.N. Share, 1974, Pharmacological studies on active bronchial anaphylaxis in the rat, Can. J. Physiol. Pharmacol. 52, 1119.
397
Short communication INHIBITION BY 5-HYDROXYTRYPTAMINE OF ANAPHYLACTIC HISTAMINE RELEASE FROM BOVINE GRANULOCYTES MARTIN C. HOLROYDE and PETER EYRE
Pharmacology Laboratory, Department of Biomedical Sciences, University of Guelph, Guelph, Ontario, Canada Received 25 March 1976, accepted 21 April 1976
M.C. HOLROYDE and P. EYRE, Inhibition by 5-hydroxytryptamine of anaphylactic histamine release from bovine granulocytes, European J. Pharmacol. 37 (1976) 397--399. A suspension of granulocytes from calves sensitised to horse plasma released histamine when exposed to the antigen. Histamine release was strongly inhibited by 5-hydroxytryptamine in a dose-related fashion. 5-HT is known to be released from bovine lungs in vitro, arid may regulate the release of histamine in vivo during anaphylaxis. Anaphylaxis
Granulocyte
Histamine
5-Hydroxytryptamine
1. Introduction The release of histamine from tissue mast cells during anaphylaxis has been described in all species so far studied. Histamine has also been shown to be released from basophil leucocytes of man (Lichtenstein and Osler, 1964), guinea pig (Greaves and Burdis, 1968) and rabbit (Greaves and Mongar, 1968). Several pharmacological agents have been shown to modulate the release process in either one or both cell types, including catecholamines, methylxanthines, E prostaglandins, carbamylcholine and histamine itself {Bourne et al., 1974; Ishizaka et al., 1971; Kaliner et al., 1972). All these agents appear to operate through the cyclic nucleotide system {Bourne et al., 1974). We have recently shown that granulocytes from calves sensitised to horse plasma release histamine when exposed to the antigen (Holroyde and Eyre, 1975), a process which is modulated by catecholamines in an unusual manner: fl-adrenoceptor stimulation results in potentiation, rather than inhibition, of histamine release, whereas a-adrenoceptor stimula-
tion results in inhibition (Holroyde and Eyre, 1976). We now present evidence that anaphylactic histamine release from bovine granulocytes is strongly inhibited by 5-hydroxytryptamine (5-HT), a substance known to be released from sensitised bovine lung upon exposure to antigen (Eyre, 1972).
2. Materials and methods As previously described (Holroyde and Eyre, 1975), a pure granulocyte fraction was isolated from the venous blood of 4 calves previously sensitised to horse plasma. Aliquots of this fraction (18 X 106 leukocytes) were incubated at 37°C for 30 min in a tris buffer together with sufficient horse plasma to release 40--60% of the available histamine. 5-HT (10-s--10 -3 M) was added to the incubate 2 min before the antigen. After incubation, the released histamine was extracted and analysed fluorometrically (Holroyde and Eyre, 1976). High concentrations of 5-HT produced a yellow discolouration of the final extract, and a slight increase in background
398
M.C. HOLROYDE, P. EYRE
fluorescence• Suitable controls allowed corrections to be made for this fluorescence where necessary.
3. Results and discussion Histamine release from the granulocytes was significantly inhibited by 5-HT in a doserelated manner over the whole concentration range tested (fig. 1). 10 -3 M 5-HT completely abolished histamine release in each calf. 5-HT is present in bovine tissues and is related both in vitro following exposure to specific antigen (Eyre, 1972), and in vivo following treatment with c o m p o u n d 48/80 (Lewis and Eyre, 1972). Its presence in bovine granulocytes has not been demonstrated, partly due to the lack of an analytical technique of sufficient sensitivity. Hence it is not known whether bovine granulocytes release 5-HT in addition to histamine when exposed to specif. ic antigen. The present results suggest that such a situation could possibly provide the basis for a feedback regulation of the histamine release process in bovine granulocytes. The resting plasma concentration of 5-HT in cattle is in the region of 10 -7 M (Lewis and Eyre, 1972), which is approximately the same
o
100,
'
as in dog and human (Garattini and Valzelli, 1965). The concentration following anaphylaxis is unknown, but presumably could approximate the normal serum concentration, due to release from platelets. In many species, including human, this concentration approaches 10 -s M (Garattini and Valzelli, 1965). Under these conditions, histamine release from bovine granulocytes would be inhibited by 50%. Indeed, the local concentration of 5-HT at the site of release could conceivably be even higher. We believe this is the first report of any response to 5-HT in an immunological system such as this. It is particularly important in view of the proven release of 5-HT from sensitised bovine lung, and its probable contribution to the development of the varied symptoms of systemic anaphylaxis in this species (Eyre et al., 1973). It is somewhat surprising that no similar study has been reported in the rat or mouse: species in which the involvement of 5-HT in inflammation and anaphylaxis is well established (Spector and Willoughby, 1964; Church et al., 1972; Stotland and Share, 1974). As 5-HT is reported to increase cyclic AMP levels in rat mast cells (Gripenberg et al., 1974}, and histamine release from many tissues is inhibited by cyclic AMP (Bourne et al., 1974), it is quite possible that 5-HT may modulate histamine release from rat and mouse tissues.
80,
Acknowledgements -~ z
60
M.C. Holroyde is the holder of an Ontario Graduate Scholarship. This investigation was supported by the Ontario Ministry of Agriculture and Food and by Grant No. A5937 of the National Research Council of Canada.
Lo 20
o-
,;-8
,0-,
(P)l
,0-5
5-HT
,;-,
,0:3
Fig. 1. Inhibition by 5-hydroxytryptamine of anaphylactic release of histamine from bovine granulocytes. Results are mean ± S.E.M., n = 4. All points are significantly different from zero (p < 0.05).
References Bourne, H.R., L.M. Lichtenstein, K.L. Melmon, C.S. Henney, Y. Weinstein and G.M. Shearer, 1974, Modulation of inflammation and immunity by cyclic AMP, Science 184, 19. Church, M.K., H.O.J. Collier and G.W.L. James,
INHIBITION OF HISTAMINE RELEASE 1972, The inhibition by dexamethasone and diso° dium cromoglycate of anaphylactic bronchoconstriction in the rat, Brit. J. Pharmacol. 46, 56. Eyre, P., 1972, Release of 5-hydroxytryptamine from sensitized calf lung by antigen and by compound 48/80, Arch. Intern. Pharmacodyn. Therap. 199, 245. Eyre, P., A.J. Lewis and P.W. Wells, 1973, Acute systemic anaphylaxis in the calf, Brit. J. Pharmacol. 47,504. Garattini, S. and L. Valzelli, 1965, Serotonin (Elsevier, Amsterdam). Greaves, M.W. and B.D. Burdis, 1968, Anaphylactic histamine release from guinea pig basophil leucocytes, Intern. Arch. Allergy Appl. Immunol. 34, 313. Greaves, M.W. and J.L. Mongar, 1968, The histamine content of rabbit leucocytes and its release during in vitro anaphylaxis, Immunology 15, 733. Gripenberg, J., M. Harkonen and S.-E. Jansson, 1974, Stimulation of adenosine 3,5,-monophosphate formation in mast cells by 5-hydroxytryptamine and guanethidine, Acta Physiol. Scand. 9 0 , 6 4 8 . Holroyde, M.C. and P. Eyre, 1975, Immunologic release of histamine from bovine polymorphonuclear l e u k o c y t e s , Amer. J. Vet. Res. 36, 1801. Holroyde, M.C. and P. Eyre, 1976, Immunological re-
399 lease of histamine from bovine leukocytes: unusual adrenergic modulation, Immunology (in press). Ishizaka, T., K. Ishizaka, R.P. Orange and K.F. Austen, 1971, Pharmacologic inhibition of the antigen-induced release of histamine and slow-reacting substance of anaphylaxis (SRS-A) from monkey lung tissues mediated by human IgE, J. Immunol. 106, 1267. Kaliner, M., R.P. Orange and K.F. Austen, 1972, Immunological release of histamine and slow reacting substance of anaphylaxis from human lung. IV. Enhancement by cholinergic and ~-adrenergic stimulation, J. Exptl. Med. 136,556. Lichtenstein, L.M. and A.G. Osler, 1964, Studies on the mechanisms of hypersensitivity phenomena. IX. Histamine release from human leukocytes by ragweed pollen antigen, J. Exptl. Med. 120, 507. Lewis, A.J. and P. Eyre, 1972, Some cardiorespiratory effects of histamine, 5-hydroxytryptamine, and compound 48/80 in the calf, Can. J. Physiol. Pharmacol. 50, 545. Spector, W.G. and D.A. Willoughby, 1964, Vasoactive amines in acute inflammation, Ann. N.Y. Acad. Sci. 116,839. Stotland, L.M. and N.N. Share, 1974, Pharmacological studies on active bronchial anaphylaxis in the rat, Can. J. Physiol. Pharmacol. 52, 1119.
