Introduction

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Interleukin (IL)13 is a cytokine known to be produced by activated Th2 cells (1) and the human mast cell line HMC-1 upon activation with either phorbol myristate acetate (PMA) or the calcium ionophore A23187 (2). We now report the expression and production of IL-13 mRNA and protein in human alveolar macrophages (AMs). The human IL-13 gene occurs as a single copy in the haploid genome and maps to human chromosome 5. It can be further localized to a region where a cluster of genes encoding other immune and hemopoietic factors, including IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-5, and IL-4, have been previously mapped (3). IL-13 shares a number of fundamental characteristics with IL-4, a key Th2-derived cytokine; however, there is an important difference, because in contrast to IL-4, IL-13 does not stimulate T cells. IL-13 has a profound effect on primary immune cells by inducing immunoglobulin production, proliferation of B cells, and the differentiation of cells of the monocytic lineage (4). It can inhibit the production of inflammatory cytokines by both macrophages and monocytes (5).

The role of IL-13 in the lung has been little explored (6). We are interested in cytokines with a potential regulatory role on tumor necrosis factor  (TNF-) production and activity. We and others have previously investigated the role of another cytokine considered to have an anti-inflammatory role, IL-10, and have shown that in the normal AM exogenous IL-10 downregulates TNF- mRNA (7), but this effect is diminished in AMs from subjects with pulmonary fibrosis (10). Some reports have suggested that exogenous IL-13 has a variable effect on inflammatory cytokine production by AMs, such as TNF-, this being dependent upon the timing of its addition after the initial inflammatory stimulus (11, 12). We hypothesized that IL-13 would be produced by human AMs with the potential to play a regulatory role in the inflammatory response and would be upregulated in lung disease with an inflammatory pathological basis, such as pulmonary fibrosis. In the current study, we have examined expression and production of IL-13 by adherence-purified AMs from healthy control subjects compared to subjects with forms of pulmonary fibrosis.

	Materials and Methods

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Subjects

IL-13 expression was studied in two groups of subjects: 13 patients with forms of pulmonary fibrosis (six with rheumatoid disease, two with idiopathic pulmonary fibrosis, three with systemic sclerosis, two with sarcoidosis) and eight normal control subjects. The study was approved by the Ethics Committee of the Southmead Health Services Trust. All patients underwent clinical evaluation, including chest radiography, lung function measurements, and thin-section (3-mm sections) computed tomography (CT) prior to fiberoptic bronchoscopy and bronchoalveolar lavage (BAL). The presence of pulmonary fibrosis was determined by CT findings and lung biopsy. The clinical data are summarized in Tables 1 and 2.

Alveolar Macrophage Preparation

AMs for this study were obtained from BAL of patients undergoing fiberoptic bronchoscopy. Subjects were injected intramuscularly with 0.6 mg of atropine and sedated with 2 mg of alfentanil and 0-10 mg of midazolam. Topical lignocaine was administered to anesthetize the airway. Four 60-ml volumes of sterile 0.9% saline buffered with 8.4% sodium bicarbonate were instilled into the right middle lobe and then aspirated into a siliconized bottle kept on ice. The bronchoalveolar lavage fluid (BALF) was strained through a single layer of coarse gauze to remove mucus clumps and the filtrate was washed twice at 500 × g in RPMI 1640 (Sigma, Poole, Dorset, UK) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.5 µg/ml amphotericin B (Fungizone; culture medium). The cell pellet was resuspended in culture medium and viability assessed with 0.1% trypan blue. The cells were adjusted to 1 × 10⁶/ml and incubated in tissue culture petri dishes for 1 h at 37°C for mRNA analysis. The nonadherent cells were discarded and the plate vigorously rinsed twice to remove residual nonadherent cells. The cells were then cultured for a further 2 h and washed twice again prior to RNA extraction. The adherent population was scraped off with a sterile cell scraper and differential staining with Diff-Quik (Baxter, Dudingen, Switzerland) revealed > 99% pure AMs. AMs cultured for the determination of IL-13 protein were incubated for 24 h at 37°C, 5% CO₂ in the presence or absence of a dose range of lipopolysaccharide (LPS) (Sigma) before removal of the supernatants. These were stored at 70°C until analysis.

RNA Extraction

RnAzol B (AMS Biotechnology, Witney, Oxon, UK) was added to 0.5-1 × 10⁶ cells for the extraction of total RNA. The method used was based on the acid guanidinium-phenol-chloroform method (13). The purity of the RNA was determined by agarose gel electrophoresis and optical density ratio.

Reverse Transcription-Polymerase Chain Reaction

Perkin-Elmer GeneAmp thermostable rTth DNA polymerase (Perkin-Elmer, Warrington, Cheshire, UK) was used in two separate buffers to enhance its reverse transcriptase or DNA polymerase activity. This allowed synthesis of cDNA through any secondary structures, because the reverse transcriptase could be performed at a higher temperature. For each reverse transcriptase reaction the following conditions were used: 10 mM Tris-HCl (pH 8.3), 90 mM KCl, 1 mM MnCl₂, 200 µM dNTPs, 750 nM "downstream" primer, 5 units of rTth DNA polymerase in a volume of 20 µl with 1 µl of sample RNA. This reaction was incubated at 70°C for 15 min and then cooled to 4°C. For each polymerase chain reaction the following conditions were used: 1.5 mM MgCl₂, 0.8% chelating buffer (4% [vol/ vol] glycerol, 8 mM Tris-HCl [pH 8.3], 80 mM KCl, 0.04% Tween 20, 600 µM EGTA), 150 nM "upstream" primer in a final volume of 100 µl with the 20-µl reverse transcriptase sample added. This reaction was incubated at 95°C for 1 min, followed by 35 cycles of 95°C for 10 s and 60°C for 15 s, and a final incubation at 60°C for 7 min. The samples were then cooled to 4°C. One-fifth of the product was analyzed on a 2% TAE gel containing ethidium bromide alongside molecular weight markers pUC18/HaeIII (Sigma). For comparative reverse transcription-polymerase chain reaction (RT-PCR), a proportion of the cDNA was also amplified with primers for -actin and the results analyzed by both densitometry and Southern hybridization. A negative control following through the same protocol in the absence of cellular RNA and a sample run without RT were also included in the studies. Examples are shown in Figure 1. Primer sequences are displayed in Table 3. All primers were synthesized by Perkin-Elmer and are taken from published sequences (14).

View larger version (50K): [in this window] [in a new window]   View larger version (46K): [in this window] [in a new window]   Figure 1.   (A) PCR analysis of IL-13 mRNA from four subjects with rheumatoid arthritis (RA, lanes 3-6), one subject with idiopathic fibrosing alveolitis (IPF1, lane 7), one subject with systemic sclerosis (SSC1, lane 8) and six normal controls (N1-6, lanes 9-14). Lane 1, molecular markers; lane 2, control lane with no reverse transcriptase added. (B) Laser densitometry of the gel shown in (A) normalizing the IL-13 mRNA to that of -actin.

Southern Blotting of PCR Products

Blotting of agarose gel-separated and denatured DNA was carried out on nylon membranes (Boehringer Mannheim, Lewes, Sussex, UK). The filters were hybridized at 50°C (according to manufacturer instructions in digoxigenin [DIG] oligonucleotide labeling kit; Boehringer Mannheim) in hybridization buffer (5× SSC, 1% block [Boehringer Mannheim], 0.1% N-lauryl sarcosine, 0.02% sodium dodecyl sulfate [SDS]), washed twice with 2× SSC (6 M sodium chloride/0.6 M sodium citrate) containing SDS, 0.1× SSC containing 0.1% SDS for 5 min at room temperature, twice with 0.5× SSC/0.1% SDS for 15 min at 50°C, and detected by chemiluminescent CSPD (Boehringer Mannheim). An end-tailed digoxigenin-labeled human IL-13 oligonucleotide probe 5'-TGGGGAAGACTGTGGCT-3' (nucleotides 3275-3291) was used.

Densitometric Analysis

Densitometric analysis was performed on a calibrated Bio-Rad imaging densitometer, using the Molecular Analyst/ PC package (Bio-Rad, Hercules, CA). The ethidium bromide-stained gels were photographed and the expression standardized to that of -actin expression from the same reverse-transcribed mRNA sample. To prove the linearity of the system, experiments were performed in which a PCR sample was serially diluted and the densitometer used to analyze each resulting band. Samples were deemed negative if no band was seen by ethidium bromide staining or Southern blotting. All positive samples were readily confirmed by agarose gel examination.

Interleukin 13 Enzyme-Linked Immunoabsorbent Assay

An IL-13 enzyme-linked immunoabsorbent assay (ELISA) method (R&D Systems, Abingdon, Oxon, UK) was used (following manufacturer instructions) to determine the concentration of IL-13 in BALF and AM supernatants. IL-13 concentrations were determined by comparison to recombinant standards run in parallel with each batch of assays. Each sample was determined in duplicate. The sensitivity of this ELISA is cited at < 32 pg/ml; however, in our hands we obtained a detection limit of < 39 pg/ml.

Statistics

ELISA data were compared by Mann-Whitney U analysis using the StatsWork package for the Apple Macintosh (Data Metrics Inc., CA). The data were presumed to be nonparametric owing to the inclusion of samples with IL-13 levels below the detection limit of the assay. A P value < 0.05 was considered significant.

	Results

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The results are summarized in Figures 1-3 and Table 4. A greater number of subjects with pulmonary fibrosis expressed IL-13 mRNA in BAL-derived AMs than control subjects (11 of 13 compared to 2 of 8, P < 0.01; Table ). Because the AMs in this study were > 99% pure and because the RT-PCR signal obtained was from the equivalent of 10,000 AM cells, we think it unlikely that IL-13 mRNA has been isolated from T cells in the BALF. Previous experiments were carried out to compare IL-13 mRNA isolated by direct lysis of freshly obtained AMs with that from adherence-purified AMs, and no upregulation was detected (data not shown). IL-13 immunoreactivity in unconcentrated BALF was detectable in 8 of 12 subjects with pulmonary fibrosis but in none of the control subjects (P < 0.005). All of the normal subjects were below the detection limit of the assay (Figure 2). Twenty-four-hour alveolar macrophage culture supernatants demonstrated increased production of IL-13 by AMs derived from subjects with systemic sclerosis compared to control subjects (Figure 3), although large variations between subjects (n = 3) prevented statistical significance. The presence of IL-13 protein in BALF correlates with the expression of IL-13 mRNA and protein production in AMs (Table ).

View larger version (9K): [in this window] [in a new window]   Figure 2.   IL-13 concentrations in BALF samples from healthy subjects and subjects with pulmonary fibrosis. Results are expressed as picograms of IL-13 per milliliter of BALF. Each point represents a different subject, and the horizontal lines are the means for each group. Abbreviations: RA = rheumatoid arthritis; SSc = systemic sclerosis; IPF = idiopathic pulmonary fibrosis; SARC = sarcoidosis.

View larger version (29K): [in this window] [in a new window]   Figure 3.   IL-13 production by AMs cultured for 24 h in the presence or absence of a dose range of LPS. Data are presented as the mean ± standard error. Black bars = control subjects; gray bars = subjects with systemic sclerosis.

	Discussion

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To our knowledge, this is the first report of the expression of IL-13 mRNA in human AMs. We considered the possibility that this finding might be due to contamination by other cells in the BALF. The AMs in this study were > 99% pure and the RT-PCR signal obtained was from the equivalent of 10,000 cells, hence only macrophages were present in sufficient numbers to produce this signal. Also, amplification of the RNA samples with CD3 primers specific for T cells was negative, ruling out T-cell contamination. Thus, it seems improbable that IL-13 mRNA has been isolated from cells other than AMs. The presence of IL-13 protein in BALF correlates with the expression of IL-13 mRNA in AMs. This would suggest that at least some of the IL-13 mRNA is being translated into protein in these samples and the IL-13 protein detected in the AM supernatants from patients with systemic sclerosis would support this conclusion. In these preliminary cases, the absence of IL-13 protein in the BALF from all of the control subjects would suggest that this may be due to lack of translation or because the amount of protein is below the detection limit of the ELISA.

The human IL-13 gene maps to human chromosome 5, to a region where a cluster of genes encoding other immune and hemopoietic factors, including IL-3, GM-CSF, IL-5, and IL-4, have been previously localized (3). IL-13 has a profound effect on primary immune cells by inducing immunoglobulin production, proliferation of B cells, and the differentiation of cells of the monocytic lineage (4), and is a monocyte chemoattractant (17). It can inhibit the production of inflammatory cytokines by both macrophages and monocytes via a protein kinase C-dependent pathway (5), and its function in this context shows some similarities to that of IL-10. Some of these properties are related to the ability of IL-13 to downregulate the expression of CD14, the major LPS receptor (18, 19). One study has suggested that exogenous IL-13 has a variable effect on inflammatory cytokine production such as LPS-induced TNF- (6). We hypothesized that IL-13 would be produced by human AMs with the potential to play a regulatory role in the inflammatory response and would be upregulated in lung disease with an inflammatory pathological basis, such as pulmonary fibrosis. Increased expression of IL-13 mRNA in AMs in subjects with pulmonary fibrosis may be involved in modulating the inflammatory response within the lung, possibly by reducing destruction and inducing repair, although our data do not address this point.

IL-13 mRNA was detected in some normal subjects using the ELISA kit described, although protein was not present in the BALF of these subjects. AMs from normal subjects suppress T-cell proliferation in vitro (20) and this constitutive expression may reflect a protective mechanism to prevent an inappropriate immune response to environmental stimuli. The increased amount of IL-13 mRNA in AMs and IL-13 protein in BALF from subjects with fibrotic lung disease would suggest that IL-13 has a functional role in this process. Further studies will be needed to evaluate this suggestion.

We and others have previously investigated the role of another cytokine considered to have an anti-inflammatory role, IL-10, and have shown that in the normal AM exogenous IL-10 downregulates TNF- mRNA (7), but this effect is diminished in AMs from subjects with interstitial pulmonary fibrosis (11). In the inflammatory disease rheumatoid arthritis, IL-10 has been shown to have an autocrine effect in synovial monocytes, and that when its effect was inhibited, inflammatory cytokine production was increased (12). This would suggest that IL-10 was having an anti-inflammatory role and it has been thought to act as an early T-cell-mediated suppressive factor. It is interesting to speculate whether inhibition of IL-13 would have a similar effect and these studies are currently ongoing in our laboratory.