LEUKOCYTE AND LYMPHOCYTE CYCLIC AMP RESPONSES IN ATOPIC ECZEMA

Transcription

1 THE JOtJllNAL OP INvESTJGATIV1! DE.IlMATOLOGY. 68: , 1977 Copyright by The Williams & Wilkins Co. VoL 68, No. 5 Printed in U.SA. LEUKOCYTE AND LYMPHOCYTE CYCLIC AMP RESPONSES IN ATOPIC ECZEMA CHARLES W. PARKER, M.D., SUSAN KENNEDY, M.D., AND ARTHUR z. EISEN, M.D. Department of Medicine, Divisions of Immnology and Dermatology, Washington, U niersity School of Medicine, St. Lois, Missori, U.S. A. Lymphocytes from sbjects with mild and severe atopic eczema were compared with normal control sbjects in regard to their camp (3';5'-cyclic adenosine monophosphate) responses to a variety of stimlatory agents. Individals in the severe eczema grop were shown to have a significant dimintion in their nstimlated lymphocyte camp levels and absolte camp responses to 0.5 mm theophylline, 0.5 mm theophylline+ 1 JJ.M epinephrine, 10 mm isoproterenol, 1 mm isoproterenol, 10 mm salbtamol, and 3 1-tM PGE,. Individals with mild eczema had a redced response to 0.5 mm theophylline. The severe eczema grops also differed in a nmber of these responses from a grop of5 sbjects with severe psoriasis. Mixed lekocyte camp responses to 10 mm isoproterenol also were examined and fond to be diminished in individals with eczema. A systemic alteration in beta-adrenergic responsiveness is observed freqently in individals with severe and persistent allergic bronchial asthma (reviewed in [l-3]), bt the mechanism is ncertain and in some individals concrrent bronchodilator therapy may be a contribtory factor [1,4-7]. Unfortnately, it often is not possible to withhold medications from asthmatic sbjects for a sfficiently long period of time to exclde residal effects of therapy. In attempting to determine whether the altered catecholamine response is a reflection of the allergic state, per se, an alternative approach is to stdy individals with atopic eczema who have not received bronchodilator therapy. This condition freqently is associated with bronchial hyperreactivity to cholinergic agents, high serm IgE levels, and, in some, the evental development of overt asthma LB-11]. We have previosly presented preliminary evidence that circlating lekocytes from individals with atopic eczema have a statistically redced camp (3';5' cyclic adenosine monophosphate) response to catecholamines in vitro [10]. The present report describes the reslts of more detailed stdies in this condition inclding serial observations in a nmber of affected individals. MATERlALS AND METHODS Methods for stdying hormonal responsiveness in isolated hman lekocytes and lymphocytes in vitro have been described in detail previosly [4,12]. Mixed Manscript received October 6, 1976: accepted for pblication December 2, This work was spported by U.S.P.H.S. Allergy Clinical Center (Al 10405) and Training (AM 05611) Grants. Reprint reqests to: Dr. C. W. Parker, Division of Immnology, Department of Medicine, Washington University School of Medicine, 660 S. Eclid, St. Lois, Missori lekocytes were obta.ined from heparinized peripheral blood by dextran sedimentation followed by 2 roomtemperatre washes in phosphate-saline (0.15 M NaCl, 0.01 M phosphate, ph 7.4) and 2 low-speed centrifgations to remove platelets. The qantity of blood sed varied from 25 to 150 ml Preparations sally contained 25-40% lymphocytes, the majority of the remai n ing cells being polymorphonclear lekocytes. Prified lymphocytes were obtained from dextran-sedimented mixed lekocytes by Ficoll-Hypaqe (Pharmacia Fine Chemicals, Piscataway, N.J.l density gradient centrifgation yielding 92-99% lymphocytes and few, if any, erythrocytes. The remaining cells were granlocytes and monocytes. The average lymphocyt e recovery was 70%. All prified cell preparations were centrifged at low speed to remove platelets. Reagents and soltions. Reagents and their sorces were described previosly [4]. Soltions of catecholamines (DL-isoproterenol HCl,! -norepinephrine HCI, and! -epinephrine H ClJ and theophylline were prepared in 0.15 M NaCl jst before se. Cyclic AMP determtnations. Mixed lekocytes were sspended in Gey's soltion, ph 7.8 [13], or Krebs Ringer bicarbonate bffer (Krebs' bffer), ph 7.5 [14], at a concentration of 4-8 x 10 mixed lekocytes/mj. Prified lymphocytes were sed at a concentration of2-5 x cells/ml Volmes of0.5 mj of the cell sspension were added to each tbe. Additions were made at room temperatre. Catecholamines, bffer, and other soltions were added in a volme of 0.05 ml and the cells were incbated for 1-60 min at 37 c. The ph of the cell sspensions was stable except at the highest isoproterenol and norepinephrine concentrations (10 mm) where there was a fall of abot 0.3 ph nit over a 30-min period (Gey's soltion only). In experiments with ph adjsted, 10 mm isoproterenol and norepinephrine soltions gave slightly lower camp vales. After incbation the cells were centrifged at 2500 rpm for 2 min at room temperatre. The spernatant soltions were decanted and the pellets frozen in ethanol-dry ice. Specimens were stored at or below - 7o c ntil assay. Cyclic AMP was determined by radioimmnoassay sing antibody to 2' -0-sccinyl-cyclic AMP and f' 25 l]2'- 0-sccinyl-cyclic AMP-tyrosine methyl ester. Details in

2 11ay 1977 regard to the sensitivity and specificity of the camp immnoassay have been presented previosly l15]. Immnoflorescent staining. Ficoll-Hypaqe prified cells were stained for srface immnogloblins by a florescence doble-antibody techniqe sing a mixtre of rabbit antibodies specific for hman lgg and [gm as the first antibody and floresceinated goat antirabbit IgG as the second antibody l4]. Individal rabbit antisera were shown to be monospecific by immnoelectrophoresis, and positive florescent staining responses were blocked almost completely by the appropriate hman Ig at a concentration of 1 mg/ml. Percentages of stained cells were corrected for the nmber of monocytes in the preparation. With Ficoll-Hypaqe prified lymphocytes the mean corrected percentage of florescent cells in 12 normal control sbjects was 19.5 (::!: 1.1, SEM). Hman sbjects. Hman adlt or teen-age volnteers were obtained from the patient poplation and employees of Barnes Hospital and Washington University School of Medicine. Almost a1l of the individals in the atopic eczema grop were private or clinic patients (of A.Z.E.), some of whom have been followed for periods in excess of 5 years. Data are available on a total of 48 sbjects with atopic eczema (inclding individals both with severe-characterized by prrits, erythema, and lichenification involving at least 15% or greater of their body srface-and mild or inactive disease) bt 7 of these were eliminated becase of concrrent therapy or incomplete historical information. Sbjects receiving systemic corticosteroids, oral contraceptives, or antiasthmatic medications were exclded from the stdy. None of the individals diagnosed as having atopic eczema had had persistent symptoms of bronchial asthma. Controls inclded 40 previosly reported sbjects covering the age, race, and sex range of the patient poplation as well as an additional 20 sbjects stdied in parallel with the eczema patients. In addition, 5 sbjects with clinically active psoriasis were stdied. In the normal control grop no consistent differences in responsiveness with respect to age, race, or sex were noted. Statistical analysis was by Stdent's t-test. RESULTS In lekocytes incbated in Gey's soltion for 30 min at 37"C, cells from individals with atopic eczema (12 sbjects, the majority with extensive disease) exhibited a significantly redced camp response to 10 mm isoproterenol, a concentration shown previosly to give a maximal response. In cells from patients with eczema, camp vales rose only 10 picomoles per 10; cells as compared with 26 picomoles in control cells (p < 0.05) (Tab. I). The TABLE I. Lekocyte camp responses in individals with eczema Mixed peripheral blood lekocytes were incbated in Gey's soltion for 30 min at 37"C as described in Materials and Methods; given as the mean ( :!: SEMJ. Picomoles per 10' cells Nmber Unstimlated 10 mm lsoprotereno! Eczema (:!: 0.78) 17.2 (± 3.54)" Control (± 0.50) 34.6 (± 5.0) Statistically different from control cells (p < 0.05). Theophyll1ne CYCLIC AMP RESPONSES 303 Normal Co ntrol a_ 400 With 0 w t hot 6 <t: "' = Q) c: 300 wo "' oc: wo 200 :::, :: Q) > - 0 C:_a 0 Q) Q.. Eczema Mtntes FIG. 1. Isoproterenol stimlation of mixed lekocytes as a fnction of time. Cells sspended in Krebs' bffer were stimlated with 10 mm isoproterenol for the indicated time periods in the absence and presence of 0.5 mm theophylline. The reslts are the mean (± SEM) of 4 experiments with normal control cells and 4 experiments with cells from individals with atopic eczema. Cyclic AMP vales in nstimlated cells remained within 10% of the initial vale over the 30-min time period. bffer sed did not affect the reslts, since when cells from an additional10 sbjects were incbated for either 15 or 30 min at 37 C in Krebs' lactate bffer, rather than Gey's soltion, similar reslts were obtained (not shown). Stdies of the time corse of isoproterenol stimlation revealed that differences in 4 sbjects (selected by previos screening as showing decreased camp responsiveness) when compared to 4 controls were demonstrable at 2 min and 5 min as well as at later times, indicating that prolonged incbation times are not needed to see differences between the two grops (Fig. 1). In the same experiment differences in camp responsiveness also were seen at lower concentrations of isoproterenol (Fig. 2). The next qestion we considered was the role of celllar heterogeneity in the altered camp response. Dring stdies of adrenergic responses in asthmatic sbjects, lymphocytes were fond to accont for most of the camp response to hormonal agents. making prified lymphocytes the most sitable cells for stdy [ 4]. When prified lymphocytes from individals with extensive eczema and normal sbjects were compared, again significant differences in the camp response to 10 mm isoproterenol were seen by comparison either with concrrently stdied control sbjects (Tab. ll) (p < 0.005) or with a considerably larger grop of control sbjects stdied nder identical conditions over a period of several years (p < 0.005) (not shown). Ths, variations in the nmber ofnetrophils, eosinophils, or monocytes are not the basis for the differences in lekocyte responsiveness. While alterations in relative proportions oft- and B-lymphocytes in eczema still had to be considered [16,17], immnoflorescence stdies in lymphocytes from 3 individals whose isoproterenol response was considerably redced revealed only

3 304 PARKER, KENNEDY, AND EISEN Vol. 68, No. 5 small changes in T-cell-B-cell distribtion (26 [± 3, SEM] % B-cells as compared with 19.5 l± 1.1, SEM] % in 10 normal controls). Differences of this magnitde wold not be expected to alter significantly hormonal responsiveness. Moreover, previos stdies sggest that, if anything, catecholamine responsiveness is greater in B-cells than in T-cells [4]. Nonetheless, it shold be kept in mind that we cannot completely exclde a more marked alteration in a sbpoplation oft-cells. 5 -o Ql + 0 :::> 4 E -.,_"' Ql 3 :: c a..- 0 <( 0 >- r+ 10,000 1, " I Isoproterenol Conc entrot1on, J.IM FIG. 2. The camp response of mixed lelilocytes to a range of concentrations of isoproterenol. Cells were incbated in Krebs' bffer for 30 min at 37"C. The reslts given are the experimental means from 4 experiments each with lekocytes from individals with severe atopic eczema (closed bars) and normal controls (open bars). They are expressed as the percent increase above 30-min bffer control vales (6.4 and 5.9 picomoles/107 cells, respectively) z SEM. No theophylline was present. Other pharmacologic stimli and lower concentrations of isoproterenol were examined with prified lymphocytes. Individals with severe eczema also showed a redced response to a 10-fold lower concentration of isoproterenol. Responses to prostaglandin E 1 (PGE> 3 J.LM), theophylline (0.5 mm), salbtamol (10 mm), a selective B 2 agonist, and epinephrine (1 J.LM) + theophylline (0.5 mm) were all decreased as were nstimlated cell camp vales, althogh not at the same level of statistical significance as the 10 mm isoproterenol response (Tab. II). The mean 1 J.LM epinephrine response also was less marked (25 vs 47 picomoles/10 7 cells, respectively) bt not to a level of statistical significance (0.05 < p < 0.10). It is therefore apparent that the alteration in camp metabolism is not limited strictly to adrenergic stimli. In contrast to the reslts in extensive eczema, less severe disease was not associated with statistically significant alterations in lymphocyte responsiveness althogh with most of the stimli some redction in the mean camp response was seen, sggesting that at least some individals in this grop are intermediate in their response (Tab. Ill. The single exception was the 0.5 mm theophylline r esponse which was significantly redced relative to control sbjects. Serial stdies condcted in lekocytes or lymphocytes on at least 3 occasions over a 4-year period are available in 3 originally severely affected individals. In 1 of the 3 sbjects, hormonal responsiveness was markedly decr eased on each of the 6 occasions on which the analysis was made. In the other 2 sbjects, lymphocyte responsiveness increased in association with marked clinical improvement. In 2 other sbjects stdied on at least 2 occasions the severity of the defect appeared to correlate with disease activity. TARLE ll. Comparison of lymphocyte camp responses in severe and mild eczema, psoriasis, and normal controls Prified lymphocytes were incbated in Gey's soltion for 30 min at 37 C. Data are given as the mean (± SEMl. Normal Severe eczema Mild eczema Psoriasis Nmber of sbjects 12" Bffer 25.3 ± ± :!: ± 12.0 Theophylline 0.5 mm 44.2 ± ± ± ± 18.7 Epinephrine 1 IJ.M 47.0 ± ± ± ± 32.0 Epinephrine 1 J.LM + theo ± ± ± ± 36.0 phylline 0.5 mm Isoproterenol 10 mm ± ± ± ± 15.8 Isoproterenol 1 mm 99.7 ± ± :!: 16.0 Salbtamol 10 mm 57.2 ± ± ± 18.7 PGE, 3 J.M :t: ± ± ± 79.0 Cyclic AMP vales (picomoles/10 7 lymphocytes) in 40 additional control sbjects stdied nder identical conditions bt not concrrently with eczema patients were: bffer control 26 (± 3), 3 J.LM PGE, 148 ( ± 20), 10 mm isoproterenol 140 (z 15). Similar statistical reslts are obtained comparing these vales with those in sbjects with atopic eczema. b The cells from patients with severe eczema were statistically different from normal control cells in bffer (p < 0.05), 0.5 mm theophylline (p < 0.05), 0.5 mm theophylline + 1 IJ.M epinephrine (p < 0.025), 10 mm isoproterenol (p < ), 1 mm isoproterenol (p < 0.025), 10 mm salbtamol (p < 0.05), and 3 J.LM PGE 1 (p < 0.05). The severe eczema grop was statistically different from the severe psoriasis grop in bffer (p < 0.025), 0.5 mm theophylline (p < 0.025), 11J.M epinephrine (p < 0.005), 0.5 IDM theophylline + 1 IJ.M epinephrine (p < 0.01), and 3 IJ.M PGE, (p < 0.005). The mild eczema grop was statistically different from the control grop in their response to theophylline (p < 0.05).

4 May 1977 To determine whether the alteration in lymphocyte responsiveness was specifically associated with atopic eczema or wold be manifest in sbjects with another skin disease in which alterations of camp have been implicated [18], 5 sbjects with generalized, long-standing psoriasis also were stdied. While apparent alterations in pharmacologic responsiveness were seen, the pattern was qite different from that of the severe eczema grop. Somewhat paradoxically, the response to epinephrine 1 IJ.M and epinephrine 1 IJ.M + theophylline was actally increased, whereas the response to isoproterenol (10 mm) was dimirshed. Unfortnately, lower concentrations of isoproterenol were not examined. It is possible that if this had been done, increased responsiveness wold have been seen to isoproterenol as well as to epinephrine (as might be tre if the 10 mm isoproterenol response was at the pper end of a bell-shaped dose-response crve). DISCUSSION The reslts of the present stdy confirm or earlier observations on the redction of catecholamine responsiveness in lekocytes from individals with severe atopic eczema flo] and show, in addition, that responses in their prified lymphocytes also are altered. The alterations in lekocyte camp metabolism seen are generally similar to those in severe bronchial asthma where nstimlated camp levels and responses to isoproterenol. epinephrine, and epinephrine or isoproterenol and theophylline are all redced [19]. While PGE, responsiveness is sally well maintained in asthmatic individals, the most severely affected sbjects sometimes exhibit redced responsiveness to PGE,. In individals with severe eczema, a statistically significant decrease in PGE, responsiveness is seen, bt the response to isoproterenol is more markedly affected. Since there is some redction in resting camp levels and PGE, and theophylline responsiveness, it is apparent the defect is not entirely limited to the beta-adrenergic system per se. Among the possibilities are decreased sbstrate (ATP) concentrations or availability to the adenylate cyclase, loss of a cofactor in the adenylate cyclase response sch as GTP, or ineffective copling between hormonal receptors and the cyclase. However, the decreased response to theophylline arges agajnst the third possibility. An increase in phosphodiesterase activity also mst be considered althogh if this is the mechanjsm the enzyme appears to be relatively resistant to theophylline. It also appears that the magnitde of the alteration is related to the clircal activity of the inflammatory process in the skin. Ths, the changes appear to be, at least in part, acqired rather than congenital, in some way reflecting the severity of the nderlying disea process in the skin. Similar conclsions have been drawn in bronchial asthma where individals with long periods of clinical inactivity have normal or near normal responses l4]. In less extensive CYCLIC AMP RESPONSES 305 stdies in individals with psoriasis, alterations in lekocyte responsiveness also occr to a more limited extent, bt the pattern is different from that seen in severe eczema, and it seems necessary to assme that different mechanisms are involved. Or findings in psoriasis of an increased lymphocyte camp response to epinephrine resemble those in skin by Voorhees and Dell [18) who fond that the response to 1 p.m isoproterenol was normal or increased. Or fmdjngs of redced catecholamjne responses in severe atopic eczema, on the other hand, are similar to those of Halprin et al l20] in psoriatic skin. Since extensive skin disease per se does not appear to explain the alteration, the occrrence of altered lekocyte responsiveness in association with severe atopic eczema strengthens the argment that the changes seen in asthmatic sbjects are not de solely to antiasthmatic therapy. Since camp was measred in isolated lymphocytes rather than whole blood it is possible that the changes in hormonal responsiveness that are seen are in part a reflection of the isolation procedre. Nonetheless, previos stdies indicate that altered responses are observable even after isolated lymphocytes have been majntained in tisse cltre for several hors. Moreover, since the cells are handled identically, some preexisting alteration in celllar metabolism mst be involved. The 10 mm isoproterenol concentration was selected becase it prodces maximal increases in camp in hman lymphocytes. While this concentration is very high relative to the concentrations of isoproterenol achieved in vivo, the responses are largely inhibited by beta-adrenergic blocking agents, indicating that beta-adrenergic receptors are involved. Moreover, similar changes are seen with epinephrine concentrations which prodce easily measrable responses at concentrations in the low p.m range. The basis for the defect in sbjects with atopic eczema will reqire frther stdy. A variety of observations over a period of several decades sggests that ctaneos vasclar activity is altered in this disease. Carr et al [21] stdied proliferative responses in vitro of skin biopsy specimens from individals with atopic eczema. Inhibition of cell replication by catecholamines was seen in normal skin in accord with earlier observations [18] bt not in eczematos skin, sggesting that atopic eczema was associated with a defect in ctaneos catecholamine responsiveness similar to that seen in lymphocytes. Later, direct measrements of the camp response in skin sggested that marimal absolte isoproterenol responses were normal, whereas nstimlated camp levels were increased 2-fold [22]. Measrements of ctaneos adenylate cyclase, camp-dependent protein kinase, and camp phosphodiesterase also have been reported both in this condition and in psoriasis [18,23-25]. Frther work is needed both in skin biopsy material and lekocytes to define more precisely the natre of the changes and elcidate

5 306 PARKER, KENNEDY, AND EISEN their mechanism. One possibility is that the altered responsiveness is secondary to release of one or several inflammatory mediators of immediate hypersensitivity sch as prostaglandins, chemotactic factors, SRS-A, or histamine. We wish to thank Ms. Mary Hber and Ms. Mary Bamann for excellent technical assistance. REFERENCES 1. Parker CW: Adrenergic responsiveness in asthma, Asthma: Phsiology, Immnopharmacology, and Treatment. Edited by LM Lichtenstein, KF Asten. New York, Academic, 1973, pp Reed CE: Beta-blockade and allergy. A progress report, New Concepts in Allergy and Clinical Immnology. Edited by U Serafini, A W Frankland, C Masala, JM Jamar. Amsterdam, Excerpts Medica, 1971, pp Middleton E Jr: Atoimmne imbalance in asthma with special reference to beta adrenergic blockade. Adv Intern Med 18: , Parker CW, Smith JW: Alterations in cyclic adenosine monophosphate metabolism in hman bronchial asthma. I. Lekocyte responsiveness to {3- adrenergic agents. J Clin Invest 52:48-59, Parker CW, Hber MG, Bamann ML: Alterations in cyclic AMP metabolism in hman bronchial asthma. III. Lekocyte and lymphocyte responses to steroids. J Clio Invest 52: , Nelson HS, Black JW, Branch LB, Pfetze B, Spalding H, Smmers R, Wood D: Sbsensttivity to epinephrine fo11owing administration of epinephrine and ephedrine to normal indi\ idals. J Allergy Clin Immnol 55: , Jenne JW, Strickland RD, Chick TW, Wall FJ: Indction of bet.a receptor tolerance by terbtaline (abstr). J Allergy Clin lmmnol 55:96, Masda T, Nato A, Kinoshita M, Harada S, Sasagawa S, Araki H, Makino S: Acetylcholine inhalation test in atopic dermatitis. J Allergy 40: , Jhlin L, Johansson SGO, Bennich H, Hagman C. Thyresson N: Immnogloblin E in dermatoses. Arch Dermatol 100:12-16, Parker CW, Eizen AZ: Altered cyclic AMP metabolism in atopic eczema (abstr). Clin Res 20:418, Stone SP, Gleich GJ, Mller SA: Atopic dermatitis and IgE. Arch Dermatol 112: , 1976 Vol. 68, No Smith JW, Steiner AL, Newberry WM Jr, Parker CW: Cyclic adenosine 3',5' mono phosphate in hman lymphocytes. Alterations after phytohemaggltinin stimlation. J Clin Invest 50: , Gey GO, Gey MK: The maintenance of hman normal cells and tmor cells in continos cltre. Preliminary report: the cltivation of mesoblastic tmors and normal tisse and notes on methods of cltivation. Am J Cancer 27:45-76, Umbreit WW, Brris RH, Staffer JF: Manometric Techniqes. Minneapolis, Brgess, 1957, p Steiner AL, Kipnis DM, Utiger R, Parker CW: Radioimmnoassay for the measrement of adenosine 3',5'-cyclic phosphate. Proc Natl Acad Sci USA 64: , Rachelefsky GS, Opelz G, Mickey MR. Kichi M, Terasaki PI, Siegel SC, Stiehm ER: Defective T cell fnction in atopic dermatitis: J Allergy Clin Immnol 57: , McGeady SJ, Bckley RH: Depression of cell-mediated immnity in atopic eczema. J AJlergy Clin lmmnol 56: , Voorhees JJ, Dell EA: imbalanced cyclic AMPcyclic GMP levels in psonasis. Advances in Cyclic Ncleotide Research 5: , Parker CW, Bamann ML, Hber MG: Alterations in cyclic AMP metabolism in hman bronchial asthma. II. Lekocyte and lymphocyte responses to prostaglandins. J Clin Invest 52: , Halprin KM, Adachi K, Yoshikawa K, Levine V, Mi MM, Hsta SL: Cyclic AMP and psoriasis. J Invest Dennatol 65: , Carr RH, Bsse WW, Reed CE: Failre of catecholamines to inhibit epidermal mitosis in vitro. J Allergy Clin lmmnol 51: , Lee TP, Bsse WW. Reed CE: Epidermal adenyl cyclase of hman and mose. J Allergy Clin Immnol 53: , Mier PD. Urselmann E: The adenyl cyclase of skin. II. Adenyl cyclase levels tn atopic eczema. Br J Dermatol 83: , Mier PD, Van den Hrk J, Rolla SWJ, Hollman EPMJ, Porters JE, Weemers MBM: Cyclic 3',5' adenosine monophosphate-dependenl protein kinase of skm. ll. Levels in atopic dermatitis and psoriasts. Br J Dermatol 87: , Rolla SWJ, Hollman EPMJ, Mier PD, Staak WJBM, Urselmann E, WaindorfT JA: Adenosine 3',5'-cyclic monophosphate phosphodiesterase in sldn. II. Levels in atopic dermatitis. Br J Dermato! 86: , 1972