Introduction

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Chronic airway inflammation plays a crucial role in the pathogenesis of asthma (1). The inflammatory infiltrate is characterized by the presence of eosinophils, activated lymphocytes, and mast cells in the airways even of subjects with clinically mild disease (2). Although eosinophils are important effector cells through their release of cationic proteins, lipid mediators, and cytokines (5) it is now recognized that CD4⁺ T cells, through the production of a variety of cytokines, regulate the events that lead to the inflammatory response in asthma. Increased numbers of activated CD4⁺ T cells are found in airways of asthmatic subjects studied both at baseline (8, 9) and after challenge (10). In humans, a subset of activated CD4⁺ T cells with a predominant Th2-like cytokine profile has been identified in airways of asthmatics and is thought to play a pivotal role in the development and maintenance of the inflammatory response in asthma (11). The role of CD4⁺ T cells as effector cells in mediating airway inflammation in asthma is also supported by animal studies. Indeed, adoptive transfer of antigen-primed specific CD4⁺ T cells to unsensitized animals resulted in antigen-induced airway bronchoconstriction and airway eosinophilia in rats (15), whereas in vivo depletion of murine CD4⁺ T cells prevented the development of antigen-induced airway hyperresponsiveness and airway eosinophilia (16, 17). The mechanisms that regulate the selective accumulation and activation of CD4⁺ T cells in the airways of asthmatic subjects are areas of active investigation. Present evidence suggests that this process involves leukocyte-endothelial interactions through adhesion molecules and local release of chemotactic factors (18). These steps appear to be modulated by local production of cytokines at sites of inflammation (19).

Interleukin (IL)-16, formerly known as "lymphocyte chemoattractant factor," is a newly characterized cytokine which has specific activities on CD4⁺ cells (20). IL-16 selectively induces migration of CD4⁺ cells, including CD4⁺ T cells (21) and CD4-bearing eosinophils (22). In addition to its effects on cell migration, IL-16 induces rises in intracellular calcium and inositol trisphosphate in CD4⁺ T cells (23). IL-16 also acts as a growth factor for CD4⁺ T cells by promoting their entry into the G₁ phase of the cell cycle and induction of IL-2R and MHC Class II molecules on resting CD4⁺ T cells (24). IL-16 was first identified as a product of peripheral blood mononuclear cells following mitogen (25, 26) and histamine (27) stimulation in vitro. Subsequent studies have shown that IL-16 is of CD8⁺ T-cell origin and is released following stimulation with histamine (28) and serotonin (29) in vitro. On the basis of these properties, this newly described cytokine may have a considerable role to play in the immune regulation of CD4-mediated processes such as those associated with asthma.

We hypothesized that IL-16 expression is upregulated in asthmatic airways and that IL-16 expression correlates with CD4⁺ cell infiltration in the bronchial submucosa. To investigate the potential role of IL-16 in the pathogenesis of asthma, we have examined the expression of IL-16 in bronchial biopsies from subjects with mild asthma. We compared both IL-16 mRNA and immunoreactivity in bronchial biopsies obtained from atopic asthmatic subjects, atopic nonasthmatic individuals, and normal control subjects.

	Materials and Methods

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Subjects

Ten atopic asthmatic subjects, 6 atopic nonasthmatic subjects, and ten age-matched normal volunteers were recruited from the outpatient clinic of Montreal General Hospital, McGill University (Montréal, PQ, Canada). Clinical and demographic characteristics of the subjects are shown in Table 1. The diagnosis of asthma was based on the criteria proposed by the American Thoracic Society (30). Asthmatic subjects were studied in stable condition and had clinically mild asthma requiring only ₂-agonist therapy. None of the asthmatics had received inhaled or oral steroid therapy in the 3 months prior to the study. Baseline forced expiratory volume in 1 s (FEV₁) was greater than 60% predicted value. All asthmatic subjects had increased bronchial reactivity to inhaled methacholine (i.e., provocative concentration required to decrease FEV₁ by 20% of its baseline value [PC₂₀FEV₁] less than 6 mg/ ml). Subjects were considered atopic if they demonstrated a positive skin-prick test (> 3-mm skin wheal) to one or more of 11 common aeroallergens. Serum IgE concentrations were obtained. The nonatopic healthy control subjects had no history of asthma and allergy, had normal bronchial reactivity, and showed negative skin-prick tests to the battery of aeroallergens. All subjects were nonsmokers, had no evidence of any other pulmonary disease, and had not experienced a respiratory tract infection during the 2 months preceding the study. The study was approved by the Ethics Commitee of Montreal General Hospital and all subjects gave written informed consent.

Bronchoscopy and Biopsy

Fiberoptic bronchoscopy was performed following American Thoracic Society guidelines (31). The procedure was undertaken if the FEV₁ > 60% and platelets and clotting parameters were within normal limits. Premedication consisted of midazolam (1- 4 mg) administered intravenously and albuterol (400 µg) by a metered dose inhaler. Topical anesthesia was acheived using a 4% lidocaine solution. Continuous oxymetry was performed and oxygen was administered throughout the procedure. Biopsies were taken from the segmental divisions of the main bronchi of the right lung using an alligator forceps (Olympus Corp., Tokyo, Japan).

Tissue Preparation

Endobronchial biopsies were prepared for immunocytochemistry and in situ hybridization procedures. Tissues were fixed in a 4% paraformaldehyde/phosphate-buffered saline (PBS) solution for 2 h, washed in 15% sucrose/PBS 3 times, embedded in OCT compound (Tissu-Tek, Miles Inc., Elkhart, IN), and snap-frozen in isopentane cooled in liquid nitrogen. Cryostat sections 10 µm thick were cut on 0.1% poly-L-lysine-coated slides, air-dried overnight at 37°C, and stored at 80°C.

Antibodies

An antihuman CD4 monoclonal antibody and an antihuman cytokeratin polyclonal antibody (antipankeratin) were obtained from Dako Canada, Inc. (Mississauga, ON, Canada). Mouse antihuman IL-16 mAb (IgG2a kappa) raised against human rIL-16 molecules was prepared by AGMED, Inc. (Bedford, MA) and has been characterized previously (20).

Preparation of Riboprobe

Radiolabelled IL-16 riboprobes were prepared as previously described (11). An IL-16-specific cRNA probe was produced from a 379-bp fragment of the human IL-16 cDNA corresponding to the bp 436 to 815 of the published sequence (20). The IL-16 cDNA inserted in a SPT19 vector was grown in Escherichia coli and linearized, then transcribed in the presence of [³⁵S]-UTP with the RNA polymerases SP6 and T7 to generate antisense (complementary to mRNA) and sense probes (identical to mRNA), respectively.

Immunocytochemistry

We used the alkaline phosphatase-antialkaline phosphatase technique as previously described (32). Sections were incubated with the primary antibodies (IL-16: 1:30 dilution; CD4: 1:10 dilution) at 4°C overnight. Sections were then incubated with the rabbit antimouse Ig secondary antibody, followed by the alkaline phosphatase-antialkaline phosphatase complex. The reaction was visualized with Fast Red alkaline phosphatase substrate. As control, sections were processed in the absence of the primary antibody.

Double Immunocytochemistry

To colocalize IL-16 to epithelial cells, double immunocytochemistry was performed on biopsy sections using a rabbit polyclonal antibody with reactivity against cytokeratin (antipankeratin) as a marker of epithelial cells and an antihuman IL-16 monoclonal antibody. The technique of double immunostaining has been described elsewhere (33). Mouse antihuman IL-16 (1:30 dilution) together with rabbit antipankeratin (1:100 dilution) were applied to cryostat sections. Cytokeratin immunoreactivity was visualized using avidin-biotin complex and diaminobenzidine (brown staining). The IL-16 immunoreactivity was visualized using alkaline phosphatase-antialkaline phosphatase and developed with Fast Red (red staining).

In situ Hybridization

In situ hybridization was performed as previously described (11). In brief, cryostat sections were first permeabilized by immersion in 0.3% Triton X-100 in PBS for 10 min, followed by exposure to proteinase K solution (1 µg/ ml in 20 mM Tris-HCl and 1 mM EDTA, pH 7.2) for 30 min at 37°C. This reaction was terminated by immersion in 4% paraformaldehyde in PBS for 5 min. After washes with PBS, the slides were treated with 10 mM iodoacetamide and N-ethylmaleimide for 30 min at 37°C and then 0.5% acetic anhydride and 0.1 M triethanolamine for 10 min before air-drying. The slides were then prehybridized with 50% formamide in 2X saline-sodium citrate (SSC) for 15 min at 37°C. Hybridization was carried out using a [³⁵S]UTP-labeled IL-16 riboprobe (either antisense or sense). Hybridization was conducted for 16 h at 42°C. Post-hybridization washes were performed with SSC (4X SSC-0.1X SSC), followed by RNase treatment to remove unhybridized single-stranded RNA. The preparations were dehydrated, immersed in emulsion, and then subjected to autoradiography for 14 days. The autoradiograms were developed and subsequently counterstained with hematoxylin. Hybridization between IL-16 mRNA and the cRNA probe was identified as dense collections of silver grains in photographic emulsion overlying cells. As a negative control, sections were hybridized with the sense probe or by treatment with RNase prior to hybridization with the antisense probe.

Counting and Data Analysis

Bronchial biopsies were coded, and evaluation of immunoreactivity and in situ hybridization signals was performed blindly. IL-16 mRNA-positive cells and IL-16 immunoreactive cells in the epithelium were counted by optical analysis and expressed as a semiquantitative score based on the percentage of the epithelium showing positive signal/total epithelium (0: no staining; 0.5:  12.5%; 1: 12.5-25%; 1.5: 25-37.5%; 2: 37.5-50%; 2.5: 50-62.5%; 3: 62.5-75%; 3.5: 75-87.5%; 4: 87.5-100%). In the subepithelial tissue, IL-16 mRNA-positive and IL-16 immuoreactive cells as well as CD4⁺ cells were counted in a depth of 115 microns and results are expressed as mean numbers of positive cells per millimeter of basement membrane (BM) (11). Two slides were processed for each immunostaining and in situ hybridization analysis. There was good reproducibility of epithelial staining and the within-observed coefficient of variation for repeated measures was less than 5% (11, 32).

Statistical comparisons of the results were performed using one-way analysis of variance with subsequent post hoc t test for multiple comparisons. Correlations between IL-16 mRNA and immunoreactivity and the numbers of infiltrating CD4⁺ cells, baseline FEV₁, and log PC₂₀FEV₁ were done using linear regression analysis. Significance was accepted at the 5% level of confidence. Statistical analyses were performed using a standard computer package (Systat version 5.03; Systat, Inc., Evanston, IL).

	Results

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IL-16 immunoreactivity was identified in bronchial biopsies obtained from normal, atopic nonasthmatic, and asthmatic individuals. In all subjects, IL-16 localized predominantly in the epithelium, although some subepithelial staining was also observed (Figure 1). Within the epithelium, basal epithelial cells showed greater IL-16 immunoreactivity than the surface epithelial cells. Epithelial IL-16 immunoreactivity was observed in all asthmatic subjects, in 5 out of 6 atopic nonasthmatics, and in 6 out of 10 nonatopic normal control subjects. However, bronchial biopsies obtained from asthmatic subjects demonstrated higher scores for epithelial IL-16 immunoreactivity (2.55 ± 0.21) as compared with those from both the atopic nonasthmatic subjects (0.50 ± 0.26, P < 0.001) and the normal controls (0.65 ± 0.26, P < 0.001) (Figure 2a). A similar degree of loss of epithelium was observed in biopsies obtained from all subjects. To determine whether IL-16 immunoreactivity was localized to epithelial cells, double immunocytochemistry was performed using a polyclonal antibody against cytokeratin as a marker for epithelial cells. Within the epithelial layer, IL-16 immunoreactivity coexpressed with cytokeratin immunoreactivity, confirming the epithelial nature of IL-16 immunoreactive cells (Figure 1).

View larger version (82K): [in this window] [in a new window]   Figure 1.   Panel A shows an example of IL-16 immunoreactivity in the epithelium (E) of an asthmatic subject. Panel B shows an example of double immunocytochemistry using antibodies against IL-16 developed with Fast Red (red) and cytokeratin developed with deaminobenzidine (brown). IL-16 immunoreactivity is coexpressed with cytokeratin immunoreactivity in the epithelium. Note IL-16 immunoreactive cells in the subepithelial area (arrow). Panel C shows a representative example of CD4 immunoreactivity in a bronchial biopsy of an asthmatic individual; arrows show representative examples of CD4 immunoreactive cells identified in the subepithelial layer (small arrow) and in close contact with the epithelium (large arrows).

View larger version (12K): [in this window] [in a new window]   Figure 2.   Epithelial IL-16 immunoreactivity (left) and mRNA (right) expression in atopic asthmatic, atopic nonasthmatic, and normal subjects. Results are expressed as a percentage of positive cells/total cells (0- 4, see MATERIALS AND METHODS). *P < 0.001 compared with atopic nonasthmatic and normal subjects (ANOVA).

The numbers of subepithelial IL-16 immunoreactive positive cells were significantly higher in bronchial biopsies obtained from asthmatic subjects (4.35 ± 0.36 cells/ mm BM) as compared with those from atopic nonasthmatic individuals (1.18 ± 0.46 cells/mm BM, P < 0.001) and normal controls (0.3 ± 0.46 cells/mm BM, P < 0.001) (Figure 4a). The specificity of the reaction with the anti-IL-16 mAb was confirmed by the lack of positive reaction following omission of the anti-IL-16 antibody.

View larger version (13K): [in this window] [in a new window]   Figure 4.   Subepithelial IL-16 immunoreactivity (left) and mRNA (right) expression in atopic asthmatic, atopic nonasthmatic, and normal subjects. Results are expressed as the numbers of positive cells per mm of basement membrane. *P < 0.001 compared with atopic nonasthmatic and normal subjects (ANOVA).

The distribution of IL-16 mRNA followed a similar pattern to the IL-16 immunoreactivity (Figure 3). Positive hybridization signals were identified mainly in the bronchial epithelial cells, although some IL-16 mRNA-positive cells could also be detected in the subepithelium. A variable degree of positive hybridization signals were identified within the airways in all subjects (Figures 3 and 4). However, the mean scores for epithelial IL-16 mRNA and the numbers of subepithelial IL-16 mRNA-positive cells were significantly greater in asthmatic subjects as compared with those from both atopic nonasthmatic and normal subjects (P < 0.001). The corresponding sense probe and RNase pretreatment were used as controls and in all cases this gave negative results.

View larger version (121K): [in this window] [in a new window]   Figure 3.   Representative autoradiographs (dark field illumination) of in situ hybridization using a 35S-labeled antisense IL-16 probe of a section from a bronchial biopsy from an asthmatic subject (A); only focal in situ hybridization signal is observed in normal subjects (C). No hybridization signal was observed using a 35S-labeled sense IL-16 probe (B).

There was an increased number of CD4 immunoreactive cells in bronchial biopsies obtained from atopic asthmatic subjects (26.18 ± 2.28 cells/mm BM) as compared with those obtained from atopic nonasthmatic (7.96 ± 2.9 cells/mm BM, P < 0.001) and normal individuals (9.6 ± 2.9 cells/mm BM, P < 0.001) (Figure 5). CD4⁺ cells were observed mainly in the subepithelial area, although a relatively good number of CD4⁺ cells could also be identified within or in close contact with the epithelium (Figure 1). The degree of epithelial IL-16 immunoreactivity and mRNA correlated with the numbers of CD4 immunoreactive cells in the submucosa (r² = 0.70, P < 0.001, and r² = 0.40, P = 0.001, respectively; Figure 6). Similarly, the numbers of subepithelial IL-16 immunoreactive cells and mRNA positive cells correlated with the numbers of infiltrating CD4⁺ cells (Figure 6).

View larger version (11K): [in this window] [in a new window]   Figure 5.   CD4 immunoreactivity of bronchial biopsies from atopic asthmatic, atopic nonasthmatic, and normal subjects. Results are expressed as the numbers of positive cells per mm of basement membrane. *P < 0.001 compared with atopic nonasthmatic and normal subjects (ANOVA).

View larger version (24K): [in this window] [in a new window]   Figure 6.   Relationships between IL-16 immunoreactivity and CD4+ cell infiltration and airway responsiveness. There were significant correlations between the epithelial IL-16 immunoreactivity score (A), the number of subepithelial IL-16  immunoreactive cells/mm BM (B), and CD4+ cell infiltration. Epithelial (C) and subepithelial (D) IL-16 immunoreactivity correlated with airway responsiveness to methacholine (log PC20FEV1). Closed circles, atopic asthma; closed triangles, atopic nonasthmatics; open circles, normal control subjects.

There was a correlation between both epithelial (r² = 0.433, P = 0.04) and subepithelial (r² = 0.794, P = 0.001) IL-16 immunoreactivity and airway responsiveness to methacholine (log PC₂₀FEV₁) in the group of asthmatic subjects. No significant association was observed between IL-16 expression and the degree of airflow obstruction (FEV₁).

	Discussion

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In this study we investigated the in vivo expression of a novel cytokine, IL-16, in bronchial biopsies of asthmatic subjects as compared with both atopic nonasthmatic and nonatopic normal control subjects. Using in situ hybridization in conjunction with immunocytochemical staining techniques, our results provide direct evidence that IL-16 is expressed both at the gene and protein levels in human airways. IL-16 expression was found to be upregulated significantly in bronchial biopsies obtained from subjects with asthma as compared with those from both atopic nonasthmatic individuals and normal controls, indicating that upregulation of IL-16 expression is not a function of atopy. IL-16 mRNA and immunoreactivity were observed mainly in the bronchial epithelium, and the degree of IL-16 expression correlated with CD4⁺ cell infiltration within the submucosa. In addition, there were significant associations between epithelial and subepithelial IL-16 immunoreactivity and airway responsiveness to methacholine. These data suggest that IL-16 may play a role in the pathogenesis of asthma and that airway epithelial cells are a major in vivo cellular source of IL-16 within the airways.

The contribution of IL-16 in the development of various inflammatory diseases, including asthma, has not been fully elucidated. Elevated concentrations of IL-16 have been found in bronchoalveolar lavage fluid obtained from asthmatic subjects following antigen challenge (34). The current study was undertaken to localize IL-16 mRNA and protein in endobronchial biopsy specimens from asthmatic subjects compared with normal controls. These experiments extend these previous observations and highlight the predominant expression of IL-16 at the tissue level in airways of subjects with stable asthma. Our results clearly indicate that there is a significant increase in the numbers of IL-16 immunoreactive cells in asthmatic airways. The strong immunoreactivity observed in tissues can be explained by the fact that we used a specific antibody that was raised against the C-terminal peptide of the molecule and that recognizes both the precursor and the secreted forms of IL-16. In order to determine whether the IL-16 immunoreactivity was the result of local protein synthesis, IL-16 mRNA expression was also determined using the technique of in situ hybridization and was also found to be enhanced in subjects with asthma.

While both peripheral blood CD8⁺ and CD4⁺ T cells are major sources of IL-16 in vitro (28), the present data suggest that bronchial epithelial cells are a main in vivo cellular source of IL-16 in both normal and asthmatic airway tissues. To confirm the epithelial origin of IL-16, double immunocytochemistry was performed using a polyclonal antibody against cytokeratin as a specific marker for epithelial cells and an anti-IL-16 monoclonal antibody. These experiments confirm the colocalization of IL-16 immunoreactivity and cytokeratin immunoreactivity in the epithelium. The epithelial cell as a source of IL-16 has also been supported by preliminary data identifying IL-16 mRNA in bronchial epithelial cell lines following stimulation with cytokine, mitogen, or histamine (35, 36). IL-16-like activity has also been detected in cell culture supernatants generated from histamine-stimulated tracheal epithelial cells obtained from asthmatic subjects in vitro (37). IL-16 thus joins the list of cytokines and chemokines known to be produced by epithelial cells in asthma, including the granulocyte/macrophage colony-stimulating factor, IL-6, IL-8, and monocyte chemoattractant protein-1 (38, 39). Although less prominent, IL-16 expression was also identified in the lamina propria. The cellular source of these IL-16 positive cells remains to be determined. The morphology of IL-16 positive cells in bronchial biopsies of asthmatic subjects in the present study is consistent with T lymphocytes, although the contribution of other inflammatory cells has not been excluded. In this regard, human eosinophils have recently been shown to produce IL-16 (40).

In this study we have identified elevated numbers of CD4⁺ in bronchial biopsies from asthmatics, as compared with atopic nonasthmatics and normal control subjects. Conflicting reports exist in the literature on the association between increased CD4⁺ cell numbers and asthma. Walker and coinvestigators found increased numbers of CD4⁺ cells in bronchoalveolar lavage from asthmatic subjects compared with normal control subjects (41). A 2-fold increase in the mean numbers of CD4⁺ cells was observed in bronchial biopsies from asthmatics compared with normal controls, although the trend to the increase in CD4⁺ cells did not reach statistical significance (3, 8). The majority of the asthmatic subjects in the current study had newly diagnosed asthma or mild disease which had not required previous use of anti-inflammatory drugs. The increase in CD4⁺ cell numbers observed in our study might be explained by the absence of recent or past glucocorticoid therapy, which can affect inflammatory cell populations in the airways. Another possible explanation for our findings might be related to improved immunocytochemical reagents and differences in techniques used for identification of CD4⁺ cells. The majority of CD4⁺ cells identified in this study are small mononuclear cells and the morphology is consistent with lymphocytes. Blood-derived eosinophils, eosinophilic cell lines, and cells of the monocyte/ macrophage lineage have been shown to express the CD4 surface antigen when cultured in presence of specific cytokines. Whether tissue eosinophils or macrophages express CD4 in vivo needs to be confirmed.

The physiologic significance of epithelial-derived IL-16 in the pathogenesis of asthma is unclear at this moment. IL-16 is known principally for its ability to induce CD4⁺ cell migration (20). Histologic studies have shown that T cells are often found within or in close contact with the epithelial layer in the bronchial mucosa (4, 42), suggesting that local release of chemotactic factors, including IL-16, by epithelial cells may contribute (at least in part) to their accumulation. The correlation observed between IL-16 expression and CD4⁺ cell infiltration supports its potential functional significance. Most CD4⁺ cells were in the submucosa rather than in the epithelium, suggesting the possibility of the contribution of nonepithelial IL-16 to the accumulation of CD4⁺ cells in the submucosa. The in vitro migratory response to IL-16 appears to be higher in memory T-cells; however, the responding lymphocyte population to IL-16 has not been fully characterized. Theoretically, there should be a correlation between IL-16 expression and the numbers of CD4-bearing macrophages and CD4-bearing eosinophils. In this study, however, we did not examine the association between IL-16 expression and the different cell types that might bear CD4 surface antigen in vivo.

Other potential mechanisms by which IL-16 may be involved in asthma are related to its capacity to reversibly induce functional anergy in CD4⁺ cells following TCR/ CD3 costimulation in vitro, suggesting that IL-16 might have important immunoregulatory properties (43). It is therefore possible that increased synthesis of IL-16 in inflamed tissues, such as in asthmatic airways, may serve to limit the ongoing immune response. It is unclear whether the immunomodulatory function of IL-16 might be relevant to or more important than its effect on cell migration with regard to asthma pathogenesis; however these two functional properties are not mutually exclusive. Rather, it is likely that the IL-16 responsive motile CD4⁺ T cell is transiently unresponsive to its own antigen specificity; a phenomenon that might explain the preponderance of CD4⁺ T cells in the airways of asthmatics to which no antigenic specificity can be attributed. Thus, while these cells might proliferate in situ following recruitment, they would not require antigen to divide. More information on the functional activities of IL-16 are needed before we can fully understand the precise role of this cytokine in various inflammatory processes. However, the present findings clearly indicate that epithelial-associated IL-16 expression is a feature of asthma.

Within the asthmatic group there was a correlation between IL-16 immunoreactivity and airway responsiveness to methacholine. Although these findings do not prove a causal association, they support the concept that IL-16 contributes to airway hyperresponsiveness in atopic asthma, presumably through recruitment of CD4⁺ T cells, or that a common proximal event induces both IL-16 expression and bronchial hyperreactivity. We found no correlation between IL-16 expression and measurement of airway obstruction, probably due to the relatively mild disease of the subjects studied. Indeed, the subjects recruited in the present study have mild asthma based on clinical history and normal FEV₁ values. Only subjects who required ₂ agonists were recruited, as glucocorticosteroid therapy may have profound effects on the expression of epithelial-derived cytokines (44).

We have demonstrated the predominant expression of IL-16 mRNA and immunoreactivity in airway epithelium in vivo in stable atopic asthmatic subjects. The present findings support the concept that epithelial cells may interact with CD4⁺ T cells by virtue of their capacity to synthesize a cytokine, IL-16, that interacts specifically with CD4-bearing cells. These experiments provide an additional mechanism by which airway epithelial cells might participate in the immunoregulation of asthma and contribute to the recruitment of CD4⁺ cells within the airways. Factors that regulate IL-16 synthesis and secretion by airway epithelial cells have yet to be ascertained.