INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Asthma is characterized as a chronic inflammatory disease of the bronchial mucosa, in which mast cells, eosinophils, and activated T cells are of considerable importance (1). Although mast cells and eosinophils are the major effector cells inducing acute bronchial constriction and chronic epithelial damage by the release of mediators, it has been suggested that activated CD4⁺ T cells play a key role in orchestrating the inflammatory process through the release of an array of cytokines. Increased numbers of CD4⁺ cells are found in airways of patients with asthma that show signs of activation (2), and depletion of CD4⁺ T cells has been shown to prevent the development of allergen-induced eosinophilia and hyperresponsiveness in a murine asthma model (3). However, the mechanisms that lead to the recruitment and activation of CD4⁺ cells into the bronchial mucosa are poorly understood. The interaction between adhesion molecules on T lymphocytes and endothelial cells together with the local production of chemotactic factors for T cells are probably the most important mechanisms for T-cell recruitment (4). Although a variety of cytokines and chemokines including interleukin 1 (IL-1), IL-2, IL-8, RANTES, macrophage inflammatory protein 1 (MIP-1), MIP-1, monocyte chemotactic protein (MCP-1), MCP-2, MCP-3, MCP-4 are chemoattractant for T cells (5), the T-cell cytokine IL-16 seems to be of particular interest. Because IL-16 is a natural ligand of the CD4 molecule it selectively induces migration of CD4⁺ T cells and eosinophils. Furthermore, IL-16 leads to an induction of IL-2 receptor and MHC class II molecules on CD4⁺ T cells and primes them for responsiveness to IL-2 (11). On this basis, IL-16 may play a crucial role in the allergic inflammation in patients with asthma.

Indeed, in a murine model of allergic asthma the involvement of IL-16 in the induction of airway hyperresponsiveness has been demonstrated (14). The only data available regarding the presence of bioactive IL-16 in humans with asthma revealed that IL-16 is present in bronchoalveolar lavage (BAL) fluid of patients with asthma 6 h after segmental allergen and histamine provocation, an early time point, which precedes the recruitment of eosinophils and lymphocytes into the airways (15, 16). Although increased expression of IL-16 has been shown in the bronchial mucosa of patients with asthma compared with control subjects (17), little is known about the bioactivity of IL-16 in the airways at later time points after allergen challenge. This is of major relevance in determining the role of IL-16 for CD4⁺ T-cell and eosinophil recruitment in vivo. We have therefore determined the concentration of IL-16 using ELISA and the T-cell chemoattractant activity using a modified Boyden chamber assay in BAL fluid from 13 patients with asthma and 9 nonatopic control subjects at baseline and 24 h after segmental allergen and saline challenge. Furthermore, our aim was to correlate these data with eosinophil and T-cell recruitment as well as with CD4⁺ cell activation. Since no data are available on the ability of pulmonary CD4⁺ T cells to produce IL-16 by themselves we further determined the percentage of IL-16-positive CD4⁺ T cells in the different BAL samples using a flow cytometric intracellular cytokine assay.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Study Subjects

Thirteen patients with mild asthma and 9 normal control subjects participated in the study (for patient characteristics see Table 1). All patients had mild allergic asthma as defined in the Consensus Report (18). Each patient had a positive skin prick test to one or more of eight common allergens (Dermatophagoides pteronyssinus, Dermatophagoides farinae, mixed grass pollen, mixed tree pollen, dog hair, feather, cat fur, Alternaria; allergen extraction from Abelló, Bornheim, Germany). The allergen extract used for segmental allergen challenge was the extract that produced the largest wheal response on skin prick testing and the concentration chosen was one-tenth the dilution in saline that elicited a 3-mm-diameter skin wheal response. Bronchial hyperresponsiveness and provocative concentration causing a 20% fall in FEV₁ (PC₂₀) were determined as described (19). Patients were using salbutamol only when required for relief of symptoms. None of the patients was treated with corticosteroids, sodium cromoglycate, or theophylline. The normal control subjects had no history of allergic or other diseases, negative skin prick tests, normal immunoglobulin E (IgE) ( 100 IU/ml), normal lung function tests, and no bronchial hyperresponsiveness (PC₂₀ > 8 mg/ml). All study subjects were nonsmokers and no subject had an acute bronchitis 4 wk before the investigations. All subjects were volunteers and gave their written consent after being fully informed about the purpose and nature of the studies, which were approved by the Ethics Committee of Hannover Medical School.

Segmental Allergen Challenge

Segmental allergen challenge was performed as previously described (19, 20). Briefly, all subjects received nebulized salbutamol (1.25 mg), atropine (0.5 mg subcutaneously) and midazolam (2-8 mg intravenously) before the bronchoscopy. Lidocaine was used to achieve local anesthesia of upper and lower airways. The bronchoscope (P30, Olympus Optical, Tokyo, Japan) was wedged into the inferior lingular bronchus and a BAL was performed with 5 × 20 ml of sterile saline. The instrument was passed into the superior lingular bronchus and 10 ml of saline solution was instilled as a control challenge. Finally, the bronchoscope was passed to the medial segment of the middle lobe and 10 ml of allergen solution was instilled. After 24 h, subjects (all patients with asthma and five control subjects) were rebronchoscoped with the same premedication, and the superior lingular bronchus and the medial middle bronchus were lavaged with 100 ml of saline.

BAL fluid samples were processed as described (19). Briely, cells were filtered through a 100-µm filter and centrifuged, and the supernatant was stored at 80° C. The total count of nucleated cells was performed using a Neubauer hemocytometer. Differential cell counts were performed from cytospin slides with 300 cells per slide being counted. An aliquot of the cells was separated for flow cytometric analysis.

Flow Cytometric Determination of T-cell Subsets and Expression of Fas Ligand on BAL T Cells

We have reported that Fas ligand is upregulated on BAL T cells after segmental allergen challenge (19). In the present study we have used the already published data for the calculation of possible correlations between IL-16 protein concentration and the T-cell expression of Fas ligand, since these data were obtained separately in nine patients with asthma and all control subjects of the same study population. Briefly, BAL cells were incubated with biotinylated anti-CD95L and one of the following antibodies: anti-CD3-fluorescein isothiocyanate (FITC), anti-CD4-FITC, or anti-CD8-FITC. Cells were washed and incubated with streptavidin-Tricolor. Negative controls consisted of isotype-matched FITC-labeled, nonspecific antibodies, and in the case of the biotinylated antibody, the primary antibody was omitted. The percentages of CD3⁺, CD4⁺, and CD8⁺ lymphocytes (for all study subjects) and the percentage of CD3⁺, CD4⁺, and CD8⁺ lymphocytes bearing the surface markers Fas ligand (for all control subjects and nine patients with asthma) were calculated.

T-cell Chemotaxis Assay

The presence of IL-16 in BAL fluid was assessed by quantification of human nylon wool nonadherent T lymphocyte migration (95% T cells) using a modified Boyden chemotaxis chamber technique (11). A total of 50 µl of T-cell suspension (10 × 10⁶ cells/ml) was placed in the upper compartments of the 48-well microchemotaxis chambers separated from 32 µl of test samples by 8-µm micropore nitrocellulose filters (Neuroprobe, Cabin John, MD) and cells were incubated at 37° C in a 5% CO₂ atmosphere for 2 h. The filters were fixed, stained with hematoxylin, dehydrated, and mounted on glass slides. Cell migration was quantified by counting the total number of cells that had migrated beyond 50 µm into the filter. Control migration was established by cells stimulated with buffer alone, and these experiments averaged 10-15 cells/high-power field. Control migration was normalized to 100% to directly compare migration in separate filters and in separate experiments. For each chemotaxis experiment six high-power fields in duplicate were calculated and expressed as percentage values of control migration. For each BAL sample, four or five individual chemotaxis experiments were performed and the mean values of lymphocyte migration were used for statistical analysis to identify differences between experimental and control conditions (paired Student's t test, level of confidence p < 0.05). A value of > 140% represents migration significantly different from control migration.

To assess specificity for IL-16, neutralizing experiments were conducted by incubating test samples for 15 min with neutralizing concentrations of affinity-purified rabbit anti-recombinant human IL-16 (rhuIL-16) antisera (5-10 µg/ml). At this concentration, the optimal chemotactic activity of 50 ng/ml rhuIL-16 is completely neutralized. There is no cross-neutralization by this antibody with the chemotactic activity induced by any of the lymphocyte chemoattractant chemokines tested thus far.

Detection of IL-16 in BAL Fluid (ELISA)

Quantitation of IL-16 protein in the BAL fluid was accomplished by ELISA as previously described (15). Briefly, anti-IL-16 monoclonal antibody was coated directly onto 96-well ELISA plates (Costar, Cambridge, MA) at a concentration of 1 µg/ml in coating buffer (0.1 M sodium bicarbonate, pH 8.8) and incubated overnight at 4° C. To eliminate nonspecific binding by the primary antibody, the plates were blocked with 300 µl of blocking buffer (phosphate-buffered saline [PBS] containing 10% fetal bovine serum [FBS] and 0.05% NaN₃) for 2 h at ambient temperature. The plates were then washed twice with PBS Tween (PBS containing 0.05% Tween 20). A standard curve was generated using serial dilutions of recombinant interleukin 16 (rIL-16). BAL fluid samples (100 µl) were incubated in duplicate in the 96-well plates (Nunc, Naperville, IL) at 37° C for 1 h. Following the 1-h incubation, the protein was removed and the wells were washed with PBS containing 0.1% Tween 20. Nonspecific binding was reduced by blocking with 1% bovine serum albumin for 1 h. After washing, 100 µl of rabbit polyclonal anti-IL-16 antibody (10 µg/ml) diluted in PBS containing 0.05% Tween 20 was added to each well. The presence of IL-16 was then detected by incubating for 1 h with biotinylated goat anti-rabbit IgG diluted 1:500 in PBS. The lower limit of detection for the ELISA is routinely 10-15 pg/ml. Linearity was in the range of 12-500 pg/ml (coefficent of 0.993 in that range).

Flow Cytometric Detection of Intracellular IL-16 in BAL T Cells

Intracellular cytokine detection of BAL-derived T cells was performed in seven patients with asthma and all control subjects as previously described (21). Briefly, BAL cells were resuspended in culture medium supplemented with 10% fetal calf serum (1 × 10⁶ cells/ml). Cells were stimulated with phorbol 12-myristate 13-acetate (PMA, 10 ng/ml) and ionomycin (1 µM) in the presence of monensin (2.5 µM). After incubation for 4 h cells were fixed in 4% paraformaldehyde for 10 min. Cells were resuspended in saponin buffer and incubated with anti-IL-16 (1 µg/ml, clone 14.1, mouse IgG₂), anti-CD4-FITC (1 µg/ ml mouse IgG₁; Coulter, Krefeld, Germany) and anti-CD8-Tricolor (1 µg/ml; mouse IgG₁; Medac, Hamburg, Germany) for 20 min. After a further wash in saponin buffer cells were incubated with 0.5 µl of PE-labeled goat anti-mouse IgG₂ (Southern Biotechnology Associates, Birmingham, AL) as a secondary antibody for 20 min. As controls nonspecific FITC- and Tricolor-labeled mouse IgG₁ and unlabeled mouse IgG₂ antibodies were used (Becton Dickinson and Medac). Flow cytometric analysis was performed with scatter gates on the lymphocyte fraction. The percentages of CD4⁺ and CD8⁺ lymphocytes staining positively for IL-16 were calculated.

Statistical Analysis

The Mann-Whitney U test was used for intergroup comparison between patients with asthma and control subjects. For comparison of paired data within the major study groups (baseline, saline, and allergen) significant variability was first established using Friedman's nonparametric test. The Wilcoxon test was then used for the individual comparisons. Correlation coefficients were obtained by the Spearman rank-order method. Probability values of p < 0.05 were accepted as significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

BAL Recovery, Differential Cell Count, and T cell Subsets

BAL recovery did not differ between baseline BAL, saline-challenged BAL, and allergen-challenged BAL (Table 2). There was an increased total cell count after saline and allergen challenge in patients with asthma and control subjects, which did achieve significance only in the patients with asthma (p < 0.05). The absolute numbers and the percentage of neutrophils were increased after saline and allergen challenge in both patients with asthma (p < 0.01) and control subjects (p < 0.05), whereas the numbers and percentage of eosinophils were elevated only after allergen challenge in patients with asthma (p < 0.01). There were no changes in the absolute numbers of lymphocytes and CD3⁺, CD4⁺, and CD8⁺ cells after allergen and saline challenge in both groups (Table 3).

T-cell Chemotactic Activity in BAL Fluid

Figure 1A shows the induced migration of peripheral T cells by unconcentrated BAL fluids obtained from patients with asthma and control subjects at baseline and 24 h after saline and allergen challenge. In BAL samples from the control subjects, with the exception of one subject, no T-cell chemotactic activity was detectable without changes after saline or allergen challenge. In comparison with control subjects the chemotactic activity in patients with asthma was increased in all three BAL samples (p < 0.01 in each case). After allergen challenge, but not after saline challenge, a significant increase in induced cell migration was detectable in the patients with asthma (p < 0.01).

View larger version (26K): [in this window] [in a new window]   Figure 1.   Induced chemotaxis of human T lymphocytes by BAL fluid from patients with asthma (n = 13) and control subjects (n = 9 for baseline, n = 5 for saline and allergen). BAL was obtained at baseline and 24 h after segmental saline and allergen challenge. Chemotaxis assay was performed as described in METHODS either (A) without or (B) with preincubation with an neutralizing anti-IL-16 antibody. The data are expressed as a percentage of cell migration in control buffer (a value of 140% represents significant different migration from control migration). Bars indicate median values and asterisks denote significant different migration between the BAL samples (p < 0.01).

Preincubation of BAL samples with a neutralizing anti-IL-16 antibody (Figure 1B) resulted in a significant reduction in induced cell migration in all three BAL samples (baseline, saline, and allergen) from the patients with asthma (p < 0.01 in each case). This demonstrates that the majority of the T-cell chemotactic activity is attributable to IL-16. However, in the BAL fluid after allergen challenge this activity was not completely blocked by anti-IL-16, resulting in a still significant elevated chemoattractant activity compared with baseline BAL (p < 0.01). In the control subjects preincubation with anti-IL-16 reduced the T-cell chemotactic activity in the subject with detectable activity.

Concentration of IL-16 Protein in BAL Fluid

In patients with asthma IL-16 protein was detectable in unconcentrated BAL fluid samples from six patients at baseline and seven patients after saline challenge without significant differences (Figure 2). After allergen challenge there was a significant increase in all patients with asthma (median, 97 pg/ml; range, 38-362 pg/ ml; p < 0.01). In the control group IL-16 was detectable only in the subject with the measurable T-cell chemotactic activity.

View larger version (13K): [in this window] [in a new window]   Figure 2.   Concentration of IL-16 protein determined by ELISA in unconcentrated BAL samples from patients with asthma (n = 13) and control subjects (n = 9 for baseline, n = 5 for saline and allergen). BAL was obtained at baseline and 24 h after segmental saline and allergen challenge. Bars indicate median values and asterisks denote significant different migration between the BAL samples (p < 0.01).

Correlations between IL-16 Protein Levels and T-cell Chemotactic Activity, Eosinophils, CD4⁺ T Cells, and Expression of Fas Ligand on CD4⁺ T Cells

When the concentration of IL-16 protein in BAL fluid after allergen challenge and the T-cell chemotactic activity in this lavage were plotted against each other (Figure 3A) a significant positive correlation was noted. This observation is consistent with the finding that most of the T-cell chemotactic activity was blocked after pretreatment with anti-IL-16. The close relationship between IL-16 concentrations and the T-cell chemotactic activity is also notable in the baseline BAL of the patients with asthma. The six patients with asthma who had detectable IL-16 concentrations were identical to the six patients who showed significant T-cell chemoattractant activities.

View larger version (19K): [in this window] [in a new window]   Figure 3.   Correlation between the concentration of IL-16 protein and (A) T-cell chemotaxis (n = 13) and (B) the percentage of Fas ligand-positive CD4+ cells (n = 9) from the BAL fluid of patients with asthma 24 h after allergen challenge.

Because IL-16 is a potent chemoattractant for eosinophils, T lymphocytes, and especially CD4⁺ T cells, a positive correlation between IL-16 protein levels and the number of these cells in the BAL after allergen challenge was expected. However, we were unable to detect any significant correlations between IL-16 protein levels and absolute cell numbers and percentages of eosinophils, T cells, or CD4⁺ cells in the BAL after allergen challenge. We further looked for correlations between IL-16 and T-cell activation, since IL-16 is known to be a selective activator of CD4⁺ cells (11). Fas ligand has been shown to be expressed on T cells after T-cell receptor-mediated activation (22, 23). We therefore used the data concerning enhanced Fas ligand expression on BAL T cells after segmental allergen challenge (19), which were obtained in nine patients with asthma and all control subjects of the same study population as the present data, for the calculation of possible correlations between levels of IL-16 and the T-cell expression of Fas ligand. We found a significant correlation between IL-16 protein levels and the percentage of Fas ligand-positive CD4⁺ but not CD3⁺ or CD8⁺ cells 24 h after allergen challenge in the BAL fluid of the patients with asthma (Figure 3B).

Intracellular Expression of IL-16 in BAL T Cells

The percentage of CD4⁺ and CD8⁺ cells staining positively for IL-16 after 4 h of stimulation with PMA and ionomycin in the presence of monensin is shown in Table 4. In patients with asthma and control subjects we found high percentages of IL-16-positive cells in CD4⁺ and CD8⁺ BAL cells without significant differences between the groups at baseline or after saline and allergen challenge.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The bronchial inflammation in patients with asthma is characterized by the presence of activated CD4⁺ cells, which orchestrate the inflammatory process by the release of several cytokines. Because IL-16 is a cytokine that uses the CD4 molecule as its receptor leading in vitro to chemotactic responses and activation of CD4⁺ T cells (11, 12) it could play an important role in the recruitment and activation of CD4⁺ cells in patients with asthma.

Elevated concentrations of bioactive IL-16 have been demonstrated previously in 100-fold concentrated BAL fluid from patients with asthma at an early time point following segmental allergen (15) and histamine challenge (16). In these studies the BAL was obtained at 6 h after challenge, a time point that precedes the infiltration of T cells and eosinophils in the lung. The current study was undertaken to extend these observations and we demonstrate here that at 24 h after segmental allergen challenge high levels of bioactive IL-16 are present even in unconcentrated BAL fluid. Because IL-16 is a potent chemoattractant for CD4-bearing leukocytes, a correlation between levels of IL-16 protein and the number of CD4⁺ T cells and eosinophils should be expected. Although a marked elevation of eosinophils was present in our study, we were unable to definitively correlate IL-16 levels to eosinophilia. Interestingly, it has been shown in a murine asthma model that eosinophilia in the BAL was not influenced by pretreatment with anti-IL-16 (14). Thus, any role for IL-16 in eosinophil recruitment probably represents a minor contribution compared with other cytokines. Regarding the recruitment of CD4⁺ lymphocytes we found a trend but no significant increase in percentage and absolute cell numbers at 24 h after allergen challenge (p = 0.11). Consequently, no significant correlations to IL-16 levels were detectable. These results are in accordance with other studies, in which no changes in CD4⁺ lymphocyte proportions were detectable in BAL fluid at 18 h (24), 24 h (25), and 48 h (26) after segmental allergen challenge. Changes in the BAL fluid might not reflect changes of cell numbers in the bronchial mucosa. Indeed, it has been shown by Laberge and coworkers (17) that in the submucosa from patients with asthma compared to control subjects there is an increased number of CD4⁺ lymphocytes with a close correlation to IL-16 immunoreactivity in epithelial and subepithelial cells. Despite unchanged proportions of CD4⁺ lymphocytes in BAL it has been shown that 24 h after allergen challenge an increased percentage of CD4⁺ cells expresses the IL-2 receptor (CD25) (24). Because IL-16 induces the expression of this activation marker on CD4⁺ T cells in vitro (11, 13) it is reasonable to speculate that the upregulation of the IL-2 receptor on BAL CD4⁺ lymphocytes is also induced by IL-16 in vivo. We did not measure the T-cell expression of CD25 in the current study, however, we have determined the T-cell expression of Fas ligand on most of the study subjects as part of an investigation (19). We demonstrated an upregulation of Fas ligand on BAL T cells 24 h after allergen challenge in patients with asthma, which is a sign of T-cell receptor (TCR)-induced T-cell activation (23, 27, 28). Interestingly we found a highly significant correlation between the level of IL-16 and the expression of Fas ligand on CD4⁺ but not on CD8⁺ lymphocytes in the BAL after allergen challenge. IL-16 has been shown to directly induce Fas antigen (CD95) on CD4⁺ cells in synergy with IL-2 (13), but the current studies are the first to show that IL-16 may regulate the expression of Fas ligand on CD4⁺ T cells.

It has been shown that a variety of cells including mast cells (29), eosinophils (30), epithelial cells (17), and T cells (31, 32) are able to produce IL-16. Although CD8⁺ T cells rapidly release stored bioactive IL-16, CD4⁺ T cells require TCR-mediated activation to process pro-IL-16 into bioactive IL-16 before the secretion of the active cytokine is possible (31). In patients with asthma the cellular source of IL-16 is largely unknown. There is only one study that has localized the expression of IL-16 mRNA and protein to epithelial and subepithelial cells, which were not further characterized (17). Thus, it is not known whether pulmonary T cells, and in particular CD4⁺ cells, express IL-16. We have shown that in the bronchoalveolar space of both normal subjects and patients with asthma there is a high percentage of CD4⁺ and CD8⁺ T cells that have the capacity to produce IL-16 on stimulation. Thus, pulmonary T cells may secrete IL-16 and induce further recruitment and activation of CD4⁺ T cells as a positive feedback mechanism. Since the anti-IL-16 antibody recognizes both bioactive IL-16 as well as inactive pro-IL-16 (32), we are unable to distinguish between intracellular precursor and mature IL-16 in the T cells. Together with the fact that the majority of unstimulated peripheral blood CD4⁺ and CD8⁺ T cells constitutively express pro-IL-16 (32) it remains unclear whether in patients with asthma BAL T cells release bioactive IL-16 in vivo.

In conclusion, bioactive IL-16 is present in the BAL fluid 24 h after allergen challenge in patients with asthma in relation to increased T cell chemoattractant activity and Fas ligand expression on CD4⁺ cells. Future studies need to elucidate the possible role of IL-16 for activation and apoptosis of CD4⁺ cells and whether Fas ligand is involved in this process. Although pulmonary T cells are a possible source of IL-16, further studies are necessary to determine their contribution to IL-16 production in the lung.