The Relationship between Airway Responsiveness Measured before and after the Allergen-Induced Late Asthmatic Response* David I. Bernstein, M.D.;t Yongyudh Ploysongsang, M.D ., EC.C.P.; Robert J Mittman, M.D.; Arkapol Piyamahunt, M.D.; and I. Leonard Bernstein, M.D.

Single blind allergen (Ag) and saline solution bronchial challenges were perfonned on two successive study days in ten asthmatic subjects. Histamine challenges were perfonned before, at approximately 2 h (or after resolution of the immediate bronchial response [IRn, and 24 h after saline solution or Ag inhalation. Specmc airway conductance (SGaw) was measured after delivery of challenge agents until a 50 percent fall in SGaw was observed. The SGaw was monitored over 8 h for immediate and late asthmatic responses (LAB). Results were expressed as provocative concentrations eliciting a 50 percent decrease in SGaw (SGawPC50IDS). No signmcant changes from baseline SGaw or SGawPC50HIS were demonstrated after saline solution. Eight subjects (dual reactors) exhibited both an IR and LAB after Ag and two had isolated IRs. Of the eight dual reactors, 6ve had greater than 50 percent decreases in SGawPC50HIS immediately after resolution of the IR and sixexhibited such decrements 24 h after Agprovocation . Mean baseline SGawPC50HIS (N = 10) on the Ag challenge day was 3.2±4.59 mg/ml and decreased to 0.92±4.56 mg/

It histamine has been recognized that airway responsiveness to and methacholine increases after allergen inhalation. Such increases in airway reactivity have been associated with the allergen-induced late asthmatic response (LAR) and have been documented to occur at 8 and 24 h after allergen challenge. Cockcroft et all suggested that increased airway responsiveness is dependent on airway inflammation and exclusively associated with the LAR. Cartier and coworkers" later demonstrated that the magnitude of decrease in FEV I during the LAR was correlated with the decrease in histamine PC20 measured at least 24 h after allergen exposure. When Cockcroft and Murdock" studied changes in airway responsiveness to histamine at various intervals after allergen challenge, they reported no significant change in histamine PC20 from baseline at 2 h. In contrast, histamine *From the Divisions of Immunology and Pulmonary and Critical Care Medicine , University of Cincinnati Medical Sciences Center, Cincinnati. tRecipient of a Special Emphasis Research Career Award Grant from the National Institute of Occupational Safety and Health. Manuscript received March 12; revision accepted June 27. Reprint requests : Dr. D. Bernstein, ML 231 Bethesda Avenue, Cincinnati 45267

ml at 102 to 187 minutes after Ag (p = 0.0009) and was signmcantly decreased from baseline at 1.47 ± 3.8 mg/ml 24 h after Ag (p=O.OOO4). One of the two patients with isolated IR also showed an early onset increase in airway responsiveness (EOR). There was a signi6cant correlation between the percentage of fall from baseline in SGawPC50IDS immediately after the IR and that at 24 h after Ag (r=0.811, p=O.OO5), There was no signi6cant correlation between the decrease in SGawPC50HIS after the IR and the magnitude of the LAR. These data suggest that (1) the early events occurring prior to the LAR may detennine changes in airway responsiveness observed at 24 h after Ag challenge, and (2) the EAR to histamine is not exclusively (Chut 1992; 101:437-41) associated with the LAR. AU= allergen unit; EOR = early onset increase in airway reactivity; m= immediate response; LAB = late asthmatic response; PBS = phosphate-buffered saline solution; SGaw = specific airway conductance ; SGawpcsomS=provocative concentrations of histamine eliciting a 50 percent decrease in SGaw.

PC20 was significantly decreased from baseline at 7 and 30 h after allergen." Subsequently, our laboratory and other investigators have demonstrated that airway responsiveness to histamine increases within 1 to 3 h or immediately after resolution of the allergen-induced immediate response (IR) and prior to the onset of the LAR in dual responding asthmatics.v" In a separate study, it was demonstrated that pretreatment with prednisone prevented the early onset increase in airway reactivity (EOR) to histamine after allergen Inhalation." Mechanisms postulated to explain the action of corticosteroids on EOR could include reduction of airway inflammation and modulation of release of bioactive mediators. The present study was undertaken to determine if a relationship exists between changes in airway responsiveness occurring between 1 and 3 h after allergen inhalation with those that occur 24 h after allergen challenge and whether such changes are related to the LAR. Subjects

METHODS

The study population consisted of ten subjects (six male, four CHEST I 101 12 I FEBRUARY, 1992

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female) who reported cough and wheezing during the grass pollinating season or immediate-onset asthma associated with exposure to cats. None of the subjects was receiving prior treatment with cromolyn sodium or corticosteroids. Subjects exhibited epicutaneous reactivity to fescue pollen allergen (Hollister-Stier; Spokane. WA)or cat hair and dander antigens (Alk, A1lergologiskLaboratories, Denmark). All studies of grass-sensitive subjects were performed at least ten weeks prior to or following the conclusion of the grass pollinating season . All subjects had absent or minimal asthmatic symptoms at the time of challenge. Treatment with all theophylline bronchodilator medications was withheld for at least 24 h and inhaled ~-2-agonists were withheld for at least 8 h prior to bronchoprovocation test days. An informed consent statement approved by the University of Cincinnati Medical Center Institutional Review Board was signed by all participants. Study Design

All challenge testing was performed in the University of Cincinnati pulmonary function laboratory. A schema is shown in Figure I outlining the single blind bronchial challenge protocol. On day I. subjects underwent a saline solution placebo challenge and an allergen challenge on day 2. Three separate histamine challenges were performed on each test day. On the saline solution control day (day I), histamine challenges were performed 1 h before. 2 h. and 24 h after saline solution challenge. On the allergen day, 3 histamine challenges were performed: 1 h before allergen; another immediately after resolution of the immediate asthmatic response (102 to 187 min); and a final challenge 24 h after allergen bronchoprovocation. Challenge Procedures

The specific airway conductance (SGaw) was measured using a constant volume. whole-body plethysmograph, and a quiet breathing technique previously described by Krell et al. 7 BrieBy, airBow was measured with a heated pneumotachograph (Fleisch) connected to a differential pressure transducer (model MP 45; Validyne, Northridge, CAl; box pressures were also measured with a pressure transducer (Validyne MP 45). Electrical outputs from both these transducers via carrier demodulators (Validyne CD 15) were amplified by a dual trace amplifier (model 5A18N; Tektronix Inc. Beaverton, OR) and displayed simultaneously on an X-Y storage oscilloscope (Tektronix 5113) so that the slope described during the initial, rapid phase of inspiration could be measured and SGaw could be calculated from it. To avoid stretching the smooth muscle of the tracheobronchial tree, the subject was instructed to breathe tidally and to avoid sighing and deep inspiration during measurements of SGaw. To perform histamine bronchoprovocation. serial twofold dilutions of histamine dihydrochloride ranging from 0.03-32 mg/ml were prepared in phosphate-buffered saline solution (PBS).' Aerosols

sruOY PROTOCOL DESIGN HIS Saline Day 1 (CONTROL)

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with particle sizes between 1 and 2 J.Lm were generated using a nebulizer (DeVilbiss No. 646). A dosimeter (Rosenthal-French) was used to deliver an aerosol at 25 psi and a subject received it for 0.6 s during each tidal inspiration. Subjects received ten tidal breaths of each incremental concentration of histamine delivered at 5-min intervals as well as the SGaw measurement until a 50 percent decrease in SGaw from saline solution baseline was detected. The concentration of inhaled histamine that produced a 50 percent decrease in SGaw from the baseline saline solution control (SGawPCSOHIS) was determined by interpolation from a doseresponse curve plotted on a semilogarithmic scale . Allergen bronchoprovocation was performed with PBS dilutions of lyophilized fescue or cat allergen (AIk). To determine each subject's starting allergen inhalation dose. intracutaneous threshold testing was performed with increasing three-fold concentrations of either fescue or cat allergen.' Allergens were prepared daily from lyophilized products and diluted from a starting concentration of 100,000 allergen units (AU) per milliliter. Of the ten subjects, eight received fescue grass pollen and two received cat allergen bronchial challenges. A previous study reported by Cockcroft et al' indicated that patterns of bronchial responses and associated changes in nonspecific airway responsiveness did not differ substantially with respect to the source of the allergens. 1 Therefore. we used different allergens dependent on the clinical sensitivities of imlividual subjects. Allergen bronchoprovocation was begun with an allergen dose that was one log., concentration lower than that of the predetermined intracutaneous threshold concentration. The PBS vehicle used for dilution of histamine and challenge antigens was the same reagent used for the control day challenge. After baseline saline solution challenge. ten tidal breaths of threefold increasing concentrations of allergen were administered at ten-minute intervals until a 50 percent decrease in SGaw below the baseline saline control was observed. SGaw was measured every 10 min until lung function returned to within 10 percent of the prechallenge baseline value . After resolution of the immediate response and completion of a second histamine challenge. measurements of SGaw were performed every 30 min for 6 to 8 h after allergen challenge to detect a possible late asthmatic response . A LAR was arbitrarily defined as a fall in SGaw of greater than 35 percent from preallergen saline solution baseline. Data Analysis

All SGawPCSOHIS values were transformed to log•• values and

analyzed by the paired Student's t test with a p value of and geometric mean SGawPC50HIS between the saline solution control and allergen test days. On the saline solution control day, there was a mean increase in SGaw from saline solution baseline (4.3± 11.7) in SGaw at 7 h after saline solution inhalation. A decrease from baseline was observed in only one subject (-14 percent) at 7 h after saline solution exposure. The latter subject exhibited a 50 percent decrease in SGaw during the LAR on the allergen test day. Following the early response to allergen on day 2, SGaw recovered completely to prechallenge saline solution baseline values in seven subjects prior to performance of histamine challenge; in the entire group the mean percentage of difference in SGaw after the IR from saline solution baseline was -1.39± 15.7. There were no apparent differences in the patterns of bronchial responses or in changes in airway responsiveness between two subjects challenged with cat allergen and eight individuals challenged with fescue antigen. On the saline solution control day (Fig 2), no significant change in the SGawPC50HIS was observed between the presaline solution baseline histamine challenge in comparison to those performed at 2 h and 24 h after saline solution challenge. On the allergen test day, there was a significant decrease in

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the mean SGawPC50HIS from baseline at approximately 2 h after allergen (p = 0.0009) (Fig 3). As shown in Figure 3, nine of ten subjects demonstrated a decrease in SGawPC50HIS immediately after resolution of the immediate response , which was measured within 102 to 187 min after the onset of the allergeninduced immediate response . As shown in Figure 3, a significant decrease in the mean SGawPC50HIS compared with preallergen baseline was also detected at 24 h after allergen (p = 0.0004). Linear regression analysis demonstrated a positive correlation between the percent change in SGawPC50HIS from baseline observed at approximately 2 h and that determined at 24 h (Fig 4) (r = 0.811 p = 0.005). Interestingly, there was no correlation between the percentage of change in SGawPC50HIS observed at 2 h and the maximal percentage offall in SGaw during the LAR (r = 0.1834 p>0.10).Similarly, no significant correlation was found between the percentage of fall in SGawPC50HIS at 24 h and the maximal percentage of fall in SGaw during the LAR. DISCUSSION

An early increase in airway responsiveness to hisCHEST I 101 12 I FEBRUARY, 1992

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tamine that precedes the LAR has been demonstrated to occur after inhalation challenge to common aeroallergens and to reactive chemicals encountered in an occupational setting.4.5·8 Early investigations in our laboratory had shown that airway responsiveness can begin as soon as 45 min after the onset of the early response to allergen Inhalation ." Durham and colleagues" subsequently reported that changes in airway responsiveness precede the LAR and that the magnitude of decrease in histamine PC20 correlated with the decrement in FEV I during the LAR.5 These authors suggested that tissue events associated with airway inflammation may precede the LAR and could be responsible for the early changes in airway responsiveness. In the present study, we did not detect a significant relationship between the percentage of fall in SGawPC50HIS from baseline and the maximal decrement in lung function during the LAR. Differences between our findings and those of Durham et al could partially be attributed to the use of different parameters of lung function (SGaw vs FEV I ) . We also found a correlation between the percentage of change in airway responsiveness observed at approximately 2 and 24 h after allergen challenge, an observation that was not noted in the aforementioned study of Durham et al. Based on this finding, it appears that the degree of airway responsiveness prior to the LAR may be an importan t determinant of the magnitude of increase in airway responsiveness that occurs during LAR. A greater than twofold decrease in SGawPC50HIS soon after resolution of the IR was detected in one subject who manifested an isolated immediate response to antigen that was not sustained and returned to baseline 24 h after allergen exposure. This raises the possibility that an increase of airway respon440

siveness is not exclusively associated with LAR as suggested by Cockcroft et al.' The occurrence ofEOR to histamine had also been documented among a group of isolated immediate responders in a previous study" Malo and coworkers" have also demonstrated that airway responsiveness may increase for at least 7 to 8 h after an isolated immediate response to diphenyl methane diisocyanate. The exact mechanism of EOR to histamine has not been defined clearly. It is possible that EOR could be related in part to airway edema and hyperemia. This mechanism is plausible in at least three of our subjects who did not recover completely after the early response prior to histamine challenge. Other investigators have demonstrated that a variety of cellular mediators are released soon after the allergen-induced bronchial response. Such mediators could include both preformed and newly synthesized chemical mediators that have been measured in bronchoalveolar lavage fluid following bronchial allergen challenge in humans. Wenzel et al lOreported local release of histamine and tryptase into subsegmental human bronchi 5 min following antigen challenge and in the absence of increased numbers of eosinophils or other cell populations. Similarly, increased levels of PGD2 and 15 HETE have been detected in bronchoalveolar lavage flurds within 9 min after local instillation of dust mite allergen." The sulfidopeptide leukotriene LTC. has been measured in human airways after allergen inhalation challenge and LTE. has been found in bronchoalveolar lavage of asthmatic subjects. 12 •13 Urinary excretion of LTE. also increases in association with the allergen-induced immediate asthmatic response. I. It is also noteworthy that LTE. is the only leukotriene that has been shown to increase airway responsiveness in asthmatics. IS Thus, it is possible that EOR to histamine could be enhanced by release of secondary bioactive mediators formed in the airways during or soon after the immediate response. Because this hypothesis is yet unproven, further pharmacologic studies will be necessary to determine whether pretreatment with specific prostanoid and leukotriene antagonists could modulate expression of EOR. In human studies, pretreatment with lipoxygenase inhibitors and leukotriene antagonists have not yet exhibited dramatic effects on allergen-induced bronchial responses.P-'? However, Kirby et al l8 demonstrated that pretreatment with indomethacin, a cyclooxygenase inhibitor, caused a reduction in the increase in airway responsiveness following the LAR after allergen challenge without affecting the LAR itself suggesting that prostaglandins and/or thromboxanes may playa significant role in elicitations of allergen-induced reactivity of the airways. 18 In summary, the current data suggest that early events of the allergic response preceding the LAR

AJrway Responsiveness before and alter AJlergen-induced Aslhmatlc Response (Bernste in st 81)

may initiate the first phase of airway reactivity and may determine subsequent changes in airway responsiveness for at least 24 h after allergen inhalation. ACKNOWLEDGMENTS: The writers thank Ms. Jim sb~ini:m and Mr. Matt Lovell for secretarial assistance in preparing the manuscript. REFERENCES

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