Clin. exp. Immunol. (1976) 25, 177-179.
BRIEF COMMUNICATION
Pmnal anaphylaxis in the mouse MEDIATING ANTIBODIES AND RHYTHMIC VARIATIONS IN THE RESPONSE P. M ILLER & M. K. CHURCH* Department of Pharmacology, Roussel Laboratories, Covingham, Swindon
(Received 9 January 1976)
SUMMARY
The anaphylactic reaction in the pinna of a mouse actively sensitized to horse serum is mediated by IgGi and IgE antibodies. Anaphylactic sensitivity is subject to diurnal variation, peak sensitivity occurring at 16.00 hours and minimum sensitivity at 10.00 hours. Anaphylactic sensitivity also varied with the season of the year, being lower during June and July than in the rest of the year, when the reaction was fairly constant.
INTRODUCTION The mouse pinna presents a readily accessible area suitable for the study of anaphylactic reactions (Church & Miller, 1975). In this report, data is presented on the characterization of the antibodies responsible for mediation of the reaction in mice actively sensitized to horse serum. It also describes how circadian rhythm and seasonal variation affect the severity ofthe reaction following antigen challenge. MATERIALS AND METHODS Animals. Male CFLP mice and CFHB rats were obtained from Anglia Laboratory Animals, Alconbury, Huntingdon. They were housed in animal rooms maintained at a temperature of 2-210C with artificial lighting supplied on a fixed schedule from 08.00 to 18.00 hours daily. The animals were allowed free access to food and water throughout. Pinnal anaphylaxis. Mice were sensitized to horse serum and challenge was carried out as previously described (Church & Miller, 1975). Estimation ofcirculating antibody level by PCA. Pooled serum was obtained from a group of twenty actively sensitized mice. Dilutions of serum in saline were injected into sites on the shaved back of either mice, two injections of 0 05 ml per mouse, or rats, four injections of 0 1 ml per rat. The animals were challenged 4 or 72 hr later by the intravenous injection of horse serum plus Evans blue dye. The PCA titre was recorded as the reciprocal of the greatest dilution of serum to evoke a definite blue weal when the skin was examined from the reverse side 30 min after antigen challenge. The titre was determined in groups of five animals.
RESULTS Characterization of anaphylactic antibodies The pooled serum taken from actively sensitized mice had a 72-hr titre of 5 in both mice and rats. The 4-hr titre in mice was 40, but in rats only neat serum produced a reaction. On heating the serum at * Present address: Department of Clinical Pharmacology, University of Southampton, Bassett Crescent East, Southampton S09 3TU. Correspondence: Dr P. Miller, Department of Pharmacology, Roussel Laboratories Limited, Kingfisher Drive, Covingham, Swindon, Wiltshire SN3 5BZ.
M
177
178
P. Miller & M. K. Church
560C for 1 hr, a procedure which destroys reaginic antibody (Mota, 1964), both the 72-hr reaction in mice and rats and the 4-hr reaction in rats were abolished. Heating the serum reduced the 4-hr titre in mice to 20. Circadian rhythm in anaphylactic sensitivity Variations in anaphylactic sensitivity over a 30-hr period were followed in an experiment in which mice were tested every 6 hr. The results (Fig. 1) show that there is a marked variation in anaphylactic sensitivity over the period of the experiment. Maximum sensitivity was observed at 16.00 hours and minimum sensitivity at 10.00 hours. 45-
40E
E
g35g30-
25 20 _
04.00 10.00 22.00 16.00 Time (hours) FIG. 1. Variation in anaphylactic sensitivity over a 30-hr period. Size of reaction is plotted against the time of day. Each result is the mean of responses in nine mice. 10.00
16.00
Histamine and 5-hydroxytryptamine (5-HT) have been identified as the pharmacological mediators of pinnal anaphylaxis and direct application ofthese substances to the pinna induces a reaction (Church & Miller, 1975). Reactions induced in the pinna by the direct application of histamine and 5-HT were not subject to circadian variation (data not shown).
Seasonal variation in anaphylactic sensitivity Seasonal variations in anaphylactic sensitivity were investigated by calculating, for each month, the mean value of the control groups in the experiments carried out during that month. Fig. 2 shows the 50t_ 6
45 _ (7)
40F_ E0 a}
35 _
c
30 ,-.c I 99R
I II Feb. March Jan.
April
Moy
July Month
June
II Aug. Sept.
Oct.
Nov.
Dec.
FIG. 2. Seasonal variation in anaphylactic sensitivity. Results are the mean of the control experiments formed in each month. Figures in parentheses show the number of experiments for that month.
per-
Pinnal anaphylaxis
179
results of these calculations. Anaphylactic sensitivity was found to be lower during June and July than in the rest of the year, when the reaction was fairly constant. The size of the reaction obtained during July was significantly (P< 0.05) smaller than during the rest of the year with the exception ofJune and August.
DISCUSSION Antibodies capable of mediating short latency (4 hr) and long latency (72 hr) PCA reactions in mice were found in serum from sensitized mice. Heating the serum at 560C for 1 hr abolished its ability to mediate a 72-hr PCA reaction. The mouse has been shown to produce two anaphylactic antibodies, heat-stable IgG1, mediating a short latency (4 hr) reaction and a heat-labile reaginic antibody, IgE, mediating a longer latency (48-72 hr) reaction (McCamish, 1967). Konig, Okudaira & Ishizaka (1974) have shown that mouse reaginic antibody, but not mouse IgG1, will bind to rat peritoneal mast cells. When mouse antiserum to horse serum was tested for its PCA titre in rats, positive reactions were only obtained with the unheated serum, indicating that only the reaginic antibody was bound to the mast cells of the rat skin. These experiments thus demonstrate the presence of both reaginic and IgG1 antibody in the serum of mice sensitized to horse serum according to the schedule used in this study. Circadian rhythms have been identified for a number of functions in mice (Halberg et al., 1958), as in other species. Diurnal variations have been reported in humans for histamine release from leucocytes (Leffert, 1974) and intradermal provocation tests (Reinberg et al., 1969). Our results show that a 24-hr rhythm also exists for anaphylactic sensitivity in mice, peak sensitivity being found at 16.00 hours with minimum sensitivity at 10.00 hours. Halberg et al. (1958) investigated the rhythm in a number of parameters in mice, among them serum corticosterone levels. With mice maintained on a fixed lighting schedule with light from 06.00 to 18.00 hours, peak serum levels of corticosterone were observed at 20.00 hours with minimum levels at 04.00-08.00 hours. Following the oral administration of corticosterone to sensitized mice, 6-12 hr are necessary between dosing and antigen challenge for the corticosterone to exert its maximal inhibitory effect upon the anaphylactic reaction (P. Miller, unpublished results). It may well be, therefore, that the observed circadian rhythm in anaphylactic sensitivity reflects the effect of the known circadian rhythm in serum corticosterone levels. Seasonal variations in the mortality rate of sensitized rats following antigen challenge have been reported by Ankier et al. (1965). They observed a marked decrease in mortality over the July-August period. We have now shown that a seasonal variation also exists for pinnal anaphylaxis in the mouse, though the variation was not as great as that observed by Ankier et al. This difference may be due to the different lighting regimens employed in the two animal houses. The rats of Ankier et al. were maintained at a constant temperature throughout the year but were exposed to natural daylight (S. I. Ankier, personal communication). Thus although they were not exposed to seasonal changes of temperature, they were aware of the seasonal changes in daylight hours. The mice used in our studies were kept at a constant temperature and were dependent upon artificial lighting which was provided on a fixed schedule from 08.00 to 18.00 hours daily. The slight change in anaphylactic sensitivity observed in these mice may be due to some inherent rhythm rather than seasonal changes in temperature or daylight hours. REFERENCES ANKIER, S.I., DAWSON, W., KARADY, S. & WEST, G.B. (1965) LEFFERT, F. (1974) The in vivo effect of theophylline on histamine release from human leucocytes. Clin. Allergy, 4, Seasonal variation in the resistance of rats. J. Pharm. Pharmac. 17, 187. 301. CHURCH, M.K. & MILLER, P. (1975) Simple models of MCCAMISH, J. (1967) A heat-labile skin sensitising activity of mouse serum. Nature (Lond.), 214, 1228. anaphylaxis and of histamine and 5-hydroxytryptamine induced inflammation using the mouse pinna. Brit.J. MOTA, I. (1964) The mechanism of anaphylaxis. I. ProPharmac. 55, 315P. duction and biological properties of 'mast cell sensitising' HALBERG, F., BARNUM, C.P., SILBER, R.H. & BITTNER, J.J. antibody. Immunology, 7, 681. (1958) 24 hour rhythms at several levels of integration in REINBERG, A., ZAGULA-MALLY, Z., GHATA, J. & HALBERG, mice on different lighting regimens. Proc. Soc. exp. F. (1969) Circadian reactivity rhythm of human skin to Biol. (N.Y.), 97, 897. house dust, penicillin, and histamine. 7. Allergy, 44, KONIG, W., OKUDAIRA, H. & ISHIZAKA, K. (1974) Specific 292. binding of mouse IgE with rat mast cells. A. Immunol. 112, 1652.
BRIEF COMMUNICATION
Pmnal anaphylaxis in the mouse MEDIATING ANTIBODIES AND RHYTHMIC VARIATIONS IN THE RESPONSE P. M ILLER & M. K. CHURCH* Department of Pharmacology, Roussel Laboratories, Covingham, Swindon
(Received 9 January 1976)
SUMMARY
The anaphylactic reaction in the pinna of a mouse actively sensitized to horse serum is mediated by IgGi and IgE antibodies. Anaphylactic sensitivity is subject to diurnal variation, peak sensitivity occurring at 16.00 hours and minimum sensitivity at 10.00 hours. Anaphylactic sensitivity also varied with the season of the year, being lower during June and July than in the rest of the year, when the reaction was fairly constant.
INTRODUCTION The mouse pinna presents a readily accessible area suitable for the study of anaphylactic reactions (Church & Miller, 1975). In this report, data is presented on the characterization of the antibodies responsible for mediation of the reaction in mice actively sensitized to horse serum. It also describes how circadian rhythm and seasonal variation affect the severity ofthe reaction following antigen challenge. MATERIALS AND METHODS Animals. Male CFLP mice and CFHB rats were obtained from Anglia Laboratory Animals, Alconbury, Huntingdon. They were housed in animal rooms maintained at a temperature of 2-210C with artificial lighting supplied on a fixed schedule from 08.00 to 18.00 hours daily. The animals were allowed free access to food and water throughout. Pinnal anaphylaxis. Mice were sensitized to horse serum and challenge was carried out as previously described (Church & Miller, 1975). Estimation ofcirculating antibody level by PCA. Pooled serum was obtained from a group of twenty actively sensitized mice. Dilutions of serum in saline were injected into sites on the shaved back of either mice, two injections of 0 05 ml per mouse, or rats, four injections of 0 1 ml per rat. The animals were challenged 4 or 72 hr later by the intravenous injection of horse serum plus Evans blue dye. The PCA titre was recorded as the reciprocal of the greatest dilution of serum to evoke a definite blue weal when the skin was examined from the reverse side 30 min after antigen challenge. The titre was determined in groups of five animals.
RESULTS Characterization of anaphylactic antibodies The pooled serum taken from actively sensitized mice had a 72-hr titre of 5 in both mice and rats. The 4-hr titre in mice was 40, but in rats only neat serum produced a reaction. On heating the serum at * Present address: Department of Clinical Pharmacology, University of Southampton, Bassett Crescent East, Southampton S09 3TU. Correspondence: Dr P. Miller, Department of Pharmacology, Roussel Laboratories Limited, Kingfisher Drive, Covingham, Swindon, Wiltshire SN3 5BZ.
M
177
178
P. Miller & M. K. Church
560C for 1 hr, a procedure which destroys reaginic antibody (Mota, 1964), both the 72-hr reaction in mice and rats and the 4-hr reaction in rats were abolished. Heating the serum reduced the 4-hr titre in mice to 20. Circadian rhythm in anaphylactic sensitivity Variations in anaphylactic sensitivity over a 30-hr period were followed in an experiment in which mice were tested every 6 hr. The results (Fig. 1) show that there is a marked variation in anaphylactic sensitivity over the period of the experiment. Maximum sensitivity was observed at 16.00 hours and minimum sensitivity at 10.00 hours. 45-
40E
E
g35g30-
25 20 _
04.00 10.00 22.00 16.00 Time (hours) FIG. 1. Variation in anaphylactic sensitivity over a 30-hr period. Size of reaction is plotted against the time of day. Each result is the mean of responses in nine mice. 10.00
16.00
Histamine and 5-hydroxytryptamine (5-HT) have been identified as the pharmacological mediators of pinnal anaphylaxis and direct application ofthese substances to the pinna induces a reaction (Church & Miller, 1975). Reactions induced in the pinna by the direct application of histamine and 5-HT were not subject to circadian variation (data not shown).
Seasonal variation in anaphylactic sensitivity Seasonal variations in anaphylactic sensitivity were investigated by calculating, for each month, the mean value of the control groups in the experiments carried out during that month. Fig. 2 shows the 50t_ 6
45 _ (7)
40F_ E0 a}
35 _
c
30 ,-.c I 99R
I II Feb. March Jan.
April
Moy
July Month
June
II Aug. Sept.
Oct.
Nov.
Dec.
FIG. 2. Seasonal variation in anaphylactic sensitivity. Results are the mean of the control experiments formed in each month. Figures in parentheses show the number of experiments for that month.
per-
Pinnal anaphylaxis
179
results of these calculations. Anaphylactic sensitivity was found to be lower during June and July than in the rest of the year, when the reaction was fairly constant. The size of the reaction obtained during July was significantly (P< 0.05) smaller than during the rest of the year with the exception ofJune and August.
DISCUSSION Antibodies capable of mediating short latency (4 hr) and long latency (72 hr) PCA reactions in mice were found in serum from sensitized mice. Heating the serum at 560C for 1 hr abolished its ability to mediate a 72-hr PCA reaction. The mouse has been shown to produce two anaphylactic antibodies, heat-stable IgG1, mediating a short latency (4 hr) reaction and a heat-labile reaginic antibody, IgE, mediating a longer latency (48-72 hr) reaction (McCamish, 1967). Konig, Okudaira & Ishizaka (1974) have shown that mouse reaginic antibody, but not mouse IgG1, will bind to rat peritoneal mast cells. When mouse antiserum to horse serum was tested for its PCA titre in rats, positive reactions were only obtained with the unheated serum, indicating that only the reaginic antibody was bound to the mast cells of the rat skin. These experiments thus demonstrate the presence of both reaginic and IgG1 antibody in the serum of mice sensitized to horse serum according to the schedule used in this study. Circadian rhythms have been identified for a number of functions in mice (Halberg et al., 1958), as in other species. Diurnal variations have been reported in humans for histamine release from leucocytes (Leffert, 1974) and intradermal provocation tests (Reinberg et al., 1969). Our results show that a 24-hr rhythm also exists for anaphylactic sensitivity in mice, peak sensitivity being found at 16.00 hours with minimum sensitivity at 10.00 hours. Halberg et al. (1958) investigated the rhythm in a number of parameters in mice, among them serum corticosterone levels. With mice maintained on a fixed lighting schedule with light from 06.00 to 18.00 hours, peak serum levels of corticosterone were observed at 20.00 hours with minimum levels at 04.00-08.00 hours. Following the oral administration of corticosterone to sensitized mice, 6-12 hr are necessary between dosing and antigen challenge for the corticosterone to exert its maximal inhibitory effect upon the anaphylactic reaction (P. Miller, unpublished results). It may well be, therefore, that the observed circadian rhythm in anaphylactic sensitivity reflects the effect of the known circadian rhythm in serum corticosterone levels. Seasonal variations in the mortality rate of sensitized rats following antigen challenge have been reported by Ankier et al. (1965). They observed a marked decrease in mortality over the July-August period. We have now shown that a seasonal variation also exists for pinnal anaphylaxis in the mouse, though the variation was not as great as that observed by Ankier et al. This difference may be due to the different lighting regimens employed in the two animal houses. The rats of Ankier et al. were maintained at a constant temperature throughout the year but were exposed to natural daylight (S. I. Ankier, personal communication). Thus although they were not exposed to seasonal changes of temperature, they were aware of the seasonal changes in daylight hours. The mice used in our studies were kept at a constant temperature and were dependent upon artificial lighting which was provided on a fixed schedule from 08.00 to 18.00 hours daily. The slight change in anaphylactic sensitivity observed in these mice may be due to some inherent rhythm rather than seasonal changes in temperature or daylight hours. REFERENCES ANKIER, S.I., DAWSON, W., KARADY, S. & WEST, G.B. (1965) LEFFERT, F. (1974) The in vivo effect of theophylline on histamine release from human leucocytes. Clin. Allergy, 4, Seasonal variation in the resistance of rats. J. Pharm. Pharmac. 17, 187. 301. CHURCH, M.K. & MILLER, P. (1975) Simple models of MCCAMISH, J. (1967) A heat-labile skin sensitising activity of mouse serum. Nature (Lond.), 214, 1228. anaphylaxis and of histamine and 5-hydroxytryptamine induced inflammation using the mouse pinna. Brit.J. MOTA, I. (1964) The mechanism of anaphylaxis. I. ProPharmac. 55, 315P. duction and biological properties of 'mast cell sensitising' HALBERG, F., BARNUM, C.P., SILBER, R.H. & BITTNER, J.J. antibody. Immunology, 7, 681. (1958) 24 hour rhythms at several levels of integration in REINBERG, A., ZAGULA-MALLY, Z., GHATA, J. & HALBERG, mice on different lighting regimens. Proc. Soc. exp. F. (1969) Circadian reactivity rhythm of human skin to Biol. (N.Y.), 97, 897. house dust, penicillin, and histamine. 7. Allergy, 44, KONIG, W., OKUDAIRA, H. & ISHIZAKA, K. (1974) Specific 292. binding of mouse IgE with rat mast cells. A. Immunol. 112, 1652.
