Urinary leukotriene E4 levels during early and late asthmatic responses Patrick J. Manning, MD,* Joshua Rokach, PhD, Jean-Luc Malo, MD, Diane Ethier, BSc, Andre Cartier, MD, Yves Girard, BSc, Stella Charleson, BSc, and Paul M. O'Byrne, MB** Hamilton, Ontario, and Montreal and Kirkland, Quebec, Canada The sulphidopeptide leukotrienes C4 and D4 (LTC4, LTD4) are p~otent bronchoconstrictor mediators, released from human lung fragments after challenge with specific allergens in vitro. The purpose of this study was to measure urinary LTE4 (metabolite of LTC4 and LTD4) in subjects undergoing inhalation challenges with allergens or occupational sensitizing agents in the laboratory. Eighteen subjects with previously documented isolated early asthmatic responses (EARs), isolated late asthmatic responses (LARs), or dual (both early and late) asthmatic responses were studied. Urinary LTE4 levels increased in subjects who developed either isolated EARs (mean fall in FEV1, 27.98%) or early responses preceding LARs (mean fall in FEVI, 15.01%). The baseline levels of LTE4 were 150.26 (SEM, 49.5)pg/mg of creatinine in the isolated responders and 66.60 (SEM, 13.5) pg/mg of creatinine in the dual responders. These levels increased to 1816 (SEM, 606.1)pg/mg of creatinine (p = 0.041) and 174.80 (SEM, 40.1) pg/mg of creatinine (p = 0.025), respectively, after the EAR. The degree of maximal bronchoconstriction during the EAR correlated with the levels of LTE4 (r = 0.68; p = 0.001). No significant increase in urinary LTE4 levels occurred during the LAR. These results suggest that the LTE4 precursors, LTC4 and LTD4, are important bronchoconstrictor mediators causing EARs after allergen inhalation. ( J ALLERGY CLtN IMMUNOL 1990;86:211-20.)

Increasing symptoms o f asthma can occur in sensitized individuals during seasonal allergen exposure,1 after inhalation o f occupational sensitizing agents in the workplace, 2 or after bronchoprovocation with allergen 3 or sensitizing agents in the laboratory. 4 Inhalation o f allergens or occupational sensitizing agents can result in three patterns of response: (1) an isolated E A R during which airway narrowing develops within 10 minutes of the inhalation, reaches a m a x i m u m within 30 minutes, and generally resolves within 1 to 3 hours, (2) a dual response during which initial airway narrowing occurs that either does not From the Department of Medicine, McMaster University, Hamilton, Ontario; Hopital du Sacre-Coeur de Montreal, Montreal, Quebec; Merck-Frosst Center for Therapeutic Research, Kirkland, Quebec, Canada. Supported by a grant from Merck-Frosst, Quebec, Canada. Received for publication Jan. 23, 1990. Revised April 6, 1990. Accepted for publication April 14, 1990. Reprint requests: P. M. O'Byme, MB, Department of Medicine, Health Sciences Center, McMaster University, 1200 Main St. West, Hamilton, Ontario, Canada L8N 3Z5. *Recipient of Medical Research Council of Canada Fellowship. **Scientist at Medical Research Council of Canada. 1/1/21727

Abbreviations used LAR: EAR: DAR: BAL: LTC,, LTD4, LTE4: PC~o: PBS: TDI: HDI: RIA: HPLC:

Late asthmatic response Early asthmatic response Dual asthmatic response Bronchoalveolar lavage Leukotrienes C4, D4, and E4 Provocative concentration causing a 20% fall in FEVj Phosphate-buffered saline Toluene diisocyanate Hexamethylene diisocyanate Radioimmunoassay High-performance liquid chromatography

return to baseline values or recurs after 3 to 4 hours, reaching a m a x i m u m between 8 to 12 hoursS; this response occurs in approximately 50% o f subjects demonstrating the E A R , and (3) an isolated L A R in which no E A R occurs but airway narrowing begins 2 to 3 hours after the inhalation and reaches a maximum between 8 to 12 hours. A i r w a y hyperresponsiveness, which can occur after

211

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TABLE I. Characteristics of subject

Subject

Sex

Age ( y r )

Atopy

FEV1 (% predl

PC=** (mg/ml)

Responsibleagent

Isolated EARs 1 2 3 4 5

M M M M M

29 36 31 29 30

+ + + +

101 97 107 78 96

10.1 1.0 3.1 0.6 3.2

Guar gum Flour HDI Tertiary amine Guar gum

Isolated LARs 6 7 8 9 10

F F M M M

37 44 45 36 38

+ + + -

105 83 75 97 I00

12.8 0.1 1.4 1.5 1.2

Tertiary amine Tertiary amine TDI HDI Western red cedar

F F M M F M F F

34 46 21 36 25 27 25 36

+ + + + + + + +

102 79 90 80 100 99 78 106

7.4 1.7 4.0 5.3 1.5 32.0 1.9 7.3

Ragweed House dust mite Mixed tree House dust mite House dust mite Ragweed Ragweed Ragweed

DARs 11 12 13 14 15 16 17 18

*Provocation concentration of inhaled agonist causing a 20% fall in FEV~.

inhalation of allergen or occupational sensitizing agents and which can last for several days or weeks, 6 is usually associated with the L A R 6 and may be caused by the presence o f inflammatory cells in the airways. 7 Studies examining the importance o f specific mediators in the pathogenesis of asthma are complicated by the lack of potent, specific mediator antagonists and synthesis inhibitors, and by the difficulties in measuring the mediator or its metabolite at the site of action in the airways. Partly for these reasons, the role of the sulphidopeptides LTC4 and LTD4 in the pathogenesis o f the asthmatic responses and in causing airway hyperresponsiveness after inhaled allergen is controversial.8 The sulphidopeptide leukotrienes are released from chopped human lung from allergic subjects in response to challenge with allergen. 9 Also, increased levels of LTE4 have been demonstrated in BAL fluid from subjects with mild to moderate, but stable, asthma, ]~ in urine from patients with acute asthma, H and during E A R s after allergen challenge. 1~ In addition, inhaled LTC 4 and LTD4 are potent bronchoconstrictors in both normal subjects and subjects with asthma. J2-,4However, to date, there have been no studies of measurements of sulphidopeptide leukotriene release during both EARs and LARs induced by al-

lergens or occupational sensitizing agents in subjects with asthma, which would implicate the leukotrienes 'in the pathogenesis o f these responses. LTC4 and LTD4 are rapidly metabolized in human plasma to the stable metabolite, LTE4, ~5 and the predominant route of excretion is in the urine. ]6 The purpose o f this study was to measure the levels o f urinary LTE4 during both E A R and LAR. This was done in subjects who were known to be sensitized to inhaled allergens or to occupational sensitizing agents and who developed either isolated EARs, isolated LARs, or DARs after inhalation challenge.

METHODS Subjects Eighteen subjects were studied in the clinical research laboratories of McMaster University Medical Center, Hamilton, and Hopital du Sacre-Coeur, Montreal, Canada. The subjects were chosen because they were known to develop asthmatic responses after inhaling either allergens or occupational sensitizers. Ten subjects were known to develop either isolated EARs or isolated LARs after inhalation of occupational sensitizers (Table I). Eight other subjects were known to develop both EARs and LARs after inhalation of environmental allergens (Table I). The histamine PC_~o in these subjects ranged from 0.1 to 32.0 mg/ml. (Subjects

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with asthma and with current symptoms usually have a PC2~, < 8 mg/ml.) Fifteen of the subjects were atopic, as determined by one or more positive responses to prick skin tests with a battery of 15 common allergens. All subjects with current or seasonal asthma were studied at a time when the asthma was mild and controlled by bronchodilators alone. No subject was taking any other medication. There was no current exposure to allergens to which subjects were sensitized (with the exception of the house dust mite), and there had been no exacerbations of asthma for at least 4 weeks. The baseline FEV, was > 7 5 % of predicted normal z7 in all subjects on all study days. The study was approved by the Ethics Committee of both hospitals, and each subject gave written informed consent.

Study design Subjects attended the laboratory on 2 study days. On the first day for studies with inhaled allergens, subjects' characteristics were documented, and an inhalation test with either the allergen or the appropriate diluent was performed. Since all subjects had inhaled allergens previously, an allergen dose-finding study was not performed. On the second study day, the alternate inhalation challenge was performed. For studies with the occupational sensitizing agents, the order of allocation of the study days was not randomized; the diluent day preceded the exposure to the sensitizing agent. All the urine produced during the following periods of time was collected for measurement of LTE~: the hour immediately before the inhalation tests, 0 to 3 hours (during the EAR), and 3 to 7 hours (during the LAR) after beginning the inhalation tests. Subjects were asked not to pass urine except to void immediately before and at 3 hours and 7 hours after the inhalation test. Inhaled 132-agonists were withheld for at least 8 hours before all inhalation tests. Subjects were asked to abstain from caffeine and vigorous exercise the morning of the inhalation tests.

Skin tests Skin testing was performed by the prick method with 15 common inhaled allergens (Hollister-Stier, Mississauga, Ontario). For subjects exposed to guar gum, prick skin tests were also carried out with this preparation diluted in PBS at a concentration of 0.1 mg/ml. Various cereal extracts (Hollister-Stier) were used for the subject sensitive to flour. A significant reaction was defined as a wheal, 3 rnm, with a negative control reaction to the diluent 10 minutes after the introduction of the antigen.

Histamine-inhalation tests Histamine-inhalation tests were carried out according to the method of Cockcroft et al. ~ with the Wright nebulizer (output, 0.13 to 0.15 ml/min; mass median aerodynamic diameter of particles, 1.3 I~m). After nebulization of PBS with 1.5% benzyl alcohol (pH 7.4), increasing doubling concentrations of histamine phosphate were inhaled until a

PC~0 was obtained. Dose-response curves were drawn on a semilogarithmic noncumulative scale. The PC2o was interpolated from the individual dose-response curves. A methacholine PC2o was obtained in all subjects in this manner.

Allergen-inhalation tests Allergen-inhalation tests were performed with a Wright nebulizer operated by oxygen at 50 psi and at a flow rate to produce an output of 0.13 ml/rain. The nose was clipped, and aerosols were inhaled through a mouthpiece during tidal breathing. The starting concentration of allergen extract for inhalation was determined according to a formula described by Cockcroft et al.~9 with the results from prick skin tests with the same extract and the histamine PC~ in the entry period. The starting concentration of allergen extract for inhalation was two doubling concentrations below the concentration predicted to cause a PC2o. Doubling concentrations of allergen were inhaled for 2 minutes, and FEV~ was measured 10 minutes after each inhalation. If the FEV, had fallen by 10% or more, the FEVt was repeated at 10-minute intervals until no further fall in FEV~ was observed. The FEV~ was then measured at 20, 30, 45, 60, 90, and 120 minutes, and at hourly intervals until 7 hours after the allergen inhalation. The allergen extracts were obtained from Miles/HollisterStier. Extracts were stored at - 7 0 ~ C and diluted for skin tests and allergen inhalation on the day they were to be used.

Diluent-inhalation tests Diluent-inhalation tests were performed with a Wright nebulizer as described above. Subjects inhaled diluent (PBS with 1.5% benzyl alcohol) three times for 2 minutes, followed by measurements of FEV, according to the same schedule as the schedule followed during the allergeninhalation tests.

Inhalation tests with occupational sensitizing agents Sensitizing agents in powder form (guar gum, flour, western red cedar) and lactose were aerosolized with an apparatus previously described. 2~This apparatus consists of a particle generator with an endless screw, an exposure room, and a photometer and cascade impactor located 1 to 2 cm from the mouth. The concentration of particles was < 10 m g / m ~ corresponding to the threshold limit value-short term exposure limit in all instances; > 5 0 % of inhaled particles had a diameter < I 0 ~m, thus allowing for deposition in the trachea and airways. Challenges with agents in vapor or liquid forms were carried out in a challenge room similar to the room originally described by Pepys and Hutchcroft. 2' TDI, available in a liquid form, was put in a recipient 30 to 40 cm away from the subject. HDI and the tertiary amine were nebulized at a distance of 30 to 40 cm from the mouth. In the case of TDI and HDI, continuous monitoring with

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1990

105-

I I

100 I 95 9O FEV 1 (%)

85 803000

75-

2500

70-

2000

65-

LTE 4 1 5 0 0 (pg/mg CreaUnlne) 1000 500 0 I

I

l

l

I

I

I

I

0

1

2

3

4

5

6

7

TIME POST INHALATION (h) FIG. 1. Change in FEV~ (percent) in five subjects w h o developed an isolated EAR after inhaled occupational sensitizing agents (e) and after inhalation of the appropriate diluent (o). Also illustrated are the levels of urinary LTE4 (mean and SEM) measured before inhalation of the diluent (open bars) or the sensitizing agent (solid bars) between 2 to 3 hours and between 6 to 7 hours after inhalation. A significant increase in urinary LTE. (p = 0.041) occurred after the EAR.

an MDA-7100 apparatus (MDA Scientific, Inc., Glenview, Ill.) insured that the concentration remained below the 20 ppb level, which does not result in toxic reactions. Progressive increasing durations of exposure (one breath, 15 and 45 seconds, 2, 5, 15, and 30 minutes) were carried out, the FEV, being assessed immediately and 10 minutes after the end of exposure. Total duration of exposure to the sensitizing agent varied from a minimum of 1 minute to a maximum of 30 minutes. Isolated late responders received the maximum exposure. Placebo exposures were carried out in a similar way with lactose in the cases of guar gum and flour, a wood dust not containingred cedar in the case of red cedar, and the organic diluent usually mixed to TDI, HDI, and tertiary amine for these specific agents. Measurements

of u r i n a r y LTE4

All urine was collected into containers containing 1 ml of 0.I mol/L of the oxygen radical scavenger, 4hydroxytetramethyl-piperidinooxy-free radical, and 0.5 ml 10 N NaOH. The samples were stored at - 7 0 ~ C. Under

these conditions, LTE4 is stable for at least 5 months. The urines were analyzed within l month after collection. After thawing the urine, a 1 ml sample was taken to measure creatinine levels, and a 10 ml sample was centrifuged at 8000 g for 10 minutes. For recovery determinations, radioactive LTC4 (14,15-[3H]LTC4) (New England Nuclear, Boston, Mass.) was added to the separated supernatant, and the pH was adjusted to 5.4 with acetic acid. The sample was then loaded onto a C18 SEP-PAK (Waters Associates, Milford, Mass.) cartridge, which was pretreated successively with 10 ml of distilled water, 10 ml of methanol, and 20 ml of 0.5% ethylenediaminetetraacetic acid buffer adjusted to pH 5.4 with acetic acid. Initial experiments demonstrated that pH 5.4 afforded optimal recoveries. If the urine is applied directly to the SEP-PAK, the leukotrienes are not retained. Acidic conditions are avoided because of the instability of leukotrienes in acidic media. The SEP-PAK was then washed with 20 ml of distilled water. The leukotrienes were eluted with 4 rnl of methanol. The methanol fraction was evaporated to dryness and reconstituted in 150 ixl of 45% aqueous methanol.

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105 100 95 FEV 1

90

(%) SS 80 75 600 70

j1

|

i

i

J I

I

I

I

I

0

1

2

3

4

5

6

7

4oo LTE4 2 0 0 (pg/mg

J 0 Creatlnlne)

TIME POST INHALATION (h)

RG. 2. Change in FEV1 (percent) in five subjects who developed an isolated LAR after inhaled occupational sensitizing agents (e) and after inhalation of the appropriate diluent (o). Also illustrated are the levels of urinary LTE, (mean and SEM) measured before inhalation of the diluent (open bars) or the sensitizing agent (solid bars) between 2 to 3 hours and between 6 to 7 hours after inhalation. Note the scale is different from scale of Fig. 1. No significant rise in LTE4 was demonstrated.

The total sample was injected onto a reverse-phase 3 ixm NOVA-PAK (Waters Associates) HPLC column, eluted with a 60: 40 mixture of methanol / water, and buffered to pH 5.4 with ammonium acetate at a flow rate of 1 ml/min. Twentyfive fractions of 1 ml were collected and evaporated to dryness. Fractions 1 to 10 were reconstituted in water and counted for percent recovery. Fractions 11 to 25 were individually reconstituted in R1A buffer for RIA. The endogenous LTE, immunoreactivity elutes in fractions 13 to 15. Recently, we have reported a semiautomated HPLC procedure for the separation of LTE, from the other peptidoleukotrienes.2: LTE4 was measured by RIA in which dextran-coated charcoal was used to separate antibody-bound LTE, from free LTE,. The antibody used in this assay was a monoclonal raised to LTC, conjugate, z3 which had good cross-reactivity to LTE,. Therefore, the antibody used cross-reacts with other peptidoleukotrienes. For this reason, HPLC was used to separate the peptidoleukotrienes before quantitation. To obviate any problems of nonparallelism of the standard curves between LTC, and LTE,, [3H]LTE4 was used as the ligand, and cold LTE4 was the displacer for the standard curve. The RIA was carried out according to the method reported by Hayes et al. 2' with some modifications. [3H]LTE, (38 to 40 Ci/mmol) was diluted in PBS per 0.1% gelatin, pH 7.2, to contain 20,000 dpm (approximately 2 • l0 -13 mol) of radiolabeled ligand per 100 I,tl. One hundred microliters of

this solution was added to 2 ml of polypropylene centrifuge tubes containing 100 txl of buffer or competing ligand and 50 I~l of appropriately diluted anti-LTC, antibody in PBS per 0.1% gelatin. The mixture was incubated 18 hours at 4 ~ C and then placed in an ice-water bath for 15 minutes before the addition of 1 ml of dextran-coated Norit (Serva, Heidelberg, West Germany) charcoal suspension in PBS per 0.1% gelatin. After 15 minutes in ice water, the charcoal was pelleted by centrifugation at 12,000 g for 2 minutes in a Beckman microfuge 12 (Beckman Instruments, Inc., Irvine, Calif.). A 0.9 ml aliquot of the supernatant was transferred to a 20 ml liquid scintillation vial, 9 ml Aquasol (New England Nuclear) was added, and the radiaoctivity was determined by liquid scintillation spectrometry in a Beckman LS 9000 counter at 38% efficiency. Recoveries of [3H]LTC4, [3H]LTE,, and exogenous LTE4 (10 to 200 pg/ml) from urine, as determined by control experiments, were 70.3% (SEM, 1.6%) (n = 21), 46.9% (SEM, 3.5%) (n = 3), and 50.9% (SEM, 11.0%) (n = 5), respectively. The measurement was corrected for recovery and related to the creatinine content of urine. Levels were expressed as picograms per milligram of creatinine. Analysis

The airway narrowing after inhaled allergen or occupational sensitizers is expressed as the maximal fall in FEVI during the EAR and during the LAR and compared to the

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105 100 95 FEV 1 (%)

9(] 85 80 -1300

75

LTE 4 -1200 (pg/mg

+ i

r'i.ot

1 0 0 Creatlnlne)

|

I

|

i

i

|

I

i

0

1

2

3

4

5

6

7

TIME POST INHALATION (h) RG. 3. Change in FEV1 (percent) in eight subjects who developed a dual response after inhaled environmental allergens (e) and after inhalation of the appropriate diluent (o). Also illustrated are the levels of urinary LTE4(mean and SEM) measured before inhalation of the diluent (open bars) or the allergen (solid bars) between 2 to 3 hours and between 6 to 7 hours after inhalation. Note the scale is different from scale of Fig. 1. A significant increase in urinary LTE+ (p = 0.025) occurred after the EAR.

same time point during the diluent inhalation. An EAR was defined as a fall in FEVt > 10% from baseline between 0 to 3 hours after inhaled allergen, and an LAR as a fall in FEV, >10% between 3 to 7 hours after inhaled allergen. The falls in FEV~ were compared with Student's t tests for paired observations. The levels of urinary LTE, measured at baseline and after inhalation of allergen, occupational sensitizers, and diluent were compared with a two-way analysis of variance. Linear regression analysis was used to examine the relationship between the maximal fall in FEV, during the EAR and the levels of urinary LTE+ during the EAR. A probability of >95% was considered significant. RESULTS

Ten subjects were studied with occupational sensitizing agents. Five of these subjects had isolated EARs and five had isolated LARs. All eight subjects studied with environmental allergens developed dual responses. In subjects developing isolated EARs, the maximal fall in FEV~ between 0 to 3 hours after inhalation of the sensitizing agent was 27.96% (SEM, 5.2%) at 10 minutes, whereas the change in FEV~ at the same time after the diluent was +0.72% (SEM, 1.6%) (p = 0.002) (Fig. 1). The maximal fall in FEV] in the same subjects between 3 to 7 hours after inhalation

of the sensitizing agent was 3.32% (SEM, 0.7%), which did not differ significantly from the change in FEV~ on the day of diluent inhalation (p = 0.08) (Fig. 1). In subjects developing isolated LARs, the maximal fall in FEVt between 0 to 3 hours after inhalation of the sensitizing agent was 7.18% (SEM, 1.4%) at 10 minutes, whereas the change in FEVt at the same time after the diluent was +0.9% (SEM, 1.1%) (p = 0.02) (Fig. 2). The maximal fall in FEV~ in the same subjects between 3 to 7 hours after inhalation of the sensitizing agent was 24.86% (SEM, 3.1%) at 6 hours, whereas the change in FEV~ at the same time on the day of diluent inhalation was + 1.5% (SEM, 1.8%) (p = 0.002) (Fig. 2). In subjects developing dual responses, the maximal fall in FEV1 between 0 to 3 hours after inhalation of allergen was 15.01% (SEM, 1.9%) at 10 minutes, whereas the change in FEV~ at the same time after the diluent was +2.80% (SEM, 1.5%) (p < 0.0001) (Fig. 3). The maximal fall in FEV~ in the same subjects between 3 to 7 hours after inhalation of allergen was 20.58% (SEM, 3.7%) at 7 hours, whereas the change in FEV~ at the same time on the diluent day was +3.21% (SEM, 0.9%) (p = 0.0005) (Fig. 3). Levels of urinary LTE4 were increased in subjects

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LTE, during asthmatic responses 217

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5000

URINARY LTE4

1000 500

DURING EARLY RESPONSE

r=0.68 0 0

oOee

lOq 50 I

I

0

10

,

I

I

I

I

2O

3O

4O

50

M A X I M U M FALL IN FEV I DURING EARLY RESPONSE FIG. 4. A linear correlation exists between the levels of urinary LTE4 measured between 2 to 3 hours after inhalation of the sensitizing agents or allergens and the m a x i m u m fall in FEV1 during the EAR in all subjects (r = 0.68; p = 0.001 ). The open circles represent subjects with isolated LARs, the closed circles are subjects with dual responses, and the closed squares are subjects with isolated EARs.

during EARs but not during LARs (Table II). In subjects with isolated EARs. the urinary LTE4 increased from baseline levels of 150.26 (SEM, 49.5) pg/mg of creatinine to 1816.18 (SEM, 606. i) pg/mg of creatinine during the first 3 hours after inhalation and returned to 240.46 (SEM, 113.7) pg/mg of creatinine between 3 to 7 hours (p = 0.04) (Fig. 1). In subjects with isolated LARs, the urinary LTE, levels were 187.04 (SEM, 28.6) pg/mg of creatinine at baseline, 283.08 (SEM, 81.0) pg/mg of creatinine during the first 3 hours after inhalation, and 331.29 (SEM, 90.17) pg/mg of creatinine between 3 to 7 hours (p = 0.65) (Fig. 2). In subjects with DARs, urinary LTE4 increased from baseline levels of 66.50 (SEM, 13.5) pg/mg of creatinine to 174.80 (SEM, 40.1) pg/mg of creatinine during the first 3 hours after inhalation and returned to 70.30 (SEM, 14.1) pg/mg of creatinine between 3 to 7 hours (p = 0.025) (Fig. 3). The levels of urinary ErE, measured during the first 3 hours after inhalation of allergens or occupational sensitizers were significantly correlated with the maximal falls in FEVj 0 to 3 hours after inhalation (r = 0.68; p = 0.001) (Fig. 4). DISCUSSION

This study has demonstrated that increased levels of urinary LTE4, the metabolite of LTCa and LTD,,

occurs after EARs to either inhaled allergens or occupational sensitizing agents. However, levels of urinary LTE4 are not significantly increased during LARs after these stimuli. These results suggest that the sulphidopeptide leukotrienes are involved in causing bronchoconstriction during the EAR. The absence of significantly increased levels during the LAR suggests either that leukotrienes are not involved in causing LARs or, alternatively, that other leukotriene metabelites, besides LTE4, might be predominant. The metabolism of peptide leukotrienes has been recently extensively studied. In animals, metabolites of LTC4, such as LTD4 and LTE,, and the subsequent oxidative metabolites, such as 20-hydroxy, 20carboxy, 18-carboxy diner, and 16-carboxy-14,15dihydro tetranor LTE4, and their corresponding Nacetyl derivatives have been identified. 25-3~Allergen challenge in allergic rats causes marked elevation of leukotriene metabolites in bile. 3~ All the biliary metabolites from LTE4 to the 20-carboxy-14,15-dihydro tetranor LTE, are N-acetylated. 32 In all other animals studied at this time, the metabolites do not appear to be N-acetylated to any significant degree. The identification of other 13-oxidant metabolites subsequent to the 16-carboxy-14,15-dihydro tetranor LTE, is being currently investigated. However, the only urinary metabolite identified at this time in man is LTE4.-9

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TABLE II. Urinary LTE, levels (picograms per milligram of creatinine) after diluent, occupational

sensitizer, and allergen challenges Baseline (SEM)

Isolated EARs Inhalation Active Diluent Isolated LARs Inhalation Active Diluent DARs Inhalation Active Diluent

2-3 hr (SEM)

6-7 hr (SEM)

150.3 (49.5) 101.8 (24.4)

1816.2 (606.05) 102.5 (20.2)

240.5 (113.7) 57.4 (13.5)

0.04

187.0 (28.6) 196.9 (27.5)

283.1 (81.0) 177.9 (56.4)

331.3 (90.2) 164.0 (46.3)

0.36

66.5 (13.5) 67.5 (15.1)

174.8 (40.1) 78.3 (22.4)

70.3 (14.1) 45.2 (16.5)

0.025

Measurements of leukotriene release in the lung are difficult to make, particularly during bronchoconstriction induced by inhalation challenges. Also, measurements made with BAL fluid reflect release during one small time period of a relatively prolonged response. Urine is easy to collect and, whereas the levels of urinary LTE4 are probably a small proportion of the total metabolite from the lung, they do reflect the release of leukotrienes during the entire EARs and LARs. We have developed methodology to measure urinary LTE4 levels in man. This methodology relies on the extractive isolation of leukotrienes followed by reverse-phase HPLC separation of LTE4 before quantitation by RIA techniques with a monoclonal antibody developed from LTC4conjugate. 23 We have assumed, however, that the urinary LTE4 has originated from leukotrienes released in the airways during the induced asthmatic responses. This assumption is partly supported by a study from Verhagen et al. t6 who demonstrated that inhaled LTD4 could be detected as LTE4 in urine. This study has included subjects sensitized to environmental allergens, all of whom developed dual responses, and subjects sensitized to occupational sensitizers with isolated EARs or LARs. This was done because isolated LARs are much more common after inhalation of occupational sensitizers than after environmental stimuli. The methods of challenge with these stimuli differs. Challenges with environmental allergens are easier to control because a specific re-

p

0.16

0.70

0.27

sponse end point (15% fall in FEVj in our laboratories) can be determined once a skin test end point and level of histamine airway responsiveness are known. By contrast, challenges with occupational sensitizers cannot be controlled in this way. For this reason, generally larger falls in FEV~ occur, as happened in this study. The EAR after inhaled allergens is likely mainly due to the effect of bronchoconstrictor mediators released from cells, such as mast cells, within the airway lumen. This is because the early response is transient, with a time course of recovery similar to the time course for the sulphidopeptide leukotriene stimulation. 33 Also, the EAR can be prevented by pretreatment with, and rapidly reversed by, inhaled 132adrenoceptor agonists, 34 as well as the responses to other inhaled bronchoconstrictor mediators, such as histamine or methacholine. Last, the EAR can be partially inhibited, in some studies, by pretreatment with antihistamines and prostaglandin synthesis inhibitors) 5 This concept is supported by the results of this study, in which the levels of urinary LTE4 after the EAR were highest in the subjects experiencing isolated EARs after inhaled occupational sensitizers. This finding was associated with greater bronchoconstriction in these subjects compared to subjects with dual responses after inhaled allergen (maximal fall in FEV~, 27.96% versus 15.01%). In addition, the significant correlation between the degree of bronchoconstriction during the EAR and the levels of urinary LTE4 (r2 = 0.46) suggests that LTC4 and/or LTD4

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are, in part, causing the bronchoconstriction. However, to fully clarify the role of these leukotrienes in the pathogenesis o f EARs, studies must be performed with leukotriene antagonists or synthesis inhibitors. By contrast to the E A R , the L A R is more prolonged, is not prevented or fully reversed by inhaled 132-adrenoceptor agonists, 36 nor influenced by prostaglandin synthesis inhibitors. 37 These studies have suggested that airway narowing occurring during the L A R is caused by mucosal edema, excess secretions, and influx of inflammatory ceils in the airways 7 rather than by the release o f bronchoconstrictor mediators. The results of the current study are consistent with this interpretation. It is unlikely that the timing of the urine collections could have influenced these results because, whereas the urine collections were made after the E A R was over (at 3 hours) and at the peak of the L A R (at 7 hours), at this time during the LAR, all subjects had experienced at least 2 hours of bronchoconstriction equivalent to that occurring during the E A R (Figs. 2 and 3). Therefore, some increase in the levels of urinary LTE4 might have been expected if LTC, and LTD4 were, in part, responsible for this bronchoconstriction. However, it remains possible that peptidoleukotriene release during the L A R did not result in increases in urinary LTE4 when the urine was collected at 7 hours after the challenge. This could occur, for example, if metabolites other than LTE4, such as the oxidative metabolites already discussed, are predominant in urine during LARs. Since these metabolites do not cross-react with the monoclonal antibody used in this study nor with any other antibody currently available, the urinary levels of these metabolites cannot be measured. The definitive role of the peptidoleukotrienes in causing these responses awaits studies with potent and selective peptidoleukotriene receptor antagonists and 5-1ipoxygenase inhibitors. LTE4 has previously been reported to be present in BAL fluid from 15 of 17 patients with mild to moderately severe, but stable, asthma ~~ and in urine in subjects wtih acute severe asthma. 23 The baseline levels of urinary LTE4 in our study were beyond the 95% confidence intervals for levels of urinary leukotrienes in 12 normal subjects without asthma (mean value, 59.4 [SD, 41.9] p g / m g of creatinine) in 10 of the 18 subjects studied. These studies raise the possibility that ongoing release of sulphidopeptide leukotrienes is occurring in some subjects with stable as well as acute asthma. In summary, this study has demonstrated that increased levels of urinary LTE4 can be measured after EARs in subjects with isolated EARs after inhaled occupational sensitizers and after EARs in subjects with dual responses after inhaled environmental al-

lergens. These results are consistent with the only other study that has examined urinary levels of LTE4 during E A R s . " By contrast, urinary LTE4 levels were not significantly elevated in subjects with isolated L A R s after inhaled occupational sensitizers or during the L A R in subjects with dual responses after inhaled allergens. These results suggest that the sulphidopeptide leukotrienes are important bronchoconstrictor mediators in causing EARs.

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