Impaired Beta Adrenergic Receptor Binding and

Function in Cystic Fibrosis Neutrophils

STANLEYP. GALANT, LYDIA NORTON,JOHNHERBST, and CAROLYNWOOD,Department of Pediatrics, University of Utah, Salt Lake City, Utah 84321

A B S T R A C T Cystic fibrosis (CF), a genetic diseasecharacterized by abnormalities of exocrine gland andmucociliary function, has recently been shown to beassociated with abnormal adrenergic and cholinergicphysiologic responses in addition to decreased betaadrenergic-induced cyclic AMPgeneration in humanleukocytes. In this study we have attempted to eluci-date the nature of this hyporesponsiveness by assess-ing beta adrenergic receptor number and affinity (KD)in the intact neutrophil using the antagonist ligand[3H]dihydroalprenolol and cyclic AMP responses toisoproterenol in addition to histamine, and prosta-glandin E, in CF subjects, CF obligate heterozygotes(CFH), and normal control subjects. CF patients hadsignificantly less (P < 0.025) cyclic AMPstimulationabove basals levels with isoproterenol (0.1 ,uM to0.1 mM), compared with control values, but no con-sistent differences between groups were noted withhistamine or PGEI. CF neutrophils had significantlyfewer (P < 0.005) beta adrenergic receptors perneutrophil (398.0+54.2 vs. 819.4+67.2) compared withcontrol neutrophils, but the KD (0.740±0.11 vs. 0.630+0.05 nM) did not differ significantly (P > 0.05). Therewas no correlation between clinical severity and eithercyclic AMPgeneration or dihydroalprenolol binding(r = 0.27 and 0.24, respectively, P > 0.05). The CFHgroup had -50% of the cyclic AMP stimulationcompared with controls, but the number (909.8+89.3)and KD (0.710+0.09 nM) of their beta adrenergicreceptors were indistinguishable from control subjects.These findings suggest "down regulation" of the betareceptor in the CF patient. The cause of this remainsunknown. Although the etiology of the decreasedcyclic AMP responses in CFH was not due to de-creased beta adrenergic receptors as assessed byantagonist ligand binding, further studies in the CFH

Address reprint requests to Dr. Galant, Memorial HospitalMedical Center of Long Beach, Calif.

Received for publication 12 August 1980 and in revisedform 23 March 1981.

group to include agonist binding, receptor-adenylatecyclase coupling, intrinsic adenylate cyclase activity,and catecholamine metabolism may help determinethe basic cause of beta adrenergic hyporesponsive-ness in both CFHand CF.

INTRODUCTION

Cystic fibrosis (CF)' is a genetic disease characterizedby abnormal exocrine secretion and mucoiliary ac-tivity (1, 2). The pathogenesis of the disease remainsunknown at present, but because of the importanceof the autonomic nervous system (ANS) in regulatingexocrine gland secretion, assessment of adrenergicand cholinergic function has become a major topic ofinvestigation. For example, chronic administration ofpharmacologic agents with ANS activity to laboratoryanimals has been shown to produce exocrine glandfunction and morphology that closely resembles thoseseen in CF (3, 4). Physiologic assessment of the ANSin CF has revealed decreased beta adrenergic, andheightened alpha and cholinergic function (5-8). Themolecular basis for an ANSabnormality in CFhas beenbest studied for the beta adrenergic system sincethis pathway, involving the generation of the secondmessenger cyclic AMP, has been most clearly eluci-dated (9). The majority of studies utilizing humantissue have used the skin fibroblast, because this cellproduces ciliostatic factor (10) and was thought to befree of drugs and other in vivo effects such as theseverity of CF. Unfortunately, data using this modelhave conflicted with several studies showing enhancedbeta adrenergic responses (11, 12), whereas othersreport no difference in isoproterenol-induced cyclicAMPgeneration between CF and control populations(13). These contradictory observations probably re-flect the large number of variables inherent in tissueculture systems.

'Abbreviations used in this paper: ANS, autonomousnervous system; CF, cystic fibrosis; CFH, cystic fibrosisheterozygote(s); DHA, dihydroalprenolol.

J. Clin. Invest. (© The American Society for Clinical Investigation, Inc. - 0021-9738/81/0710253/06 $1.00Volume 68 July 1981 253-258

253

The human neutrophil offers a number of advantagesin this regard. This cell type can be highly purified(>95%) in relatively large numbers (2 x 108/60 ml)without the necessity to establish a cell line in culture.Regulation of neutrophil lysosomal enzyme secretion iscontrolled by the same neurotransmitters as are foundin exocrine tissues. Secretion is inhibited by mediatorsthat increase intracellular cyclic AMP(isoproterenol,histamine, and prostaglandin E1, [PGE1]) and is en-hanced by agents that increase cyclic guanosine 5'-monophosphate (GMP) (carbachol) in addition to beingcalcium dependent as well (14, 15). Another similaritybetween these two tissues is the importance of micro-tubules and microfilaments in macromolecular secre-tion (16). Thus, defective ANS regulation of exocrinetissue could be manifest in the CF neutrophil. Usinghuman leukocytes, Davis et al. (17) reported decreasedcyclic AMP generation with isoproterenol, but notPGE1 stimulation in CF and in some CF heterozygote(CFH) subjects. The nature of this selective defectiveresponse was not elucidated.

We have previously characterized the beta adren-ergic receptor in neutrophil sonicates (18). Morerecently, Dulis and Wilson (19), as well as ourselves(20), have successfully established the presence ofthese receptors in the intact cell, using the beta adren-ergic antagonist ligand [3H]dihydroalprenolol (DHA).DHAbinding in our system is rapid, reversible, andstereoselective, and demonstrates beta-2 rank orderpotency. Wehave applied this technique to measureneutrophil beta adrenergic receptor binding in addi-tion to cyclic AMPgeneration in CFand obligate CFHsubjects to determine whether previous observationscould have been based on defective beta adrenergicreceptors.

METHODS

Subjects. For this study the CF population consisted of23 subjects, 14 males and 9 females, with a mean age of 18.5± 1.3 yr (±SEM). Two patients were receiving theophyl-line, one patient received occasional beta adrenergicaerosol, and one received terbutaline tablets. Beta adrener-gic agonists, methylxanthines, and aspirin were prohibitedwithin 24 h of the study period. 53% of the patients werereceiving antibiotics during the study period. Clinical assess-ment was made using the Shwachman-Kulczycki system(21), whereby a score of 100 indicates no disease and a scoreof 45 indicates severe disease. 79% of the subjects hadpancreatic insufficiency, and 26% had atopic disease withhistories consistent with allergic rhinitis and bronchialasthma. There were 10 CFH parents of the CF patients: 5males and 5 females, with a mean age of 38.1±2.9 yr (±SEM),and 36 healthy control subjects without personal or familyhistories of asthma or cystic fibrosis. The latter group con-sisted of 28 females and 8 males, with a mean age of 25.5+1.0 yr (±SEM). The control and CFH subjects were notreceiving medication at the time of study.

Cell preparation. Neutrophils were prepared and assayedfor cyclic AMPby the method of Davis et al. (17). Whole

blood was collected in nonheparinized glass tubes contain-ing a dextran (mol wt 170,000, Sigma Chemical Co., St. Louis,Mo.)-heparin (sodium, preservative-free, Sigma ChemicalCo.) mixture in a proportion 5:1), and allowed to settle for25-30 min at room temperature. The leukocyte-rich frac-tion was then underlayered with Ficoll-Hypaque and spunat 400 g for 30 min at 20°C (22), and the resulting pelletwashed three times with physiologic saline. The neutro-phils in the pellet constituted >95% of the nucleated cellsin this fraction. The percentage of band forms (immatureneutrophils) was determined by counting 100 nucleatedcells stained with Wright stain. CF patients had 11.5±0.87%band forms, whereas control subjects had 3.6±0.5% bandforms.

Cyclic AMP assay. For the cyclic AMP assay 5 x 106neutrophils per tube were preincubated for 5 min at roomtemperature in incubation buffer (50 mMTris, pH 7.5, and8 mMtheophylline in physiologic saline) and then exposedto media alone or to hormones (isoproterenol, histamine,and PGE,) at 37°C in a total volume of 100 ,ul for 5 min.Accumulation of cyclic AMP was terminated by boilingfor 5 min and freezing for -30 min. The samples werethawed and assayed for cyclic AMPby the saturation methodof Brown et al. (23). This method permitted quantitativerecovery of exogenous [3H]cyclic AMP. The results are ex-pressed as picomoles per 106 cells. The change in neutrophilcyclic AMPafter incubation with agonists is expressed as thepercentage increase over basal cyclic AMPlevels.

DHA binding assay. The binding assay used the sameincubation condition as for cyclic AMP. [3H]DHA (0.1-10 nM)was incubated with 5 x 106 neutrophils per tube containingan incubation volume of 180 ,ul 1 mMascorbic acid, 0.1mMphentolamine, and 0.3 mMcatechol (which are thoughtto block nonreceptor binding sites) (24, 25) for 15 min at37°C, and stopped with 5 ml of Tris, 50 mMHCI, and 10 mMMgCl2 for 30 s. The mixture was then rapidly filteredthrough a Gelman A-E glass fiber filter presoaked with stopbuffer containing 0.1 mMdl propranolol (Gelman Sciences,Inc., Ann Arbor, Mich.). The filters were rapidly washed with25 ml incubation buffer and counted in a liquid scintillationsystem. Specific binding represents the total number of DHAcounts minus DHAbinding in the presence of 0.3 ,IM dlpropranolol, and was 50-80% of the total amount bound at allDHAconcentrations. Wehave recently demonstrated the im-portance of phentolamine (0.1 mM) in reducing nonspecificDHA binding (19). At this concentration phentolaminereduces nonspecific binding (from 69 to 33%) without af-fecting binding to specific receptor sites. All samples wererun in duplicate and differed from each other by - 15%.Specific binding was linear with the cell number used forthese experiments.

Calculations. (a) Receptor number was calculated fromthe expression: Receptors/cell =

mol of DHAbound 1 mgproteinxmgprotein 1.8 x 107 cells

6.02 x 1023 moleculesmol

(b) Statistical analysis utilized nonpaired Student's t test andcorrelation coefficient. (c) Percentage of cyclic AMPstimula-tion = stimulated cyclic AMPminus basal cyclic AMP/basalcyclic AMPx 100%.

Materials. Most reagents were obtained from sources pre-viouisly listed (18). Components for the cyclic AMPassaywere purchased from Amersham Corp., Arlington Heights, Ill.

254 S. P. Galant, L. Norton, J. Herbst, and C. Wood

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E

*1 /

)o /)0

8 7 6 5 4 8 7 6 5 4-Log [Isoproterenol]M -Log [Histamine] M

8 7 6 5 4-Log [PGE I] M

FIGURE 1 Comparison of CF (0) and control subjects' (0)agonist stimulation of neutrophil cyclic AMP. Each agonistwas incubated with 5 x 106 cells for 5 min at 37°C after a 5-minpreincubation in incubation buffer consisting of 50 mMTris (pH 7.5) in physiologic saline and 8 mMtheophyl-line (17). Shown is the percentage of cyclic AMPstimulationabove basal values (mean+SEM) for each agonist (10 nM to0.1 mM). The only consistently significant differences (*)between the two study populations occurred with isopro-terenol stimulation (P < 0.025). For isoproterenol there were37 experiments in the control, 33 in CF group; for histamineand PGEI the number of experiments was 7 and 6 for controland CF groups, respectively.

RESULTS

Cyclic AMPresponses. Fig. 1 illustrates a compari-son of isoproterenol, histamine, and PGE,-inducedcyclic AMP generation of CF and control subjects'neutrophils, expressed as percentage of stimulationover basal levels. CF patients had significantly less(P < 0.025) beta adrenergic induction of adenylatecyclase than controls at 0.1 uM to 0.1 mMisoproterenol

concentrations, as shown by nonpaired Student's ttest analysis. There were no consistent differences incyclic AMPgeneration induced by either histamine orPGE,, although 10 nM histamine and 0.1 ,uM PGE,were more potent in control subjects (P < 0.025).In Table I the data are expressed as absolute values(pmol/106 cells). CFHare also analyzed. The absolutequantity of cyclic AMPin CF neutrophils was signifi-cantly lower for basal (P < 0.025) and for all isopro-terenol concentrations used compared with controllevels (P < 0.005), whereas CFH were significantlylower than normal controls at isoproterenol concen-trations of 1 ,uM to 0.1 mM(P < 0.05). CFH cyclicAMPlevels were significantly greater (P < 0.05) thanthose found in CF subjects for basal and all beta ad-renergic hormone responses. Histamine and PGE1were again not consistently different in CFand normalcontrols.

DHA binding. A DHA concentration curve com-paring control subjects with CF and CFH subjectsshows that there is no difference between control andCFH populations, but both groups have significantlymore (P < 0.01) DHA binding at all ligand concen-trations than the CF group (Fig. 2A). A Scatchard plotanalysis of the mean data for each group shows thatDHAbound to a single class of high affinity sites on theneutrophil membrane (Fig. 2B). The meantSEM forBmax and KD calculated from Scatchard analysis of allsubjects is shown in Table II. From these data thetotal number of receptors/neutrophil was determined.These data reveal that both the control with 819.4 +67.2(mean +SEM) receptors/neutrophil and the CFHgroup

TABLE IAbsolute Cyclic AMPLevels in CF, CFH, and Control Subjects after Agonist Stimulation

Agonist

Subject group Basal 8 7 6 5 4

(-log M) (pmol/106 cells)

Isoproterenol

Control (n = 37) 1.30±0.17 1.30+0.17 1.86+0.23 2.35±0.22 2.70±0.24 3.24±0.25CF (n = 33) 0.87±0.18* 0.55±0.09* 0.88±0.12* 1.12±0.13* 1.26±0.15* 1.38±0.15*CFH (n = 8) 1.44±0.08 1.45+0.07 1.75±0.10 1.89±0.09t 2.04±0.11t 2.14±0.08t

Histamine

Control (n = 7) 1.62±0.35 1.52±0.30 1.70±0.37 1.95±0.45 2.25±0.45 3.44±0.48CF (n = 6) 0.99±0.22 0.76±0.27 0.94+0.26 1.19±0.26 1.39±0.26 1.79±0.31*

PGE,Control (n = 7) 1.62±0.35 1.59±0.35 1.89±0.41 2.18±0.43 3.51±0.51 4.38±0.84CF (n = 6) 0.99+0.22 0.88±0.20 0.96±0.23 1.42±0.24 2.23±0.32* 3.09±0.50

* P < 0.025, comparing control and CF subjects.t P < 0.05, comparing control and CFHsubjects.

Cystic Fibrosis Beta Adrenergic Receptors 255

\1a,.

.0 .\. l.

10 20 30 40 50D,hyd,oolprenolol (pM)

FIGURE 2 (A) [3H]DHA concentration curve for CF patients(0), CFH (C), and control subjects (0). The binding assayutilized the same incubation conditions as for cyclic AMP.DHA (0.1-10 nM) was incubated with 5 x 106 cells/180 ,ulincubation volume containinig 1 mMascorbic acid, 0.1 mMphentolamine, and 0.3 mMcatechol for 15 mIi at 37°C. Detailsof the procedure are given in Methods. Specific binding (finol/mg) represents the total number of DHAcounts minus DHAbinding in the presence of 0.3 ,M dl propranolol. 2 x 107neutrophils is equivalent to 1 mg protein (18). Shown is themean+SEMDHAbinding in 11 CF experiments, comparedwith 10 CFH and 21 control experiments. *, indicates sig-nificant differences (P < 0.01) between CF patients and bothcontrol or CFH subjects at each DHAconcentration. Onlyspecific DHAbinding is shown. (B) Scatchard plot analysisof the mean data from (A). This shows that DHAbinds to asingle class of high affinity receptor. The total ntumber ofreceptors is markedly decreased in CF (13.04 fmol/mg),whereas the dissociation constant (KD) is similar: 0.66 nMfor CFcompared with 0.58 and 0.43 nM for CFHanid controls,respectively.

with 909.8+±89.3 receptors/neuitrophil had significantlymore (P< 0.005) receptors than the CF patients with398.0±54.2 receptors/nieuitrophil. The Kn values of0.630±0.05 nM, 0.740+0. 11 nM, and 0.710 ±0.9 nM for

TABLE IIBeta Adretnergic Recep)tor Bitnditng in CF

CF patiernt Ba,x* K,t Clinical score§

fmollntg protein

1 11.46 1.31 352 11.20 1.20 353 7.81 0.37 354 4.35 0.92 735 14.29 0.99 656 14.29 0.93 357 10.56 0.35 958 17.20 0.44 659 18.32 0.53 90

10 15.63 0.31 95Mean+SEM 12.51± 1.23 0.74+(0.11CFH" (n = 10) 27.2±2.69 0.71±0.09Controls (it = 21) 24.8±+1.60 0.63±0.05P (vs. con1trols) <0.005 >0.05P (vs. CFH) <0.005 >0.05

* Maximtum number of bindinvg sites.Dissociation constant.

§ Shwachman-Ktilczycki system (20).CFHare the CF heterozygote parents.

control, CF, and CFH groups, respectively, did notdiffer significantly (P > 0.05).

The severity of CF (Shwachman-Kulczycki score) didnot correlate with either the maximum percentage ofisoproterenol (0.1 mM)-induced cyclic AMPstimula-tion or DHA binding at saturation (5 nM DHAconcentration). There also appeared to be no correla-tion l)etween cyclic AMP responsiveness or betaadrenergic receptor binding annd the presence ofpancreatic insufficiency, atopy, or the admiinistration ofantibiotics or prior bronichodilator therapy (data notshown). The percentage of neuitrophil band formns didnot correlate with either the percentage of cyclic AMPstimulation (r = 0.35, P > 0.05), or the absolutequiantity of cyclic AMPgenerated (r = -0.16, P > 0.05)after maximal isoproterenol (0.1 mM) stimullation(Table III). Fturthermore, altlhouigh the CF patientswere somewhat younger (18.5 +1.3 yr) than controlsubjects (25.5±1.0 yr), this is not a likely explanation forthe differences noted, becauise there was no significantcorrelation between age and DHAbinding or cyclicAMPstimulation in our study popuilation.

DISCUSSION

Wehave confirmed the observation in CF neutrophilsreported by Davis et al. (17) that suggests defective

TABLE IIIRelationiship of Imnmature Neutrophil Forms ancl Cyclic

AMPResponses in CF Subject.s

Cyclic AMP

NeutrophilCF subjects Stimulationi Cells bandl fomis

% p11101lO/10, %

1 78 0.91 112 166 0.77 103 100 2.20 104 24 0.31 105 20 0.60 96 64 0.54 127 178 0.75 178 111 3.80 189 79 1.(0 15

10 93 1.16 711 630 0.73 1512 265 0.73 1513 58 2.26 914 68 3.30 215 396 1.34 1316 56 2.00 1417 100 2.00 818 500 0.60 12

Correlation coefficient (r) for netitrophil cyclic AMPstinm-lation (second column) and for absoluite cyclic AMP levels(third column) with 0.1 mMisoproterenol wvas 0.35 (P > 0.05)ail(I -0.16 (P > 0.05), respectively.

256 S. P. Galant, L. Norton, J. Her-bst, anid C. Wood

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cyclic AMPgeneration that is quite specific for thebeta adrenergic pathway since neither histaimine norPGEl-induced adenylate cyclase activation appearsabnormal, and since the intrinsic activity of adenylatecyclase itself is normal (26). In contrast to those in-vestigators who report normal receptor number (27),however, we find a marked (50%) decrease in thenumber of beta adrenergic receptors in CF neutrophilswithout a change in receptor affinity, as assessed byDHAbinding. The reason for this discrepaincy is notclear. Davis and Hill (27), who measured DHAbindingin mononuclear membrane preparations, reported-250 fmol/mg membrane protein, which is consider-ably greater DHAbinding thani is reported by eitherour previous study with neutrophil sonicates (20) orthat of Davies and Lefkowitz (28). Details of theirmethodology are not provided (27). Furthermore,since our study deals with DHAbinding in the intactneutrophil, a direct comparison of our data with thoseof Davis and Hill (27) may not be appropriate. Wemust add that although a significant reduction in DHAbinding was not found in CFH, it is conceivable thata small (-25%) reduction in receptor sites might bemissed because of lack of this degree of sensitivityof our assay system.

These findings, suggestive of down regulation of thebeta adrenergic receptor associated with the desen-sitization phenomeienoi, are generally produiced bychronic beta adrenergic stimulation (29). We havepreviously demonstrated down regulation in neutro-phil sonicates with administration of the beta-2agonist terbutaline (30), and others have reported thisphenomenon in patients with pheochromocytoma (31),and after administration of antidepressants (32), whichblock reuptake of catecholamines. The CF patients,in general, were not receiving drugs known to causethis effect, but they could have increased endogenouscatecholamine secretion as a result of chronic stress.This is not entirely consistent with the lack of corre-lation between severity of disease and beta adrener-gic responses, or with the presence of decreased cyclicAMPgeneration in the CFHnot under stress. Further-more, we were unable to demonstrate an effect of pan-creatic function, atopy, or neutrophil maturity on betaadrenergic function in the CF population. These find-ings are suggestive of a primary genetic defect in thebeta adrenergic system itself. The cause of the de-creased cyclic AMPresponse in CFH is not clear, sinceit is not associated with reduction in receptor nuin-bers, at least as measured by antagonist ligaind bind-ing. Perhaps the desensitization model nmay prove uise-fuil in explaining this finding. Decreased cyclic AMPresponses following chronic exposture to isoprotereinolmay be due to several mechanisms (lepenclent on thecell source, durationi of exposure, and concenttrationiof agonist (33, 34). Thus, decreased responsivenesswithout a chanige in receptor number measured by

antagonist ligands, such as DHA, could be due to de-creased agonist binding (34) or ain uncoupling ofreceptor to the enzyme adenylate cyclase (33). A moreprofound effect, usually with high agonist concen-tration or long duration of exposure in some cell types,is a decrease in receptor number (measured by antago-nist binding) consistent with diminished adenylatecyclase responsiveness (33). Desensitization is fre-quently agonist specific (35), so that beta adrenergicdown regulation is not usually associated with de-creased responses to histamine or PGE1.

The beta adrenergic defect in CF could be a conl-tinuum with the CFH populationi having normiialantagonist binding but abnormal atgonist bindingor abnormial receptor-enzyme coupling, so that theresult is decreased cyclic AMPgeneration, whereasthe CF patient has a more severe defect consistiingof decreased aintagonist binding and adenylate cyclaseactivation. If a desensitizationi phenomenon is thebasic mechanism behind our observation, we are stillwithout an explanation for this effect. One mightconsider an intrinsic cellular defect with an increasedpropensity to desensitize; circulating catecholaminelevels that are chronically evaluated, owing to in-creased synthesis or decreased uptake; or inhibitorvsubstances produced by the CF cell (11). Other thanfor the last nianmed, there is nio evidence for thesehypotheses in the literature, and in fact one inivesti-gation reports decreased norepinephrine levels in CF,although it presents Ino data (13). Physiologic sttudiesin CFH (5-8), however, clearly demonstrate ANSimbalance, and this could be associated with dis-ordered catecholaminie metabolism. Study of CFHmayhold the key to the primary defect in CF.

Finally, the relationship of the beta adr-energicdefect to the disease process deserves coimmiiient. Thefact that several investigators (5, 13), as wvell as our-selves, have fouind evidence of ANS abnormnalities inthe asymptomatic CFHsubject makes such phenomnenaless likely to be important in the pathogenesis of CF.Nevertheless, the CFH population must be fiurtherstudied to define clearly such beta adrenergic dif-ferences between themn and the CF group as receptorbinding, coupling, activration of adenylate cylase, andcatecholamine metabolism. This might not only pro-vide a useful marker of the heterozygote state, butmay also shed light oni the relationship between betaadrenergic fuinction and pathogenesis of the diseasestate.

ACKNOWXLEDGlIMENTS

'fIis StUdy was sup1)porte(d b)y a granllt froirJ the Cystic FibrosisFowl nd(alti on.

Dr. Gatlaint is at recil)ielnt of ani AllergXic Diseatses AcademleicAwarcl (A100304) f'roim tbie National Iistituite of' Allergiy andInfectious Diseatses.

Cystic Fibrosis Beta Adrenecr-ic Receptors 257

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