Cyclic AMP Agonist Inhibition Increases at Low Levels of Histamine Release from Human Basophils1

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1 /81/ $2OO/O THE Jourai OF PHARMACOLOGY AND EXPERMENTAL ThERAPEUTCS Vol 218, No3 Copyright 1981 by The American Society for Pharmacology and Experimental Therapeutics p,j,,jj j,, Cyclic AMP Agonist nhibition ncreases at Lo Levels of Histamine Release from Human Basophils1 RONALD S TUNG and LAWRENCE M LCHTENSTEN Division of Clinical mmunology, Johns Hopkins University School of Medicine at Good Samaritan Hospital, Baltimore, Maryland Accepted for publication June 1, 1981 ABSTRACT Ronald S Tung and Larence M Lichtenstein: Cyclic AMP cyclic AMP, all act by increasing the cyclic AMP level Each agonist inhibition increases at lo levels of histamine release agonist as 1 - to 1 -fold more potent (relative D5) at lo from human basophils J Pharmacol Exp Ther 218: 642- levels of histamine release (5-1 % of total histamine) than at 646, high levels (5-8%) Thus, the inhibitory potential of a drug is a function of the concentration of antigen used to initiate the The relationship beteen the intensity of the signal for antigen- response Our results are no more in accord ith the inhibiinduced immunoglobulin E-mediated histamine release from tory profile of these drugs in human lung tissue t is suggested human basophils and the concentration of agonist needed to that in vivo release is likely to be lo and that this is the level inhibit release has been determined The agonists, prostaglan- at hich to evaluate drugs in vitro din E1, dimaprit, fenoterol, isobutylmethylxanthine and dibutyryl Histamine release from the basophils of mixed leukocytes dose required to exert the same result in leukocytes is greater and from the mast cells of chopped lung preparations are ell than 1 M studied in vitro models of allergic phenomena (Lichtenstein Consequently, the present study asked hether there as a and Osler, 1964; Kaliner and Austen, 1975) Release in both relationship beteen the inhibition by camp active drugs and systems can be accomplished similarly, for example, ith anti- the magnitude of histamine release The potency of these drugs immunoglobulin E antibody or ith an appropriate antigen to as examined both at high, conventionally studied levels of hich the target cells are sensitied Moreover, inhibition of release from human leukocytes and at loer levels of release histamine release occurs in both systems in the presence of previously not studied By using more precise measurements of drugs hich act on adenylate cyclase, such as beta agonists and lo percentages of release, e have shon that the inhibitory prostaglandins, and drugs hich inhibit phosphodiesterase such potential of agonists is directly related to the level of histamine as methylxanthines (Lichtenstein et at, 1972; Bourne et at, release, ith significantly greater inhibition of release at loer 1971; Orange et al, 1971; Tauber et at, 1973) levels of stimulation n spite of this, certain qualitative and quantitative differences beteen the leukocyte and lung models have been found Materials and Methods For example, hen challenged ith antigen, chopped lung Reagents BMX, dibutyryl camp and piperaine-n-n -bis(2-ethmaximally releases only 1 to 3% of total histamine, compared anesulfonic) acid ere purchased from Sigma Chemical Company (St ith 7 to 1% in mixed leukocytes (Osler et at, 1968) n Louis, MO) Human serum albumin as obtained from Miles Laboraaddition, lung fragments appear to be more sensitive to certain tories, nc (Elkhart, N) 6% Dextran 7 in 9% NaC1 as supplied by agonists, in that a loer concentration of drug achieves the McGa Laboratories (rvine, CA) EDTA as purchased from Fisher same inhibition of histamine release in lung as higher concentrations in basophils For example the concentration of isopro- Scientific Company (Fairlan, NJ) The folloing ere generously donated: dimaprit from Dr M Par- Sons, Smith Kline and French Laboratories (Herts, England); cimetiterenol necessary to inhibit 5% of histamine release from lung 7 dine from Mr J G Paul, Smith Kline and French Laboratones (Philadelphia, PA); fenoterol from Boehringer ngeiheim (Ridgefield, is beteen 1 and 1 M (Butchers et at, 1979), hereas the CT); Received for publication October 1, 198 rageed antigen E from Dr T P King (Ne York, NY); rye group antigen from Dr D Marsh (Baltimore, MD); PGE1 from Dr J Pike, The Upjohn Company (Kalamaoo, M); and sodium cromoglycate from Ms Diane Srnski, Fisons Corporation (Bedford, MA) Buffers 1) PA as used to ash leukocytes and 2) PACM as used 1 This ork as supported in part by Grant Al 729, National nstitute of Allergy and nfectious Diseases, and Grant HL 23586, National Heart, Lung and Blood nstitute, National nstitutes of Health Publication 439, O Neill Research Laboratories, The Good Samaritan Hospital for release studies The buffers ere adjusted to ph 74 Donloaded from jpetaspetjournalsorg at ASPET Journals on March 6, 16 ABBREVATONS: camp, cyclic adenosine 3 :5 -monophosphate; BMX, 3-isobutyl-1-methylxanthine; PGE,, prostaglandin E,; PA, buffer containing 25 mm piperaine-n-n -bis(2-ethanesulfonic) acid, 1 1 mm NaC, 5 mm KC and 3% human serum albumin; PACM, buffer containing PA, 6 mm CaCl2 and 1 mm MgCl2 642

2 1981 Histamine release assay Donors ere adult, rageed- or grassallergic volunteers ho had given informed consent Blood as dran into dextran-edta, alloed to sediment 9 mis and the leukocyte-rich upper layer as dran off and centrifuged The resultant leukocyte pellet as ashed tice in PA and resuspended in PA or PACM before challenge ndividual assay tubes contained the leukocytes from 3 to 3 ml of hole blood as required (see belo) n experiments involving BMX, PGE1, diinaprit and dibutyryl camp, a 1-nun preincubation of cells ith drug in PACM as alloed before addition of antigen (either rageed antigen E or rye group antigen), after hich the release process as alloed to proceed for 6 miii at 37#{176}CAntigen concentrations in each experiment ere chosen to release a ide range of total cellular histamine, from barely detectable release to near-maximal liberation, and ere never sufficient to cause antigen-excess inhibition (Lichtenstein and Osler, 1964) The total reaction volume as 1 ml per sample All experiments ere carried out in duplicate or triplicate, using three or more different donors for each experimental design When the to stages of histamine release ere studied (Lichtenstein and DeBernardo, 1971), as as necessary hen using fenoterol, cells ere preincubated for 1 mis ith drug in PA, then challenged for 2 mis ith antigen, folloed by to ashes in cold PA and final resuspension in PACM for 1 hr at 37#{176}CAfter the release reaction, the leukocytes ere removed by centrifugation (1 x g for 2 ml) and the supernatants ere assayed for histamine content by using an automated fluorometric technique (Siraganian, 1974) The difference beteen replicate histamine measurements as less than 5% Aliquots of cells in the same reaction volume ere lysed ith 2% perchlonc acid to evaluate total histamine Cells in buffer only ere incubated to determine spontaneous liberation (2-5%), hich as subtracted from all values The histamine released as then calculated as a percentage of the total histamine content None of the drugs used interfered ith the histamine assay nhibition as calculated as the percentage decrease in release vs release by antigen alone E - C V) Results n order to increase precision of the measurement of lo percentages of histamine release, the number of cells in the appropriate samples as increased by up to 1-fold the normal number of leukocytes used (usually io basophils per mffliliter of incubation tube) Figure 1 shos the histamine release from Total Histamine 6% Release -S 12%Release Ml Blood/Tube Baso xl3/tube Fig 1 Relationship beteen concentration of cells and histamine release at to concentrations of antigen Circles sho the challenge of a small concentration of antigen (1 _2 g/ml) and the squares sho a higher concentration of antigen (1 g/ml) The dashed lines represent the amount of histamine released and the solid line portrays the total histamine present The upper abscissa scale shos the milliliters of blood yielding the basophils (Baso) used in the corresponding experimental tubes and the loer scale shos the number of Baso (in thousands) per tube :i: a: a- Stimulus-nhibitor Relationshps 643 PERCENT RELEASE OF HSTAMNE DMPT lo H BMX lo4n Fig 2 Relationship beteen antigen-induced histamine release and inhibition by various camp agents The leukocytes ere preincubated ith drug for 1 mm, folloed by antigenic challenge The antigen concentrations ranged from 3 x 1 O to 1 x 1 O tg/ml DMPT, dimaprit varying numbers of leukocytes at to levels of antigen stimulation: that required to release a small percentage (12%), depicted by circles, and a high percentage (6%), represented by squares As the number of basophils increased from /mi to 165,/mi, a constant percentage of histamine as released at each level of stimulation At the loer level of stimulation, the absolute concentration of histamine liberated as only 1 to 2 ng/ml hen the normal number of basophils (1,) as used, but increased to 12 ng/ml hen 1, basophils ere employed Similarly, the histamine concentration resulting from an even smaller stimulps (such as that leading to 2% histamine release) as undetectable hen 1, basophils ere used, but became 2 ng/ml hen their number as increased to 1, Since the limit of sensitivity of the histamine assay used is 5-1 ng/ml (Siraganian, 1975), e used 2% release as the loer limit of our histamine release determinations The amount of inhibition by agonists as a function of percentage of histamine released is depicted in figure 2 For each inhibitor, in the experiments shon, there as a marked inverse relationship beteen the amount of stimulation (by antigen), expressed as the control (no inhibitor) percentage of histamine release, and the degree of inhibition by a fixed concentration of drug, in this case, BMX, PGE1 and dimaprit At lo levels of antigen stimulation, hich cause a small percentage of release, there as complete inhibition of histamine release; ith more usual antigen concentrations and the release of 5% or more of total histamine, there as less inhibition The greater inhibitory effect at lo release as not a function of delayed release, since, in separate time course experiments (not shon), release as complete by the end of incubation Next, the concentratiots of inhibitors, as ell as the level of histamine release, ere varied t as reasoned that ith concentrations of agonists previously reported to be ineffective (at high release), significant effects might still be observed at lo release Shon in figure 3, A, B, C and D are the relationships beteen antigen-induced histamine release and inhibition by varying concentrations of dimaprit, PGE1, BMX and dibutyryl cyclic AMP, respectively Figure 3A demonstrates, among other things, a marked effect of dimaprit at a concentration of 1_6 M; this causes virtually no effect at 85% release, hereas greater POE, Donloaded from jpetaspetjournalsorg at ASPET Journals on March 6, 16

3 644 Tung and Lichtenstein Vol C so a: a PERCENT RELEASE 1BMX ii5m a i OC Sc so c_4 3 i PERCENT RELEASE OM 7 5 DBcANP N to PER CENT RELEASE Fig 3 Relationship beteen antigen-induced histamine release and inhibition by camp agents at various concentrations Cells ere preincubated ith drug for 1 mm, folloed by antigenic challenge A, the effect of dimaprit (DMPT) on histamine release Antigen concentrations ranged from 5 x io-5 to 5 x i4 tg/ml Data points represent mean ± one SD of triplicate samples B, the effect of PGE, on histamine release Antigen concentrations ranged from 1 x 1 to x 1 o- ig/ml C, the effect of BMX on histamine release Antigen concentrations ranged from 4 x 1 O to 25 x 1_2 ig/ml D, the effect of dibutyryl camp (DBcAMP) on histamine release Antigen concentrations ranged from x 1 O to 6 x 1 O- ig/ml Note slight scale variation among the panels Donloaded from jpetaspetjournalsorg at ASPET Journals on March 6, 16 than 5% inhibition is observed at small (less than 1%) levels of release Furthermore, since 5% inhibition is observed at io M hen the release is 7% and at 1 M hen the release is %, it is apparent that comparable inhibition by the drug can be studied at a full order of magnitude loer if the degree of control response (histamine release) is decreased Figure 3B depicts the inhibition by several doses of PGE1 at various levels of antigen stimulation Again, using the concentration 1 M as an example in this experiment, only 13% inhibition as achieved at 75% release, hereas 4% inhibition as observed at 15% release n addition, the difference in drug concentrations needed for a comparable effect is even more strikingly ifiustrated in this figure At 7% histamine release, i M PGE1 as required for 25% inhibition; at % release, 1-times less drug (18 M) as needed to achieve the same response Similar, although not as marked, effects are seen in figure 3C ith the phosphodiesterase inhibitor BMX A concentration of io M caused only 3% suppression at 5% release, hereas 64% inhibition as observed at lo (less than 1%) degrees of release Finally, that these effects are mediated by camp is supported by the studies ith dibutyryl camp, shon in figure 3D Whereas io M dibutyryl camp led to almost no inhibition at 85% release, the same concentration caused greater than 6% inhibition at control release of less than 1% Similar results (not shon) ere obtained for the beta-2 agonist, fenoterol, in that at least a 1-fold difference in drug concentration as required for comparable inhibition of lo and of high release levels of histamine Hoever, inhibition as high as 5% as difficult to achieve at higher percentages of release (as ith PGE1 ) At lo percentages of release, the

4 1981 Stimulus-nhibitor Relationships 645 concentration necessary to block 5% (C) averaged beteen io and 1_8 M The inhibition of histamine release by histamine is antagonied competitively by H2-antihistamines (Lichtenstein and Gillespie, 1975), as is that by dimaprit, a specific H2-agonist (R S Tung and L M Lichtenstein, unpublished observation) Although inhibition of histamine release by dimaprit in the present studies as highly dependent on the degree of release, the ability of cimetidine, a specific H2-antagonist, to block this inhibition as found to be constant regardless of magnitude of release n three experiments, the KB of cimetidine vs dimaprit as determined by standard techniques (Tallarida et at, 1979) simultaneously at lo (8 ± 5) and at high (66 ± 74) release At lo release, the KB averaged 24 ± 7 x j#{216}7 M and at high levels 31 ± 14 X i7 M (P = NS) Sodium cromoglycate, an agent hich inhibits histamine release from rat mast cells (Garland, 1973) and human mast cells (Assem and Mongar, 197) but hich as previously considered not to affect that from human basophils (Lichtenstein et a!, 1979), as tested at lo antigen concentrations to see hether effects could be seen at lo percentages of release Hoever, at concentrations up to 1 M, no inhibition by sodium cromoglycate as observed regardless of the degree of control histamine release Discussion These results indicate that a different potency of agonists hich inhibit histamine release by acting on adenylate cyclase to increase camp ill be appreciated, depending on the control amount of histamine release hich is examined At lo levels of release, much loer concentrations of agonists ere required hen compared ith those needed at high levels of release This difference, at least 1-fold for all drugs and as much as 1-fold in one case (PGE1 ), held for all agonists tested (PGE1, dimaprit and fenoterol), as ell as for the phosphodiesterase inhibitor BMX, and the camp analogue, dibutyryl camp Cimetidine, a specific H2-antihistamine, antagonied the inhibition by dimaprit, but its potency (as measured by KB values) as independent of degree of release, consistent ith classical receptor theory These results are of interest for several reasons As mentioned previously, lung mast cells are usually capable of releasing only 1 to 3% of their histamine, no matter ho large the antigenic challenge, hereas basophils are able to release much greater percentages (The reason for this difference is not clear) To use basophils as a model for the pathological site of disease (in this case, the mast cells of the lung), it seems reasonable to study those proportions of histamine release in basophils comparable ith those likely to be occurring in vivo in mast cells, ie, no greater than 1 to 3% Additional support for the importance of lo levels of histamine release from lung mast cells comes from to findings: 1) maximal guinea-pig tracheal contraction from anaphylactic challenge occurs ith only 7% histamine release (Adams and Lichtenstein, 1979) and 2) antigen levels achieved in vivo are at best very small (ie, extremely unlikely to cause more than 1-3% histamine release even in basophils) (Marsh, 1975) Thus, if the mixed leukocyte assay is to be used to assess the potential in vivo utility of a drug, inhibition of lo levels of histamine release ould appear to be more pertinent n this study, a ide range ofhistamine release from basophils has been examined by varying the intensity of stimulus t has been shon that the degree of inhibition by camp drugs in basophils is highly dependent on the magnitude of histamine release Whereas a given concentration of drug may effectively inhibit a (conventional) release of 5 to 8%, at least a 1-fold loer concentration ill achieve the same result at lo (ca 5-1%) release, hich is probably of greater clinical relevance When our drug inhibition data of basophil histamine release are compared ith published data regarding the effects of comparable drugs in human lung tissue, some interesting points emerge First, hereas concentrations of 5 to 75 x 1 M isoproterenol and salbutamol, to beta adrenergic agonists, inhibit 5% of histamine release (Co) in human lung (Tauber et at, 1973; Butchers et at, 1979), the concentration of fenoterol (another beta agonist) needed to net the same inhibition is greater than 1_6 M hen the histamine release from basophils is greater than 5% Hoever, the Co is much less, beteen io- and io M, hen the release is only 1% The similarity in C among these three drugs in analogous (lo) percentages of histamine release is striking Second, a like comparison can be dran beteen the inhibition by PGE1 of histamine release from basophils vs mast cells The published Co of PGE1 in human lung tissue, 5 x i M (Tauber et at, 1973), closely matches the Co e have found in basophils at lo levels of release (1 M, fig 3B) Finally, although inhibition by dimaprit in basophils is also markedly dependent on base-line histamine release, this is still at variance ith the results reported from mast cell studies, in hich stimulation by H2-agonists does not change lung mast cell histamine release (Kaliner, 1978) The reason is unknon, but possible explanations include baseline release by the tissue ofhistamine (leading to desensitiation of H2-receptors) and/or significant metabolic breakdon by lung tissue t is clear, hoever, that histamine release by basophils or mast cells is inhibited by similar concentrations of most drugs tested hen similar levels of histamine release are examined This strengthens the analogy beteen the to cell systems and supports the usefulness of basophils in evaluation of allergic processes Drug studies of purified lung mast cells (Patterson et at, 1976) to test the analogy more critically ill be very interesting Although examination ofdrug effects at lo histamine release brought out subtle inhibition not seen previously, sodium cromoglycate still proved ineffective in the basophil system, regardless of the base-line release level The explanation for the disparate effects of this drug on mast cells vs basophils remains elusive n addition, the present studies point out the balance beteen stimulus and inhibitor This agonist-antagonist relationship, pointed out by Ari#{235}ns(1964), has more recently been shon to exist in other systems For example, in both calf tracheal muscle and guinea-pig ileum, greater relaxation is achieved by isoprenaline, a beta agonist, if the control amount of contraction by methacholine or histamine is reduced (Van den Brink, 1973) Furthermore, dibutyryl camp leads to greater inhibition of compound 48/8-induced histamine release from rat mast cells at the loest concentrations of releasing agent (Pearce et al, 1979) n our study, the same relationship has been found: the less stimulus present (leading to decreased histamine release), the less inhibitor required to suppress it This has important pharmacological implications in that the inhibitory potential such as C25 or Co of a given drug (in this case, an inhibitor of histamine release) is highly dependent on the intensity of the event being studied (in this case, control histamine release) Donloaded from jpetaspetjournalsorg at ASPET Journals on March 6, 16

5 646 lung and Lichtenstein Vol 218 References ADAMS, G K AND LCHTENSTEN, L M: n vitro studies of antigen-induced bronchospasm: Effect of antihistamine and 8118-A antagonist on response of sensitied guinea pig and human airays to antigen J mmunol 122: , i979 ARENS, E J, SMONS, A M AND VAN ROSSUM, J M: Drug-receptor interaction: nteraction of one or more drugs ith different receptor systems n Molecular Pharmacology, ed by E J Ariens, vol, p 287, Academic Press, Ne York, 1964 ASSEM, E S K AND MONGAR, J L: nhibition of allergic reactions in man and other species by cromoglycate mt Arch Allergy App! mmunol 38: 68-77, 197 BOURNE, H R, MELMON, K L AND LCHTENSTEN, L M: Histamine augments leukocyte cyclic AMP and blocks antigenic histamine release Science (Wash DC) 173: , 1971 BUTCHERS, P R, FULLARTON, J R, SKDMORE, F, THOMPSON, L E, VARDEY, C J AND WHEELDON, A: A comparison of the anti-anaphylactic activities of salbutamol and disodium cromoglycate in the rat, the rat mast cell, and in human lung tissue Br J Pharmacol 67: 23-32, 1979 GARLAND, L G: Effect of cromoglycate on anaphylactic histamine release from rat peritoneal mast cells Br J Pharmacol 49: , 1973 KALNER, M: Human lung tissue and anaphylaxis: The effects of histamine on the immunologic release of mediators Annu Rev Respir Dis 118: , 1978 KALNER, M AND AUSTEN, K F: mmunologic release of chemical mediators from human tissues Am Rev Pharmacol : , 1975 LCHTENSTEN, L M AND DEBERNARDO, R: ge-mediated histamine release: n vitro separation into to phases mt Arch Allergy App mmunol 41: 56-71, 1971 LcHTENSTEN, L M, FOREMAN, J C, CONROY, M C, MARONE, G *x m NE- BALL, H H: Differences beteen histamine release from rat mast cells and human basophils and mast cells n The Mast Cell ts Role in Health and Disease, ed by J Pepys and A M Edards, Pitman Medical, Kent, England, pp 83-96, 1979 LcHTENSTEN, L M AND GLLESPE, E: The effects of the H and H2 antihistamines on allergic histamine release and its inhibition by histamine J Pharmacol Exp Ther 192: , 1975 LCHTENSTEN, L M, GLLESPE, E, BOURNE, H R AND HENNEY, C S: The effects of a series of prostaglandins on in vitro models of the allergic response and cellular immunity Prostaglandins 12: , 1972 LcHTEN5TEN, L M AND OSLER, A G: Studies on the mechanisms of hypersensitivity phenomena X Histamine release from human leukocytes by rageed pollen antigen J EEl) Med 1: , 1964 MARSH, D G: Allergens and the genetics of allergy n The Antigens, ed by M Sela, vol, pp 27i-359, Academic Press, Ne York, 1975 ORANGE, R P, KAUNER, M A, LARAA, P J AND AUSTEN, K F: mmunological release of histamine and slo reacting substance of anaphylaxis from human lung nfluence of cellular levels of cyclic AMP Fed Proc 3: , 1971 OSLER, A G, LcHTENSTEN, L M AND LEVY, D A: n vitro studies of human reaginic allergy Adv mmunol 8: , 1968 PATTERSON, N A M, WASSERMAN, S, SAD, J W AND AUSTEN, K P: Release of chemical mediators from partially purified human lung mast cells J m munol 117: , 1976 PEARcE, F L, ATKNSON, G, ENNS, M, ThuNcH, A, WESTON, P M AND WHTE, J R: Effect of anti-allergic compounds on histamine release induced by basic agents, antigen, and the calcium ionophore A23187 n The Mast Cell ts Role in Health and Disease, ed by J Pepys and A M Edards, pp 69-75, Pitman Medical, Kent, England, 1979 SRAGANAN, R P: An automated continuous-flo system for the extraction and fluorometric analysis of histamine Anal Biochem 57: , 1974 SRAGANAN, R P: Refinements in the automated fluorometric histamine analysis system J mmunol Methods 7: , 1975 TALLARDA, R J, COWAN, A AND ADLER, M W: pa2 and receptor differentiation: A statistical analysis of competitive antagonism Life Sci 25: , 1979 TAUBER, A, KALNER, M, STECHSCHULTE, D J AND AUSTEN, K F: mmunologic release of histamine and slo reacting substance of anaphylaxis from human lung V Effects of prostaglandins on release of histamine J mmunol 111: 27-32, 1973 VAN DEN BRNK, F G: The model of functional interaction H Experimental verification of a ne model: The antagonism of fl-adrenoceptor stimulants and other agonists Eur J Pharmacol 22: , 1973 Send reprint requests to: Dr Larence M Lichtenstein, Division of Clinical mmunology, Good Samaritan Hospital, 561 Loch Raven Boulevard, Baltimore, MD Donloaded from jpetaspetjournalsorg at ASPET Journals on March 6, 16