Cytokines in symptomatic asthma airways

Cytokines

in symptomatic

asthma

David H. Broide, MBChB,* Martin Lotz, MD,** Anthony David A. Coburn, MD,*** Edward C. Federman, MD,*** Stephen I. Wasserman, MD* San Diego, Caltj?

airways J. Cuomo, and

MD,***

To determine whether cytokines are generated in vivo in subjects with asthma, we have measured cytokine levels (tumor necrosis factor [TNF], granulocyte-macrophage-colony-stimulating factor [GM-CSF], interleukin [IL]-la, IL-l& IL-2, IL-4, and IL-6) in the airways of subjects with symptomatic (N = 24) and asymptomatic (N = 9) asthma with immunoassays (GM-CSF, IL-la, IL-l/3, IL-2, and IL-4) or bioassays (TNF and IL-6) and the polymerase chain reaction (IL-Ip and TNF). Significant levels of TNF (578 + 917pglml versus 24 ? 29pglml) (p = 0.01) GM-CSF (24 ? 41 pglml versus <8 pglml) (p = 0.02), and IL-6 (225 ? 327pglml versus 7 5 I2 pglml) (p = O.Ol), but not IL-la or IL-4, were detected in the bronchoalveolar lavage Juid (BALF) of patients with symptomatic compared with BALF of patients with asymptomatic asthma. Levels of IL-I/3 (266 5 270 pgiml versus <20pglml) (p = 0.001) and IL-2 (1.4 + 2.8 nglml versus CO.3 nglml) (p = 0.05) in BALF in patients with symptomatic compared with that in BALF levels in patients with asymptomatic asthma suggested activation of alveolar macrophages and T cells. Thus, in episodes of asthma, several cytokines, including TNF, GM-CSF, IL-l/S, IL-2, and IL-6 are detectable in BALF. (.I ALLERGY CLIN IMMUNOL I992;89:958-67.) Key words: Asthma, cytokines, IL-lp,

IL-2, IL-6, GM-CSF,

Current theories of the pathogenesis of asthma suggest that airway inflammation is an important determinant of bronchial hyperreactivity.’ Several airway inflammatory cells, I-3 including mast cells, eosinophils, T-lymphocytes, alveolar macrophages, neutrophils, and epithelial cells, have been postulated to participate in the inflammatory response to an inhaled antigen. Cytokines produced by these airway inflammatory cells could provide a local mechanism to amplify or modulate the initial inflammatory response to an inhaled antigen. In vitro inflammatory cells, in-

TNF

Abbreviations GM-CSF: LPS: TNF: IL: BAL: BALF: UCSD: IFN-?I: RNA-PCR: FEF,,:

From the Department of Medicine, Division of *Allergy, Immunology, and **Rheumatology, University of California-San Diego, San Diego, Calif., and ***Department of Medicine, Division of Pulmonary Medicine, Alvarado Hospital, San Diego, Calif. Supported in part by National Institutes of Health Grants AI 29974, AI 17268, and AI 20476. Presented in part at the Annual Meeting of The American Tboracic Society, Anaheim, Calif., May 1991. Received for publication June 17, 1991. Revised Jan. 2, 1992. Accepted for publication Jan. 7, 1992. Reprint requests: David H. Broide, MB, ChB, UCSD Medical Center, 8417, 225 Dickinson St., San Diego, CA 92103. 1/1/360!?1

958

used Granulocyte-macrophage-colonystimulating factor Lipopolysaccharide Tumor necrosis factor Interieukin Bronchoalveolar lavage Bronchoalveolar lavage fluid University of California-San Diego Interferon-y Ribonucleic acid-polymerase chain reaction Forced expiratory flow at 50%

eluding T cells435 and alveolar macrophages6, ’ are able to produce cytokines in response to antigen stimulation, whereas epithelial cells produce GM-CSF’ in response to LPS stimulation. The potential functional significance of cytokines generated in the airways to clinical asthma is suggested from in vitro studies in which cytokines have been demonstrated to activate eosinophils (TNF, GM-CSF, IL-5, and IL3)9-13and regulate IgE synthesis (IL-4). I4 We have thus sought to determine whether cytokines are generated

Cvtokines and asthma

VOLUME 89 NUMBER 5

in the airways of subjects with symptomatic asthma

and whether the profile of cytokines generated would suggest particular cellular source(s). MATERIAL AND METHODS Study subjects We have previously reported the clinical characteristics, technique of BAL, pulmonary function studies, BAL cell differential counts, and airway levels of mast cell (histamine and tryptase)- and eosinophil (major basic protein and eosinophil-derived neurotoxin)-derived mediators in patients with asymptomatic and symptomatic asthma.15. lb We have used the BALFs derived from these two groups of subjects with asthma for cytokine analysis. Subjects were defined as having asthma (according to the definition of the American Thoracic Society)” on the basis of clinical symptoms (history of either wheezing, chest tightness, and/or shortness of breath relieved by an inhaled B-agonist), physical examination, and pulmonary function studies demonstrating reversible airway obstruction. Each study subject with asthma had pulmonary function studies demonstrating reversible airway obstruction as manifested by either (1) a postbronchodilator increase in FEV, ?15%, (2) a posttherapy (of acute symptomatic asthma exacerbation) increase in FEV, 2 15%. or (3) an exercise-induced decrease in FEV, of 2 15%. Subjects with symptomatic asthma had daily wheezing by history and physical examination for days to several weeks before BAL. The BALF from subjects with symptomatic asthma (N = 24) was obtained from 10 previously reported patientsI as well as 14 previously unreported patients with symptomatic asthma. The BALF from patients with symptomatic asthma (six men and 18 women; aged 46 i 19 years) was obtained from subjects with asthma who underwent bronchoscopy and BAL (right lower lobe) for diagnostic or therapeutic purposes in the pulmonary department at Alvarado Hospital, San Diego, Calif. No patients with symptomatic asthma were recruited for the study, and the use of the otherwise discarded BALF was approved by the UCSD Human Subjects Committee. The use of bronchoscopy and lavage in this setting has previously been safely performed.” lo Before bronchoscopy, all patients with symptomatic asthma were treated as outpatients with various combinations of inhaled B-agonists, inhaled corticosteroids, inhaled cromolyn, inhaled ipratropium bromide (Atrovent: Boehringer Ingelheim, Inc., Ridgefield, Conn.). and oral theophylline, as well as oral or intravenous corticosteroids. All patients with symptomatic asthma received at least one dose of intravenous methylprednisolone (40 IO 125 mg, depending on body size) 4 hours before BAL. The BALF from patients with asymptomatic asthma (four men and tive women; aged 21 * 2 years) was obtained before exercise in a study of airway levels of mast cell-derived mediators in patients with exercise-induced asthma performed at UCSD. ii Medications used by patients with asymptomatic asthma included oral theophylline and inhaled B-agonists on an as needed basis. No patient with asymptomatic asthma had required treatment for an exac-

959

erbation of asthma with an inhaled, oral, or intravenous corticosteroid for at least a year before BAL

Processing

of BALF

BALF was concentrated tenfold with an Amicnn Centriprep 10 concentrator (Amicon Corp., Danvers, Mass.) before cytokine analysis. Cytokine levels are expressed per milliliter of concentrated BALF. Because several BALES had undetectable levels of cytokines, they were spiked after concentration with a known amount of cytokine equivalent to the midrange value of the particular cytokine assay. Results of these experiments are expressed as the percent of cytokine recovered in the cytokine assay.

TNF assay TNF was assayed with a standard L929 cytotoxicity assay’ in which the bioactivity of recombinant human TNF (Endogen, Boston, Mass.) was compared with TNF present in BALF. Confluent adherent monolayers of I.929 hbroblasts were trypsinized and resuspended at a concentration of 6 X 10’ cells per milliliter in media containing I pg!ml of actinomycin o-mannitol. Aliquots of 100 JLI of cells (6 X 10’ cells) were placed into each well of a 96-well. flat-bottom microtiter plate, and the cells were allowed to attach during a 2-hour incubation at 37” C. Either a 100 PI of test sample. TNF standard (10 to 1000 pgiml). or BAL lavage medium (negative control) was then added to the wells containing L9:!9 fibroblasts. Sample dilutions were prepared directly in the microtiter wells. and after an IXhour incubation at 37” C, dead or adherent cells were stained with 50 p,l of a solution containing 0.2% (wti-vol) crystal violet and 10% (vol i vol) of a 37% solution of formaldehyde prepared in 0.01 mol/L of phosphate buffer (pH Y.3). After a 5-minute incubation, dye was washed out with distilled water, the monolayer was dried, and optical density. 570 nm, was measured on a Dynatech micro-ELISA autoreadcr (Dynatech Laboratories, Inc.. Alexandria, Va. I

GM-CSF GM-CSF was assayed with an ELISA (Genzyme Corn., Boston, Mass.) with a monoclonal antihuman GM -CSF antibody and a rabbit polyvalent antibody to human GM-CSF. The assay can detect 8 to 1000 pgiml of GM-CSF, and there is no cross-reartivity in the assay with G.-CSF. MCSF, IL-3, IL-6, TNF-IX, or INF-y. Addition of known amounts of GM-CSF to BALF of patients with symptomatic asthma resulted in recovery of 103% of the added GM-CSF.

IL-4 IL-4 assays were performed with an ‘*‘l-labeled lL-4 immunoassay by Dr. Hans Spiegelberg (UCSD). A mouse monoclonal antihuman IL-4 antibody and an “‘l-labeled goat antihuman IL-4 polyclonal antibody was used in the IL-4 assay. The assay is sensitive to 200 pgiml of R-4.

IL-2 IL-2 was assayed with an IL-2 immunoassay (,Genzyme Corp.) solid-phase IELISA with a mouse monoclonal anti-

960

Broide

et al.

human 11-2 antibody, as well as a polyclonal rabbit antihuman IL-2 antibody. The assay is sensitive to 0.3 rig/ml of IL-2 and does not cross-react with IL-l (o or B), IL-3, IL-4, IL-6, G-CSF, M-CSF, GM-CSF, or INF-y. Addition of known amounts of IL-2 to BALF of patients with symptomatic asthma resulted in recovery of 98% of the added IL-2.

IL-la IL-la was assayed with an ELISA (Endogen) with a mouse monoclonal antibody specific for human IL-la as well as rabbit polyclonal antibody to human IL-la. The assay is sensitive to 75 pg/ml of IL-la and does not crossreact with IL-lB, IL-2, INF-7, IL-6, GM-CSF, or TNF. Addition of known amounts of IL-la to BALF of patients with symptomatic asthma resulted in recovery of 85% of the added IL- 1(Y.

IL-l/II IL-l B was assayed with an IL-1B ELISA (Cistron Biotechnology, Inc., Pinebrook, N.J.) with a mouse monoclonal antibody specific for human IL-1B and a polyclonal antibody to human IL-l B. The assay is sensitive to 100 pg/ml of IL-1B and does not cross-react with IL-la, TNF-a, TNF-B, IL-3, IL-4, or IL-6. Addition of known amounts of IL-1B to BALF of patients with symptomatic asthma resulted in recovery of 93% of the added IL-l B.

IL-6 IL-6 was assayed with the B9.9 hybridoma proliferation assay as previously described.20 The assay is sensitive to 0.1 pg/ml of recombinant IL-6.

RNA-PCR To determine whether cytokines detected by immunoassay or bioassay were generated by BAL cells or had diffused into the airways, RNA-PCR was performed on BAL cells derived from three patients with symptomatic asthma who each had elevated immunoassay or bioassay levels of both IL-l B and TNF, as well as on BAL cells from two patients with asymptomatic asthma with undetectable levels of immunoreactive IL-1B. Total cellular RNA was isolated from 10’ BAL cells of patients with symptomatic asthma (80.2% ? 2.4% alveolar macrophages, 10.3% 2 1.5% eosinophils, 2.7% 5 1.2% neutrophils, 5.7% +- 0.7% lymphocytes, and 1.2% ? 0.4% mast cells) or from lo5 BAL cells of patients with asymptomatic asthma (92.3% 2 0.8% alveolar macrophages, 1.3% ? 0.1% eosinophils, 0.4% 2 0.1% neutrophils, 5.9% +- 0.6% lymphocytes, and 0.1% 2 0.1% mast cells) with a modification of the single-step acid guanidium thiocyanate-phenol-chloroform procedure (RNA2,,L; CinnalBiotecx, Friendswood, Texas), followed by chloroform extraction and isopropanol precipitation.*’ Total cellular RNA was reverse transcribed (MMLV, reverse transcriptase; Bethesda Research Laboratories, Gaithersburg, Md.) to provide cDNA targets for the subsequent 40-cycle PCR amplification. Oligonucleotide primers (synthesized by the UCSD Molecu-

J. ALLERGY CLIN. IMMUNOL. MAY 1992

lar Core Laboratory) for TNF (5’ primer: 5’CAGAGGGAAGAGTTCCCCG-3’ and 3’ primer: 5’ CCTTGGTCTGGTAGGAGACG-3’) and IL-1B (5’ primer: 5’-AACAGATGAAGTGCTCCTTCCAGG-3’ and 3’ primer: 5’-TGGAGAACACCACTTGTTGCTCCA-3’) were the same as described by Wang et al.*’ Amplification conditions included heat denaturing at 94” C for 1 minute. primer annealing at 55” C for 2 minutes, and primer extension at 72” C for 6 minutes in an automated thermal cycler with a thermostable DNA polymerase (Thermus aquaticus; Perkin Elmer-Cetus, Emeryville, Calif.). PCR products were run on 2% agarose gels and visualized by ethidium bromide staining. DNA molecular weight markers and LPS (30 kg/ ml)-stimulated THP- 1 cells*’ (a monocyte leukemic cell line that produces TNF and IL-1B in response to LPS stimulation) (see Figs. 6 and 7) allowed for the identification of the appropriate-sized TNF (325 base pairs) and IL-1B (388 base pairs) PCR products. In all experiments, a negative control (PCR reagents without cells) was included to detect possible reagent contaminants.

Statistical

analysis

Comparison between cytokine levels in patients with symptomatic and asymptomatic asthma were performed with the Mann-Whitney U test for unpaired samples. A p value of CO.05 was considered to be statistically significant.

RESULTS Pulmonary

function

and BALF analysis

The FEV, (1.56 +- 0.45 L; 59% + 14% of predicted FEV,) and FEF,, (1.34 ? 0.92 L/set; 31% ? 17% of predicted FEF& in the patients with symptomatic asthma were significantly lower than the corresponding value for FEV, (3.16 t 0.45 L; 86% 2 10% of predicted FEV,) (p = 0.05) and FEF,, (4.04 + 1.54 L/set; 71% -t 25% of predicted FEFSO) (p = 0.05) in the patients with asymptomatic asthma. The BALF differential cell count in patients with symptomatic asthma (74.5% +7.3% macrophages, 8.7% -+ 1.5% lymphocytes, 11 .O% ? 4.5% eosinophils, 5.0% + 1.9% neutrophils, and 0.8% + 0.5% mast cells) revealed a statistically significant (p < 0.05) increase in BAL eosinophils and neutrophils compared with BAL cells recovered from patients with asymptomatic asthma (89.8% ? 3.6% macrophages, 7.1% 2 2.3% lymphocytes, 1.1% + 0.9% eosinophils, 0.6% -I: 0.4% neutrophils, and 1.3% ? 0.4% mast cells). The BAL lavage volume recovered (from the 100 ml of saline instilled) was 37 t 8 ml in patients with symptomatic asthma and 45 + 6 ml in patients with asymptomatic asthma.

TNF and GM-CSF Levels of TNF (578 +- 917 pg/ml versus 24 r 29 pg/ml) (p = 0.01) (Fig. 1) and GM-CSF (24 ‘-t 41

VOLUME 89 NUMBER 5

Cytokines

and

asthma

961

0 3000

I 0

/

0 2000

;

1000

1

:

lOOO-

30 i

0

/

----

_--.-..

0

l

8

0 O-

l

--~__ CDOEb 0 0

ASYMPTOMATIC SYMPTOMATIC

ASTHMA ASTHMA

Fffi. 1. TNF levels were significantly patients with symptomatic asthma with that of patients with asymptomatic (p = 0.01).

elevated in BALF of (N = 24) compared (N = 9) asthma

pg/ml versus <8 pg/ml) (p = 0.02) (Fig. 2) were significantly elevated in patients with symptomatic compared with levels in patients with asymptomatic asthma. Thus, at least two (TNF and GM-CSF) of the four cytokines (IL-3, IL-5, TNF, and GM-CSF)9*13 currently known to up regulate eosinophil effector function are present in airways of patients with symptomatic asthma. IL-1 and IL-2

To determine whether the profile of cytokines generated would suggest a particular cellular source, we determined levels of IL-l (as evidence of alveolar

. ..-

.-

ASYMPTOMATIC ASTHMA SYMPTOMATIC ASTHMA

FIG. 2. GM-CSF levels were significantly of patients with symptomatic asthma with that of patients with asymptomatic (p = 0.02).

50

_

elevated in BALF (N = 24) compared (N = 9) asthma

macrophage activation) and IL-2 (as evidence of T cell activation). Since IL- 1 is encoded by two separate genes (a and p),*’ we have measured both subtypes of IL-l with sensitive immunoassays (IL- lp represents > 80% of IL- 1 activity released by human mononuclear phagocytes).6 Levels of IL-l@ (266 it 270 pg/ml versus <20 pg/ml) (p = 0.001) (Fig. 3) but not IL-la (109 IL: 119 pg/ml versus <75 pgiml) were significantly elevated in patients with symptomatic compared with that of patients with asymptomatic asthma. Addition of known amounts of IL- 1(Yand ILlp to BALF of patients with symptomatic asthma before assay resulted in recovery of 85% aud 93%1of the added IL-l in the IL-la and IL-l@ assays. Statistically significant increased levels of IL-2 in airways of patients with symptomatic compamd with that of patients with asymptomatic asthma if .4 + 2.8 rig/ml versus CO.3 rig/ml) (p = 0.05) (Fig. 4) were also noted. Addition of known Amounts &f ZL-2 to BALF of patients with symptomatic asthma resulted in recovery of 98% of the added IL-2. IL-4 and IL-6

Since all our patients with asymptomatic asthma (N = 9) and 16 of 24 patients with symptomatic

962

Broide

J. ALLERGY CLIN. IMMUNOL. MAY 1992

et al.

16 lOOO-

400 ~~ 2 \ k?

0

i 200 200 _

ii

0

I =!

0

0

150-0 loo-0

a

50 --

l ooo.0. 0 0 0

FIG. 3. IL-lp levels were significantly patients with symptomatic asthma with that of patients with asymptomatic (p = 0.001).

TABLE

I. Clinical

elevated in BALF of (N = 24) compared (N = 9) asthma

24 6/18 46 + 19 16124 O/24 T 21124 22124 P AT 3124 CR 9124 CS (I) lo/24 CS (0) 19/24

Asymptomatic asthma

9 415 21 k 2 919 o/9 T 3/9 919 P

T, Theophylline; p, inhaled P-agonist; AT, Atrovent metered-dose inhaler; CR, cromolyn metered-dose inhaler; CS (I), inhaled corticosteroids; C.S(O), oral corticosteroids.

were known to be atopic (Table I) on the basis of either an immediate hypersensitivity skin test or an IgE RAST to inhaled environmental allergens,15 we measured levels of IL-4 in BALF to determine its

asthma

ASYMPTOMATIC ASTHMA SYMPTOMATIC ASTHMA

FIG. 4. IL-2 levels were significantly patients with symptomatic asthma with that of patients with asymptomatic

elevated in BALF of (N = 24) compared (N = 9) asthma

(p = 0.051.

characteristics Symptomatic asthma

No. Sex (M/F) 4s WI Atopic Smokers Medications

0 0

ASYMPTOMATIC ASTHMA SYMPTOMATIC ASTHMA

relationship to IgE synthesis. Levels of IL-4 in all the patients with symptomatic and asymptomatic asthma were <200 pg / ml (the sensitivity of the assay). Levels of IL-6 (225 +- 327 pg/ml versus 7 & 12 pg/ml) (p = 0.01) (Fig. 5) were significantly elevated in airways of patients with symptomatic compared with that of patients with asymptomatic asthma. Increased levels of cytokines were noted in atopic as well as nonatopic patients with symptomatic asthma (Table II).

RNA-PCR TNF (Fig. 6) and IL-lb (Fig. 7) mRNA was detected in lo5 BAL cells derived from each of three patients with symptomatic asthma with elevated BALF, TNF, and IL- 1p levels, suggesting that a proportion of the TNF and IL- 1p detected by immunoassay and bioassay was derived from BAL cells. In two patients with asymptomatic asthma with undetectable levels of IL-lp by immunoassay, only faint IL-l p bands were detected by PCR (Fig. 7, C). Similar-sized IL-lp and TNF PCR products were noted in lanes containing 105 LPS-stimulated THP-1 cells

Cytokines

VOLUME 89 NUMBER :I

1600

a11d a:
963

-

1200t

0 0

ASYMPTOMATIC ASTHMA SYMPTOMATIC ASTHMA

FIG. 5. IL-6 levels were significantly patients with symptomatic asthma with that of patients with asymptomatic (p = 0.01).

elevated in BALF of (N = 24) compared (N = 9) asthma

c

(positive control), whereas lanes containing lo5 unstimulated THP-1 cells (negative control) had faintly detectable IL- 1p and TNF PCR products. The absence of detectable PCR products in the lanes containing PCR reagents without addition of cells ensured that reagent contaminants were not responsible for the asthma BAL cell-cytokine PCR products detected. DlsClJSSION To determine whether cytokines are generated in episodes of asthma, we have measured cytokine levels (TNF, GM-CSF, IL-la, IL-l& IL-2, IL-4, and IL6) in the airways of patients with symptomatic and asymptomatic asthma and determined whether the profile of cytokines generated would suggest a particular cellular source(s). Significant elevations of TNF, GM-CSF, IL-l& IL-2, and IL-6 levels (but not IL-la or IL-4) were noted in airways of patients with symptomatic compared with that of patients with asymptomatic asthma. The relevance of the detection of TNF and GM-CSF in airways of patients with symptomatic asthma can be inferred from studies demonstrating that TNF and GM-CSF are both

FIG. 6. RNA-PCR: TNF. Ethidium bromide staining of 2% agarose gel depictilig negative control (PCR reagents, no cells) in lane 7, positive control (IO5 LPS-stimulated THP1 cells) in lane 2, DNA molecular weight markers in lane 3, and IO5 BAL cells from patients with symptomatic asthma in lanes 4, 5, and 6, respectively; arrow indicates 325 base pair size of TNF PCR products.

able to up regu,late eosinophil effector function in vitro.‘. I’. “. ” Preincubation of eosinophils with TNF increases both their production of superoxidez4 and the eosinophil peroxidase-derived oxidant hypobromous acid,‘4 as well as the cytotoxicity of’ the eosinophil to endotheliumzJ and parasites.” Similarly,

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Broide et al.

TABLE

II. BAL cytokine IL-1p h/ml)

No. BAL

Mean

levels

cytokine 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 C SD

levels

266

IL-2 hg/ml)

in symptomatic 87 <20 <20 <20 310 693 20 873 <20 513 24 53 <20 623 322 20 661 127 671 368 180 298 288 161 k

270

TNF (pdml)

asthma* CO.3 CO.3 1.5 CO.3 CO.3 CO.3 13.7 3.1 CO.3 CO.3 1.9 CO.3 0.7 CO.3 CO.3 CO.3 4.0 1.0 CO.3 CO.3 2.0 CO.3 CO.3 CO.3 1.4

+

2.8

GM-CSF (pglml)

91 280 199 487 30 163 2330 145 1136 3389 319 61 55 150 56 116 2604 71 86 213 139 182 1273 303 578

k

917

<8 <8 <8 72 <8 <8 10 <8 194 9 <8 19 <8 <8 9 <8 8 <8 <8 <8 70 28 48 <8 24

k 41

FEV, (% of predicted)

IL-6 (pglml)

74
+

327

FEho (% of predicted)

60 80 61 61 74 74 52 43 48 60 77 40 40 92 56 46 63 55 42 59 60 45 52 74 59 2

56 38 15 23 32 49 20 25 12 22 60 12 13 50 18 18 45 22 11 26 54 24 40 59 14%

31

2

17%

BAL cytokine levels in asymptomatic asthma 1 2 3 4 5 6 7 8 9

<20 <20 <20 <20 <20 <20 <20 <20 <20

CO.3 CO.3 CO.3 CO.3 CO.3 CO.3 CO.3 CO.3 CO.3

Mean -C SD

<20

CO.3

*Subjects

Nos.

1 through

16 were known

to be atopic,

95


29 subjects

eosinophils incubated with GM-CSF synthesize increased amounts of leukotriene C, and exhibit enhanced cytotoxicity to parasite targets.” Thus, the generation of TNF and GM-CSF in the airways of patients with symptomatic asthma could influence recruited eosinophil effector function. In addition, since TNF a!d IL- 1 are potent inducers on endothelial cells of endothelial-leukocyte adhesion molecule 1 and intercellular adhesion molecule 1,” the production of these cytokines in airways of patients with symptom-

<8 <8 <8 <8 <8 <8 <8 <8 <8 <8 17 through

7 *

32
88 69


85 88 72 68 113 73 91

12

86

r

40 89 58 98 42 108 52 70 83 10%

71

e

25%

24 were not atopic.

atic asthma could promote adhesion of circulating leukocytes to activated pulmonary endothelial cells and thus direct accumulation of inflammatory cells in the antigen-stimulated airways. Although IL-4 is an important positive regulator of IgE synthesis in vitro,‘4 neither patients with symptomatic (16/24 known to be atopic) nor with asymptomatic asthma (9/9 known to be atopic) had detectable levels of IL-4 in BALF. Several potential explanations for the inability to detect IL-4 in BALF,

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FIG. 7. RNA-PCR:

IL-18. Ethidium bromide staining of 2% agarose ge’i depicting A, lane 7, DNA molecular weight marker; lane 2, negative control (PCR reagents, no cells); lanes 3, 4, and 5, lo5 BAL cells from patients with symptomatic asthma, respectively; arrow, 388 base pair size of IL-18 PCR product, B, lane 7, DNA molecular weight marker; lane 2, negative control (PCR reagents, no cells), lo5 unstimulated THP-1 cells; lanes 3 and 4 (duplicate analysis), lanes 5 and 6, lo5 LPs stimulated THP-1 cells (duplicate analysis); arrow, 388 base pair size of IL-18 PCR products, and C, lanes 1 and 2, IO5 BAL cells from patients with symptomatic asthma; lanes 3 and 4, lo5 BAL cells from patients with asymptomatic asthma; lane 5, IO5 LPS stimulated THP-1 cells; lane 6, PCR reagents with no cells; lane 7, DNA molecular weight marker; arrow, 388 base pair size of IL-18 PCR product.

including dilution of IL-4 in the 100 ml saline lavage, binding of secreted IL-4 to target receptors, and thus not “free” to be recovered in BALF, degradation of IL-4 by proteolytic enzymes in BALF in symptomatic asthma, IL-4 assay sensitivity, the use of antiasthma medications (including corticosteroids) by our asthmastudy subjects, duration of asthma symptoms (varied from days to weeks in our study subjects), as well as the possibility that detectable amounts of IL-4 are not produced in the airways of patients with symptomatic asthma, need to be considered. The potential cellular source of cytokines detected

in the airways of patients with asthma include cytokines derived from cells resident or recruited into the airways (alveolar macrophages, T cells, epithelial cells, mast cells, and neutrophils) as well as cytokines that have diffused into the airways and are derived from nonairway cells (i.e., endothelium and fibroblast). The detection of IL-l p and TNF mRNA in BAL cells with RNA-PCR suggests that a proportion of these cytokines detected by immunoassay and bioassay is derived from airway cells. However, since RNA-PCR is an extremely sensitive method in detecting cytokine mRNA, the relative proportion of

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cytokines derived from BAL and non-BAL cells cannot be addressed from our study. Although the mean age of the patients with symptomatic asthma (46 ? 19 years) was older than the age of patients with asymptomatic asthma (21 ? 2 years), age alone is unlikely to account for elevations in cytokine levels. This conclusion is suggested from the subset of patients with symptomatic asthma less than 35 years of age (mean age, 24 + 9 years; N = 12) who had elevations in cytokine levels similar in range to the group with symptomatic asthma as a whole (data not presented). We were unable to address in this study whether the symptomatic state or corticosteroid treatment associated with the symptomatic state (each patient with symptomatic asthma received at least one dose of corticosteroid before BAL) was responsible for the generation of cytokines that were detected in BALF. However, an analogous study of six nonallergic patients with symptomatic asthma (FEV,, 72% ? 7% of predicted) indicated that the symptomatic state (no patient had received corticosteroid therapy before BAL) was associated with increased levels of immunoreactive IL-I B, GM-CSF, and IL-6 in BALF.26 Thus, the detection of cytokines in the BAL fluid of patients with symptomatic asthma not treated with corticosteroids,26 as well as the ability of corticosteroids to inhibit the generation of cytokines in vitro,27 would suggest that the symptomatic state and not corticosteroid therapy associated with the symptomatic state was responsible for the presence of cytokines detected in BALF. Corticosteroid therapy may also account for the absence of detectable cytokines in BALF of several patients with symptomatic asthma in this study. In patients with asthma, bronchial hyperresponsiveness to an inhaled antigen may be generated by an airway inflammatory cascade of mast cell degranulation, activation of T cells and alveolar macrophages, cytokine production, and activation of recruited airway eosinophils and resultant epithelial desquamation. Although our study demonstrates the presence of several cytokines in airways of patients with symptomatic asthma, additional studies, including the use of cell separation, RNA-PCR, and in situ hybridization techniques, will be required to determine the cellular source of such cytokines detected in the airways. We thank Cindy Beck and Dr. Hans Spiegelberg for performing IL-4 assays, Dr. Bara Racou for assistancewith RNA-PCR preparation

studies, and Crystal of this manuscript.

Goodman

for

her

skillful

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AVAILABLE NOW! The PROCEEDINGS OF THE INTERNATIONAL CONGRESS OF ALLERGOLOGY AND CLINICAL IMMUNOLOGY can be purchased from the Publisher. This collection of “state-of-the-art” presentations from the XII Congress held October 2O2.5, 1985, in Washington, D.C., brings together the current advances in basic and applied aspects of allergy and allergic diseases. It includes 528 pages covering such topics as IgE, roles of the different cell types and their products, clinical problems, asthma, rhinitis, and reactions to foods and drugs and occupational agents, collected and reviewed by Editor Charles E. Reed, MD (USA) and Associate Editors Joseph Bellanti.. MD (USA), Robert J. Davies, MD (UK), Sidney Friedlaender, MD (USA), Albert Oehling, MD (Spain), and Raymond G. Slavin, MD (USA). To purchase, call or write: Mosby-Year Book, Inc., 11830 Westline Industrial Dr.. St. Louis, MO 63146-3318, or telephone FREE l-800-325-4177, Journal Fulfillment, ext. 4351 (in Missouri call collect at 314-872-8370, Journal Fulfillment, fext. 4351). Prepayment required. Make checks payable to Mosby-Year Book, Inc. (All payments must be in US funds drawn on a US bank.) Price: $36.50 in the US, $40.50 in Canada, and $41.50 international (surface shipping charges included).