Arachidonic acid metabolism in monocytes of aspirin-sensitive asthmatic patients before and after oral aspirin challenge

Al-Rayes et al of leukocyte markers in elderly individuals. Aging Immunol Infect Dis 1990:3 1. 42. Wierenga EA, Snoek M, de Groot C, et al. Evidence for compartimentalization of functional subsets of CD4’ T lymphocytes in atopic patients. J Immunol 1990;144:4651-6. 43. Wierenga EA, Snoek M, Bos JD, Jansen HM, Kapsenberg ML. Comparison of diversity and function of house dust mitespecific T-lymphocyte clones from atopic and non-atopic donors. Eur J Immunol 1990;20: 15 19-26. 44. Parronchi P, Macchia D, Piccinni M-P, et al. Allergen- and bacterial-antigen-specific T-cell clones established from atopic donors show a different profile of cytokine production. Proc Natl Acad Sci U S A 1991;88:4538-42.

J ALLERGY

CLIN IMMUNOL OCTOBER 1992

45. Kapsenberg ML, Wierenga EA, Bos JD, Jansen HM. Functional subsets of allergen-reactive human CD4 + T cells. Immunol Today 1991;12:392-5. 46. Raynal M-C, Liu Z, Hirano T, Mayer L, Kishimoto T, Chenkiang S. Interleukin-6 induces secretion of IgG 1 by coordinated transcriptional activation and differential mRNA accumulation. Proc Nat1 Acad Sci U S A 1989;86:8024-8. 47. Roper RL, Conrad DH, Brown DM, Warner GL, Phipps RP. Prostaglandin E7 promotes IL-4-induced IgE and IgGl synthesis. J Immunol 1990;145:2644-51. 48. Deryckx S , de Waal Malefyt R, et al. Immunoregulatory functions of paf-acether. VIII. Inhibition of IL-4-induced human IgE synthesis in vitro. J Immunol 1992;148:1465-70.

Arachidonic acid metabolism in monocytes aspirin-sensitive asthmatic patients before and after oral aspirin challenge

of

Uwe R. Juergens, MD, Sandra C. Christiansen, MD, Donald D. Stevenson, MD, and Bruce L. Zuraw, MD La Jolla, Calif. Aspirin and nonsteroidal antiinjiammatory drugs induce bronchospastic reactions in patients with aspirin-sensitive respiratory disease. Although the mechanism of this reaction is unknown, all drugs that induce the respiratory reaction also inhibit the cyclooxygenase enzyme. The ensuing changes in arachidonate metabolism are presumed to play a role in the pathogenesis of the reaction. We measured generation of leukotrienes and thromboxane by calcium ionophore stimulated blood monocytes. Before aspirin challenge, monocytes released significantly more thromboxane B, in patients with aspirin sensitivity than in patients without aspirin sensitivity or in healthy control subjects (p < 0.02). During aspirin-induced bronchospasm, release of leukotriene B, increased significantly (45.5%, p = 0.018) whereas release of thromboxane BZ decreased f-46.9%, p = 0.028). Two hours after ingestion of 60 mg aspirin, normal monocyte release of thromboxane B, did not drop, whereas leukotriene B, release increased. Monocytes formed only minimal amounts of leukotriene C,. We conclude that the profile of released eicosanoids from aspirin-sensitive monocytes is distinct from non-aspirin-sensitive subjects, and that these differences could contribute to the development of bronchospasm after aspirin ingestion. (J ALLERGY CLINIMMUNOL1992;90:636-45.) Key words: Aspirin-sensitive asthma, arachidonic acid metabolism, monocytes, aspirin challenge, thromboxane B, , leukotriene B,

From the Molecular and Experimental Medicine Research Institute of Scripps Clinic, La Jolla. Supported in part by grants RR00833 and AI10386 from the National Institutes of Health and grant 1990-14 from the Department of Medicine of Scripps Clinic. Dr. Juergens supported in part by the German Society of Internal Medicine and the Alexander von Humboldt Foundation, Bonn, Germany. Received for publication Dec. 17, 1991. Revised June 9, 1992. Accepted for publication June 15, 1992. Publication no. 6786-MEM from The Scripps Research Institute. Reprint requests: Bruce L. Zuraw, MD, The Scripps Research Institute, 10666 N. Torrey Pines Rd., La Jolla, CA 92037. l/1/40235

636

Approximately 10% to 20% of the asthmatic population is sensitive to aspirin. After ingestion of aspirin or structurally unrelated nonsteroidal antiinflammatory drugs (NSAIDs), asthmatic reactions with or without accompanying nasoocular symptoms characteristically develop in patients with aspirin-sensitive respiratory disease (ASRD). I4 The potency of these compounds in causing bronchospasm is closely associated with the degree of inhibition of the cyclooxygenase enzyme, giving rise to the hypothesis that NSAIDs and aspirin induce these reactions through an alteration in arachidonic acid (AA) me-

VOLUME NUMBER

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Arachidonic acid metabolism in aspirin-sensitive

taholism. ‘, ” Previous studies have demonstrated that NSAIDs can also enhance IgE-mediated release of leukotrienes (LT) and histamine from passively sensitized human lung tissue.7-1” Several in vivo studies lend additional support to the hypothesis that AA metabolites play a pathogenic role in the ASRD asthmatic reaction. In patients with ASRD, LTC, was present in nasal secretions after cyclooxygenase blockade, whereas it was not detectable in nasal secretions of healthy subjects or in patients with ASRD after desensitization to aspirin. ” More recently it was reported that airways of patients with ASRD are more sensitive to LTE, but not to histamine compared with patients without ASRD. ” After desensitization to aspirin, airway responsiveness to inhaled LTE, decreased-suggesting downregulation of LTE, receptors.” AA is formed from phospholipids by phospholipases AZ and C, and further metabolized through either the cyclooxygenase pathway to yield prostaglandins (PGJ and thromboxane (TX) or the 5lipoxygenase pathway to yield mono-HETE, di-HETE, and the leukotrienes. ” The cyclooxygenase products PGD2, TXB!, and PGF,,, are bronchoconstrictors, whereas PGlz and PGE, are bronchodilators.” The sulfidopeptide leukotrienes C,. D,, E, have been shown to be potent mediators of bronchoconstriction.‘5 ” LTB,, a potent chernotactic factor, is believed to be involved in airway inflammation and late allergy responses by attracting effector cells such as neutrophils and eosinophils into the airways. ‘-’ To test the hypothesis that aspirin sensitivity is related to altered AA metabolism, we compared the in vitro formation of eicosanoids by peripheral blood monocytes of asthmatic patients with ASRD to the eicosanoid release by peripheral blood monocytes of non-aspirin-sensitive asthmatic patients and healthy controls. .4lveolar macrophages are thought to be an important component of the lung inflammatory response and are a major source of LTB, in human airways. ” Peripheral blood monocytes are circulating precursors of macrophages, release LTB,“’ and TXB,“” in vitro, and represent a more accessible model to study patients with ASRD. This report shows that monocytes of patients with ASRD have a significantly different AA metabolism at baseline when compared with either non-aspirinsensitive asthmatic patients or healthy controls. At the time of aspirin-induced bronchospasm after oral aspirin challenge. monocyte AA metabolism is significantly altered. MATERIAL AND METHODS Patient selection Three groups of nonsmoking volunteer subjects,ages30 to 58 years. were recruited into the study. All subjects gave

Ahhrevitrtions

patients

537

used

AA: Arachidonic acid ASRD: Aspirin-sensitive respiratory drw,~-c CFPF: Cell-free and platelet-fret EIA: FEV,: HBSS: LT: NSAID: SEM: TX: PC;: EDTA: HPLC:

Enzyme immunoassay Forced expiratory volume m ! pond Hank’s balanced salt solutiori Leukotriene Nonsteroidal antiinflammatory tirtl? Standard error of the mean Thromboxane Prostaglandin Ethylenediamine tetraacetlc acid High-performance liquid chromatography

informed consent, which was approved by the tiuman Research Committee of Scripps Clinic and Research Foundation. Patients with a history of prior asthmatic reactions to aspirin were admitted to the Clinical Research Center at Scripps Clinic to undergo oral aspirin challenge as previously described. ’ ‘. Clinical asthma and bronchial hyperreactivity were defined according to the American Thoraclc Society guidelines of reversible airway obstruction. denronstrated either by methacholine challenge or inhaled PIagonist bronchodilation. Lung function tests by use ot flow/volume measurements were performed on all patients. Serial forced expiratory volume in I second (FE%,) values were obtained at least every hour between 6 451 and 9 PM during the entire study. Antihistamines. cromolqn sodium, and &agonists were discontinued on admission (at least 24 hours before aspirin challenge), and were held throughout the hospitalization except for nebulized &agonist treatments for asthmatic reactions. Other regular medications were continued. Table I shows the medications of the subjects at the time of challenge. All patients wcrc receiving topical andi or systemic corticosteroids, and both groups received comparable amounts of these drugs. Baseline blood samples were drawn on the day before aspirin challenge or immediately before ingestion of the first aspirin-dose. Fifteen asthmatic subjects were investigated (Table II). The first group consisted of nine asthmatic patients who were shown to have definite ASRD. Bronchospahrn developed in each of these patients (fall of FEV, ?20%) during oral aspirin challenge. Seven of these patients were receiving long-term oral treatment with prednisone (Table 111. The second group consisted of six asthmatic patients who were aspirin insensitive. Four patients in this group gave d history of possible aspirin sensitivity but did not react to oral aspirin challenge. One of these patients (nl:. 13) underwent repeat aspirin challenge at a time when she was taking only IO mg every other day of prednisone. The repeat challenge was also negative. Her monocyte AA metabolism was not studied during the repeat challenge. The remaining two aspirin-insensitive asthmatic patients (patlcnts II and 15) gave no history of aspirin sensitivity. had ingested 325 to 650 mg aspirin without ill effect withir! the prc~ou~ h months, and had not ingested NSAIDs for JS lcai! 3 wccl\

638

Juergens

TABLE

J ALLERGY

et al.

I. Medications Subject

of study

subjects Other medications

prednisone*

Aspirin-sensitive asthmatic patients Uniphyl 400 mg q.d., Azmacort 2 b.i.d., Vancenase 2 b.i.d., Atrovent 2 b.i.d., Zantac 150 mg q.h.s. Theo-Dur 300 mg b.i.d. Theo-Dur 450 mg b.i.d., Vanceril 2 t.i.d., Nasalide 1 q.i.d. Theo-Dur 200 mg b.i.d., Beconase AQ 1 b.i.d., Zantac 150 mg q.h.s., Verapamil 80 mg q 8 hr Theo-Dur 450 mg b. i.d., Beclovent 3 t.i.d., Beconase 1 b.i.d. Vancenase 1 b.i.d. Uniphyl 400 mg q.d., Vancenase 1 b.i.d., Vanceril 2 q.i.d. Theo-Dur 300 b.i.d. Theo-Dur 400 b.i.d., Vancenase 1 b.i.d., Entex LA b.i.d., Proventil repetabs 4 mg b.i.d.

20 2 3 4

24 60 12 0 0 40 12 40

mean ? SD

23 2 19t

10 11 12 13 14 15 mean ? SD

0 40 0 30 20 0 15 ‘- 16

CLIN IMMUNOL OCTOBER 1992

Non-aspirin-sensitive asthmatic patients Beclovent 4 t.i.d., Nasalide 2 t.i.d., Proventil repetabs 4 mg b.i.d. Theodur 300 mg b.i.d., Vanceril 2 t.i.d., Vancenase 1 b.i.d. Azmacort 2 q.i.d., Vancenase AQ 1 q.i.d. Vanceril 2 t.i.d. Slo-Phyllin gyrocaps 250 mg b.i.d., Vanceril 2 t.i.d., Nasalide 1 b.i.d. Beclovent 3 t.i.d.

*Milligrams of prednisone equivalent/day. tp > 0.4 compared with non-aspirin-sensitive asthmatic patients. Uniphyl (Purdue Frederick); Azmacort (Rhone-Poulenc Rorer); Vancenase (Schering); Atrovent (Boehringer Sugelheim); Zantac (Glaxo); Theo-Dur (Key); Vanceril (Schering); Nasalide (Syntex); Beconase (Glaxo); Beclovent (Glaxo); Entex (Norwich Eaton); Proventil (Schering); Slo-Phyllin (Rarer).

before the study. Three of the six aspirin-insensitive asthmatic patients studied were receiving prednisone. The third group consisted of eight healthy nonasthmatic subjects (ages 23 +- 2 years) who were nonsmokers, had not ingested NSAIDs for at least 4 weeks before the study, and were not taking any medications. Three of these subjects underwent oral challenge with 60 mg aspirin without showing any adverse reactions.

Aspirin

challenge

Aspirin challenges were performed in 13 asthmatic subjects as described by McDonald et al.’ and Stevenson.*’ After 1 day of single-blind placebo challenges to establish baseline FEV, values and airway stability, subjects were given increasing oral doses of aspirin starting at 6 to 30 mg. Challenges were continued at 3-hour intervals with increasing doses until the subject reacted or tolerated 650 mg aspirin. The FEV, was measured every hour, and the patient’s symptoms were monitored. Positive asthmatic response to aspirin was defined by a drop of FEV, 220%. In addition nasoocular responses were seen during aspirin challenge in eight of nine aspirin-sensitive patients, as defined by profound nasal congestion, rhinorrhea, and/or ocular injection. The subject not manifesting

a nasoocular

reaction

was patient 9

(Tables I, II, III). Reaction blood samples were obtained at the time of the first aspirin-provoked bronchospasm. The

reaction bloods were drawn after spirometry but before administration of additional medication. In all cases the reaction blood was obtained within 20 to 30 minutes after the onset of bronchospasm.

Analytic

methods

Isolation of monocytes. Monocytes were isolated according to the method described by Boyurn.‘* Fifteen milliliters of venous blood was collected in vacuum-sealed containers containing ethylenediamine tetraacetic acid (EDTA) (Be&on Dickinson, Rutherford, N.J.). Leukocyte-rich plasma was prepared by dextran sedimentation (dextran 500, 6% dissolved in 0.9% NaCl; Pharmacia, Uppsala, Sweden) and layered over 3 ml of Nycodenz monocytes (density 1.068 gm/ml, Robbins Scientific, Calif.). After centrifugation (600 g for 15 minutes at 22” C), the top plasma and interface layers were separately recovered. The plasma was centrifuged at 2500 g for 20 minutes to prepare cell-free and platelet-free (CFPF) plasma. The interface containing the monocytes was suspended into 1 ml of 0.9% NaCl and stored at 4” C. Washing solutions were prepared with use of 1 part autologous CFPF-plasma mixed with 9 parts 0.9% NaCl. The interface was suspended into 10 ml of washing solution and centrifuged at 400 g for 7 minutes at 4” C. To remove contaminating platelets the cell pellet was resuspended in Ca+ +-, MG+ +-free Hank’s balanced salt solution

VOLUME NUMBER

Arachidonic

90 4, PART 1

TABLE II. Clinical characteristics Subject

1 2 3 4 5 6 7 8 9

Sex

Age (yrl

F

44 54 49 41 49 32 29 58 54

M

M F F M F M F

mean 2 SD 45.5 ? 9 10 11 12 13 14 IS

F F F F M M mean t SD 44

30 32 50 51 48 42 & 4.5

acid metabolism

in aspirin-sensrtive

paCent?

639

of study subjects Atopic

rhinitislsinusitis

Polyps

Severity score+

asthmatic patients 2 Yes 2 Yes 1 Yes 2 Yes 1 Yes 0 Yes I Yes 2 Yes I Yes 1.3 419 919 9/9 Non-aspirin-sensitive asthmatic patients I Yes Yes Yes 2 No Yes Yes I No Yes Yes 1 No Yes Yes 2 Yes Yes Yes 1 Yes Yes No 1.3 316 616 516 Yes No No Yes Yes No Yes No No

%FEV, of pfedkted

Aspirin-sensitive Yes Yes Yes Yes Yes Yes Yes Yes Yes

75 73 71 x5 100 85 79 61 81 79 k lot 82 56 92 59 53 40 64 L 18

Aspirin (WI)

%Dr0p

FEV,

f> 60 60

26 15 >? _.

1O(1

d-7 c

60 6(! hi! 60 txi

a) 27 .l4 Xl 2’3

58”“22

32i’Ilt

6SO 6Si hS0 6511 05( 1 650 ...--.__-

* i ‘.S - 5 .s ND ND _.

.-..,_

ND, not determined. *Severity score: 0. no systemic steroids; 1, occasional systemic steroids; 2, steroids dependent. tp > 0.07 compared with non-aspirin-sensitiveasthmatic patients tp < 0.003 compared with basline.

(HBSS, Irvine Scientific, Santa Ana, Calif.), layered over 3 ml of CFPF-autologous EDTA plasma, and centrifuged at 50 g for 10 minutes. After resuspension of the pellet in HBSS, the cells were washed again with HBSS (400 g for 10 minutes at 4” C), counted in a hemocytometer, and resuspended in 1 ml Ca ++- and Mg + +-free HBSS. Monocytes were platelet free and >95% pure as assessed by light microscopy. The absence of contaminating platelet was confirmed by fluorescence-activated cell sorting by use of the anti-GPIIb-IIIa monoclonal antibody (LJ-W), which is directed against platelets.‘j Viability was >99% by trypan blue exclusion. The average yield of purified and viable monocytes was 0.5 to 1 x IO6 cells/ 15 ml EDTA blood. Stimulation of monocytes in suspension. Aliquots of 5 x 10’ monocytes in 1 ml of HBSS containing 10 pmol/L Ca++ were placed in polypropylene tubes (Falcon, 10 x 7.5 mm) and cultured in a waterbath at 37” C. They were stimulated by 10 pmol/L Ca++ ionophore A23187 (Sigma Chemical Co., St. Louis, MO.), which was dissolved in dimethylsulfoxide and diluted to 0.1% final (vol/vol) in culture medium. After 30 minutes in the culture medium, the cells were pelleted by centrifugation (500 g for 5 minutes at 4” C) and the supematants harvested. The supematants were immediately frozen in liquid nitrogen and stored in Eppendorf tubes at -80” C until assayed by enzyme immunoassay (EIA). The amount of Ca++, ionophore A23187, and the duration of stimulation were experimentally determined to provide optimal TXB,, LTB,, and LTC, release. When the monocytes were cultured in suspension, less

than 5% of the monocytes became adherent after 30 minutes. In preliminary studies, plastic adherent monocytes were cultured in the absence of fetal calf serum, and eicosanoid release was determined. After 3 hours of adherence, ionophore A23 187-stimulated monocytes released elevated amounts of TXB, but decreased amounts of LTB, compared with monocytes cultured for 30 minutes in suspension. After 20 hours of adherence, the ratio of TXB,/LTB, was similar to that seen in short-term cultures in suspension; however, the total level of mediator release was only approximately 55% that of monocytes in suspension (data not shown). Therefore all subsequent experiments used short-term cultures in suspension. QuantiJication of AA metabolites. LTB,, UC,, and TXB, (the stable product of TXA,) were measured in the culture supematants by direct EIA.14 Tracers linked to acetylcholinesterase, antisera, mouse monocional antirabbit IgG, lyophilized synthetic standards, and the enzyme substrate (Ellman’s reagent) were purchased from Cayman Chemical Corp., Ann Arbor, Mich. Certified B&well nticrotiter plates were purchased from Nunc, Kamstrup. Denmark. EIA for the measurement of the monitored eicosanoids was performed according to the instructions of the vendor, and as recently reported.25 Standards and samples (50 ~1 per well) were assayed in duplicate, and the maximum binding in the absence of eicosanoids was determined in quadruplicate. Serial dilutions of stimulated culture supematants were assayed so that samples would fall close to 50% B / Bo of the standard curve. After overnight incubation

640 Juergens et al.

J ALLERGY

TABLE III. Monocytes

from aspirin-sensitive

asthmatic

subjects

Subject

Aspirin-sensitive

1 2 3 4 5 6 7 8 9

asthmatic patients

Non-aspirin-sensitive

mean SE asthmatic patients

10 11 12 13 14 15

mean SE

secrete increased

CLIN IMMUNOL OCTOBER 1992

eicosanoids*

TXB,

LTB,

LTC,

2599 460 692 1255 210 240 647 1787 878 974t 262 246 196 225 264 117 144 198 23

1625 578 336 1142 355 723 888 886 903 826$ 133 0 1315 429 909 402 250 551 195

123 133 101 223 66 55 325 132 49 134 29 62 540 43 30 23 53 138 90

320 50

458 69

92 14

Healthy subjects mean SE

16 - 23

*A23187-stimulated monocyte supematant measured by EIA (pg/5 tp < 0.02. #p < 0.03 compared with healthy subjects

(18 to 22 hours) at room temperature, plates were washed, 200 ~1 of enzyme substrate was added to each well, and the absorbance at 412 nm of each well was measured in a MR600 Microplate Reader (Dynatech Laboratories, Alexandria, Va.) after 30 minutes to 3 to 4 hours. The concentration of eicosanoid (pg/ml) was calculated by interpolation from a standard curve (v > 0.98) in a linear range of 20% to 80% B /Bo by least squares regression analysis. The sensitivity of the assays (at 80% B/Be) varied as follows: LTB,, 4.5 to 10 pg/ml; TXB,, 7.8 to 18 pg/ml; and LTC,, 15 to 38 pg/ml. Cross-reactivity of the antisera as reported by the manufacturer are: antisera to LTC, (100%) crossreact with LTD, (46%), LTE, (2%), and LTB, (0.01%); antisera to LTB, (100%) cross-react with S(S), 12(S), monoand di-HETE (0.3%) LTC, and LTD, (O.Ol%), and antisera to TXB, (100%) cross-react with 2,3 dinor TXB, (8.2%), prostaglandins (<0.44%), and LTB, (0.01%). The specificity of the LTB, EIA was confirmed by reversephase high-performance liquid chromatography (HPLC) with use of previously described methods.z6,27Baseline and reaction samples containing a total of 504 pg and 4300 pg of LTB,, respectively, were spiked with 1 nCi of ‘H-LTB, (DuPont, Wilmington, Del.) and loaded onto conditioned C,,-SepPak cartridges (Waters, Milford, Mass.). The samples were eluted with methanol, evaporated in a Speedvac, (Sevart Instruments, Hicksville, N.Y.) dissolved in methanol, and applied to a Nucleosil C,, 5 p.m 4.6 x 250 mm HPLC column (Alltech, Deerfield, Ill.). The HPLC column

x

lo4 cells).

was run with use of buffer A (65% methanol/O.l% AcOH12.5 mmol/L pentanesulfonic acid) and buffer B (100% methanol/O.l% AcOH12.5 mmol/L pentanesulfonic acid) at a flow rate of 0.8 ml/ min. LTB, concentration was estimated from cold LTB, standards, and the reaction sample gave a result (-3.1 ng) in good agreement with the EIA result. The baseline sample was below the sensitivity of the method (1 to 2 ng) and was not detected.

Statistical

analysis

All data are expressed as the mean +- standard error of the mean (SEM) for triplicate cultures of 5 x 104 cells, each assayed in duplicate. The two-tailed t test was used for statistical calculations on normally distributed data. The Mann Whitney and Wilcoxon signed-rank tests were used for the comparison between groups of subjects. P values CO.05 were considered significant. All analyses were performed on a Macintosh computer (Apple Computer, Inc., Cupertino, Calif.) with use of StatView II (Abacus Concepts, Berkeley, Calif.).

RESULTS Clinical characteristics Thirteen asthmatic patients with history of adverse reactions to aspirin were challenged with aspirin as previously described. The clinical profile of these patients is shown in Table II. Bronchospasm developed

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Arachidonic

TABLE IV. Spontaneous secretion

eicosanoid

j g

Subject

No.

TXB,

LTB,

LTC,

i-\sptrin sensitive Non-aspirin sensitive Control

8

38 2 7

11 * 5

<15*

5

45 f

8

38 -ir 5 picograms

10

per

5+1

<15*

<4*

NDI

5 X IO’

eicosanoid

12007

monocytes

in nine patients after aspirin administration, and they were considered to be aspirin sensitive. In this group the percent FEV, predicted at baseline (78.7% 2 10%) dropped significantly (p < 0.003) on reaction (57.8% + 9%). Seven of these aspirinsensitive asthmatic patients reacted after administration of 60 mg aspirin, one after 6 mg and one after 100 mg aspirin. The elapsed time from administration of the provoking dose to reaction correlated (7 = 0.82) with the amount of prednisone taken. Four patients (patients 10 to 13) did not react to aspirin and were considered to be aspirin-insensitive (Table II). Patients 10 and 12 had mild asthma and were not on systemic corticosteroids. Two other patients (patient 11 and patient 13) had moderate asthma and were on long-term treatment with prednisone. The aspirin-sensitive asthmatic subjects (n = 9, group 1) and the aspirin-insensitive asthmatic subjects (n = 6. group 2) were similar in sex and age. The two patient groups did not differ significantly in mean prednisone dose (group 1, 23 + 19 mglday; group 2, 15 t 16 mgiday; p > 0.4) or percent FEV, predicted at baseline (group 1, 78.2% t 10%; group 2, 63.7% k 17.7%;p > 0.07). Baseline

in aspirin-sensitive

p.;tiepts

641

1400-

at baseline

Results reported in (mem z SEM). *QB’Bo >XOR, ‘rNI>. not determined.

acid metabolism

production

Spontaneous release of LTB4, LTC, and TXB, was measured in the supematants of monocytes cultured in HBSS for 30 minutes. The unstimulated monocytes released only small amounts of the monitored eicosanoids, and no differences between the three groups of subjects were found (Table IV). Because of the minimal spontaneous release, all further experiments used in vitro stimulation with calcium ionophore A23187. Eicosanoid release was measured from ionophorestimulated monocytes from nine patients with ASRD, six patients without ASRD, and eight healthy subjects (Table III). No correlation was found between prednisone dosage and mediator release. Fig. 1 shows the

m

ix52

LTB4

Control

Non-ASA sensitive

ASA gar,srtive

FIG. 1. Monocytes of aspirin-sensitive asthmatic patients release increased amounts of TXB? and LTB, after stimulation with A23187 in vitro. Eicosanoid release measured from the monocytes of eight healthy voiunteers, six nonaspirin-sensitive asthmatic patients, and nine aspirin-sensitive asthmatic patients. TXB, (*p =. 0.021 and LTB, (**p < 0.04) release (pg!5 x IO4 cells, mean t SEM) were significantly higher from ASRD monocytes than from normal monocytes. ASRD monocytes from aspirinsensitive patients released significantly more TXB, than monocytes from non-aspirin-sensitive asthm&ic patients.

profile of released TXB2, LTB4, and LTC., in all three groups. TXB, release was significantly greater from monocytes of patients with ASRD (974.2 it 262.5 pg/ml) than monocytes from patients without ASRD (198.6 t 23.7 pgiml, p < 0.01) and healthy subjects (319.9 -t 50.2 pgiml, p < 0.02). LTB, release by monocytes from aspirin-insensitive asthmatic patients (550.8 t 195 pg/ml, II = 6) was not significantly different (p > 0.3) compared with patients with ASRD (826.2 i: 133.6 pg/mi, tz --: 9). However, LTB, release was greater in monocytes of both asthmatic populations compared with healthy controls (p < 0.03). LTC, release was considerably lower than LTB, or TXB, release and did not differ among the three groups. Although no single eicosanoid product correlated with FEV,, the ratio of TXB, to LTB,, did correlate (r = 0.72) with the baseline FEV, as a percentage of predicted. Eicosanoid

profile

after aspirin

chalenge

Since aspirin-induced bronchospasm has been proposed to result from alterations in AA metabolism, eicosanoid release was determined in peripheral blood monocytes obtained at the time of aspirin-induced bronchospasm. We obtained monocytes from seven aspirin-sensitive asthmatic patients within 30 minutes of the development of bronchospastic reactions after ingestion of aspirin. The provoking dose\ of aspirin in these seven patients were: 6 mg 07 =. I ?. 60 mg (n = 5). and 100 mg (n = 1B.The mean&psed time

642

Juergens

g $

1400 1200

2 0w

1000

E g

600

:: El

J ALLERGY

et al.

CLIN IMMUNOL OCTOBER 1992

TxBp LTB4 LTC4

800

400 200 0 Baseline

Reaction

FIG. 2. Changes in monocyte AA metabolism after aspirin challenge. Seven patients with ASRD were challenged with increasing doses of aspirin. Monocytes were obtained before aspirin challenge (baseline) and at the time of aspirin-induced bronchospasm (reaction). After stimulation with A23187, TXB,, LTB,, and LTC, release was measured (pg/5 x IO* cells, mean f SEMI. At the time of reaction, monocytes released significantly less TXB, (*p = 0.028) and more LTB, (**p = 0.018) than at baseline.

between aspirin ingestion and bronchospasm was 125 + 48 minutes. Two patients with ASRD were excluded: one (patient 6) because of too low a recovery of monocytes from the aspirin reaction blood; the other (patient 7) because of a protracted delay in collecting the reaction blood (50 minutes after bronchospasm compared with 20 to 30 minutes for all the other patients with ASRD). The percent change in eicosanoid release after aspirin was compared with matched samples obtained before aspirin and analyzed by Wilcoxon signed rank test. At the time of reaction, release of LTB, by A23 187stimulated monocytes increased significantly from 832.1 + 174 pg/ml at baseline to 1211.4 + 213 pg/ml. All seven patients with ASRD demonstrated increased LTB, release at reaction (p = 0.0 18). LTC, release increased in three of seven patients with ASRD, going from 132 ? 22 pg/ml at baseline to 174.1 -+ 34.1 pg/ml at reaction (p = 0.398) (Fig. 2). The release of TXB, dropped from 1125.8 +- 314.5 pg/ml at baseline to 597.9 r 407.6 pg/ml at reaction. Six of seven patients with ASRD showed a decrease in TXB, release after aspirin (p = 0.028), the one exception being the patient who reacted to only 6 mg of aspirin. After aspirin-induced bronchospasm, monocyte LTB, release increased 45.5% (equivalent to 379 pg/S X lo4 cells), whereas TXB, release decreased 46.9% (equivalent to 528 pg/5 X lo4 cells). To determine whether these changes were unique to patients with ASRD, monocyte AA metabolism was

Before Aspirin

After Aspirin

Before Aspirin

AftCU Aspirin

FIG. 3. Effects of 60 mg aspirin on TXB, and LTB, release by normal and ASRD monocytes. Bronchospasm developed in five patients with ASRD after challenge with 60 mg aspirin. Three healthy volunteers were also given 60 mg aspirin. Monocytes were obtained before challenge and at the time of bronchospasm in the patients with ASRD (90 to 205 minutes) or 2 hours after aspirin ingestion in the healthy volunteers. A23187stimulated TXB, and LTB, release was measured (pg/5 x 10’ cells, mean 2 SEM). Both groups showed increased LTB4 release after aspirin, however, only the patients with ASRD showed decreased TXB, release (*p = 0.0431; MannWhitney). The increase in LTB, was significant for the patients with ASRD (*p = 0.0431; Mann-Whitney) but not for the healthv volunteers.

studied in three healthy subjects 2 hours after ingestion of 60 mg aspirin. Ingestion of the aspirin was not associated with any adverse reaction. The baseline production of TXBl by ionophore-stimulated normal monocytes was 380.3 + 104.1 pg/ml and was not significantly suppressed 2 hours after 60 mg aspirin (331.3 +- 20.1 pg/ml, p > 0.8). In comparison, the five aspirin-sensitive asthmatic patients who reacted to 60 mg aspirin demonstrated a significant fall in TXBz from (805.4 + 269.7 pglml) at baseline to (227.68 + 105 pg/ml) at reaction (Fig. 3). However, as in the patients with ASRD, the release of LTB, by normal monocytes increased from 369 pg / ml at baseline to 6 12 pg / ml 2 hours after ingesting 60 mg aspirin (Fig. 3). DISCUSSION The underlying mechanism of bronchospasm in aspirin-sensitive asthmatic patients after cyclooxygenase inhibition is not known. It is generally accepted that bronchospasm may result from changes in the profile of AA metabolites formed after NSAID administration. Several hypotheses have been proposed to account for the occurrence of asthmatic reactions in patients with aspirin sensitivity. One explanation is that this group of asthmatic patients differ in their basal bronchomotor regulatory mechanism such that

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depletion of bronchoprotective prostaglandins results in imbalance between bronchodilatory and bronchoconstrictive mediators.6 Another hypothesis postulates an increased metabolism of AA into the 5-lipoxygenase pathway after cyclooxygenase blockade, resulting in the increased formation of potent bronchoconstrictive mediators, such as LTC4, LTD, and LTE4.28 Alternatively, AA may be diverted into the 12- and 15lipoxygenase pathway.29 We sought to determine whether there were underlying differences in AA metabolism in patients with ASRD compared with asthmatic patients without aspirin sensitivity and healthy controls, and whether shunting of AA into the lipoxygenase pathway after aspirin could be demonstrated. Because of their easy accessibility, ability to release eicosanoids, and developmental relationship to fixed tissue macrophages such as alveolar macrophages , peripheral blood monocytes were used to study the AA metabolism. The profile of AA metabolites released from A23 i87-stimulated monocytes in this study agrees with prior reports. 19.“. 30.3’ AA, the major membrane fatty acid component of human mononuclear phagocytes, is predominantly converted into TXBz by the cyclooxygenase pathway and 5-HETE and LTB, by the 5-lipoxygenase pathway.“. 32Mononuclear phagocytes have been reported to release only small amounts of PGE2, PGF,,, and 15-HETE after stimulation with ionophore A23 187.‘? We chose to study short-term monocyte cultures in suspension rather than monocytes cultured while adherent to plastic for several reasons. Adherence to plastic has been reported to cause nonspecific cell activation. 34 Furthermore, in preliminary experiments we found the eicosanoid release from adherent monocytes to be somewhat variable and to change depending on the duration of the adherence step. In contrast, AA metabolite release from monocytes cultured in suspension gave excellent reproducibility, varying less than 10% between triplicate cultures as determined by direct EIA of the culture supematants.‘4 This highlights the importance of culture conditions when comparing results of monocyte AA metabolism. After ionophore stimulation, monocytes of nine patients with ASRD released significantly more TXB, than asthmatic patients without ASRD (p < 0.0095) or healthy subjects (p < 0.015) (Table III, Fig. 1). This suggests that there is an ongoing abnormality of AA metabolism in patients with ASRD, involving an increased liberation of AA by the action of phospholipases and/ or an elevation of the cyclooxygenase enzyme activity. Two patients (5 and 6) were found to have normal levels of TXB, despite being aspirin sensitive. These patients were also distinguished by hav-

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ing the mildest asthma in the group with ASRD and being the only patients with ASRD not taking systemic steroids. Monocytes from patients with ASRD released significantly (p < 0.03) mote XSTB, than healthy controls. In accord with previousi? published observations that the major lipoxygenase products of monocytes and alveolar macrophages are 5-HETE and LTB‘,,” we found that monocytes released considerably less LTC, than LTB, or TXB,. The UK4 release did not vary significantly among any oi’ the three groups. We can not entirely exclude further enzymatic metabolism of LTC, into LTD, and LTE, as well as nonenzymatic degradation into leukotriene-sulfoxids. However. the antisera used in the EIA partially crossreacted with LTD., (46%) and LTE, 12%), and thus we do not believe this to be a likely source of error. The relatively low amount of LTC, released was close to the detection limit of the assay (38 pg,, and may have prevented our discerning any differences between the groups. After in vivo provocation with aspirin. the FEV, dropped significantly (p < 0.003; range, 23% to 59%) in all patients with ASRD. LTB, release increased in all seven patients with ASRD (mean increase, 45.5% compared with the before aspirin release; p = 0.0 18). Monocyte LTC., release increased in only three of seven patients with ASRD after aspirin-provoked bronchospasm (mean increase, 29.9%; p = 0.398). The mean release of TXB, dropped in six of seven patients with ASRD (mean decrease, 46.9%; p = 0.028) compared with before aspirin release. The only patient who failed to show a fall in monocyte TXB, release had a partial reaction to only 6 mg of aspirin. No blood was obtained when this patient had her full reaction to a higher dose of aspirin. It is possible that in vitro stimulation with calcium ionophore may have obscured a partial in vivo cyclooxygenase blockade caused by such a low dose of aspirin. The observed fall in TXB, release and increase in LTB, release in patients with ASRD after in vivo ingestion of aspirin is consistent with shunting of AA into the 5-lipoxygenase pathway. However, the increase in LTB, release after aspirin ingestion was not unique to the patients with ASRD. Normal subjects also demonstrated increased monocyte LTB, release after aspirin challenge compared with baseline levels. Unlike the patients with ASRD, the nomlal subjects did not demonstrate a corresporiding fall in TXB, release after aspirin. Aspirin is known to be a relatively weak cyclooxygenase inhibitor compared with ot.her NSAfDs. Although patient one did not show a substantial depression of TXB, release after 6 mg of aspirin, ingestion of 60 to 100 mg of aspirin led to a significant decrease

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of TXB, release in the other subjects with ASRD. This is in marked contrast to healthy subjects who had significantly lower TXB, release before aspirin and did not show any change in TXB, release after 60 mg of aspirin. Since TXB2 has been reported to be the major cyclooxygenase metabolite of human monocytes ,‘O these data suggest the possibility of an increased susceptibility of the cyclooxygenase enzyme to NSAIDs in patients with ASRD. To test this possibility, we are in the process of determining the sensitivity of monocyte cyclooxygenase to in vitro indomethacin exposure. Ferreri et al.” reported that the release of PGE, into nasal secretions did not change after low-dose aspirin challenge but decreased after ingestion of 650 mg aspirin in desensitized aspirinsensitive asthmatic patients and healthy subjects. TXB, levels were not measured in the nasal lavage fluid during that study. In addition, the differences between these two studies may reflect the different methodologic approaches. The lavage model measured release of PGE, from nasal tissue that included a variety of normal and inflammatory cell types, whereas our study measured release of TXB, from calcium ionophore-stimulated monocytes that had been purified to homogeneity, making the results only partially comparable. The relationship between these changes in AA metabolism and the onset of bronchospasm in patients with ASRD is uncertain. Although our data suggest shunting of AA metabolites from the cyclooxygenase to the 5-lipoxygenase pathway at the time of aspirininduced bronchospasm in monocytes of patients with ASRD, the increase in LTB, release without a corresponding fall in TXB2 release in normal monocytes is troubling. The data obtained with normal monocytes could be reconciled with the shunting theory if another cyclooxygenase product decreased after aspirin administration. Since TXB, was the only cyclooxygenase product measured in our current study, this issue is not resolved. Another possibility is that shunting does not occur at the time of aspirin-induced bronchospasm. Instead, one could postulate that the precipitous drop in monocyte TXB, release (uniquely seen in patients with ASRD) was unrelated to the increase in LTB, release (seen in all subjects). The cause of bronchospasm, therefore, might be related to a rapid shift in the balance between bronchoconstrictive and bronchodilatory AA products. In this hypothesis, the apparent susceptibility of monocyte cyclooxygenase to inhibition by low-dose aspirin could be an important determinant of aspirin sensitivity because such inhibition would have a rapid and profound effect on the availability of PGI, and PGE,. It will be important to determine whether cells other than mono-

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cytes also show an increased susceptibility to cyclooxygenase inhibition in patients with ASRD. Our data show that even small doses of aspirin may increase the formation of leukotrienes by ionophorestimulated monocytes from patients with ASRD and healthy subjects. Since patients with ASRD are more sensitive to inhaled LTE, than are aspirin-insensitive asthmatic patients or healthy subjects,12increased target organ sensitivity to leukotrienes may be an important distinguishing feature in ASRD. Although monocytes and alveolar macrophages do not form large amounts of sulfidopeptide leukotrienes, a variety of other cells such as bronchial epithelial cells, mast cells, eosinophils, and platelets do form sulfidopeptide leukotrienes and may contribute to the generation of leukotrienes in the lung during aspirin-induced bronchospasm. Transcellular synthesis of eicosanoids through export of LTA, from one cell and its catalytic conversion by LTA, hydrolase of another cell could also increase the production of sulfidopeptide leukotrienes in bronchial epithelial cells. Both mononuclear phagocytes and polymorphonuclear leukocytes have been shown to export LTA,.35 Therefore increased LTB, formation by monocytes or alveolar macrophages after ingestion of aspirin could also indirectly lead to increased LTC, generation in the airways. In summary, calcium ionophore-stimulated monocytes of aspirin-sensitive asthmatic patients release increased amounts of TXB2 before administration of aspirin, have cyclooxygenase enzymes that appear to be highly susceptible to the inhibitory effects of aspirin, and demonstrate a marked decrease in TXB,, release as well as an increase in LTB, release during aspirin-induced bronchospasm. These abnormalities appear to distinguish patients with ASRD from both healthy controls and non-aspirin-sensitive asthmatic patients. Although these data do not resolve the question of how aspirin ingestion results in the development of bronchospasm in sensitive individuals, it does provide novel information that supports the hypothesis that bronchospasm develops in patients with ASRD because of the effect of aspirin on AA metabolism. The authors thank Dr. Philippe Pfeifer for his assistance in performing the HPLC, and Drs. Eng M. Tan and Charles G. Cochrane for their advice and encouragement. REFERENCES

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