Abstract
Persistent polymorphonuclear neutrophil (PMN) recruitment to airway is thought to be an important component of continuing inflammation and progression of chronic destructive lung diseases. Although chemoattractants are required for the PMN to migrate, the nature of the chemoattractants in the airways has not yet been clarified. We therefore investigated the contribution of interleukin-8 (IL-8) and leukotriene-B4 (LTB4) to the chemotactic activity of lung secretions by inhibiting their activity using a monoclonal antibody to IL-8 and an LTB4 receptor antagonist (LY293111 sodium). Fifty-nine sputum samples obtained from 19 patients with bronchiectasis were studied. In preliminary studies the chemotactic responses to IL-8 and LTB4 were found to be additive, and we were able to remove their contribution independently with the appropriate antibody and antagonist. The chemotactic activity of the secretions was related to the macroscopic appearance (mucoid, mucopurulent, and purulent), and this appeared to be related to an increase in IL-8 contribution. Chemotactic activity was reduced by antibiotic therapy and again that seemed to relate to a reduction in the IL-8 contribution. The contributions of LTB4 were similar among the three types of sputum in varying clinical states. These data suggest that LTB4 and IL-8 are important chemotactic factors in lung secretions from such patients, although IL-8 appears to play a more important role during acute exacerbations. These results may be useful in determining therapeutic strategies for chronic destructive lung diseases in the future.
Bronchial secretions obtained from patients with bronchiectasis or chronic bronchitis contain varying numbers of neutrophils and their products (1), and the purulent nature of sputum usually reflects its neutrophil content due to the presence of myeloperoxidase (2). Previous studies have indicated that the secretions obtained from such patients contain chemoattractant activity, which may be due to several different individual factors (3). The chemoattractant activity of sputum increases with the degree of sputum purulence (3), and it is likely that the contribution of individual factors will change during different clinical states.
Understanding the chemoattractant nature of sputum may be of critical importance in the development of new therapeutic strategies. For instance, in some patients with stable bronchial disease the secretions are permanently purulent and contain neutrophil proteinases that play a role in the pathogenesis of the disease and may perpetuate the ongoing inflammation by interfering with critical host defenses, such as immunoglobulins (4) and opsonophagocytosis (5). Strategies that reduce neutrophil traffic and thus the delivery of neutrophil proteinases may prove beneficial by reducing the release of proteinases in the airways and hence their detrimental effect on host defenses.
Studies in patients with bronchial disease associated with cystic fibrosis have implicated several chemotactic factors. Recently, it has been reported that 75–98% of the chemotactic activity is due to the presence of interleukin-8 (IL-8) from patients with cystic fibrosis, bronchiectasis, and chronic bronchitis (6). However, others have suggested that leukotriene B4 (LTB4) is important in cystic fibrosis (7). In addition, there are a variety of other potential chemoattractants in the airways, including C5a (8) and modified alpha1-antitrypsin (9). Thus, the nature of the chemoattractants in lung secretions is a subject of some uncertainty. In order to investigate this in more detail, we have carried out a preliminary study of the contributions of IL-8 and LTB4 to the chemotactic activity of sputum obtained from patients with bronchiectasis. The results have been compared between secretions of different natures and between subjects in the stable clinical state and those with acute exacerbations. In addition, the response to effective antibiotic therapy has also been assessed.
Polymorphonuclear neutrophils (PMNs) were isolated using the method of Jepsen and Skottun (10), described by us previously (9). The PMNs (> 96% pure, > 98% viable, by exclusion of trypan blue) were resuspended at required concentrations in RPMI 1640 medium (Flow Laboratories, Rickmansworth, UK) containing 2 mg/ml bovine serum albumin. All reagents were confirmed to contain less than 20 ng/L of endotoxin activity using the KabiVitrum Coat Test (Flow Laboratories).
For verification of the methodology, a sputum sol phase pool was obtained using samples from patients with bronchiectasis who expectorated mucopurulent sputum (MP) (n = 8) or those with chronic bronchitis who expectorated mucoid sputum (M) (n = 8). None were taking corticosteroids during the 4 wk prior to collection. The diagnosis of bronchiectasis or chronic bronchitis and emphysema was based on clinical history, radiological findings consistent with the diagnosis by chest X-ray and computed tomography (CT) scan or bronchogram (for bronchiectasis), and compatible pulmonary function tests (airflow obstruction, air trapping, and low gas transfer for the emphysema patients). Sputum sol phase was obtained by ultracentrifugation (at 50,000 × g for 90 min at 4° C). The sample pool was stored in aliquots at −70° C until used (up to 6 mo).
For the subsequent studies to assess the contributions of IL-8 and LTB4 to chemotactic activity in sputum, a 4-h sputum sample was collected from rising. Fifty-nine samples were processed to obtain the sol phase from 19 nonsmoking patients with bronchiectasis, confirmed radiologically (male: 7; female: 12; age: 60.2 ± 3.3 yr old). None had cystic fibrosis, immunodeficiency, or α1-antitrypsin deficiency. The sputum samples were classified by their macroscopic appearance into three types: 22 mucoid (clear or white), 24 mucopurulent (pale yellow or pale green), and 13 purulent samples (dark yellow or dark green), as described previously (11). In nine patients sputum samples were also obtained at presentation with an acute exacerbation of their lung disease (increased sputum production and purulence together with a change in well-being and/or breathlessness), at the end of treatment with antibiotics for 2 wk and in the stable clinical state 2 wk later.
The chemotaxis assay was performed using the 48-well microchemotaxis chamber (12), as described previously (13). In order to determine the optimal dilution of sputum for the chemotaxis assay, PMN chemotaxis was assessed to sputum sol phase from the pools, neat and at 1:2, 1:4, 1:5, 1:6, 1:8, 1:10, 1:12, 1:50, 1:100, 1:500, and 1:1,000 dilution. Subsequent chemotactic studies were performed using the optimal dilution.
First, studies were carried out to determine whether chemotactic activity could be appropriately suppressed by a monoclonal antibody to IL-8 (anti-hIL-8 monoclonal antibody; R&D Systems, Abingdon, UK) or the LTB4 receptor antagonist (LY 2293111 Sodium; Eli Lilly, Basingstoke, UK). Increasing concentrations of the IL-8 antibody and LTB4 antagonist were used to assess their effect on chemotactic response to optimal concentration of pure IL-8 (R&D Systems) and pure LTB4 (Sigma Chemicals, Poole, UK).
Each concentration of IL-8 antibody (10−9 to 10−1 mg/ml) was preincubated with 10 nM pure IL-8 suspended in RPMI solution containing 2 mg/ml bovine serum albumin at 37° C for 1 h prior to performing the chemotaxis assay. The LTB4 receptor antagonist at each concentration (10−11 to 10−5 M) was pre-incubated with PMN (suspended in RPMI solution containing bovine serum albumin) at 37° C for 1 h and washed twice prior to the chemotaxis assay.
Second, we assessed the effect of these inhibiting strategies with a mixture of the chemoattractants to ensure that their individual contributions could be determined. The chemotactic response to the mixture of optimal concentrations of IL-8 and LTB4 (5 and 10 nM, respectively) was determined and compared to the subsequent response with each antagonist alone. Following these studies IL-8 antibody (10 μg/ml) was added to the mixture, and chemotactic response was reassessed after 1-h incubation at 37° C. At the same time the chemotactic response to the mixture was reassessed using PMNs preincubated with 10 μM of LTB4 antagonist at 37° C for 1 h.
In order to determine whether the IL-8 antibody and LTB4 antagonist could appropriately suppress the chemotactic activity of sputum, the dose–response effect of IL-8 antibody (10−7 to 10−1 mg/ml) and LTB4 antagonist (10−9 to 10−5 M) on PMN chemotaxis to the M or MP sputum pools was investigated in a similar way to that described above.
The contribution of IL-8 and LTB4 in sputum samples from patients with bronchiectasis was then determined to assess the relationship to sputum characteristics and clinical status, using diluted sputum samples and the optimal concentrations of IL-8 antibody and the LTB4 antagonist as determined by the experiments described above. The suppression of chemotaxis by IL-8 antibody or LTB4 antagonist was taken as the contribution of IL-8 or LTB4 to the total chemotactic activity of the sample.
Absolute IL-8 concentrations were measured in the 59 sputum sol phase samples by ELISA using Quantikine IL-8 immunoassay kit (R&D Systems). Mucoid samples were diluted 1 in 100 in reaction buffer (Calibrator Diluent RD5) and MP or P samples 1 in 500 prior to measurement. One hundred microliters of IL-8 standard or duplicate diluted samples were added to the wells of the microtiter plate with 100 μl reaction buffer (Assay Diluent RD1A). After incubation at room temperature for 2 h, each well was aspirated and washed three times with the kit wash buffer. After the last wash, any remaining wash buffer was removed by inverting the plate and tapping against absorbent paper toweling. Following this, 200 μl of IL-8 conjugate was added to each well. The plate was then covered with an adhesive strip and incubated for 2 h at room temperature. The aspiration and washing was repeated as above, and 200 μl of substrate solution was added and incubated for 20 min at room temperature. Finally, 50 μl of stopping solution was added to each well and the optical density was determined using a spectrophotometer at 450 nm with a 570 nm reference subtracted. A standard curve was obtained by linear regression and the IL-8 concentration in each sample was obtained by interpolation, corrected for sample dilution, and the mean value for the duplicate samples was then taken as the result.
Statistical analyses of the effects of IL-8 antibody or LTB4 antagonist on PMN chemotaxis to pure IL-8, pure LTB4, and sputum samples were performed using Wilcoxon test for paired data. All other results were assessed by Wilcoxon test for paired and unpaired data (where appropriate).
Figure 1A shows a dose-dependent inhibition of chemotaxis to IL-8 by the antibody from a control mean of 18.9 (mean) ± 1.6 (SEM) to 2.3 ± 0.5 cells/field at 100 μg/ml of antibody. Figure 1B summarizes the suppression of chemotaxis to LTB4 by the LTB4 antagonist from a control mean of 9.8 ± 2.2 to 0.6 ± 0.1 cells/field at 10 μM of antagonist. At the above concentrations of IL-8 antibody and LTB4 antagonist, there was no detectable effect on cell viability as assessed by trypan blue exclusion. Following these initial results, the experiments were repeated to determine whether the chemotactic responses could be abrogated in the same way in a mixture of the chemoattractants (as might be found in secretions).
Fig. 1. Inhibition of chemotactic response to IL-8 and LTB4. (a) indicates chemotactic response to 10 nM IL-8 and the effect of increasing concentrations of IL-8 antibody. Histograms are mean of five experiments ± standard error. (b) shows chemotactic response to LTB4 with the effect of pre-incubation of neutrophils with increasing concentrations of LTB4 antagonist. Histograms are mean ± standard error for five experiments. Asterisks indicate significant suppression from control.
[More] [Minimize]When optimal concentrations of IL-8 and LTB4 were mixed, the chemotactic response increased in an additive manner (23.2 ± 2.8 cells/field for 5 nM IL-8 alone, 11.1 ± 2.7 cells/ field for 10 nM LTB4 alone, and 32.7 ± 5.9 cells/field for the mixture). When IL-8 antibody (10 μg/ml) was added to the mixture or the LTB4 antagonist (10 μM) was preincubated with PMN, the chemotaxis to the mixture was suppressed appropriately to 13.3 ± 3.4 cells/field for IL-8 antibody and 22.8 ± 2.7 cells/field for the LTB4 antagonist (n = 6 for all experiments).
The dose response of varying dilutions of sputum sol phase on neutrophil chemotaxis is summarized in Figure 2. The PMN chemotaxis was maximal at 1 in 6 dilution for mucopurulent sputum (similar results were obtained with a purulent sputum pool; data not shown) obtained from bronchiectatic patients and 1 in 8 dilution for mucoid sputum obtained from bronchitic patients. These optimal dilutions were used for the further validation of the effects of IL-8 antibody and LTB4 antagonist on chemotactic response to sputum.
Fig. 2. The chemotactic response to increasing dilutions of mucopurulent sputum (closed bars) and mucoid sputum (open bars). Histograms are mean ± standard error.
[More] [Minimize]Figure 3A shows that chemotaxis to diluted MP sputum was suppressed in a dose-dependent manner after preincubation with IL-8 antibody from the control value (reaching a plateau) to 69.2 ± 7.6% of control at 100 μg/ml antibody (p < 0.04). Chemotaxis to diluted M sputum was also suppressed in a similar manner to 62.2 ± 10.2% of the control value at 100 μg/ml antibody (p < 0.04).
Fig. 3. The effect of IL-8 antibody and LTB4 antagonist on chemotaxis to diluted sputum. (a) shows the effect of IL-8 antibody and (b) the effect of the LTB4 antagonist on the chemotactic response to mucopurulent (closed bars) and mucoid (open bars) sputum (diluted 1 in 6 and 1 in 8, respectively). Histograms are mean ± standard error of five experiments. Asterisks indicate significant reduction below control. Control values are normalized to 100%.
[More] [Minimize]Figure 3B shows the result for the LTB4 antagonist. Chemotaxis to diluted MP sputum was suppressed in a dose-dependent manner from the control value to 52.8 ± 4.9% at 10 μM antagonist (p < 0.04). Chemotaxis to diluted M sputum was suppressed in a similar manner to 63.7 ± 4.8% at 10 μM antagonist (p < 0.04). The suppression of both chemotactic response curves reached a plateau at 1 μM of LTB4 antagonist. The reduction in chemotactic activity from the control value by an antibody or antagonist was taken as the contribution of IL-8 and LTB4, respectively, for the subsequent experiments, but the concentration of each antagonist was always one or two orders of magnitude greater than those that achieved maximal suppression in these preliminary studies.
The results of chemotaxis to diluted sputum and the contribution of IL-8 and LTB4 with samples that were macroscopically different are shown in Figure 4. The average chemotactic response to MP and P sputum were 33.4 ± 1.6 and 35.4 ± 1.5 cells/field, respectively, which was significantly (p < 0.05) higher than that to M sputum (30.5 ± 2.3 cells/field). The contribution of IL-8 to the chemotactic activity of both MP and P sputum was also significantly (p < 0.01) greater (13.1 ± 1.3 and 15.3 ± 1.9 cells/field, respectively) than that of M (7.9 ± 0.5 cells/field). However, there were no significant differences in the contribution of LTB4 among these three sputum types (7.7 ± 0.7 cells/field for M sputum, 9.2 ± 0.9 for MP, and 9.2 ± 1 for P).
Fig. 4. The contribution of IL-8 and LTB4 to the chemotactic activity of mucoid (M), mucopurulent (MP), and purulent (P) sputum is shown. The vertical axis is the chemotactic response to diluted sputum control; the contribution removed by IL-8 antibody and LTB4 is shown. Histograms are mean ± standard error (see text for details). The significance of difference from the results for mucoid secretions are shown (*p < 0.05; **p < 0.01).
[More] [Minimize]When the contribution of IL-8 and LTB4 were evaluated as the percent of the total chemotactic activity, the contribution of IL-8 and LTB4 in diluted P sputum was 42.8 ± 4.7 and 26.5 ± 2.8%, respectively, and 38.1 ± 2.8 and 27.5 ± 2.2%, respectively, in diluted MP samples, whereas that of diluted M sputum was 27.2 ± 2.5 and 26.3 ± 2.2%, respectively.
Figure 5 shows the contribution of IL-8 and LTB4 total chemotactic activity in different clinical states. Of the nine patients studied, only six showed a clear clinical response with their sputum becoming mucoid, and thus these were analyzed separately. The total chemotactic activity of sputum was significantly decreased from 34.2 ± 2 cells/field at the start of an exacerbation of 26.4 ± 1.9 (p < 0.05) at the end of successful treatment (when sputum had changed from purulent to mucoid) and 24.5 ± 1.9 (p < 0.01) cells/field in the subsequent stable state. The contribution of IL-8 at the end of treatment and in the stable state also showed significant reduction (p < 0.05 and p < 0.01, respectively, compared to that at the start of an exacerbation: 17.5 ± 2 cells/field with an exacerbation; 9.3 ± 0.7 at the end of treatment; 7.9 ± 0.7 in the stable state).
Fig. 5. Contribution of IL-8 and LTB4 to the chemotactic activity of diluted sputum from patients during an acute exacerbation (Ex), at the end of 2 wk successful antibiotic therapy (End Tx), and in the subsequent stable state are shown. The average result for sputum alone is shown with the contribution of IL-8 and LTB4. Significance is indicated (*p < 0.05; **p < 0.01).
[More] [Minimize]The absolute contribution of LTB4 at the end of treatment (8.9 ± 1.5 cells/field) was not significantly different from that at the start of an exacerbation (11.4 ± 1.4 cells/field), although the contribution in stable state after treatment (7.1 ± 1 cells/ field) showed a significant (p < 0.05) decrease compared to the contribution at the start of an exacerbation. When these results were evaluated as percent of the total chemotactic activity, the proportion contribution by IL-8 decreased after treatment and in the stable clinical state (from 50.4 ± 4.6% to 35.5 ± 2.2, p < 0.05 and 32.2 ± 1.5%, p < 0.01, respectively). However, the proportion of the total chemotactic activity contributed by LTB4 was similar (33.6 ± 3.8% at the start of the exacerbation, 33.5 ± 4.9 at the end of the treatment, and 28.6 ± 2.5 in the subsequent stable state).
The average levels in P samples (19.1 ± 3.9 nM; p < 0.01) and MP (17.9 ± 3.0 nM; p < 0.02) were significantly higher than that in M sputum (9.4 ± 1.4 nM). Similarly, the IL-8 levels in sputum in the six patients with acute exacerbations who showed a clear response with improvement of sputum from purulent to mucoid showed a significant (p < 0.04) decrease from 12.0 ± 1.6 nM at the start of the exacerbation to 6.2 ± 2.0 nM after treatment.
The present studies were designed to assess the role of IL-8 and LTB4 as chemoattractants in the bronchial secretions of patients with established bronchial disease in varying clinical states and at different stages of pulmonary inflammation. The preliminary experiments were carried out to confirm the methodology. The data show that the monoclonal antibody to IL-8 and the LTB4 antagonist LY293111 are able to prevent the chemotactic response to these agents. The studies confirm that IL-8 and LTB4 were additive in terms of their short-term chemotactic response using the Boyden chamber and neutrophils purified from healthy individuals. In addition, the monoclonal antibody and the LTB4 antagonist were able to separately remove the appropriate contribution of each agent from combined chemotactic response. On the basis of these preliminary studies, it was felt that the use of the antibody and antagonist would provide a valid assessment of the contributions of IL-8 and LTB4, respectively, in complex biological fluids.
Studies using secretions collected from patients with bronchiectasis confirmed that all such secretions contained chemoattractants with a wide range of activity among individual samples. The average data for the secretions showed a relationship to the macroscopic nature as might be expected. Previous studies from our laboratory have confirmed that patients with bronchiectasis in the stable clinical state usually cough up secretions that are macroscopically mucoid, mucopurulent, or frankly purulent in nature (11). The degree of purulence relates to the neutrophil influx and reflects the concentration of myeloperoxidase in these secretions, the presence and concentrations of neutrophil elastase, and the degree of bronchial inflammation as reflected by albumin leakage from the plasma (2).
Mucoid secretions from patients with bronchial disease do contain small numbers of neutrophils, and bronchial biopsies from patients with established stable bronchitis show the presence of neutrophil infiltration (14), suggesting that even in such patients continued neutrophil recruitment exists. In mucopurulent and purulent secretions, neutrophil traffic is clearly much enhanced despite very little change in total chemotactic activity demonstrated in the current studies. The reason for this major change in neutrophil influx with relatively little change in total chemotactic activity is currently uncertain. It may reflect a threshold effect or the chemotactic activity may show clearer differences between secretions of a different character in a more physiological system, such as transendothelial migration (compared to the short-term Boyden chamber assay used here). Alternative reasons might include enhanced chemotactic response in vivo due to PMN activation, although previous studies from our laboratory suggest that such activation may decrease chemotaxis (15). Finally, upregulation of adhesion molecules on vascular endothelium by local cytokine release may also result in enhanced recruitment to purulent secretions in vivo. Clearly, further studies will be required to clarify these possibilities and/or their relative contribution.
The inhibition studies have confirmed that IL-8 is an important contributor to the chemotactic activity of secretions and its contribution increases with the degree of purulence (as reflected by the macroscopic appearance). The contribution is not as great as that reported by other workers (6), although the reasons are currently uncertain. However, our validation experiments do suggest that the results presented here are a true reflection of the contribution of IL-8. Thus, why the results are different from that reported by others currently remains unresolved. It may reflect differences in the patient population, mechanisms of secretion collection, or even the methodology for assessing chemotactic response. Nevertheless, it does seem that in a complex biological fluid such as bronchial secretions there are likely to be other chemoattractants present and active, and the current results would support this hypothesis. The direct measurements of IL-8 confirm that the values obtained are similar to those reported by others (6, 16). In addition, the concentrations of IL-8 is related to the nature of the secretions being higher in the purulent samples, and these data would be consistent with the increased contribution of IL-8 to the chemotaxis observed in purulent secretions.
The source of the IL-8 is uncertain. Potentially this may come from macrophages, the neutrophils themselves, or the epithelial cells. In these bronchial secretions it is likely that the epithelial cells are the major source, since significant concentrations of IL-8 are present in the mucoid secretions when neutrophil recruitment and activation is likely to be relatively low. Indeed, these secretions often contain bacteria (17), and bacterial products (18) have been shown to stimulate epithelial cell production of IL-8. However, further studies will be necessary to confirm the contribution of each potential source to the overall amount of IL-8 within the secretions studied here.
Based on the studies reported here, the contribution of LTB4 to total chemotaxis of the sputum samples is less than that of IL-8. Furthermore, there is no difference between the contribution attributed to LTB4 seen in sputum samples of different macroscopic appearance. The source of the LTB4 is also currently unknown. Studies have shown that both bovine and canine bronchial epithelial cells are able to produce LTB4 (19, 20). However, similar studies with human bronchial epithelium tissues have failed to demonstrate production of LTB4 (19), although a recent abstract suggests this may not be the case (21). On the other hand, neutrophils when activated can produce LTB4 (22), but the current studies there was no relationship to the presence of sputum purulence when excess activated neutrophils are likely to have been recruited to the airway.
We have been unable to measure LTB4 levels in the samples studied here, and the concentrations have been obtained by inference only from their contribution to chemotaxis. It is therefore possible that the results may be partly artifactual. Clearly, our studies have shown that IL-8 and LTB4 do not account for all the chemotactic activity seen in the secretions. Although our preliminary studies showed that IL-8 and LTB4 were additive (total equal to the sum of both individually), the same may not be true for the other chemoattractants that have not yet been identified. Thus, it is possible that removal of the LTB4 effect on chemotaxis may be partially offset by a greater response of the control neutrophils to the other unquantified chemoattractants. Further studies, including the identification of the remaining chemotactic activity and direct measurement of LTB4, will need to be undertaken to clarify this possibility.
The studies on samples obtained from patients receiving treatment for acute exacerbations of bronchiectasis show that the total chemotactic activity of the sputum decreases significantly, providing the sputum changes from purulent to mucoid. This observation would be entirely consistent with a reduction in the excessive neutrophil recruitment in purulent samples compared to that of mucoid samples and was associated with a significant reduction in the IL-8 concentration. The results presented here are at variance with those presented previously by Eller and colleagues (16). However, it should be noted that the IL-8 concentrations are high in both mucopurulent and purulent sputum secretions and only significantly lower in those that are clearly mucoid. In view of this we have been careful only to report the results of sputum samples in patients with a purulent exacerbation of their disease that have shown a clear change to mucoid following treatment. This situation may be confused in the ill-defined clinical features that describe an exacerbation and response (increased breathlessness, increased sputum production, and increased sputum purulence). Indeed, in our study if the three remaining patients who had an acute exacerbation but in whom the sputum remained purulent at the end of antibiotic treatment were included, no significant differences were found in IL-8 concentrations or chemotactic activity. Thus, the specificity of defining a clinical exacerbation and its response is clearly critical in understanding changes in secretion chemotaxis.
In summary, our data show that there are several chemotactic factors present in secretions and that the chemotactic activity of purulent secretions is higher than that of mucoid ones. This is largely related to an increase in IL-8 levels, whereas the contribution of LTB4 remains relatively stable. This observation has important implications in deciding future therapeutic strategies since continued neutrophil recruitment is thought to be important to the pathogenesis and progression of chronic bronchial disease (23). Removal or reduction of the chemotactic drive may present an attractive therapeutic strategy. However, neutrophil chemotaxis is clearly required during episodes of acute infective exacerbations of bronchial disease. Prevention of this normal response may therefore prove harmful. For this reason measures aimed at removing the IL-8 drive may prove counterproductive, whereas an approach in abrogating the LTB4 drive may be more successful since this would remove continued background drive, still leaving a regulatable IL-8 response. Clearly, further studies are required with particular reference to the regulation of these and other chemoattractant presence in bronchial disease.
The authors wish to thank Ms. J. Mitchell for technical assistance and Mrs. E. P. Ford for typing the manuscript.
Supported by funding from Glaxo (UK).
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