Agents and Actions, vol. 30, 1/2 (1990)
0065-4299/90/020137-03 $1.50+0.20/0 9 1990 BirkhfiuserVerlag, Basel
Influence of histamine H3-antagonists on human leukocytes J. Kleine-Tebbe, J. Schramm, M. Bolz, R. Lipp 1, W. Schunack i and G. Kunkel Department of Clinical Immunology and Asthma-Policlinic,University Hospital Rudolf Virchow, Free University Berlin, 1000 Berlin 65, FRG, and 1 Institute of Pharmacy, Free University Berlin, 1000 Berlin 33, FRG
Abstract
To determine the role o f the histamine H3-receptor on basophils, different specific H3-antagonists were investigated. Incubation of washed leukocytes with N~-acylated histamine-derivatives (N~-ahd) induced elevated histamine levels. This process turned out to be dependent on dose, time and temperature, but independent of Ca 2 + and Mg 2+ ions. IgE-mcdiated histamine release was not modulated. [3H]-L-histidine was not decarboxylated into [3H]-histamine in spite of the observed histamine increase. Highly purified basophils did not show any histamine elevation but purified neutrophils and eosinophils were found to have increased histamine levels even after disintegration and subsequent incubation with N~-ahd. It seems that the increased histamine levels result from the cleavage of the applied histamine amides. Other potent H 3-antagonists (e.g. thioperamidc) neither produced increased histamine levels nor influenced lgE-mediated release from basophil leukocytes. The existence of H3-receptors on h u m a n basophils therefore seems unlikely.
Materials and methods
basophils was performed with Percoll gradients and elutriation, and purification of eosinophils and neutrophils with a five step Percoll gradient, as previously described [4 6]. A cell disruptor with a microtip was used for ultrasonic disintegration. Histamine was measured in duplicates by an automated fluorometric method [4], and in some cases with two different histamine-radioimmunoassays (Pharmacia '"~and Immunotech "~) and by thin layer c h r o m a t o g r a p h y . Specific antigens (timothy-SQ, birchpollen SQ, 10 100 SQ/ml, d o n a t e d by A L K Laboratories, D e n m a r k ) and anti-lgE (Behring, F R G , 100 IU/ml) were used to trigger IgE-mediated histamine release [4].
Leukocytes were prepared by dextran sedimentation o f human whole blood. Purification o f
Chemicals
This work was supported by Grant No. K1 622/1-1 from the Deutsche Forschungsgemeinschaft,Bonn, FRG.
Histidine decarboxylase inhibitors: L-histidinemethyl ester and c~-fluoromethyl-histidine (donat-
Introduction
Stimulation of presynaptic H3-receptors has been shown to inhibit histamine synthesis and release through autofeedback in histaminergic neurons [1]. IgE-mediated histamine release from h u m a n basophils can be inhibited by stimulation of El 2-receptors on the cell surface, which acts by an elevation of c A M P [2]. Because the existence of H3-receptors had also been shown in peripheral tissues [3], we evaluated the role of this new receptor on h u m a n basophils.
138
Agents and Actions, vol. 30, I/2 (1990)
ed by Merrel Dow, Strassbourgh France); adenylate cyclase stimulating agent: forskolin (Calbiochem/FRG); H3-agonist: (R)-c~-methyl-histamine; H3-antagonists: (1) N~-ahd [7]: no. 1 (N-[2-(1H-imidazol-4-yl)ethyl]-4-cyclohexylbutanamide), no. 2 (N-[2-(1H-imidazol-4-yl)ethyl]-4phenylbutanamide, and no. 3 (N-[2-(1H-imidazol4-yl)-ethyl]-4-(2-pyridyl)butanamide), (2) with different structures: no. 4, thioperamide.
histamine [ n g / I 06 cMls] 50--
n=4
30--
20--
Results
Incubation (37 ~ of washed leukocytes with N ~ahd (>_ 0.1 gM) caused an increase in their histamine content. This effect was maximal with the H3-antagonist no. 1 (20 gM) which produced a 6 to 10-fold elevation in the content of the amine. Using several concentrations (10-9-10-43//) of the H3-antagonists 2 and 3, similar but smaller increases (1.5-fold and 3-fold) were found at drug concentrations of 10 -4 M. Time-course experiments showed a rather rapid elevation of the whole histamine (2 5 h), which was measured after disintegration and removal of the cell fragments. In parallel, a linear increase of extracellular histamine (after 4 8 h) was observed by investigating the supernatants without previous disintegration. The histamine elevation proved to be independent of Ca =+ ( 2 x 1 0 - 5 - 2 x 1 0 aM, n = 4 ) and Mg =+ (5 x 10 -6 5 x 10 -4 M, n=4). The maximum histamine increase induced by compound no. 1 ( 1 0 - 5 M ) was found at a temperature of 37~ (0 ~ no histamine detectable, 20 ~ 10 _+ 1 ng/ml, 37~ 27_+2ng/ml). The presence of I 0 - 3 M L-histidine did not potentiate this process and [3H]-histamine was not found after the addition of [3H]-L-histidine. Preincubation (2h, 37 ~ with the two specific histidine-decarboxylase-inhibitors L-histidine-methyl ester and c~-fluoromethyl-histidine (10-12_ 10 .6 M, n = 8 ) did not change the histamine increase. Preincubation with the H3-agonist (R)-c~methyl-histamine (10 9 - 1 0 - 6 M , n = 8 ) or the adenylate cyclase stimulating agent forskolin ( 1 0 - v - 1 0 -4 M, n = 8 ) also did not modulate the observed histamine increase. IgE-mediated histamine release was neither enhanced nor inhibited in spite of the increased histamine levels: the maximal releases ( • before and after the histamine increases were 44_+ 12% and 41 _+15%, respectively. Highly purified basophils (83_+12%)
cell fractions:
40--
eosinopN}s
1095 %
87 %
5%
0 - - 13% grl
--
neutrophiis
gr
Percoll gradients (gr)
Figure 1
1.085 < gr 1 < 1.090 g/ml 1.090 < gr 2 < 1.095 g/ml
Histamine levels of purified neutrophils and eosinophils incubated with the histamine Ha-antagonist no. 1. Discontinuous Percoll gradients were used to prepare purified neutrophils (1.085 ~ grl < 1.090 g/ml) and eosinophils (1.090 ~ gr2 ~ 1.095 g/ ml). Purity values are depicted as averages beside the columns, which represent the amount of histamine after incubation with 10- s M of N-[2-(1H-imidazol-4-yl)ethyl]-4-cyclohexylbutanamide
(no. 1).
histamine release [~l (HRMAx A= 100%) 120 --
100 9
80 9 9
no. 4 (n = 5) thioperamide (n = 8)
J_
60-110 8
Figure 2
110. 7
[
10"6
110-5
-
I
10 .4
H3 - antagonist I MI
Influence of H3-antagonists (no. 4, thioperamide) on anti-IgE induced histamine release from peripheral human leukocytes.
did not show any histamine elevation after incubation with N~-ahd ( 1 0 - 9 - 1 0 4 M, n=4). The histamine increase appeared in the presence of highly purified eosinophils (95_+3%) as well as in the presence of highly purified neutrophils (87 + 4 % ) (Fig. 1). Cell fragments of washed leukocytes,
139
Agents and Actions, voh 30, 1/2 (1990)
which were ultrasonically disintegrated (2 min) before being incubated with N%ahd (3x 10 -v M, n = 4), produced similar elevated histamine levels (32_+ 9.5 ng/ml) to untreated cells (24.9-+ 8.7 ng/ ml). H3-antagonists of other structure (thioperamide, 4; 1 0 - 9 - 1 0 - 4 M , n = 8 ) neither increased histamine levels nor influenced IgE-mediated antigen-induced histamine release from washed leukocytes. Only the highest concentration of thioperamide (10 4 M) caused a 25_+4.5% inhibition of leukocyte histamine release (Fig. 2). Discussion
Specific H 3-antagonists (thioperamide and N % ahd) have been used to attempt to antagonize the histaminc-induccd inhibition of its own synthesis and release via HB-receptors [8]. Our earlier finding of increasing histamine levels after incubation of human mixed leukocytes with N%ahd indicated an involvement of Ha-receptors in this process [9]. However, the histamine containing basophils did not show any histamine elevation after purification and incubation with N%ahd. These substances, which possess an histamine moiety and an alnido group, must therefore have been clcaved into fragments by neutrophil and eosinophil enzymes and measured as histamine in our assay. Because H3-antagonists without an histamine moiety failed to modulate IgE-mediated histamine release, the existence of H3-receptors on human basophils seems rather unlikely. While investigating substances which modulate histamine release from peripheral leukocytes in vitro, direct interfer-
ence (i.e. fluorescence) with the assay has to be excluded but also the possibility of degradation of the molecules and the consecutive interference of the resulting fragments (i.e. histamine) with the applied method should be considered.
References [1] J.-M. Arrang, M. Garbarg and J.-C. Schwartz, Auto-inhibition (d brain histamine reh>ase mediated by a novel class (H3) o f histamine receplor. Nature (London) 302, 832- 837 (1983). [2] L. M. Lichtenstein and E. Gillespie, The el]bets q/the H 1- and H2-anlihistamines on "allergic" histamine reh, ase and its inhibition hy histamine. J. Pharmacol. exp. Ther. 192, 441 450 (1975). [3] S. Ishikawa and N. Sperelakis, A novelela
0065-4299/90/020137-03 $1.50+0.20/0 9 1990 BirkhfiuserVerlag, Basel
Influence of histamine H3-antagonists on human leukocytes J. Kleine-Tebbe, J. Schramm, M. Bolz, R. Lipp 1, W. Schunack i and G. Kunkel Department of Clinical Immunology and Asthma-Policlinic,University Hospital Rudolf Virchow, Free University Berlin, 1000 Berlin 65, FRG, and 1 Institute of Pharmacy, Free University Berlin, 1000 Berlin 33, FRG
Abstract
To determine the role o f the histamine H3-receptor on basophils, different specific H3-antagonists were investigated. Incubation of washed leukocytes with N~-acylated histamine-derivatives (N~-ahd) induced elevated histamine levels. This process turned out to be dependent on dose, time and temperature, but independent of Ca 2 + and Mg 2+ ions. IgE-mcdiated histamine release was not modulated. [3H]-L-histidine was not decarboxylated into [3H]-histamine in spite of the observed histamine increase. Highly purified basophils did not show any histamine elevation but purified neutrophils and eosinophils were found to have increased histamine levels even after disintegration and subsequent incubation with N~-ahd. It seems that the increased histamine levels result from the cleavage of the applied histamine amides. Other potent H 3-antagonists (e.g. thioperamidc) neither produced increased histamine levels nor influenced lgE-mediated release from basophil leukocytes. The existence of H3-receptors on h u m a n basophils therefore seems unlikely.
Materials and methods
basophils was performed with Percoll gradients and elutriation, and purification of eosinophils and neutrophils with a five step Percoll gradient, as previously described [4 6]. A cell disruptor with a microtip was used for ultrasonic disintegration. Histamine was measured in duplicates by an automated fluorometric method [4], and in some cases with two different histamine-radioimmunoassays (Pharmacia '"~and Immunotech "~) and by thin layer c h r o m a t o g r a p h y . Specific antigens (timothy-SQ, birchpollen SQ, 10 100 SQ/ml, d o n a t e d by A L K Laboratories, D e n m a r k ) and anti-lgE (Behring, F R G , 100 IU/ml) were used to trigger IgE-mediated histamine release [4].
Leukocytes were prepared by dextran sedimentation o f human whole blood. Purification o f
Chemicals
This work was supported by Grant No. K1 622/1-1 from the Deutsche Forschungsgemeinschaft,Bonn, FRG.
Histidine decarboxylase inhibitors: L-histidinemethyl ester and c~-fluoromethyl-histidine (donat-
Introduction
Stimulation of presynaptic H3-receptors has been shown to inhibit histamine synthesis and release through autofeedback in histaminergic neurons [1]. IgE-mediated histamine release from h u m a n basophils can be inhibited by stimulation of El 2-receptors on the cell surface, which acts by an elevation of c A M P [2]. Because the existence of H3-receptors had also been shown in peripheral tissues [3], we evaluated the role of this new receptor on h u m a n basophils.
138
Agents and Actions, vol. 30, I/2 (1990)
ed by Merrel Dow, Strassbourgh France); adenylate cyclase stimulating agent: forskolin (Calbiochem/FRG); H3-agonist: (R)-c~-methyl-histamine; H3-antagonists: (1) N~-ahd [7]: no. 1 (N-[2-(1H-imidazol-4-yl)ethyl]-4-cyclohexylbutanamide), no. 2 (N-[2-(1H-imidazol-4-yl)ethyl]-4phenylbutanamide, and no. 3 (N-[2-(1H-imidazol4-yl)-ethyl]-4-(2-pyridyl)butanamide), (2) with different structures: no. 4, thioperamide.
histamine [ n g / I 06 cMls] 50--
n=4
30--
20--
Results
Incubation (37 ~ of washed leukocytes with N ~ahd (>_ 0.1 gM) caused an increase in their histamine content. This effect was maximal with the H3-antagonist no. 1 (20 gM) which produced a 6 to 10-fold elevation in the content of the amine. Using several concentrations (10-9-10-43//) of the H3-antagonists 2 and 3, similar but smaller increases (1.5-fold and 3-fold) were found at drug concentrations of 10 -4 M. Time-course experiments showed a rather rapid elevation of the whole histamine (2 5 h), which was measured after disintegration and removal of the cell fragments. In parallel, a linear increase of extracellular histamine (after 4 8 h) was observed by investigating the supernatants without previous disintegration. The histamine elevation proved to be independent of Ca =+ ( 2 x 1 0 - 5 - 2 x 1 0 aM, n = 4 ) and Mg =+ (5 x 10 -6 5 x 10 -4 M, n=4). The maximum histamine increase induced by compound no. 1 ( 1 0 - 5 M ) was found at a temperature of 37~ (0 ~ no histamine detectable, 20 ~ 10 _+ 1 ng/ml, 37~ 27_+2ng/ml). The presence of I 0 - 3 M L-histidine did not potentiate this process and [3H]-histamine was not found after the addition of [3H]-L-histidine. Preincubation (2h, 37 ~ with the two specific histidine-decarboxylase-inhibitors L-histidine-methyl ester and c~-fluoromethyl-histidine (10-12_ 10 .6 M, n = 8 ) did not change the histamine increase. Preincubation with the H3-agonist (R)-c~methyl-histamine (10 9 - 1 0 - 6 M , n = 8 ) or the adenylate cyclase stimulating agent forskolin ( 1 0 - v - 1 0 -4 M, n = 8 ) also did not modulate the observed histamine increase. IgE-mediated histamine release was neither enhanced nor inhibited in spite of the increased histamine levels: the maximal releases ( • before and after the histamine increases were 44_+ 12% and 41 _+15%, respectively. Highly purified basophils (83_+12%)
cell fractions:
40--
eosinopN}s
1095 %
87 %
5%
0 - - 13% grl
--
neutrophiis
gr
Percoll gradients (gr)
Figure 1
1.085 < gr 1 < 1.090 g/ml 1.090 < gr 2 < 1.095 g/ml
Histamine levels of purified neutrophils and eosinophils incubated with the histamine Ha-antagonist no. 1. Discontinuous Percoll gradients were used to prepare purified neutrophils (1.085 ~ grl < 1.090 g/ml) and eosinophils (1.090 ~ gr2 ~ 1.095 g/ ml). Purity values are depicted as averages beside the columns, which represent the amount of histamine after incubation with 10- s M of N-[2-(1H-imidazol-4-yl)ethyl]-4-cyclohexylbutanamide
(no. 1).
histamine release [~l (HRMAx A= 100%) 120 --
100 9
80 9 9
no. 4 (n = 5) thioperamide (n = 8)
J_
60-110 8
Figure 2
110. 7
[
10"6
110-5
-
I
10 .4
H3 - antagonist I MI
Influence of H3-antagonists (no. 4, thioperamide) on anti-IgE induced histamine release from peripheral human leukocytes.
did not show any histamine elevation after incubation with N~-ahd ( 1 0 - 9 - 1 0 4 M, n=4). The histamine increase appeared in the presence of highly purified eosinophils (95_+3%) as well as in the presence of highly purified neutrophils (87 + 4 % ) (Fig. 1). Cell fragments of washed leukocytes,
139
Agents and Actions, voh 30, 1/2 (1990)
which were ultrasonically disintegrated (2 min) before being incubated with N%ahd (3x 10 -v M, n = 4), produced similar elevated histamine levels (32_+ 9.5 ng/ml) to untreated cells (24.9-+ 8.7 ng/ ml). H3-antagonists of other structure (thioperamide, 4; 1 0 - 9 - 1 0 - 4 M , n = 8 ) neither increased histamine levels nor influenced IgE-mediated antigen-induced histamine release from washed leukocytes. Only the highest concentration of thioperamide (10 4 M) caused a 25_+4.5% inhibition of leukocyte histamine release (Fig. 2). Discussion
Specific H 3-antagonists (thioperamide and N % ahd) have been used to attempt to antagonize the histaminc-induccd inhibition of its own synthesis and release via HB-receptors [8]. Our earlier finding of increasing histamine levels after incubation of human mixed leukocytes with N%ahd indicated an involvement of Ha-receptors in this process [9]. However, the histamine containing basophils did not show any histamine elevation after purification and incubation with N%ahd. These substances, which possess an histamine moiety and an alnido group, must therefore have been clcaved into fragments by neutrophil and eosinophil enzymes and measured as histamine in our assay. Because H3-antagonists without an histamine moiety failed to modulate IgE-mediated histamine release, the existence of H3-receptors on human basophils seems rather unlikely. While investigating substances which modulate histamine release from peripheral leukocytes in vitro, direct interfer-
ence (i.e. fluorescence) with the assay has to be excluded but also the possibility of degradation of the molecules and the consecutive interference of the resulting fragments (i.e. histamine) with the applied method should be considered.
References [1] J.-M. Arrang, M. Garbarg and J.-C. Schwartz, Auto-inhibition (d brain histamine reh>ase mediated by a novel class (H3) o f histamine receplor. Nature (London) 302, 832- 837 (1983). [2] L. M. Lichtenstein and E. Gillespie, The el]bets q/the H 1- and H2-anlihistamines on "allergic" histamine reh, ase and its inhibition hy histamine. J. Pharmacol. exp. Ther. 192, 441 450 (1975). [3] S. Ishikawa and N. Sperelakis, A novelela
