Introduction

Abstract Introduction Materials and Methods Results Discussion References

Atopic asthma is characterized by bronchial inflammation and airway hyperresponsiveness to a variety of stimuli including inhaled allergens (1). The infiltration of activated T lymphocytes, mast cells, and eosinophils into the bronchial mucosa typify the inflammation (2). T cells are thought to orchestrate the airway inflammatory response that is associated with an increase in Th2 cytokine expression (4), suggesting activation of a gene cluster on human chromosome 5 that includes interleukin (IL)-3, IL-4, IL-5, IL-9, IL-13, and granulocyte macrophage colony-stimulating factor (GM-CSF).

A number of studies have demonstrated the presence of several cytokines in bronchial biopsies and bronchoalveolar lavage fluid (BALF) of asthmatic patients. When in situ hybridization is used, increases in the expression of mRNAs for IL-2, IL-3, IL-4, IL-5, and GM-CSF have been found in the asthmatic airways (4, 5). More recently, when reverse transcription-polymerase chain reaction (RT-PCR) analysis was used, an enhanced expression of both IL-5 (6) and IL-13 (7) was detected in BALF after allergen challenge of lung segments of asthmatic patients. IL-5 is of particular relevance to bronchial inflammation, because it is crucial to the activation, differentiation, recruitment, and survival of eosinophils (8). Human IL-4 and interferon- (IFN-) play important roles in the regulation of immunoglobulin E (IgE) synthesis and allergic inflammation (11). IFN- exerts a downregulatory effect on Th2 responses and consequently inhibits IgE production, whereas IL-4 enhances IgE production and favors the commitment of naive T cells to the Th2 type. More recently, IL-13 has also been shown to promote the isotype switching of B cells toward IgE production (12).

The cytokines expressed in the asthmatic airways probably do not act in isolation but form a cascade in which the induction of one influences the expression of others. Further understanding of the underlying disease process requires identifying which cytokines are produced and their roles in promoting the inflammatory response. For the purpose of dissecting cellular interactions required for cytokine expression and allergen reactivity, we have developed an ex vivo bronchial explant model. The main advantages of using this approach are that (1) the production of cytokine proteins can be quantified in the culture supernatants; (2) the possibility of cellular recruitment can be circumvented and the responses of leukocytes resident in the bronchial mucosa can be examined; and (3) the effects of inhibiting specific immunological processes can be monitored and immune intervention protocols developed. In this report we describe the application of the bronchial explant model to examine cytokine expression and allergen reactivity of airway mucosal T cells in asthma. In addition to T-cell receptor ligation with specific antigen, activation of T lymphocytes and cytokine production requires costimulatory signals (13, 14). The contribution of T-cell costimulation to the pathogenesis of allergic asthma is unknown. We have therefore investigated the role of CD28 and its ligands (CD80 and CD86) on T-cell cytokine production in asthmatic bronchial tissue.

	Materials and Methods

Abstract Introduction Materials and Methods Results Discussion References

Subjects

Twelve mild atopic asthmatic (forced expiratory volume in 1 s [FEV₁] > 80% predicted) and 7 healthy nonatopic control subjects participated in the study. The asthmatic subjects were selected on the basis of having positive skinprick tests to house dust mite extract, Dermatophagoides pteronyssinus (Der p) and demonstration of increased airway responsiveness to methacholine, that is, cumulative concentration producing a fall in FEV₁ of 20% from baseline (PC₂₀) < 16 mg/ml. They had not experienced an exacerbation of their asthma or upper respiratory tract infection for at least 6 wk before participation in the study and were using only inhaled, short-acting ₂-agonist medication as required for relief of symptoms. The asthmatic subjects were not newly diagnosed and had onset of asthma during childhood. The normal control volunteers had no history of asthma or other allergic disease, normal serum total IgE concentrations, negative skinprick tests to a panel of common allergens (including house dust mite, grass pollen, tree pollen, feather, aspergillus, cat, dog) normal FEV₁ values and a methacholine PC₂₀ > 32 mg/ml. The clinical characteristics of all study subjects are shown in Table 1. All volunteers were nonsmokers. Informed written consent was obtained from the subjects before participation and the study was approved by the joint Ethics Committee of Southampton University and General Hospital.

Endobronchial Biopsy Samples

Fiberoptic bronchoscopy was performed by a standard technique conforming to published guidelines (15). Briefly, subjects were premedicated with nebulized salbutamol (2.5 mg), ipratropium bromide (0.5 mg), and intravenous midazolam (5 to 10 mg). Topical lidocaine 2% (upper airways) or 1% (lower airways) was used to obtain local anesthesia. Using alligator forceps, six to eight endobronchial mucosal biopsies were obtained from subcarinae separating the basal segmental bronchi of the right lower lobe and placed in culture media.

Culture Protocol

Bronchial biopsies (each 1 to 2 mm in diameter) were cultured for 24 h in serum-free medium alone (500 µl; AIM V, Life Technologies, Paisley, UK), in the presence of phytohemagglutinin ([PHA], 10 µg/ml), or medium supplemented with Der p allergen (5,000 U/ml or 0.35 µg/ml; ALK, Horsholm, Denmark). Two biopsies were used per culture condition to provide sufficient RNA for extraction and minimize effects due to tissue heterogeneity. After culture, supernatants were harvested and stored at 80°C, and biopsies were kept in liquid nitrogen pending analysis. In addition, two biopsies were not cultured but stored immediately in liquid nitrogen. The biopsies used in these experiments were obtained from asthmatic subjects 1 to 8 and control subjects 13 to 19 (Table 1). A defined culture medium (AIM V) was used throughout this study to preclude the possibility of stimulation of the tissue with serum components. The medium was supplemented with N-2- hydroxyethylpiperazine-N'-ethane sulfonic acid (10 mM), glutamine (1 mM), and 2-mercaptoethanol (2 µM). It is noteworthy that the ALK allergen extract was tested using an E-Toxate kit (Sigma, Poole, UK) and found to be free of endotoxins.

Human cytotoxic T lymphocyte antigen (CTLA)-4Ig is a chimeric fusion protein of human CTLA4/human IgG₁Fc, which has been described previously (16). To inhibit allergen-induced cytokine expression by T cells, hCTLA-4Ig (25 µg/ml; Bristol-Myers Squibb Institute, Seattle, WA) was added simultaneously with the allergen to bronchial explants from four more asthmatic subjects (Patients 9 to 12, Table 1). Human IgG₁ (Binding Site Ltd., Birmingham, UK) was used as the control in these experiments. To examine cytokine production by mast cells, biopsies from two atopic asthmatic subjects (Patients 7 and 8, Table 1) were stimulated with anti-human IgE (3 µg/ml; Serotec, Oxford, UK).

RT-PCR Detection of Cytokine mRNA

RNA was extracted from bronchial biopsies using the RNAzol B (Ams Biotechnology, Oxon, UK) technique. Briefly, tissue was homogenized in the presence of RNAzol B and chloroform, and the RNA was precipitated at 20°C in isopropanol overnight. The RNA pellet was recovered by centrifugation at 4°C, washed in 80% ethanol, air-dried, and suspended in diethylpyrocarbonate-treated water. The resulting RNA was quantified using spectrophotometry. One microgram total cellular RNA (in some experiments < 1 µg was obtained) was then reverse transcribed by avian myeloblastosis virus reverse transcriptase (RT System; Promega, Southampton, UK) at 42°C for 1 h using poly d(T)₁₅ as a primer. The cDNA was amplified by PCR in the presence of a master mix containing PCR buffer, MgCl₂ (under optimal concentrations), 1 U Taq DNA polymerase (Promega), 0.2 mM dNTPs, and specific primer pairs (Table 2). PCR was conducted for 40 cycles under the following conditions: denaturation at 94°C for 20 s, annealing at optimal temperature for each primer pair for 30 s, and extension at 72°C for 60 s in a thermocycler. Final extension was at 72°C for 10 min. PCR-amplified products (10 µl) were electrophoresed through 2% agarose gels (Bio-Rad, Hemel Hempstead, UK) containing 0.5 µg/ml ethidium bromide and compared with DNA reference markers. Products were visualized by ultraviolet illumination. All oligonucleotide primers (Table 2) were synthesized by the Department of Microbiology, Southampton General Hospital. In addition to cytokine specific primers, mRNA for adenine phosphoribosyltransferase (APRT) was measured as a positive control, and CD4 was used to detect presence of CD4⁺ T cells in the sample.

Cytokine Protein Measurement

The level of cytokine proteins in the culture supernatants of biopsy samples was determined with commercially available enzyme-linked immunosorbent assay (ELISA) kits (IL-4, IL-5, and IFN- with Quantikine [R&D Systems, Abingdon, UK]; and IL-13 with Cytoscreen [BioSource International, Camarilla, CA]), according to the manufacturer's instructions. In general, samples and standards were diluted with assay diluent and added to a 96-well microtiter plate precoated with antibody against the appropriate cytokine. The plate was sealed and incubated at room temperature for 1.25 h on a plate shaker. After washing the plate four times, the appropriate conjugated antibody was added and incubated for an additional 1.25 h, followed by four washings. Finally, substrate solution was added to the wells and color development stopped after 20 min incubation. The color reaction was read by an ELISA plate reader at 450 nm. A standard curve was plotted and cytokine concentration (pg/ml) of the samples read. Cytokine levels of biopsy supernatants were normalized and expressed in picogram per milligram wet weight of tissue. The range of cytokines measured was limited by the volume of biopsy culture supernatant available. It is important to note that on average, approximately 60 to 80 pg/ml of IL-5 (the lowest level measured was approximately 10 pg/ml and the highest 250 pg/ml) was detected in supernatants of allergen-stimulated asthmatic biopsies, and this value was divided by the tissue weight, which was approximately 5 to 10 mg for two biopsies.

Statistical Analysis

Cytokine protein levels were compared between study groups using the Mann-Whitney U test. The Wilcoxon signed-rank test for paired data was used for within-group comparisons. Analysis was performed with StatView 4.02 for Macintosh (Abacus Concepts, Berkeley, CA). Values of P < 0.05 were accepted as statistically significant.

	Results

Abstract Introduction Materials and Methods Results Discussion References

Cytokine mRNA Expression

We used the RT-PCR technique to analyze the spectrum of cytokines expressed by bronchial biopsies from six atopic asthmatics and six normal volunteers after 24 h culture. One advantage of RT-PCR analysis is that the expression of a large panel of cytokines can be evaluated in a single sample, which is not possible using other methodologies. The RT-PCR technique was used as a screen for which cytokines are expressed, and those of interest were subsequently quantified by measuring protein production in the culture supernatants through ELISA.

Figure 1 shows representative RT-PCR data from two asthmatic and two control subjects. Our analysis revealed that without any overt stimulation, mRNA transcripts for IL-5 and IL-13 were produced by asthmatic but not normal bronchial tissue (four of six versus zero of six, respectively; Table 3). In contrast, the expression of IFN- was observed in a higher proportion of cultured bronchial biopsies from the control subjects when compared with asthmatic subjects (five of six versus three of six, respectively). Both asthmatic and normal biopsies showed strong signals for IL-6 and IL-8 but not for IL-2 or IL-4 after culture (Figure 1 and Table 3).

View larger version (50K): [in this window] [in a new window]   Figure 1.   Cytokine mRNA expression by two atopic asthmatic (A) and two normal control (B) bronchial biopsies in culture. Biopsies were cultured for 24 h in medium alone, in the presence of Der p allergen (5,000 U/ml), or in the presence of PHA (10 µg/ml). Total tissue RNA was extracted and subjected to RT-PCR amplification using cytokine-specific primers. PCR products were electrophoresed and visualized by ethidium bromide staining. The data shown are representative of a total of six asthmatic and six control subjects.

The cytokine profile for control bronchial tissue was largely unchanged when tissue was challenged with Der p allergen. In contrast, explants from asthmatic subjects expressed strong signals for IL-5 mRNA after allergen stimulation (biopsies from two of six control subjects expressed only weak signals for IL-5, whereas six of six from asthmatic subjects expressed pronounced signals; Figure 2). Maximal responses of the T-cell component of the tissue was induced by stimulation with the T-cell mitogen PHA. Typically, PHA stimulation induced the pronounced expression of most cytokines including IL-2, IL-4, and IL-9 in lung biopsies from both study groups (Figure 1). Bands of lower size than that predicted for IL-4 (449 bp) were observed in some PHA-induced biopsies (Figure 1). A recent report has shown that a naturally occurring variant of human IL-4 exists in which the second exon is omitted by alternative splicing (17). This variant, IL-42, which has a similar size to our additional bands, has been shown to inhibit IL-4-stimulated T cell proliferation.

View larger version (35K): [in this window] [in a new window]   Figure 2.   IL-5 mRNA expression in atopic asthmatic (A) and normal control (B) bronchial explants stimulated with Der p allergen. See Figure 1 for methods.

For comparison, cytokine mRNA profile of biopsies before culture from both study groups was also evaluated. Pre-culture tissue expressed weaker signals for IL-6 and IL-8 than cultured biopsies. Similarly, a higher proportion of baseline tissue from asthmatic subjects expressed IL-5 and IL-13 mRNA compared with control tissue, whereas IFN- was expressed in a higher proportion of control samples (data not shown).

Cytokine Protein Secretion by Bronchial Explants

A major advantage of the explant system is that cytokine proteins produced by the bronchial mucosa are secreted into the culture supernatants and can therefore be quantified. To confirm that the expression of mRNA for IL-5 and IL-13 in the asthmatic bronchial tissue and IFN- in the normal lung was correlated with secretion of the protein, we measured the levels of these cytokines that accumulated in the supernatants. The results show that there was a significant increase in IL-5 protein in the supernatants of both Der p and PHA-stimulated biopsies from asthmatic but not control subjects (Figure 3, P < 0.02). Addition of Der p or PHA also augmented the production of IL-13 in the majority of asthmatic biopsies, although this did not attain statistical significance (Figure 4). It is possible that basal expression of this cytokine in some biopsies masked a clear response after allergen or mitogen stimulation. IFN- protein was only detected in the culture supernatants after PHA stimulation of biopsies from both study groups. The levels of IFN- were significantly higher in supernates from control samples compared with asthmatic samples (Figure 4, P < 0.02, Mann-Whitney U test). Only low levels of IL-4 protein were produced by asthmatic and normal bronchial explants. The levels were unchanged after stimulation of the tissue with Der p or PHA and were not significantly different between the two study groups (Figure 4). The response to the allergen observed in bronchial tissue from asthmatic subjects differed from those seen in peripheral blood mononuclear cells (PBMCs) of the same subjects. PBMCs from asthmatic subjects cultured for 24 h (under similar conditions to the lung tissue) did not respond to allergen, producing higher levels of IL-5 protein compared with control samples only after stimulation with PHA (data not shown).

View larger version (20K): [in this window] [in a new window]   Figure 3.   IL-5 protein secretion by asthmatic and control bronchial explants. Biopsies were cultured for 24 h in medium alone or stimulated with either Der p (A) or PHA (B) as described in the legend of Figure 1. The level of IL-5 protein in the supernatants was determined by ELISA, normalized for tissue weight, and expressed as picogram per milligram of tissue. Bars denote means of 9 to 12 asthmatic and 7 control subjects. Statistically significant increases in IL-5 production within the asthmatic group are shown (Wilcoxon rank test). Differences between the two study groups in the production of IL-5 induced by Der p or PHA were significant (P < 0.02, Mann-Whitney U test).

View larger version (8K): [in this window] [in a new window]   Figure 4.   Production of IL-13, IL-4, and IFN- by asthmatic and control bronchial explants. Cytokine proteins present in the culture supernatants were quantified by ELISA, normalized for tissue weight, and expressed as picogram per milligram of tissue. Bars denote means of 9 to 12 asthmatic and 6 control subjects for IL-13, 8 asthmatic and 5 control subjects for IL-4, and 8 asthmatic and 6 control subjects for IFN-.

Bronchial biopsies of two atopic asthmatics were also stimulated with anti-IgE and compared with Der p-challenged tissue. The production of IL-5 and IL-13 was augmented by allergen but not by anti-IgE stimulation (anti-IgE did, however, induce the secretion of histamine) (data not shown). These results indicate that the cytokines induced by the allergen were not derived from mast cells.

Inhibition of Allergen-Induced Cytokine Expression

To determine the role of T-cell costimulatory molecules in allergen-augmented IL-5 and IL-13 production, we examined the effect of CTLA-4Ig on Der p-stimulated asthmatic bronchial biopsies (Patients 9 to 12, Table 1). CTLA-4Ig, a fusion protein of the extracellular domain of CTLA-4 and Ig C1 chain (16), significantly inhibited the allergen-induced increase in IL-5 and IL-13 production (Figures 5 and 6, P < 0.05), reflecting a requirement of B7 costimulation for the expression of the cytokine. Figure 5A shows that IL-6 and IL-8 mRNA expression was not affected by CTLA-4Ig, and thus the inhibition appears to be selective to the allergen-induced cytokine expression. In addition, this preparation did not prevent the T-cell proliferative response to plate-bound anti-CD3 where the requirement for costimulatory events are less stringent (data not shown).

View larger version (16K): [in this window] [in a new window]   Figure 5.   CTLA-4Ig inhibits allergen-induced IL-5 expression by bronchial explants from four atopic asthmatic subjects. Biopsies were cultured for 24 h with Der p alone or Der p + CTLA-4Ig (25 µg/ml). (A) RT-PCR analysis of cytokine IL-5, IL-6, and IL-8 transcripts in tissue, and (B) IL-5 protein in the culture supernatants were quantified by ELISA and normalized for tissue weight and expressed as picogram per milligram of tissue. Der p + control Ig induced secretion of 8.3 ± 3.4 pg/mg IL-5. ELISA data are means ± SEM (n = 4). The percentage inhibition of the allergen-induced IL-5 production by CTLA-4Ig ranged from 57.0% to 86.1%. *P < 0.05.

View larger version (11K): [in this window] [in a new window]   Figure 6.   CTLA-4Ig inhibits allergen-induced IL-13 secretion by bronchial tissue from four atopic asthmatics (*P < 0.05). Der p + control Ig secreted 5.1 ± 2.3 pg/mg IL-13. See Figure 5 for methods.

	Discussion

Abstract Introduction Materials and Methods Results Discussion References

We have developed a bronchial explant system to gain further insight into which cytokines are involved in perpetuating allergen-induced inflammatory processes in the asthmatic airways. Our results demonstrate that bronchial biopsy tissue maintained in culture for 24 h actively transcribes cytokine mRNA without any overt stimulation. Moreover, clear differences exist in the spectrum of cytokine expression between atopic asthmatic and normal control tissue. In particular, mRNA transcripts for IL-5 and IL-13 were produced by asthmatic but not normal bronchial tissue. In contrast, the expression of IFN- was observed in a higher proportion of cultured bronchial biopsies from the control subjects compared with those from asthmatic subjects. Stimulation with Der p allergen did not alter the cytokine profile of biopsies from control individuals, but elevated the expression of IL-5 mRNA and significantly induced the secretion of the protein by the asthmatic airway tissue. The production of IL-13 was also augmented after allergen stimulation by the majority of asthmatic biopsies. To our knowledge, this is the first demonstration of IL-5 and IL-13 protein secretion by asthmatic endobronchial musosal tissue following ex vivo challenge with specific allergen. It is noteworthy that such pronounced cellular responses can be elicited by bronchial tissue obtained from asthmatic subjects with only a mild form of the disease. Both asthmatic and normal explants showed prominent mRNA signals for IL-6 and IL-8, possibly by the bronchial epithelium or macrophages present in tissue.

The primary target cell for IL-5 is the eosinophil. This cytokine has been shown to induce eosinophil maturation, chemotaxis, and transition to the activated, hypodense phenotype (8, 9, 18, 19). An in vivo role for IL-5 in eosinophil infiltration is suggested by the correlation of IL-5 mRNA expression with the number of these cells in bronchial biopsies of asthmatics (5). Activated T cells are the principal cellular source of IL-5. However, other cell types shown to express IL-5 include mast cells (20) and eosinophils (21, 22). Recently, Krishnaswamy and colleagues (6) have identified infiltrating mononuclear cells rather than eosinophils as the source of IL-5 mRNA in BALF of allergen-challenged asthmatics. Whether the mononuclear cells are resident or recruited to the airways after challenge was unresolved. However, our study demonstrates the production of IL-5 in response to ex vivo allergen stimulation of bronchial biopsies, and shows that cells resident in the bronchial mucosa can be the source of the cytokine.

To date, T cells and mast cells have been shown to produce both IL-5 and IL-13 (12, 20, 23). One major difference between the two cell types is the requirement for costimulation by T cells to trigger cytokine production (14). To investigate the contribution of T-cell costimulation to the pathogenesis of asthma, and whether IL-5 and IL-13, produced in the asthmatic bronchial mucosa after allergen stimulation, may be T-cell-derived, the effect of CTLA-4Ig was examined on Der p-challenged tissue. We found that CTLA-4Ig significantly inhibited the allergen-induced increase in IL-5 and IL-13 secretion. This is a consequence of the fusion protein binding to the costimulatory molecules CD80 (B7-1) and CD86 (B7-2) on antigen-presenting cells (APC), thereby blocking their interaction with CD28 and/or CTLA-4. The importance of interactions between B7 molecules and their counterreceptors, CD28 and/or CTLA-4, in regulating T-lymphocyte responses have been well documented (14, 16, 24). The requirement for B7 costimulation to induce cytokine expression implies that allergen-specific T cells in the bronchial tissue may be the major source of IL-5 and IL-13, as this interaction is not required for mast cell or eosinophil production of cytokines. This is the first demonstration that T cells residing in the bronchial mucosa of asthmatic individuals require B7 costimulation to trigger the production of Th2 cytokines after allergen stimulation. The rapid response to Der p allergen in the asthmatic bronchial mucosa may be a consequence of efficient presentation of the allergen by resident APC such as dendritic cells, which are found closely associated with the bronchial epithelium (27), or the presence of a high frequency of antigen specific T cells of memory phenotype. In contrast to IL-5, there is basal expression and secretion of IL-13 in unstimulated explants from asthmatics that may reflect an ongoing inflammatory response in the bronchial tissue. Our results complement those of Huang and coworkers (7) who have also detected basal production of IL-13 protein in asthmatic BALF and identified mononuclear cells as the source of the cytokine.

Consistent with findings in BALF cells (28), we detected low levels of IL-4 expression by bronchial biopsies from atopic asthmatic and control subjects, and there was no difference in the levels measured between the two study groups. IL-4 and IL-13 have similar biologic activities that include growth and differentiation of haematopoietic progenitor cells (29), modulation of human monocyte function (30), selective expression of vascular cell adhesion molecule-1 in endothelial cells (31), and promoting B-cell proliferation and Ig class switching to IgE (12, 32, 33). However, unlike IL-4, IL-13 cannot promote Th2 cell differentiation and its activation kinetics are characterized by a rapid onset and prolonged expression (34). The high levels of secreted IL-13 by asthmatic bronchial biopsies observed in the present study suggest that IL-13 plays an important role in allergen-induced inflammmatory responses of the airways.

In the course of this study, measurement of the cytokine protein in the supernates of cultured biopsies provided a means to quantify cytokine expression and corroborate the mRNA transcripts analysis. However, there was not always a direct correlation between the levels of mRNA and proteins. This is illustrated by the detection of pronounced IFN- transcripts in cultured lung biopsies of both asthmatic and normal subjects but the absence of protein, except after stimulation with PHA. Such discrepancies are not unexpected and could be accounted for by either storage of the cytokine protein products or failure to translate mRNA.

In summary, our data demonstrate that there are clear differences in the cytokine profile expressed by bronchial mucosal explants from atopic asthmatic compared to healthy nonasthmatic individuals. More importantly, Der p stimulation induced the secretion of IL-5 and IL-13 from asthmatic but not normal bronchial tissue. The allergen-induced production of these cytokines was inhibited by CTLA-4Ig, indicating a requirement for CD80 and/or CD86 T-cell costimulation in Th2 cytokine expression. This requirement for B7/CD28 costimulation suggests that these cytokines are derived from allergen-specific T cells residing in the bronchial mucosa of asthmatics. Biopsies from the control subjects appeared quiescent, expressing certain cytokines that are thought to play important roles in lung homeostasis, such as GM-CSF (35). Our observations extend into a disease setting previous findings using murine models (36), and identify T-cell costimulation as being a prerequisite for the production of Th2 cytokines in human bronchial asthma. Blockade of the interaction of B7 costimulatory molecules with CD28 on T cells using specific immunological agents may provide a novel therapeutic intervention in the treatment of allergic inflammatory diseases such as asthma.