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Potentiation of histamine release by sodium cromoglycate
Life Sciences, Vol . 23, pp . 1899-1904 Printed in the U.S .A .
Pergamon Press
POTENTIATION OF HISTAMINE RELEASE BY SODIUM CROMOGLYCATE Martin K. Church and Carolyn F. Gradidge Clinical Pharmacology, Faculty of Medicine, Universit y of Southampton, Southampton General Hospital, Tremona Road, Southampton S09 4XY, U. K. (Received in final form September 12, 1978) SUMMARY Human lung slices passively sensitized with allergic serum released histamine when incubated with specific antigen and anti-IgE but anti-IgG had no effect . Sodium cromoglycate (SCG) inhibited antigen induced histamine release but the doseresponse curve was bell-shaped. Inhibition of anti-IgE induced release was linearly related to dose, whereas that induced by anti-IgG was potentiated by increasing doses of SCG. After sensitization with allergic serum in which IgE had been inactivated by heating, specific antigen released little or no histamine but this was potentiated by SCG. It is concluded that SCG inhibits IgE mediated but potentiates IgG mediated allergic reactions thus explaining its characteristic doseresponse curve in vitro. Sodium cromoglycate (SCG) has been shown to relieve human bronchial asthma but it is effective only in 30-70°x, of patients(1). Also, it has been reported that SCG is ineffective in patients with low levels of IgE in whom asthma ie thought to be mediated by IgG(2) . Although SCG inhibits IgE mediated passive cutaneous anaphylaxis in the rat in a linear dose-related manner(3), "bell-shaped" dose-response curves are seen in many in vitro experiments which include histamine release from human lung(4). The present study examines the reasons for this "bell-shaped" dose-response relationship . METHODS Human lung obtained from lobectomy specimens was chopped finely with scissors, divided into 200mg replicates and sensitized with 0. 2ml serum, from an allergic donor, in 20m1 Krebs solution for 18 hours at room temperature followed by 1 hour at 37oC . Samples were then washed, resuspended in oxygenated Krebs solution, brought to 37oC and challenged with antigen in a final volune of 2m1. After 15 minutes incubation, the supernatant was removed and the tissue frozen then thawed to release the remaining histamine. Histamine in the two solutions was assayed epectrofluorimetrically(5) and that released by antigen expressed as a percentage of the original histamine content of the tissue sample . Samples incubated in the absence of antigen were used to correct for spontaneous 0300-9653/78/1106-1899$02 .00/0 Copyright (c) 1978 Pergamon Press
1900
Cromoglycate and Human Lung
Vol . 23, No . 19, 1978
histamine release. Antigens used were timothy pollen extract (donated by Beechams Ltd. ), anti-IgE (goat anti-human IgE, Miles Yeda) and anti-IgG (goat anti-human IgG, Miles Yeda). Each antiserum was tested for specificity using micro-precipitin tests on Ouchterlony plates, The antisera, tested against IgA, IgE, IgG, IgM and free light chains, gave positive reactions only with immunoglobulin of their respective homologous classes . In the case of anti-IgG serum, the addition of human IgM to lmg/ml had no effect on the equivalence point for precipitation with IgG (0 . 22mg/ml antiserum) . Our stocks of IgE did not permit this precipitin-inhibition test tobe carried out with anti-IgE serum, SCG (donated by Filons Ltd. ) dissolved in Krebs solution, was added 30 seconds before antigen, Each estimate of histamine release is the mean of those obtained from quadruplicate tissue samples. RESULTS In five experiments, SCG inhibited histamine release induced by timothy pollen extract (100~,g/ml) in a bell-shaped dose-related manner with a peak inhibitory activity at 25~g/ml (Fig, la). By contrast, release induced by anti-IgE (1 :1000 dilution) was inhibited in a linear dose-related way (Fig . lb). Therefore, the possibility that the inflexion of the timothy pollen curve with higher doses of SCG was initiated by an antibody other than IgE was inve stigated. In two pilot experiments, lung challenged with various concentrations of anti-IgG released little histamine . When incubated with SCG (50~,g/ml), histamine release was significantly increased at all concentrations of anti IgG (Table 1) . Similarly, anti-IgG (1 :100 dilution) released little histamine in four further experiments. The addition of SCG in increasing doses potentiated histamine release by this concentration of anti-IgG in a linear dose-related manner, theroleaee at 100~.g/ml of SCG being comparable with that induced by anti-IgE in the absence of drug (Fig . 1C). As expected, the effect of SCG on histamine released by a mixture of anti-IgE and antiIgG showed features characteristic of both antibodies, a bell-shaped doseresponse relationship being obtained (Fig, ld), To demonstrate that the results observed with anti-IgG were due neither to the aggregation of non-mast cell bound IgG remaining within the chopped tissue nor a non-specific action of the commercial preparation of antigen, five additional experiments were performed. In these, chopped lung was sensitized with allergic serum as before, allergic serum heated to 56°C for 1 hour to destroy its IgE(6), or not sensitized . Challenge was with timothy pollen extract (100Wg/ml) . In lung eeneitized with unheated serum, pollen extract caused release of 20-30°fo of tissue histamine. This was inhibited by SCG in a "bell-shaped" dose-related manner as before (Fig . 2) . In lung sensitized with heated serum, pollen extract induced less than 1°Jo histamine release . Addition of SCG caused a linear dose-related potentiation of histamine release, similar to that observed with anti-IgG in lung eeneitized with unheated serum (Fig . 2) . In non-sensitized lung, pollen extract caused no histamine release irrespective of SCG .
Vol . 23, No . 19, 1978
Cromoglycate and Human Lung
e)TPE(100N0/ml)
1901
b) Anti-IOE(1 :1000)
30
v 0 ib é
0~
y 0
. 5
10
. 20 2S
SO
100
C) Anti-IOG (1:100)
y . 0 5
10
20 ô
â0
1011
SO
100
d ) Anti-IpEHflOM) t Anti-IOG(1~10~)
20
10
O
S
10
SO 100 0 5 aD 2S Gomoplycato conc .(p0/ml)
10
20 2S
FIG. 1 The effect of SCG on histamine release from human lung in vitro induced by : (a) timothy pollen extract, 100~.g/ml; (b) anti-IgE, 1 :1000 dilution ; (c) anti-IgG, 1 :100 dilution ; (d) anti-IgE, 1 :1000 + anti-IgG, 1 :100 . Each point is the mean of quadruplicate lung samples in one experiment . At no point did the standard error of the mean exceed 2°fo histamine release.
1902
Cromoglycate and Human Lung
Vol . 23, No . 19, 1978
TABLE 1 Potentiation of Anti-IgG Induced Histamine Releaee from Human Chopped Lung in vitro Histamine release Dilution of Anti-IgG 1 :10 1 :50 1 ;100 1 ;1000
Untreated 15 . 13 . 3. 1.
1 b 5 0
(°J,)
Sodium Cromoglycate (50~.g/ml) 23, 15 . 8. 5.
q 6 0 7
Each result is the mean of duplicate samples in two experiments
30
20
m0 "E 10
S 0
Z 0
5
10 20 25 Cromoglycate conc.(Nq/ml) FIG. 2
The effect of SCG on histamine release from human lung in vitro induced by timothy pollen extract, 100~g/ml, after sensitization with allergic serum (o----o), allergic serum heated at 56°C for 1 hour to destroy IgE (~-~) or not sensitized (~-"). Each point is the mean of quadruplicate lung samples in one experiment . At no point did the standard error of the mean exceed 2% histamine release.
Vol . 23, No . 19, 1978
Cromoglycate and Hwnan Lung
1903
DLSCUSSION From these experiments it ie concluded that histamine may be released from human lung by two different mechanisms . IgE induced release, the dominant mechanism in the absence of drug, ie inhibited by SCG. The second mechanism, probably IgG induced release, ie of minor importance in this model in the absence of drug but ie potentiated by SCG. This potentiation of histamine release from human lung by SCG may explain the failure of the drug to protect patients whose asthma is thought to be mediated predominantly by IgG(2). The different effects of SCG on IgE and IgG induced histamine release may also explain the wide variability in the efficacy of the drug in the treatment of asthma . Furthermore, the finding that newer cromoglycate-like drugs, which are superior to the parent compound in animal models of IgE mediated allergic reactions, also inhibit IgE, but potentiate IgG mediated reactions in human lung (Church, preliminary unpublished results), may explain their comparative failure in man. This work was supported in part by Cilag-Chemie Foundation for Therapeutic Research. We would like to acknowledge the assistance of Professor G. T . Steveneon in testing for antiserum specificity. REFERENCES 1. 2. 3. 4. 5. 6.
R. N. BROGDEN, T. M. SPEIGHT, and G. S. AVERY, Drugs _7 164 (1974) . D. H. BRYANT, M. W. BURNS, and L. LAZARUS, Br . med. J. iv, 589 (1973). J. GOOSE and A. M. J. N. BLAIR, Immunology 16 749 (1969) . K. F. AUSTEN, Asthma : Physiology, Lmmunopharmacology and Treatment, (ede . Austen, K. F. and Lichtenetein, L. M. ), p292 Academic Preee, New York (1973). D. P. EVANS, J. A . LEWIS and D. S . THOMSON, Life Sciences 12 327 (1973). W. E . PARISH, Asthma : Physiology, Immunopharmacology and Treatment (eds . Austen, K. F. and Lichtenetein, L. M. ), pp71-90, Academic Press, New York (1973) .
Pergamon Press
POTENTIATION OF HISTAMINE RELEASE BY SODIUM CROMOGLYCATE Martin K. Church and Carolyn F. Gradidge Clinical Pharmacology, Faculty of Medicine, Universit y of Southampton, Southampton General Hospital, Tremona Road, Southampton S09 4XY, U. K. (Received in final form September 12, 1978) SUMMARY Human lung slices passively sensitized with allergic serum released histamine when incubated with specific antigen and anti-IgE but anti-IgG had no effect . Sodium cromoglycate (SCG) inhibited antigen induced histamine release but the doseresponse curve was bell-shaped. Inhibition of anti-IgE induced release was linearly related to dose, whereas that induced by anti-IgG was potentiated by increasing doses of SCG. After sensitization with allergic serum in which IgE had been inactivated by heating, specific antigen released little or no histamine but this was potentiated by SCG. It is concluded that SCG inhibits IgE mediated but potentiates IgG mediated allergic reactions thus explaining its characteristic doseresponse curve in vitro. Sodium cromoglycate (SCG) has been shown to relieve human bronchial asthma but it is effective only in 30-70°x, of patients(1). Also, it has been reported that SCG is ineffective in patients with low levels of IgE in whom asthma ie thought to be mediated by IgG(2) . Although SCG inhibits IgE mediated passive cutaneous anaphylaxis in the rat in a linear dose-related manner(3), "bell-shaped" dose-response curves are seen in many in vitro experiments which include histamine release from human lung(4). The present study examines the reasons for this "bell-shaped" dose-response relationship . METHODS Human lung obtained from lobectomy specimens was chopped finely with scissors, divided into 200mg replicates and sensitized with 0. 2ml serum, from an allergic donor, in 20m1 Krebs solution for 18 hours at room temperature followed by 1 hour at 37oC . Samples were then washed, resuspended in oxygenated Krebs solution, brought to 37oC and challenged with antigen in a final volune of 2m1. After 15 minutes incubation, the supernatant was removed and the tissue frozen then thawed to release the remaining histamine. Histamine in the two solutions was assayed epectrofluorimetrically(5) and that released by antigen expressed as a percentage of the original histamine content of the tissue sample . Samples incubated in the absence of antigen were used to correct for spontaneous 0300-9653/78/1106-1899$02 .00/0 Copyright (c) 1978 Pergamon Press
1900
Cromoglycate and Human Lung
Vol . 23, No . 19, 1978
histamine release. Antigens used were timothy pollen extract (donated by Beechams Ltd. ), anti-IgE (goat anti-human IgE, Miles Yeda) and anti-IgG (goat anti-human IgG, Miles Yeda). Each antiserum was tested for specificity using micro-precipitin tests on Ouchterlony plates, The antisera, tested against IgA, IgE, IgG, IgM and free light chains, gave positive reactions only with immunoglobulin of their respective homologous classes . In the case of anti-IgG serum, the addition of human IgM to lmg/ml had no effect on the equivalence point for precipitation with IgG (0 . 22mg/ml antiserum) . Our stocks of IgE did not permit this precipitin-inhibition test tobe carried out with anti-IgE serum, SCG (donated by Filons Ltd. ) dissolved in Krebs solution, was added 30 seconds before antigen, Each estimate of histamine release is the mean of those obtained from quadruplicate tissue samples. RESULTS In five experiments, SCG inhibited histamine release induced by timothy pollen extract (100~,g/ml) in a bell-shaped dose-related manner with a peak inhibitory activity at 25~g/ml (Fig, la). By contrast, release induced by anti-IgE (1 :1000 dilution) was inhibited in a linear dose-related way (Fig . lb). Therefore, the possibility that the inflexion of the timothy pollen curve with higher doses of SCG was initiated by an antibody other than IgE was inve stigated. In two pilot experiments, lung challenged with various concentrations of anti-IgG released little histamine . When incubated with SCG (50~,g/ml), histamine release was significantly increased at all concentrations of anti IgG (Table 1) . Similarly, anti-IgG (1 :100 dilution) released little histamine in four further experiments. The addition of SCG in increasing doses potentiated histamine release by this concentration of anti-IgG in a linear dose-related manner, theroleaee at 100~.g/ml of SCG being comparable with that induced by anti-IgE in the absence of drug (Fig . 1C). As expected, the effect of SCG on histamine released by a mixture of anti-IgE and antiIgG showed features characteristic of both antibodies, a bell-shaped doseresponse relationship being obtained (Fig, ld), To demonstrate that the results observed with anti-IgG were due neither to the aggregation of non-mast cell bound IgG remaining within the chopped tissue nor a non-specific action of the commercial preparation of antigen, five additional experiments were performed. In these, chopped lung was sensitized with allergic serum as before, allergic serum heated to 56°C for 1 hour to destroy its IgE(6), or not sensitized . Challenge was with timothy pollen extract (100Wg/ml) . In lung eeneitized with unheated serum, pollen extract caused release of 20-30°fo of tissue histamine. This was inhibited by SCG in a "bell-shaped" dose-related manner as before (Fig . 2) . In lung sensitized with heated serum, pollen extract induced less than 1°Jo histamine release . Addition of SCG caused a linear dose-related potentiation of histamine release, similar to that observed with anti-IgG in lung eeneitized with unheated serum (Fig . 2) . In non-sensitized lung, pollen extract caused no histamine release irrespective of SCG .
Vol . 23, No . 19, 1978
Cromoglycate and Human Lung
e)TPE(100N0/ml)
1901
b) Anti-IOE(1 :1000)
30
v 0 ib é
0~
y 0
. 5
10
. 20 2S
SO
100
C) Anti-IOG (1:100)
y . 0 5
10
20 ô
â0
1011
SO
100
d ) Anti-IpEHflOM) t Anti-IOG(1~10~)
20
10
O
S
10
SO 100 0 5 aD 2S Gomoplycato conc .(p0/ml)
10
20 2S
FIG. 1 The effect of SCG on histamine release from human lung in vitro induced by : (a) timothy pollen extract, 100~.g/ml; (b) anti-IgE, 1 :1000 dilution ; (c) anti-IgG, 1 :100 dilution ; (d) anti-IgE, 1 :1000 + anti-IgG, 1 :100 . Each point is the mean of quadruplicate lung samples in one experiment . At no point did the standard error of the mean exceed 2°fo histamine release.
1902
Cromoglycate and Human Lung
Vol . 23, No . 19, 1978
TABLE 1 Potentiation of Anti-IgG Induced Histamine Releaee from Human Chopped Lung in vitro Histamine release Dilution of Anti-IgG 1 :10 1 :50 1 ;100 1 ;1000
Untreated 15 . 13 . 3. 1.
1 b 5 0
(°J,)
Sodium Cromoglycate (50~.g/ml) 23, 15 . 8. 5.
q 6 0 7
Each result is the mean of duplicate samples in two experiments
30
20
m0 "E 10
S 0
Z 0
5
10 20 25 Cromoglycate conc.(Nq/ml) FIG. 2
The effect of SCG on histamine release from human lung in vitro induced by timothy pollen extract, 100~g/ml, after sensitization with allergic serum (o----o), allergic serum heated at 56°C for 1 hour to destroy IgE (~-~) or not sensitized (~-"). Each point is the mean of quadruplicate lung samples in one experiment . At no point did the standard error of the mean exceed 2% histamine release.
Vol . 23, No . 19, 1978
Cromoglycate and Hwnan Lung
1903
DLSCUSSION From these experiments it ie concluded that histamine may be released from human lung by two different mechanisms . IgE induced release, the dominant mechanism in the absence of drug, ie inhibited by SCG. The second mechanism, probably IgG induced release, ie of minor importance in this model in the absence of drug but ie potentiated by SCG. This potentiation of histamine release from human lung by SCG may explain the failure of the drug to protect patients whose asthma is thought to be mediated predominantly by IgG(2). The different effects of SCG on IgE and IgG induced histamine release may also explain the wide variability in the efficacy of the drug in the treatment of asthma . Furthermore, the finding that newer cromoglycate-like drugs, which are superior to the parent compound in animal models of IgE mediated allergic reactions, also inhibit IgE, but potentiate IgG mediated reactions in human lung (Church, preliminary unpublished results), may explain their comparative failure in man. This work was supported in part by Cilag-Chemie Foundation for Therapeutic Research. We would like to acknowledge the assistance of Professor G. T . Steveneon in testing for antiserum specificity. REFERENCES 1. 2. 3. 4. 5. 6.
R. N. BROGDEN, T. M. SPEIGHT, and G. S. AVERY, Drugs _7 164 (1974) . D. H. BRYANT, M. W. BURNS, and L. LAZARUS, Br . med. J. iv, 589 (1973). J. GOOSE and A. M. J. N. BLAIR, Immunology 16 749 (1969) . K. F. AUSTEN, Asthma : Physiology, Lmmunopharmacology and Treatment, (ede . Austen, K. F. and Lichtenetein, L. M. ), p292 Academic Preee, New York (1973). D. P. EVANS, J. A . LEWIS and D. S . THOMSON, Life Sciences 12 327 (1973). W. E . PARISH, Asthma : Physiology, Immunopharmacology and Treatment (eds . Austen, K. F. and Lichtenetein, L. M. ), pp71-90, Academic Press, New York (1973) .