Agents Actions,35 (1992)
0065-4299/92/040185-07$1.50+ 0.20/0 9 t992 Birkh~iuserVerlag,Basel
Modulation of IgE-mediated histamine release from human leukocytes by a new class of histamine H2-agonists* J. Kleine-Tebbe, A. Buschauer 1, A. Friese 1, W. Schunack 1, G. Kunkel z Clin. Immunologyand Asthma OPD, Free UniversityBerlin,Germany 1Dept. of Pharmacy,Free UniversityBerlin,Germany
Abstract
A new class of phenyl (pyridylalcyl) guanidines, acting as potent histamine H2-agonists, inhibits IgE-mediated human basophil histamine release in a nanomolar range. IC3o-level of three substitutes of this group (arpromidine, BUA-75, and FRA-19) were found to be 0.02, 0.015 and 0.008 I~M. The inhibition appeared with a fast onset (plateau after 10 min. preincubation) and claimed its maximum (60 _+2.9%, 6 3 _ 1.8%, and 61 _+3.1%, n = 7 ) with 10 ~tM of the compounds. H2-mediated inhibition was totally blocked by 10 ~tM famotidine, a potent histamine H2-antagonist. The amount of anti-IgE or antigen for the initiation of the immunological release influenced the strength of inhibition of H2-agonist FRA-19 ( p < 0.05). Combined preincubation of FRA-19 with zardaverine, a cAMP-specific phosphodiesterase III/IV inhibitor, produced a synergistical inhibitory effect of leukocyte histamine release, which might explained by their different sites of action on intracellular cAMP levels. The capability of histamine to inhibit its own release is mediated by H2-receptors exclusively. New, potent H2-receptor stimulating compounds with positive inotropic effects possess additional potent anti-allergic properties.
Introduction
The release of proinflammatory mediators from basophils initiated by IgE-crosslinking agents is a useful in vitro model for the investigation of allergic reactions and inflammatory processes. The preformed mediator histamine evokes a variety of responses through its binding to Hi-, HE- , and~the recently described H3-receptors [1, 2]. Activation of the histamine H2-receptor has been shown to be one of the mechanisms inhibiting basophil histamine release induced by IgE crosslinking agents 2 Author for correspondence:G. Kunkel, KlinischeImmunologic und Asthma Poliklinik,Universit/itsklinikumRudolfVirchow, AugustenburgerPlatz 1, 1000Berlin65, Germany * This work was supported by grant K1622/1-1 from the Deutsche Forschungsgemeinschaft.
[3, 4, 5]. The receptor binding by H2-agonists initiates an elevation of adenylate cyclase activity and a consecutive increase of cyclic adenosine monophosphate (cAMP) [6, 7]. Recently, a new generation of potent Hz-agonists has been synthesized and suggested to be a potential new class of positive inotropic drugs. These phenyl (pyridylalcyl) guanidines were created by a replacement of the cymetidine moiety in impromidine by more lipophylic pheniramine-like structures [8]. Super oxyde production by chemotactic peptides from human neutrophils was inhibited by these substances via activation of the H2-receptor [8, 9]. Inhibitory effects of H2-receptor directed compounds have also been shown in the immunologic secretion of histamine from human lung mast cells and basophils [10]. We studied the modulation of
186 the new class of H2-agonists on immunological, IgE-mediated histamine release from peripheral, basophil containing human leukocyte preparations. Methods and materials
Material
The following materials were purchased: Piperazine-N,N'-bis 2-ethanesulphonic acid (PIPES) (Serva, Germany). Human serum albumin (Red Cross, Germany), 6% dextrane in physiological saline (Knoll, Germany), 50% glucose (Braun, Germany), forskolin (Calbiochem, Germany), dimethylsulfoxyde (DMSO) (Sigma, Germany), anti-IgE antiserum (Behring, Germany), lyophylized timothy grasspollen allergen extract SQ aquagen 225 (ALK, Denmark). Histamine H 2 agonists: Arpromidine: NI-[3-(4fluorophenyt)-3-(pyridine-2-yl)propyl]-N2-[3-(1Himidazole-4-yl)propyl]guanidine-trihydrochloride; BUA-75: Nl-[3-(3,4-difluorophenyl)-3-(pyridine2-yl)propyl]-N2-[3-(IH-immidazole-4-yl)propyl]guanidine-trihydrochloride; FR-A-19: N1-[3,3 bis (4 - fluorophenyl) propyl] - N 2 - 3 [- (1 H - immidazole-4-yl) propyl]guandine-trihydrochloride; impromidine: Nl-[3-(1H-imidazole-4-yl)propyl]N2[2- [[(5-methyl] thio] ethyl]guanidine- trihydrochloride; dimaprit. Histamine H2-antagonist famotidine. Phosphodiesterase isoenzyme III/IV inhibitor zardaverine: 6-(4-difluoromethoxy-3-methoxyphenyl)-3[2H]-pyridazinone. Buffers. PIPES-albumin-glucose buffer (PAG) contained 25 m M of PIPES, 110 m M of NaC1, 5 m M of KC1, 0.003% of human serum albumin, and 0.1% glucose. PAG supplemented with 1 m M of CaCI2 and 1 m M of MgC12 (PAG-CM) was used during the release experiments. Arpromidine (10mM), BUA-75 (10mM), FRA-19 (10mM), impromidine (10 mM), dimaprit (10 mM), and famotidine were dissolved in demineralized water and kept frozen in small aliquots. Zardaverine (100 mM) was dissolved in 100% DMSO and stored frozen in apropriate aliquots. Dilutions of all pharmacological active reagents were prepared fresh daily in PAG-CM. In preliminary experiments, the concentrations of DMSO in working dilutions used experimentally had no effect on histamine release.
Agents Actions, 35 (1992) Cell isolation
Venous blood was collected from normal and atopic volunteers who had given informed consent. Mixed leukocyte preparations were obtained, employing dextrane sedimentation [11]. In brief, 20 ml of blood were mixed with a solution, containing 5ml 6% dextrane, 2rot 0 . 1 m l m M ethylene diamine tetraacetic acid and 0.8 ml 50 ml glucose. After sedimentation of red cells and polynuclear leukocytes (90-120 min.) the basophil containing supernatant was carefully sampled and centrifuged for 8 minutes with 400 x g at room temperature. The cell pellet was washed twice with PAG by centrifugation (8 min., 400 x g, 4 ~ Finally the cells were resuspended in PAG-CM to a volume, which was required for the following experiments. Challenge
The cells were preincubated with either the drug or PAG-CM as control and then challenged with either 100 IU/ml anti-IgE or 100 SQ/ml of grasspollen antigen for 40 min and 37 ~ in a waterbath. The release process was finished by centrifugation (800 x g, 15 min, 4 ~ The supernatants were removed for histamine analysis. Total histamine content was determined by lysing aliquots of the cells with 2% of HC104. Cells incubated in PAG-CM alone served as a measure of spontaneous histamine release ( < 5 % of the total histamine content). Histamine release was thus expressed as a percentage of the total histamine content after subtracting the spontaneous release. All experiments were performed in duplicate. Histamine assay
Histamine release was estimated according to the automated fluorometric method, which has been extensively described [11]. The diluted drugs did not fluoresce, nor did they effect the fluorescence of known amounts of histamine. Statistical analysis
The statistical significance of drug-related effects was analysed by control and treated cells using wilcoxon signed rank test for paired data. Values were considered significant at p < 0.05 level.
Agents Actions, 35 (1992)
Experimental Protocol 1. Histamine H2-agonists were preincubated in different concentrations (10-9_ 10-5 M), to investigate dose related inhibitory effects. 2. For time course experiments the time was varied: 0, 2, 5, 10, 15, 20, 30, 40 min. 3. The inhibitory effect of H2-agonist FR-A-19 (1 ~tM) was evaluated after preincubation with several concentrations (10- 9, 10- 7, 10- 5 M) of the histamine H2-antagonist famotidine. 4. Cells were preincubated with FRA-19 (10 - 9 1 0 - 6 M ) in combination with zardaverine, an inhibitor of cAMP specific phosphodiesterase type III and IV in different concentrations ( 1 0 - 7 - 1 0 - - 5 M) 5. FRA-19 was preincubated with the cells in different concentrations (10- 9_ 10- 5 N), before immunological histamine release was triggered by 10, 30, and 100 IE/ml anti-IgE. 6. Leukocytes of allergic donors containing sensitized basophiles were preincubated with different concentrations of FRA-19 (10- 9_ 10- 6 M), before the stimulation with the appropriate specific antigen in different concentrations (1, 10, 100 SQ/ml timothy grass pollen).
Results
1. Anti-IgE-induced histamine release from human basophils was inhibited by all H2-agonists in a dose dependent fashion (Fig. 1). All compounds demonstrated an inhibitory effect in the nanomolar range, reaching a plateau with a maximum inhibition of approximately 60% (Table 1). The inhibitory potency of the new generation of histamine H2-agonists was found to be quite similar to each other, only dimaprit - an older substance - created a weaker response. Its dose response curve was shifted to higher inhibition concentrations (approx. 30-fold compared to the inhibition concentration of FR-A-19). 2. The onset of inhibition of dimaprit (10-5 M) and BUA-75 (10 -6 M) was found to be quite similar; both substances produced the same inhibition profile, claiming 50% of inhibition after approx. 5 min., reaching a maximum after 20 minutes (Fig. 2). Interestingly, an inhibition above 25% was found even without any time of preincubation with the receptoractive drugs.
187 Inhibition [%1 70--
~'!I
(n=7)
llw
so
30"
-r
/;.t
IT ~ / ' i '
]~ ~ / ~ #
10-
/
1
/
~ Arpromidine
V
I
/
_
9BU-A-TB O bimaprit
/O
v FR-A-19 O Impromidine
~
1~/~
o-
,'0-9
,~8
,;7
,'o-o
,'o5
Figure I
H2-agonist [M] Inhibition of anti-IgE induced histamine release (means___SD) from human, peripheral leukocytes after preincubation with different histamine H2-agonists. Maximum of inhibition and inhibition concentrations (IC30) are depicted in Table 1.
[%[ ] _=5)~ ( n
Inhibition 80--
.~
60 T I / /~
40 ~
/
20
1 ,
;, 2
5
09 Dimaprit (10"5) Bu-A-75(10 6 M)
~ 0
.
.
15
20
.
. 30
40
Figure 2 time Iminl Timekinetic of H2-agonist mediated inhibition of IgE-mediated histamine release. After different times of preincubation, the cells were stimulated with 100 IU/ml anti-IgE for 40 min.
Table 1 Inhibition of anti-IgE induce histamine release from human peripheral leukocytes. Concentration of histamine H2-agonists for 30% and 50% inhibition (IC30 , IC50) are given besides maximum of inhibition (InhMAx: means_ SEM) Histamine Hz-agonists Arpromidine BUA-75 Dimaprit FRA-19 Impromidine
IC3o [I-tM] IC~0 [IxM] (median) (median) 0.02 0.015 0.3 0.008 0.01
0.2 0.4 3 0.1 0.5
Inhr~x [%] (means)
(SEM)
60 63 57 61 55
2.9 1.8 2.7 3.1 3.4
188
Agents Actions, 35 (1992) H2 agonlst/antagonist
histaminerelease[%]
anti-lgE [IU/ml]
Inhibition [%1 80--
10 30 60-=_
*
*
" 10-6
|
(n = 6)
TT
~
100
10-7 10-9
o i
i0
3
I
i
30
i
i
100
0--
i
300
1000
Figure 3 anti-lgE [IU/rnl] Influence of 10-9, 10-7, and 10-5 M famotidine - a potent H 2antagonist - on the histamine release inhibiting effects of H2agonist FRA-19 (10-6 M). First preineubation (10 rain) with famotidine, second(10 rain) with FRA-19,stimulation of mediator release (40 rain) with anti-IgE Inhibition [%1
PDE(IV) inhibitor
90
~ (n = 5)
~
T ~
A
10s [M]
e
10 -6
[M]
o
buffer Figu]'e 4
1;-9
108
10.7 10-6 H2~gonist FR-A-19 [M]
Inhibition of IgE-mediated histamine release after combined preincubation of H2-agonist FRA-19 and cAMP specificphosphodiesterase (IV) inhibitor zardaverine. The release processwas initiated by 100 IU/ml anti-IgE.
3. The inhibition of immunological histamine release by FRA-19, a substitute of this class o f substances, was blocked with specific histamine H 2antagonists in a dose dependent manner (Fig. 3). Preincubation with 10 ~tM famotidine were sufficient to block the inhibition o f the H2-agonist FR-A-19 (10-6 M) totally. 4. In another experiment we tried to enhance the inhibititory effects o f the H2-agonists by combined incubation with a cAMP-specific P D E III/IV inhibitor zardaverine. The inhibitory effect of F R A 19 in nanomolar concentrations was significantly
I
10-9
I
10-8
I
1~0-7 10-6 1=0-5 H2-egonist FR-A-19 [M]
Figure 5 Influenceof different anti-IgE concentrationson the inhibition of histamine release by H2-agonist FRA-19.
enhanced by increasing doses o f the P D E IV inhibitor (Fig. 4). The combined inhibition was more pronounced than the effect of both compounds after single preincubation indicating a synergism in the modulation of IgE-mediated histamine release." 5. In another series o f experiments, we investigated the FRA-19 induced inhibition dependent on the dose for stimulation and its subsequent signal to release the preformed mediator. N o t only the maximum of Hz-receptor linked inhibition o f histamine release appeared as a function of the strength of the immunological signal, also IC50 levels o f F R A - I 9 were shifted in a dose dependent manner: 10, 30, and 100 IE/ml anti-IgE corresponded with 0.04, 0.1, and 0.7 g M FR-A-19 for 50% inhibition (Fig. 5). Similar experiments were also obtained with sensitized basophiles from allergic donors, using the appropriate antigen to trigger IgE mediated histamine release. Antigen-induced mediator responses o f different individuals vary in a wide range: in 12 o f 20 cases antigen-induced histamine release peaked at 10 SQ/ml grass pollen extract (Fig. 6), while 8 o f 20 claimed the maxim u m of release at 100 SQ/ml (Fig. 7). The results of both groups were depicted in different curves, to s(udy the influence of the signal on the inhibitory effect o f the Hz-agonist FRA-19. According to the individual histamine release curves and the antigen dose required for the maximum release, different inhibition curves could be obtained. Significant differences were detectable between the dose-
189
Agents Actions, 35 (1992) Inhibition
[%]
/
"
I
o. .oj
J ,ooso,., Discussion
I/
...i.o, i;; r o,
o
1...,-.
.... ..... &,
~0
tion by stimulation of H2-receptor, no significant difference was found in regard to the inhibition concentration.
I
~
o
i
Fig. 6
Inhibition
J'r
buffer
i
........
10-~
i
10-s
........
J
........
i
10`7 10-6 H2-agonist FR-Ad9 [M]
[ %)
~0-- 80
/ ~
10SQ/m[
oo
Fig. 7
oo_
t
buffer
.7
1
10-9
......
t
10-a
10-7
10-s
Figure 6/7 H2-agonist FR-A-19 [ a ] Influenceof the amountof antigen on the H2-1inkedinhibitionof IgE-mediatedhistaminereleasefromsensitizedbasophilsofallergic donors. Accordingto the individual histamine releasecurves, the resultsweredivideddue to the maximumof release(10 SQ/ml vs. 100 SQ/rnl): Fig. 6 summarizesthe dose response curves of those donors, who claimed the maximumof release at 10 SQ/ml of the allergen extract (upper left pannel). The results in Fig. 7 were obtained from sensitized donors too; their antigen induced release peaked at 100 SQ/ml (upper left pannel).
dependent inhibition curves induced by 1 and 10 SQ/ml antigen (Fig. 6), but also between 1 and 100 SQ/ml (see Fig. 7); nevertheless some of these results can only be described as trends: the lower the mediator response to a defined allergen dose, the more pronounced the inhibition of IgE mediated histamine release. Comparing anti-IgE- and antigen-induced histamine release and their inhibi-
It has been known for many years, that the preformed mediator histamine possesses the capability to inhibit its own release from human basophils [3]. This effect was blocked by histamine Hz-antagonist cimetidine, indicating an involvement of histamine Hz-receptors [12]. In the meantime, inhibition of immunological histamine release of purified basophils after activation of the H2-receptor has been proven; these effects of an auto-feedback mechanism appeared to be related to the activation of adenylate cyclase and subsequent increase of cAMP [5, 7]. A new generation of potent histamine HE-receptor agonists allowed us to reevaluate the potential inhibitory effects of these compounds. Whereas dimaprit inhibits histamine release in the micromolar range [5], the new class of phenyl (pyridylalcyl) guanidines with increased H 2 agonistic potency inhibit IgE mediated histamine release at nanomolar concentrations and are at least 10-30 more potent than dimaprit. Superoxide metabolism - triggered by the chemotactic peptide FLMP in neutrophils - was also inhibited by the H2-agonist impromidine with a IC50 value of 2 IxM [13]. This effect was competitively reversed by a specific histamine Hz-antagonist which underlines the involvement of the H2-receptor. However, quite lower inhibition concentrations to reduce basophil histamine release might indicate a more important functional role of these receptor type in the histamine containing blood cells [9]. Another effect of the activation of Hz-receptor has been shown in peripheral blood mononuclear cells: histamine, but also impromidine are able to inhibit the colony formation of phorbol ester stimulated T-cells [14]. In the guinea pig atrium system some of the new compounds (BUA-75, arpromidine, FRA-19) are full H2-agonists with potencies of up to 125-fold of that of histamine [8]. A fast onset of inhibition of super oxide formation in neutrophils was detected after a short preincubation with these cells with arpromidine [13], however positive inotropic effects of this compound were found to be slow in onset in the guinea pig atrium [8]. The dose related inhibitory effect of H2-agonist FRA-19 was more pronounced, when histamine release was
190 triggered by a suboptimal dose for IgE-mediated immunological stimulation of the basophil leukocytes. These observations were proven by anti-IgE and antigen as IgE-mediated stimuli. The differences in the inhibition of anti-IgE and antigen-induced stimuli by H 2 agonist dimaprit - demonstrated in a similar study [4] - could not be confirmed by our results, as the potent H 2 agonist FRA-19 posessed the same capability to inhibit anti-IgE- as antigen-induced histamine release. Attempts to inhibit IgE mediated histamine release with histamine H 1 antagonists as diphenhydramine failed [12]. No effect on basophil histamine release could be demonstrated after preincubation with new, recently developed histamine H1agonist in concentrations up to 10 - 6 M [15]. Similarly, neither by potent histamine H3-receptor antagonists nor by a H3-agonist (up to 10 - 6 M ) IgE mediated histamine release from peripheral leucocytes was modulated [15, 16]. Therefore, stimulation of H2-receptors on basophils might be of predominant responsibility for the inhibitory effects of the preformed mediator on its own release [17]. An additional insight into the mechanism of inhibition was possible with the cAMP specific PDE III/IV inhibitor zardaverine, known to prevent cleavage of intracellular cAMP in various cell types and tissues [18]. The synergistic effect on the inhibition of histamine release after combined inhibition of H2-agonist FRA-19 with zardaverine demonstrate two different sites of action on the cAMP second messenger system [7, 19]. Indication for a therapeutic use of H2-agonist to inhibit allergic reactions is probably limited because activation of H2-receptor in other cells and tissues might cause considerable undesired side effects.
Acknowledgement We gratefully acknowledge Heidrun Gagnr, Christa Jostles, and Gabriel Frank for their excellent technical assistance. Received 3 June 1991, accepted by W Lorenz, 25 September 1991
References [1] J. M. Arrang, M. Garbarg and J. C. Schwartz, Auto-inhibition of brain histamine release mediated by a novel class (113) of histamine receptor. Nature (London) 302, 832-837 (1983). '[2] J. M. Arrang, M. Garbarg, J. C. Lancelot, J. M. Lecomte, H. Pollard, M. Robba, W Sehunack and J. C. Schwartz, Highly
Agents Actions, 35 (1992) potent and selective ligands for histamine H3-receptors. Nature (London) 327, 117 123 (1987). [3] L. M. Lichtenstein and E. Gillespie, Inhibition of histamine release by histamine is controlled by an H 2 receptor. Nature (London) 244, 287-288 (1973). [4] G. Marone, A. Kagey-Sobotka and L. M. Lichtenstein, IgEmediated histamine release from human basophils: differences between antigen E and anti-lgE-induced secretion. Int. Arch. Allergy Appl. Immunol. 65, 339-348 (1981). [5] J. A. Warner, D. W. MaeGlashan, S. P. Peters, A. KageySobotka and L. M. Lichtenstein, The pharmacologic modulation of mediator release from human basophils. J. All. Clin. Immunol. 82, 432-438 (1988). [6] C. M. Winslow and K. G. Austen, Role of cyclic nucleotides in the activation-secretion response. Prog. Allergy 34, 236270 (1984). [7] P. T. Peachell, D. W. MacGlashan Jr., L. M. Lichtenstein and R. P. Schleimer, Regulation of human basophil and lung mast cell function by cyclic adenosine monophosphate. J. Immunol. 140, 571-579 (1988). [8] A. Buschauer, Synthesis and in vitro pharmacology of arpromidine and related phenyl (pyridylalkyl) guanidines, a potential new class of positive inotropic drugs. J. Med. Chem. 32, 1963-1970 (1989). [9] R. Burde, R. Seifert, A. Buschauer and G. Schultz, Histamine inhibits activation of haman neutrophils and HL-60 leukemic cells via H2-receptors. Naunyn Schmiedebergs Arch. Pharmacol. 340, 671-678 (1989). [10] H. Y. Lau and E L. Pearce, Effects of antihistamines on isolated human lung mast cells, basophil leukocytes and erythrocytes. Agents and Actions 27, 83-85 (1989). [11] R. P. Siraganian and W A. Hook, Histamine release and assay methods for the study of human allergy. In Manual of Clinical Immunology (Eds. N. R. Rose and H. Friedmann) pp. 808-821, American Society of Microbiology, Washington 1980. [12] L. M. Lichtenstein and E. Gillespie, The effects of the H~ and H 2 antihistamines on "gllergic" histmaine release and its inhibition by histamine. J. Pharmacol Exp. Ther. 192, 441-450 (1975). [13] R. Burde, A. Buschauer and R. Seifert, Characterization of histamine H2-receptors in human neutrophils with a series of guanidine analogues of impromidine: Are cell type-specific Hz-receptors involved in the regulation of NADPH oxidase? Naunyn Schmiedebergs Arch. Pharmacol. 341, 455-461 (1990). [14] K. Meretey, Chien HD, Falus A and Walcz E, Effect of histamine on the T-cell colony formation of PHA-stimulated cells. Agents and Actions 27, 215-217 (1989). [15] J. Kleine-Tebbe, M. Bolz, J. Schramm, H. Gagnr, C. Josties, R. Lipp, A. Freise, H. Stark, V. Zingel, A. Buschauer, W Schunack and G. Kunkel, Influence of histamine Hi-, H2-, H s- agonists and antagonists on lgE-mediated histamine release from human basophils. In New trends in allergy III (Eds. J. Ring and O. Braun-Falco) Springer-Verlag Berlin Heidelberg New York (1991) [16] J. Kleine-Tebbe, J. Schramm, M. Bolz, R. Lipp, W. Schunack and G. Kunkel, Influence of histamine H3-amagonists on human leukocytes. Agents and Actions 30, 137-139 (1990). [17] J. Ring, E Sedlmeier, D. vonder Helm, T. Mayr, V. Walz, H. Ibel, H. Riepel, B. Przybilla, H. I. Reimann and W Dorsch, Histamine and allergic diseases. In New trends in allergy H
Agents Actions, 35 (1992) (Eds. J. Ring and G. Burg) pp. 44-77, Springer-Verlag Berlin Heidelberg New York (1986). [18] Ch. Schudt, S. Winder, B. Miiller and D. Ukena, Zardaverine as a selective inhibitor of phosphodiesterase isoenzymes. Biochem. Pharmaco1., in press, (1991).
191 [19] P. T. Peachell, B. J. Undem, R. P. Schleimer, D. W. MacGlashan Jr., L. M. Lichtenstein, L. B. Cieslinksi and T. J. Torphy, Characterization and role of phosphodiesterase isoenzymes in human basophils. J. Immunol., in press (1991).
0065-4299/92/040185-07$1.50+ 0.20/0 9 t992 Birkh~iuserVerlag,Basel
Modulation of IgE-mediated histamine release from human leukocytes by a new class of histamine H2-agonists* J. Kleine-Tebbe, A. Buschauer 1, A. Friese 1, W. Schunack 1, G. Kunkel z Clin. Immunologyand Asthma OPD, Free UniversityBerlin,Germany 1Dept. of Pharmacy,Free UniversityBerlin,Germany
Abstract
A new class of phenyl (pyridylalcyl) guanidines, acting as potent histamine H2-agonists, inhibits IgE-mediated human basophil histamine release in a nanomolar range. IC3o-level of three substitutes of this group (arpromidine, BUA-75, and FRA-19) were found to be 0.02, 0.015 and 0.008 I~M. The inhibition appeared with a fast onset (plateau after 10 min. preincubation) and claimed its maximum (60 _+2.9%, 6 3 _ 1.8%, and 61 _+3.1%, n = 7 ) with 10 ~tM of the compounds. H2-mediated inhibition was totally blocked by 10 ~tM famotidine, a potent histamine H2-antagonist. The amount of anti-IgE or antigen for the initiation of the immunological release influenced the strength of inhibition of H2-agonist FRA-19 ( p < 0.05). Combined preincubation of FRA-19 with zardaverine, a cAMP-specific phosphodiesterase III/IV inhibitor, produced a synergistical inhibitory effect of leukocyte histamine release, which might explained by their different sites of action on intracellular cAMP levels. The capability of histamine to inhibit its own release is mediated by H2-receptors exclusively. New, potent H2-receptor stimulating compounds with positive inotropic effects possess additional potent anti-allergic properties.
Introduction
The release of proinflammatory mediators from basophils initiated by IgE-crosslinking agents is a useful in vitro model for the investigation of allergic reactions and inflammatory processes. The preformed mediator histamine evokes a variety of responses through its binding to Hi-, HE- , and~the recently described H3-receptors [1, 2]. Activation of the histamine H2-receptor has been shown to be one of the mechanisms inhibiting basophil histamine release induced by IgE crosslinking agents 2 Author for correspondence:G. Kunkel, KlinischeImmunologic und Asthma Poliklinik,Universit/itsklinikumRudolfVirchow, AugustenburgerPlatz 1, 1000Berlin65, Germany * This work was supported by grant K1622/1-1 from the Deutsche Forschungsgemeinschaft.
[3, 4, 5]. The receptor binding by H2-agonists initiates an elevation of adenylate cyclase activity and a consecutive increase of cyclic adenosine monophosphate (cAMP) [6, 7]. Recently, a new generation of potent Hz-agonists has been synthesized and suggested to be a potential new class of positive inotropic drugs. These phenyl (pyridylalcyl) guanidines were created by a replacement of the cymetidine moiety in impromidine by more lipophylic pheniramine-like structures [8]. Super oxyde production by chemotactic peptides from human neutrophils was inhibited by these substances via activation of the H2-receptor [8, 9]. Inhibitory effects of H2-receptor directed compounds have also been shown in the immunologic secretion of histamine from human lung mast cells and basophils [10]. We studied the modulation of
186 the new class of H2-agonists on immunological, IgE-mediated histamine release from peripheral, basophil containing human leukocyte preparations. Methods and materials
Material
The following materials were purchased: Piperazine-N,N'-bis 2-ethanesulphonic acid (PIPES) (Serva, Germany). Human serum albumin (Red Cross, Germany), 6% dextrane in physiological saline (Knoll, Germany), 50% glucose (Braun, Germany), forskolin (Calbiochem, Germany), dimethylsulfoxyde (DMSO) (Sigma, Germany), anti-IgE antiserum (Behring, Germany), lyophylized timothy grasspollen allergen extract SQ aquagen 225 (ALK, Denmark). Histamine H 2 agonists: Arpromidine: NI-[3-(4fluorophenyt)-3-(pyridine-2-yl)propyl]-N2-[3-(1Himidazole-4-yl)propyl]guanidine-trihydrochloride; BUA-75: Nl-[3-(3,4-difluorophenyl)-3-(pyridine2-yl)propyl]-N2-[3-(IH-immidazole-4-yl)propyl]guanidine-trihydrochloride; FR-A-19: N1-[3,3 bis (4 - fluorophenyl) propyl] - N 2 - 3 [- (1 H - immidazole-4-yl) propyl]guandine-trihydrochloride; impromidine: Nl-[3-(1H-imidazole-4-yl)propyl]N2[2- [[(5-methyl] thio] ethyl]guanidine- trihydrochloride; dimaprit. Histamine H2-antagonist famotidine. Phosphodiesterase isoenzyme III/IV inhibitor zardaverine: 6-(4-difluoromethoxy-3-methoxyphenyl)-3[2H]-pyridazinone. Buffers. PIPES-albumin-glucose buffer (PAG) contained 25 m M of PIPES, 110 m M of NaC1, 5 m M of KC1, 0.003% of human serum albumin, and 0.1% glucose. PAG supplemented with 1 m M of CaCI2 and 1 m M of MgC12 (PAG-CM) was used during the release experiments. Arpromidine (10mM), BUA-75 (10mM), FRA-19 (10mM), impromidine (10 mM), dimaprit (10 mM), and famotidine were dissolved in demineralized water and kept frozen in small aliquots. Zardaverine (100 mM) was dissolved in 100% DMSO and stored frozen in apropriate aliquots. Dilutions of all pharmacological active reagents were prepared fresh daily in PAG-CM. In preliminary experiments, the concentrations of DMSO in working dilutions used experimentally had no effect on histamine release.
Agents Actions, 35 (1992) Cell isolation
Venous blood was collected from normal and atopic volunteers who had given informed consent. Mixed leukocyte preparations were obtained, employing dextrane sedimentation [11]. In brief, 20 ml of blood were mixed with a solution, containing 5ml 6% dextrane, 2rot 0 . 1 m l m M ethylene diamine tetraacetic acid and 0.8 ml 50 ml glucose. After sedimentation of red cells and polynuclear leukocytes (90-120 min.) the basophil containing supernatant was carefully sampled and centrifuged for 8 minutes with 400 x g at room temperature. The cell pellet was washed twice with PAG by centrifugation (8 min., 400 x g, 4 ~ Finally the cells were resuspended in PAG-CM to a volume, which was required for the following experiments. Challenge
The cells were preincubated with either the drug or PAG-CM as control and then challenged with either 100 IU/ml anti-IgE or 100 SQ/ml of grasspollen antigen for 40 min and 37 ~ in a waterbath. The release process was finished by centrifugation (800 x g, 15 min, 4 ~ The supernatants were removed for histamine analysis. Total histamine content was determined by lysing aliquots of the cells with 2% of HC104. Cells incubated in PAG-CM alone served as a measure of spontaneous histamine release ( < 5 % of the total histamine content). Histamine release was thus expressed as a percentage of the total histamine content after subtracting the spontaneous release. All experiments were performed in duplicate. Histamine assay
Histamine release was estimated according to the automated fluorometric method, which has been extensively described [11]. The diluted drugs did not fluoresce, nor did they effect the fluorescence of known amounts of histamine. Statistical analysis
The statistical significance of drug-related effects was analysed by control and treated cells using wilcoxon signed rank test for paired data. Values were considered significant at p < 0.05 level.
Agents Actions, 35 (1992)
Experimental Protocol 1. Histamine H2-agonists were preincubated in different concentrations (10-9_ 10-5 M), to investigate dose related inhibitory effects. 2. For time course experiments the time was varied: 0, 2, 5, 10, 15, 20, 30, 40 min. 3. The inhibitory effect of H2-agonist FR-A-19 (1 ~tM) was evaluated after preincubation with several concentrations (10- 9, 10- 7, 10- 5 M) of the histamine H2-antagonist famotidine. 4. Cells were preincubated with FRA-19 (10 - 9 1 0 - 6 M ) in combination with zardaverine, an inhibitor of cAMP specific phosphodiesterase type III and IV in different concentrations ( 1 0 - 7 - 1 0 - - 5 M) 5. FRA-19 was preincubated with the cells in different concentrations (10- 9_ 10- 5 N), before immunological histamine release was triggered by 10, 30, and 100 IE/ml anti-IgE. 6. Leukocytes of allergic donors containing sensitized basophiles were preincubated with different concentrations of FRA-19 (10- 9_ 10- 6 M), before the stimulation with the appropriate specific antigen in different concentrations (1, 10, 100 SQ/ml timothy grass pollen).
Results
1. Anti-IgE-induced histamine release from human basophils was inhibited by all H2-agonists in a dose dependent fashion (Fig. 1). All compounds demonstrated an inhibitory effect in the nanomolar range, reaching a plateau with a maximum inhibition of approximately 60% (Table 1). The inhibitory potency of the new generation of histamine H2-agonists was found to be quite similar to each other, only dimaprit - an older substance - created a weaker response. Its dose response curve was shifted to higher inhibition concentrations (approx. 30-fold compared to the inhibition concentration of FR-A-19). 2. The onset of inhibition of dimaprit (10-5 M) and BUA-75 (10 -6 M) was found to be quite similar; both substances produced the same inhibition profile, claiming 50% of inhibition after approx. 5 min., reaching a maximum after 20 minutes (Fig. 2). Interestingly, an inhibition above 25% was found even without any time of preincubation with the receptoractive drugs.
187 Inhibition [%1 70--
~'!I
(n=7)
llw
so
30"
-r
/;.t
IT ~ / ' i '
]~ ~ / ~ #
10-
/
1
/
~ Arpromidine
V
I
/
_
9BU-A-TB O bimaprit
/O
v FR-A-19 O Impromidine
~
1~/~
o-
,'0-9
,~8
,;7
,'o-o
,'o5
Figure I
H2-agonist [M] Inhibition of anti-IgE induced histamine release (means___SD) from human, peripheral leukocytes after preincubation with different histamine H2-agonists. Maximum of inhibition and inhibition concentrations (IC30) are depicted in Table 1.
[%[ ] _=5)~ ( n
Inhibition 80--
.~
60 T I / /~
40 ~
/
20
1 ,
;, 2
5
09 Dimaprit (10"5) Bu-A-75(10 6 M)
~ 0
.
.
15
20
.
. 30
40
Figure 2 time Iminl Timekinetic of H2-agonist mediated inhibition of IgE-mediated histamine release. After different times of preincubation, the cells were stimulated with 100 IU/ml anti-IgE for 40 min.
Table 1 Inhibition of anti-IgE induce histamine release from human peripheral leukocytes. Concentration of histamine H2-agonists for 30% and 50% inhibition (IC30 , IC50) are given besides maximum of inhibition (InhMAx: means_ SEM) Histamine Hz-agonists Arpromidine BUA-75 Dimaprit FRA-19 Impromidine
IC3o [I-tM] IC~0 [IxM] (median) (median) 0.02 0.015 0.3 0.008 0.01
0.2 0.4 3 0.1 0.5
Inhr~x [%] (means)
(SEM)
60 63 57 61 55
2.9 1.8 2.7 3.1 3.4
188
Agents Actions, 35 (1992) H2 agonlst/antagonist
histaminerelease[%]
anti-lgE [IU/ml]
Inhibition [%1 80--
10 30 60-=_
*
*
" 10-6
|
(n = 6)
TT
~
100
10-7 10-9
o i
i0
3
I
i
30
i
i
100
0--
i
300
1000
Figure 3 anti-lgE [IU/rnl] Influence of 10-9, 10-7, and 10-5 M famotidine - a potent H 2antagonist - on the histamine release inhibiting effects of H2agonist FRA-19 (10-6 M). First preineubation (10 rain) with famotidine, second(10 rain) with FRA-19,stimulation of mediator release (40 rain) with anti-IgE Inhibition [%1
PDE(IV) inhibitor
90
~ (n = 5)
~
T ~
A
10s [M]
e
10 -6
[M]
o
buffer Figu]'e 4
1;-9
108
10.7 10-6 H2~gonist FR-A-19 [M]
Inhibition of IgE-mediated histamine release after combined preincubation of H2-agonist FRA-19 and cAMP specificphosphodiesterase (IV) inhibitor zardaverine. The release processwas initiated by 100 IU/ml anti-IgE.
3. The inhibition of immunological histamine release by FRA-19, a substitute of this class o f substances, was blocked with specific histamine H 2antagonists in a dose dependent manner (Fig. 3). Preincubation with 10 ~tM famotidine were sufficient to block the inhibition o f the H2-agonist FR-A-19 (10-6 M) totally. 4. In another experiment we tried to enhance the inhibititory effects o f the H2-agonists by combined incubation with a cAMP-specific P D E III/IV inhibitor zardaverine. The inhibitory effect of F R A 19 in nanomolar concentrations was significantly
I
10-9
I
10-8
I
1~0-7 10-6 1=0-5 H2-egonist FR-A-19 [M]
Figure 5 Influenceof different anti-IgE concentrationson the inhibition of histamine release by H2-agonist FRA-19.
enhanced by increasing doses o f the P D E IV inhibitor (Fig. 4). The combined inhibition was more pronounced than the effect of both compounds after single preincubation indicating a synergism in the modulation of IgE-mediated histamine release." 5. In another series o f experiments, we investigated the FRA-19 induced inhibition dependent on the dose for stimulation and its subsequent signal to release the preformed mediator. N o t only the maximum of Hz-receptor linked inhibition o f histamine release appeared as a function of the strength of the immunological signal, also IC50 levels o f F R A - I 9 were shifted in a dose dependent manner: 10, 30, and 100 IE/ml anti-IgE corresponded with 0.04, 0.1, and 0.7 g M FR-A-19 for 50% inhibition (Fig. 5). Similar experiments were also obtained with sensitized basophiles from allergic donors, using the appropriate antigen to trigger IgE mediated histamine release. Antigen-induced mediator responses o f different individuals vary in a wide range: in 12 o f 20 cases antigen-induced histamine release peaked at 10 SQ/ml grass pollen extract (Fig. 6), while 8 o f 20 claimed the maxim u m of release at 100 SQ/ml (Fig. 7). The results of both groups were depicted in different curves, to s(udy the influence of the signal on the inhibitory effect o f the Hz-agonist FRA-19. According to the individual histamine release curves and the antigen dose required for the maximum release, different inhibition curves could be obtained. Significant differences were detectable between the dose-
189
Agents Actions, 35 (1992) Inhibition
[%]
/
"
I
o. .oj
J ,ooso,., Discussion
I/
...i.o, i;; r o,
o
1...,-.
.... ..... &,
~0
tion by stimulation of H2-receptor, no significant difference was found in regard to the inhibition concentration.
I
~
o
i
Fig. 6
Inhibition
J'r
buffer
i
........
10-~
i
10-s
........
J
........
i
10`7 10-6 H2-agonist FR-Ad9 [M]
[ %)
~0-- 80
/ ~
10SQ/m[
oo
Fig. 7
oo_
t
buffer
.7
1
10-9
......
t
10-a
10-7
10-s
Figure 6/7 H2-agonist FR-A-19 [ a ] Influenceof the amountof antigen on the H2-1inkedinhibitionof IgE-mediatedhistaminereleasefromsensitizedbasophilsofallergic donors. Accordingto the individual histamine releasecurves, the resultsweredivideddue to the maximumof release(10 SQ/ml vs. 100 SQ/rnl): Fig. 6 summarizesthe dose response curves of those donors, who claimed the maximumof release at 10 SQ/ml of the allergen extract (upper left pannel). The results in Fig. 7 were obtained from sensitized donors too; their antigen induced release peaked at 100 SQ/ml (upper left pannel).
dependent inhibition curves induced by 1 and 10 SQ/ml antigen (Fig. 6), but also between 1 and 100 SQ/ml (see Fig. 7); nevertheless some of these results can only be described as trends: the lower the mediator response to a defined allergen dose, the more pronounced the inhibition of IgE mediated histamine release. Comparing anti-IgE- and antigen-induced histamine release and their inhibi-
It has been known for many years, that the preformed mediator histamine possesses the capability to inhibit its own release from human basophils [3]. This effect was blocked by histamine Hz-antagonist cimetidine, indicating an involvement of histamine Hz-receptors [12]. In the meantime, inhibition of immunological histamine release of purified basophils after activation of the H2-receptor has been proven; these effects of an auto-feedback mechanism appeared to be related to the activation of adenylate cyclase and subsequent increase of cAMP [5, 7]. A new generation of potent histamine HE-receptor agonists allowed us to reevaluate the potential inhibitory effects of these compounds. Whereas dimaprit inhibits histamine release in the micromolar range [5], the new class of phenyl (pyridylalcyl) guanidines with increased H 2 agonistic potency inhibit IgE mediated histamine release at nanomolar concentrations and are at least 10-30 more potent than dimaprit. Superoxide metabolism - triggered by the chemotactic peptide FLMP in neutrophils - was also inhibited by the H2-agonist impromidine with a IC50 value of 2 IxM [13]. This effect was competitively reversed by a specific histamine Hz-antagonist which underlines the involvement of the H2-receptor. However, quite lower inhibition concentrations to reduce basophil histamine release might indicate a more important functional role of these receptor type in the histamine containing blood cells [9]. Another effect of the activation of Hz-receptor has been shown in peripheral blood mononuclear cells: histamine, but also impromidine are able to inhibit the colony formation of phorbol ester stimulated T-cells [14]. In the guinea pig atrium system some of the new compounds (BUA-75, arpromidine, FRA-19) are full H2-agonists with potencies of up to 125-fold of that of histamine [8]. A fast onset of inhibition of super oxide formation in neutrophils was detected after a short preincubation with these cells with arpromidine [13], however positive inotropic effects of this compound were found to be slow in onset in the guinea pig atrium [8]. The dose related inhibitory effect of H2-agonist FRA-19 was more pronounced, when histamine release was
190 triggered by a suboptimal dose for IgE-mediated immunological stimulation of the basophil leukocytes. These observations were proven by anti-IgE and antigen as IgE-mediated stimuli. The differences in the inhibition of anti-IgE and antigen-induced stimuli by H 2 agonist dimaprit - demonstrated in a similar study [4] - could not be confirmed by our results, as the potent H 2 agonist FRA-19 posessed the same capability to inhibit anti-IgE- as antigen-induced histamine release. Attempts to inhibit IgE mediated histamine release with histamine H 1 antagonists as diphenhydramine failed [12]. No effect on basophil histamine release could be demonstrated after preincubation with new, recently developed histamine H1agonist in concentrations up to 10 - 6 M [15]. Similarly, neither by potent histamine H3-receptor antagonists nor by a H3-agonist (up to 10 - 6 M ) IgE mediated histamine release from peripheral leucocytes was modulated [15, 16]. Therefore, stimulation of H2-receptors on basophils might be of predominant responsibility for the inhibitory effects of the preformed mediator on its own release [17]. An additional insight into the mechanism of inhibition was possible with the cAMP specific PDE III/IV inhibitor zardaverine, known to prevent cleavage of intracellular cAMP in various cell types and tissues [18]. The synergistic effect on the inhibition of histamine release after combined inhibition of H2-agonist FRA-19 with zardaverine demonstrate two different sites of action on the cAMP second messenger system [7, 19]. Indication for a therapeutic use of H2-agonist to inhibit allergic reactions is probably limited because activation of H2-receptor in other cells and tissues might cause considerable undesired side effects.
Acknowledgement We gratefully acknowledge Heidrun Gagnr, Christa Jostles, and Gabriel Frank for their excellent technical assistance. Received 3 June 1991, accepted by W Lorenz, 25 September 1991
References [1] J. M. Arrang, M. Garbarg and J. C. Schwartz, Auto-inhibition of brain histamine release mediated by a novel class (113) of histamine receptor. Nature (London) 302, 832-837 (1983). '[2] J. M. Arrang, M. Garbarg, J. C. Lancelot, J. M. Lecomte, H. Pollard, M. Robba, W Sehunack and J. C. Schwartz, Highly
Agents Actions, 35 (1992) potent and selective ligands for histamine H3-receptors. Nature (London) 327, 117 123 (1987). [3] L. M. Lichtenstein and E. Gillespie, Inhibition of histamine release by histamine is controlled by an H 2 receptor. Nature (London) 244, 287-288 (1973). [4] G. Marone, A. Kagey-Sobotka and L. M. Lichtenstein, IgEmediated histamine release from human basophils: differences between antigen E and anti-lgE-induced secretion. Int. Arch. Allergy Appl. Immunol. 65, 339-348 (1981). [5] J. A. Warner, D. W. MaeGlashan, S. P. Peters, A. KageySobotka and L. M. Lichtenstein, The pharmacologic modulation of mediator release from human basophils. J. All. Clin. Immunol. 82, 432-438 (1988). [6] C. M. Winslow and K. G. Austen, Role of cyclic nucleotides in the activation-secretion response. Prog. Allergy 34, 236270 (1984). [7] P. T. Peachell, D. W. MacGlashan Jr., L. M. Lichtenstein and R. P. Schleimer, Regulation of human basophil and lung mast cell function by cyclic adenosine monophosphate. J. Immunol. 140, 571-579 (1988). [8] A. Buschauer, Synthesis and in vitro pharmacology of arpromidine and related phenyl (pyridylalkyl) guanidines, a potential new class of positive inotropic drugs. J. Med. Chem. 32, 1963-1970 (1989). [9] R. Burde, R. Seifert, A. Buschauer and G. Schultz, Histamine inhibits activation of haman neutrophils and HL-60 leukemic cells via H2-receptors. Naunyn Schmiedebergs Arch. Pharmacol. 340, 671-678 (1989). [10] H. Y. Lau and E L. Pearce, Effects of antihistamines on isolated human lung mast cells, basophil leukocytes and erythrocytes. Agents and Actions 27, 83-85 (1989). [11] R. P. Siraganian and W A. Hook, Histamine release and assay methods for the study of human allergy. In Manual of Clinical Immunology (Eds. N. R. Rose and H. Friedmann) pp. 808-821, American Society of Microbiology, Washington 1980. [12] L. M. Lichtenstein and E. Gillespie, The effects of the H~ and H 2 antihistamines on "gllergic" histmaine release and its inhibition by histamine. J. Pharmacol Exp. Ther. 192, 441-450 (1975). [13] R. Burde, A. Buschauer and R. Seifert, Characterization of histamine H2-receptors in human neutrophils with a series of guanidine analogues of impromidine: Are cell type-specific Hz-receptors involved in the regulation of NADPH oxidase? Naunyn Schmiedebergs Arch. Pharmacol. 341, 455-461 (1990). [14] K. Meretey, Chien HD, Falus A and Walcz E, Effect of histamine on the T-cell colony formation of PHA-stimulated cells. Agents and Actions 27, 215-217 (1989). [15] J. Kleine-Tebbe, M. Bolz, J. Schramm, H. Gagnr, C. Josties, R. Lipp, A. Freise, H. Stark, V. Zingel, A. Buschauer, W Schunack and G. Kunkel, Influence of histamine Hi-, H2-, H s- agonists and antagonists on lgE-mediated histamine release from human basophils. In New trends in allergy III (Eds. J. Ring and O. Braun-Falco) Springer-Verlag Berlin Heidelberg New York (1991) [16] J. Kleine-Tebbe, J. Schramm, M. Bolz, R. Lipp, W. Schunack and G. Kunkel, Influence of histamine H3-amagonists on human leukocytes. Agents and Actions 30, 137-139 (1990). [17] J. Ring, E Sedlmeier, D. vonder Helm, T. Mayr, V. Walz, H. Ibel, H. Riepel, B. Przybilla, H. I. Reimann and W Dorsch, Histamine and allergic diseases. In New trends in allergy H
Agents Actions, 35 (1992) (Eds. J. Ring and G. Burg) pp. 44-77, Springer-Verlag Berlin Heidelberg New York (1986). [18] Ch. Schudt, S. Winder, B. Miiller and D. Ukena, Zardaverine as a selective inhibitor of phosphodiesterase isoenzymes. Biochem. Pharmaco1., in press, (1991).
191 [19] P. T. Peachell, B. J. Undem, R. P. Schleimer, D. W. MacGlashan Jr., L. M. Lichtenstein, L. B. Cieslinksi and T. J. Torphy, Characterization and role of phosphodiesterase isoenzymes in human basophils. J. Immunol., in press (1991).
