Plasma histamine and catecholamines in stable asthmatic subjects

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1 Clinicl Science (1982) 62, Plsm histmine nd ctecholmines in stble sthmtic subjects P. J. BARNES, P. W. IND AND M. J. BROWN Deprtments of Medicine nd Clinicl Phrmcology, Royl Postgrdute Medicl School, Hmmersmith Hospitl, London (Received 19 June 1981; ccepted 8 December 1981) Summry 1. Venous plsm histmine nd ctecholmines were mesured in stble sthmtic subjects by recently developed specific nd sensitive rdioenzymtic ssy. 2. Plsm histmine concentrtions were significntly elevted in both extrinsic nd intrinsic sthmtic subjects, compred with both norml controls nd ptients with chronic obstructive irwys disese. There ws no correltion between histmine concentrtion nd severity of irwys obstruction, however. 3. Elevted plsm histmine concentrtions t rest my indicte incresed relese of meditors from leky mst cells in sthm. 4. Plsm ctecholmine concentrtions in sthmtic subjects did not differ from norml nd there ws no correltion with severity of bronchoconstriction or with plsm histmine concentrtion. Key words: sthm, ctecholmines, histmine, mst cell meditors. Introduction Histmine is relesed from sensitized humn lung 11, 21 nd from bsophils [31 on ntigen chllenge in vitro. Histmine cuses bronchoconstriction by direct ction on bronchil smooth muscle nd indirectly by vgl reflex mechnisms. Whether histmine relesed from pulmonry mst cells cn be detected in the circultion is less certin, however. Fluorimetric ssys for histmine, which hve been vilble Correspondence: Dr Peter Brnes, School of Medicine, Crdiovsculr Reserch Institute, University of Cliforni, Sn Frncisco, CA 94143, U.S.A. for mny yers [41, re too insensitive to mesure the low concentrtions of histmine in humn plsm. More recently rdioenzymtic ssys with gretly improved sensitivity hve been used to mesure histmine in plsm; with such ssys incresed concentrtions of plsm histmine hve been reported in sthmtic subjects during ntigen chllenge [SI nd during spontneous ttcks [6, 71, lthough resting vlues were not significntly different from norml. Using rdioenzymtic ssy for plsm histmine with incresed sensitivity nd precision, we hve exmined the resting concentrtions of plsm histmine in sthmtic, chronic bronchitic nd non-sthmtic control subjects. Mst cells contin other bronchoconstrictor meditors, including prostglndin D,, leucotrienes nd brdykinin, which my ll contribute to bronchospsm in sthm [81. Since there is no evidence tht these meditors my be differentilly relesed it is possible to use plsm histmine s mrker of meditor relese in uivo. Adrenergic gonists re the most effective nd widely used bronchodiltors in the tretment of sthm. Conversely b-drenoceptor ntgonists cuse incresed bronchoconstriction in sthmtic [9-111 but not in norml subjects [12]. This suggests tht there my be incresed pdrenergic stimultion of sthmtic irwys. Since there is no convincing evidence for direct sympthetic innervtion of humn irwys [131, it is likely tht circulting endogenous ctecholmines, prticulrly drenline, ply n importnt role in the regultion of irwy tone in sthm. As with histmine, it is not possible to mesure plsm ctecholmines, prticulrly drenline by fluorimetric ssy, but the introduction of sensitive rdio-enzymtic ssys [14, 151 hs llowed the mesurement of endogenous ctecholmines in The Biochemicl Society nd the Medicl Reserch Society

2 662 P. J. Brnes, P. W. Ind nd M. J. Brown humn plsm. We hve used modifiction of these ssys with improved specificity nd sensitivity to study resting drenline nd nordrenline concentrtions in cliniclly stble sthmtic subjects. Ctecholmines my ct directly on irwy /.-receptors to produce bronchodilttion, or my ct on mst cell /.-receptors to reduce the relese of bronchoconstrictor meditors [16, 171. We hve therefore lso exmined the reltionship between drenline nd histmine concentrtions in plsm. Methods Subjects We studied ptients with sthm demonstrted by > 20% increse in forced expirtory volume in 1 s (FEV,) fter inhled bronchodiltor, nd sputum or blood eosinophili (0.3 x lo9 cells/l). They were clssified into extrinsic [multiple positive immedite rections to skin prick tests with bttery of common llergens, nd elevted serum immunoglobulin E (IgE) concentrtions1 or intrinsic, with negtive skin tests nd norml serum IgE concentrtions. All sthmtic subjects were in stble clinicl stte with no excerbtion within 4 weeks of study. We lso studied ptients with symptomtic chronic bronchitis nd irreversible irflow obstruction who hd negtive skin tests. All ptients were selected from the Chest Clinic, Hmmersmith Hospitl, nd were normlly mbulnt before blood smpling. Norml subjects with no history of sthm were selected from mongst medicl nd lbortory personnel, nd were clssified into topic (multiple positive skin tests) or non-topic (Tble 1). Norml subjects were mtched in ge to the extrinsic sthmtic group, so tht n effect of ge on plsm ctecholmine concentrtions ws voided. We hve found no effect of ge on plsm histmine concentrtions (unpublished observtions). None of the ptients ws tking orl mediction, nd none hd tken orl steroids within 2 months of smpling. All use of inhlers ws stopped t lest 4 h before smpling. In ll subjects FEV,., ws mesured by dry spirometry (McDermott, Grw Instruments, Glmorgn, U.K.) nd the highest of three consecutive forced expirtory mnoeuvres recorded. Blood ws smpled betwen hours nd hours in ll subjects, so tht the effect of circdin vritions in plsm histmine nd ctecholmine concentrtions ws not significnt. A smpling cnnul (Butterfly 19 g, Abbott Lbortories) ws inserted into forerm vein nd the subject rested supine for t lest 15 min before blood ws drwn. Blood ws collected into pre-chilled plstic tubes contining 5 ml of ethylenediminetetr-cetic cid (0.5 mol/l) for histmine estimtion nd into lithium-heprin tubes (5 ml) for ctecholmine ssy. Plsm ws seprted by centrifugtion t 4OC within 30 min of collection, nd stored t -8OOC until nlysed. Assys Plsm ctecholmines were mesured by recently developed double-isotope technique modifiction of the ctechol 0-methyltrnsferse (COMT) ssy, which llows both high precision nd sensitivity [181. Seprte liquots of ech smple were incubted with COMT (prepred from rt liver) nd either S-[3H ldenosylmethionine or S-[ 14Cldenosylmethionine (The Rdiochemicl Centre, Amershm, Bucks, U.K.). Nordrenline nd drenline stndrds (100 ng/ml) were lso dded to the ltter liquots nd converted into I 14Clmetnephrines, which were dded s trcers to the 3H tubes fter incubtion. The finl recovery of 14C corrects both for incomplete methyltion nd for losses during extrction nd during thin-lyer chromtogrphy. The sensitivity of this ssy ws 0.06 TABLE 1. Detils of subjects before mesuremen1 of plsm ctecholmines nd histmine Norml Asthmtic Chronic bronchitic Non-topic Atopic Extrinsic Intrinsic No I2 10 Sex All mle All mle 21 mle 5 mle 6 mle Age (yers) Men & SEM 27.2 i i & i f 2.1 Rnge ) predicted FEV, Men t SEM 99.3 C C C f 7.5 Rnge 89- I I5 88- I

3 Histmine nd ctecholmines in sthm 663 nmol/l for both nordrenline nd drenline. Intr- nd inter-ssy coefficients of vrition were 4.6% nd 9.8% respectively. Plsm histmine ws mesured by similr double-isotope modifiction of our previously described rdioenzymtic method [ 191. Histmine N-methyltrnsferse ws prepred from rt kidney. Sensitivity ws 0.2 nmol/l nd intr- nd inter-ssy coefficients of vrition were 6.3% nd 13.4% respectively. This ssy hd sensitivity much greter thn ny previously reported method Results were nlysed by Student's t-test for unpired smples, nd by Spermn's rnk correltion test. Results In norml subjects (n = 26) plsm histmine ws 2.42 f 0.17 nmol/l (men +_ SEM) nd there ws no significnt difference between non-topic (n = 20) nd topic (n = 6) subjects (Fig. 1). In ptients with extrinsic.sthm (n = 26) plsm histmine ws significntly higher thn in gemtched norml controls (5.72 f 0.38 nmol/l; P < 0.01). There ws no significnt correltion between plsm histmine concentrtion nd severity of sthm s mesured by % predicted FEV,., (r = 0.004). Similrly, in ptients with intrinsic sthm (n = 12) plsm histmine concentrtions were significntly higher thn in norml subjects (6.2 f 0.29 nmol/l; P < 0.01) nd did not differ significntly from vlues in extrinsic sthmtic subjects. By contrst, in chronic bronchitic ptients (n = 10) plsm histmine did not differ significntly from norml (2.05 f 0.32 nmol/l), lthough the severity of irflow obstruction ws not significntly different from tht in the intrinsic sthmtic group (Tble 1). Plsm drenline (0.45 f 0.03 nmol/l) nd nordrengline (1-76 +_ 0.12 nmol/l) in extrinsic sthmtic subjects (n = 20) did not significntly differ from vlues in ge-mtched control subjects (drenline 0.45 f 0.05 nmol/l; nordrenline 2.12 f 0.19 nmol/l; n = 20) (Fig. 2). There ws no significnt correltion between % predicted FEV,., nd plsm concentrtion of either drenline (r = 0.21) or nordrenline (r = 0.24). There ws no significnt correltion between plsm drenline nd plsm histmine (r = 0.023). Discussion Resting plsm histmine concentrtions were significntly higher in both extrinsic nd intrinsic sthmtic subjects compred with either norml subjects or chronic bronchitic subjects who hd similr degree of irwy obstruction. In previous studies with rdioenzymtic ssys lthough "1 P<O.Ol PC0.01 ' Non-:topic ** I i f # m % & "p m I Atkic Exirksic lntrkic Norml Asthmtic Chronic bronchitic FIG. 1. Venous plsm histmine in non-topic norml, topic non-sthmtic, extrinsic sthmtic, intrinsic sthmtic nd chronic bronchitic subjects. Mens f SEM for ech group re significndy higher (P < 0-01) thn those for norml or chronic bronchitic groups. 0' NAD A NAD A Norml Asthmtic FIG. 2. Venous plsm nordrenline (NAD, 0) nd drenline (A, 0) in norml subjects (n = 20, men ge 28.1 yers) nd extrinsic sthmtic subjects (men ge 23.5 yers). Mens f SEM vlues re lso shown.

4 664 P. J. Brnes, P. W. Ind nd M. J. Brown elevted concentrtions of plsm histmine hve been reported fter ntigen [51 nd spirin chllenge [211, nd during cute excerbtions of sthm [6, 71, elevted plsm histmine concentrtions hve not been found during remission. By using n ssy for plsm histmine with gretly incresed sensitivity it hs been possible to differentite sthmtic from norml subjects even during remission. As there is no good evidence for ltered clernce of plsm histmine in sthmtic compred with norml subjects (P. W. Ind, unpublished observtions), this implies n incresed secretion of histmine into the circultion either from mst cells or from circulting bsophils. There is no evidence tht mst cell meditors my be relesed differentilly, nd the elevted plsm concentrtions of histmine therefore suggest incresed relese of mst cell meditors t rest in sthmtic subjects. There ws no difference in plsm histmine between extrinsic nd intrinsic sthmtic subjects, demonstrting tht meditor relese is not solely relted to IgE-dependent mechnisms. The primry defect in sthm my therefore be incresed relese of meditors by vriety of stimuli, which my not be immunologicl, such s exercise [22]. This is supported by the finding tht bsophils from extrinsic nd intrinsic sthmtic subjects show incresed relese of histmine to vriety of non-immunologicl stimuli [231. If the primry disorder in sthm is incresed lekiness of mst cells, this might led to the bronchil hyper-rectivity which is so chrcteristic of sthm. The lck of correltion between plsm histmine concentrtion nd bronchoconstriction suggests either tht concentrtions of histmine in venous blood do not closely reflect the mount of histmine relesed loclly in the vicinity of the irwys, or tht other fctors such s vgl tone or drenergic mechnisms my modulte bronchil tone. The wide inter-individul rnge in plsm histmine concentrtion my hve obscured ny reltionship to severity of bronchoconstriction, wheres in sequentil mesurements in n individul significnt reltionship between plsm histmine concentrtion nd bronchoconstriction ws found [241. There ws no bnormlity in plsm ctecholmines in sthmtic subjects, nor ny reltionship between plsm ctecholmine concentrtion nd bronchoconstriction. This is surprising since p-drenoceptor-blockers cuse incresed bronchospsm in sthmtic subjects [9-111, nd the lck of direct sympthetic innervtion of the irwys [ 131 might suggest tht endogenous ctecholmines, prticulrly d- renline, my be elevted in these subjects. There ws no significnt correltion between plsm histmine nd plsm drenline. In previous study the cirdin vrition in plsm drenline ws closely correlted with plsm histmine concentrtions, suggesting tht circulting drenline modulted meditor relese (241. Such reltionship ws lmost certinly obscured in the present study by the lrge rnge in plsm histmine concentrtions in the sthmtic group. These results show incresed plsm histmine concentrtions in both extrinsic nd intrinsic sthmtic subjects t rest, suggesting incresed resting relese of mst cell meditors in sthm. Resting plsm ctecholmine concentrtions in sthmtic subjects re not different from vlues in ge-mtched norml controls, however. References I I I SCHILD, H.O., HAWKINS, D.F.. MONGAR, J.L. & HERxHEIMEn. H. (I95 I) Rections of isolted humn sthmtic lung nd bronchil tissue to specific ntigen. Histmine relese nd musculr contrction. Lncer, li, I21 BROCKLEHURST. W.E. (1960) The relese of histmine nd formtion of slow recting substnce (SRS-A) during nphylctic shock. JournlofPhysiologv (London), 151, LICHTENSTEIN, L.M. & OSLER, A.G. (1964) Studies on the mechnisms of hypersensitivity phenomen. IX. Histmine relese from humn leucocytes by rgweed pollen ntigen. Journl of Experimentl Medicine. 120, SHORE. P.A.. BURKHALTER, A. & COHN, V.H. (1959) A method for the fluorometric ssy of histmine in tissues. Journl of Phrmcology nd Experimenrl Therpeurics. 127, BHAT. K.N.. ARROYAVE. C.M., MARNEY. S.R., STEVENSON. D.D. & TAN, E.M. (1976) Plsm histmine chnges during provoked bronchospsm in sthmtic ptients. Journl of Allergy nd Clinicl Immunology, 58, SIMON. R.A.. STEVENSON, D.D., ARROYAVE, C.M. & TAN, E.M. (1977) The reltionship of plsm histmine to the ctivity of bronchil sthm. Journl of Allergy nd Clinicl Immunology, 60, CHARLES, T.J., WILLIAMS. S.J.. SEATON, A,. BRUCE, C. & TAYLOR, W.H. (1979) Histmine, bsophils nd eosinophils in severe sthm. Clinicl Science, I81 AusTEN. K.F. (1979) Biologicl implictions of the structurl nd functionl chrcteristics of the chemicl meditors of immedite-type hypersensitivity. Hmev Lecture Series 73, pp Acdemic Press, New York. 191 MCNEILL. R.S. (1964) EtTect of P-drenergic blocking gent. proprnolol, on sthmtics. Lncet, 11, I101 MCNEILL. R.S. & INGRAM, C.G. (1966) Effect of proprnolol on ventiltory function. Americn Journl of Crdiology, 18, ZAID. G., & BEALL, G.N. (1966) Bronchil response to bet-drenergic blockde. New Englnd Journl of Medicine. 275,58&584. I121 TATTERSFIELD. A.E.. LEAVER, D.G. & PRIDE, N.B. (1973) Effect of 8-drenergic blockde nd stimultion on norml humn irwys. Journl of Applied Physiology, I 131 RICHARDSON, J.B. (1979) Nerve supply to the lungs. Americn Review ojrespirtory Disese, I 19, DA PRADA, M. & ZURCHER, G. (1979) Simultneous rdioenzymtic determintion of plsm nd tissue drenline. nordrenline nd dopmine within the femtomole rnge. Lve Sriences. 19,

5 Histmine nd ctecholmines in sthm PEULER. J.D. & JOHNSON. G.A. (1977) Simultneous single isotope rdio-enzymtic ssy of plsm norepinephrine, epinephrine nd dopmine. Lfe Sciences, 21, I161 ASSEM. E.S.K. & SCHILD, H.O. (1969) Bet-drenergic receptors concerned with the nphylctic mechnism. Interntionl Archives of Allergy. 45, ORANGE. R.P.. AIJSTEN, W.G. & AUSTEN, K.F. (1971) Immunologicl relese of histmine nd slow recting substnce of nphylxis from humn lung. 1. Modultion by gents influencing cellulr levels of cyclic 3 3-denosine monophosphte. Journl of Experimenrl Medicine. 134, I181 BROWN. M.J. & JENNER, D.A. (1981) Novel double-isotope technique for enzymtic ssy of plsm ctecholmines, permitting high precision, sensitivity nd smple cpcity. Clinicl Science, BROWN. M.J., IND. P.W., BARNES, P.J., JENNER D.A. & DOLLERY, C.T. (1980) A sensitive nd specific rdiometric method for the mesurement of plsm histmine in norml individuls. Anlylicl Biochemistry, 109, I201 GLEICH, G.J. & HULL, W.M. (1980) Mesurement of histmine: qulity control study. Journl of Allergy nd Clinicl Immunology, 66, STEVENSON, P.D., ARROYAVE, C.M., BHAT, K.N. & TAN, E.M. (1976) Orl ASA chllenges in sthmtic ptients: study of plsm histmine. Clinicl Allergy, 6, BARNES, P.J. & BROWN, M.J. (1981) Venous plsm in histmine in exercise nd hyperventiltion-induced sthm in mn. Clinicl Science, 61, FINDLAY, S.R. & LICHTENSTEIN, L.M. (1980) Bsophil relesbility in ptients with sthm. Americn Review oj Respirtory Disese, 122, BARNES, P., F~GERALD, G., BROWN, M. & DOLLERY, C. (1980) Nocturnl sthm nd chnges in circulting epinephrine, histmine nd cortisol. New Englnd Journl oj Medicine, 303,