Induction of IL-15 mRNA and Protein in A549 Cells by Pro-inflammatory Cytokines

Immunobiol., vol. 199, pp. 14-22 (1998)

Byk Gulden, Konstanz, Germany

Induction of IL-15 mRNA and Protein in A549 Cells by Pro-inflammatory Cytokines MICHAEL STOECK, WOLFGANG KROMER and VOLKER GEKELER Received July 2, 1997· Accepted in revised form November 6, 1997

Abstract Interleukin-1S is a recently discovered cytokine which is functionally similar to IL-2. In order to learn more about possible targets for modulation of the expression of IL-1S we investigated the expression of IL-1S mRNA and protein in the AS49 (human lung carcinoma) cell line. Constitutive expression of IL-1S mRNA was detected in AS49 cells. Treatment with TNF-a or IL1~ (10 ng/ml each) induced an about 2-fold increase of IL-1S mRNA; IFN-y induced significant effects only at 100 ng/m!. Stimulation with a combination of TNF-a and IFN-y was not superior to stimulation with TNF-a alone. EGF, KGF and the combination thereof were without effects. IL-1S protein was detected in cellular lysates of unstimulated cells and was increased by stimulation with TNF-a or IL-1~. No significant amounts of IL-1S protein were detected in cellular supernatants.

Introduction The recently discovered cytokine IL-1S is a member of the «four-helix-bundle» family of cytokines (1,2). IL-1S is functionally similar to IL-2 in in vitro and in vivo systems although it has no sequence similarities with IL-2 (1, 3, 4, S). ILlS-mediated signal transduction involves the 'Yc-chain of the IL-2 receptor, in addition the ~-chain of the IL-2 receptor and a particular a-chain are part of the IL-1S receptor complex (6, 7, 8). A further IL-1S binding protein has been reported on mast cells (9). IL-1S mRNA can be detected in monocytes/ macrophages, dendritic and Langerhans cells, muscle cells, keratinocytes, epithelial and fibroblast cell lines (1, 10). IL-1S protein is not easily detected in the supernatants of normal cells which suggests that the secretion of IL-1S protein is strictly controlled. The capability of many non-lymphoid cells to produce IL-1S mRNA and (intracellular) protein together with its IL-2-like activities point to IL-1S as a possible candidate for therapeutic intervention in chronic inflammatory or autoimmune diseases. The expression of IL-15mRNA in human lung tissue has been described (1, 11). We therefore investigated the stimulatory requirements of IL-15 induction in the human A549 cell line which 01998 by Gustav Fischer Verlag

A549 and IL-15 . 15

may serve as an in vitro model for epithelial cell function in chronic inflammatory disease.

Materials and Methods Reagents and materials

Human recombinant interferon y (IFN-y) and tumor necrosis factor a (TNF-a) were obtained from Peptrotech (London, UK), human recombinant interleukin-1 ~ (IL-1~) was from Genzyme (Genzyme Virotech, Riisselsheim, Germany). PMSF, Benzamidine, Aprotinin and Pepstatin were obtained from Sigma (Sigma-Aldrich Chemie, Deisenhofen, Germany). PBS, DMEM, media supplements and FCS were obtained from Life Technologies (Eggenstein, Germany). DMEM was supplemented with 100 U/ml:100 llg/ml penicillin/streptomycin, 2 mM glutamine, 50 llM 2-mercaptoethanol, 1 mM pyruvate and 1% nonessential amino acids and will be referred to as complete medium. Tissue culture plastic was from Costar (Technomara, Fernwald, Germany). Cell culture

A549 (ATCC#CCL185) cells were grown in complete medium in the presence of 5% FCS. Cultures which had reached confluency were stimulated as indicated. For the preparation of mRNA, cells were cultured in 6 cm 0 Petri dishes in 2.5 ml of complete medium and stimulated as indicated. Stimulation was performed in presence of 0.1 % FCS. Total cellular RNA was harvested after 4 h. For the production of cellular lysates, cells were stimulated in complete medium with 0.1 % FCS in 75 cm2 tissue culture flasks for 6-7 h, the last 4 h in the presence of 1 llM Brefeldin A (Sigma). Complementary DNA-PCR (eDNA-PCR)

Total cellular RNA was obtained after lysis of cells with Gua-SCN followed by centrifugation on a cesium chloride gradient as described (12). In some experiments the InViSorb™ RNA Kit II was used (InViTek, Berlin, Germany). cDNA was synthesized using random hexanucleotide primers (Boehringer Mannheim, Germany) and RAV2 reverse transcriptase (Amersham, Braunschweig, Germany) (12). In most cases three independent cDNAs were synthesized from every RNA sample. PCR was performed according to BECK et al. (12) using cDNA equivalent to 200 ng RNA. PCR conditions: 15 sec 95°C / 30 sec 55°C / 90 sec 72 °C; IL-15: 28 cycles, GAPDH: 21 cycles. PCR products were separated on a 10% polyacrylamide gel, stained with ethidium bromide and analyzed using the CS-1 videoimager and WINCAM densitometry software (Cybertech, Berlin, Germany). The IL-15 primers were deduced from the sequence of the human IL-15 cDNA deposited at EMBL (accession no. U 14407): IL-15 sense: 5'-TAA AAC AGA AGC CAA CTG-3'; IL-15 antisense 5'-TCT ACT GTA TCA TGA ATA C-3'. This pair of primers does not distinguish, however, between the two splice variants which have been described recently by MEAZZA et al. (11). GAPDH sense 5'-CGG GAA GCT TGT GAT CAA TGG-3', GAPDH antisense: 5'-GGC AGT GAT GGC ATG GAC TG-3'. Expected size of amplified material: 204 bp (IL-15, position 448 to 652), or 358 bp (GAPDH). Digestion of the 204 bp IL-15 PCR product with Bgi II or HinfI yielded fragments of 52 bp + 152 bp and 34 bp + 170 bp, respectively, which is in accordance with the published cDNA sequence. Since the amplimers were deduced from different exons of the IL-15 gene according to KRAUSE et al. (13), eventual traces of DNA in the RNA preparation would not generate a 204 bp fragment by PCR. Signal intensities assigned to the IL-15 gene were normalized to the corresponding signal produced by the amplimers for the GAPDH gene using the same cDNA specimen.

16 . M. STOECK, W. KROMER and v. GEKELER Production of intracellular IL-15 After stimulation of cells with different cytokines for 6-7 h, the supernatant was removed the adherent A549 cells were washed with PBS and detached with 10 mM EDTA. Cells were sedimented by centrifuge, washed and the pellets were resuspended in 0.5 ml of lysis buffer (PBS with 1 mM EDTA, 0.1 mM PMSF, 2 mM Benzamidine, 0.4 Units/ml Aprotinin, 0.7 pg/ml Pepstatin). Cells were lysed by repeated shock freezing in liquid N 2• Lysates were centrifuged at 14 000 g and the content of IL-15 in the supernatant was measured with a commercial IL-15 Elisa (Genzyme) using the high sensitivity protocol provided by the manufacturer. Lysates were tested in duplicate. The content of IL-15 in the lysates was normalized to soluble protein. Protein was determined with the BCA Protein Assay Kit (Pierce; Bender & Hobein, Bruchsal, Germany) using BSA as a standard.

Results Induction of IL-15 mRNA Figure 1 depicts that unstimulated A549 cells contained IL-15 mRNA, the amount of which could be increased by stimulation with TNF-a or IL-l~. The cytokineinduced increase of IL-15 mRNA expression was reproducible in a series of independent experiments (Table 1). Cells were generally grown in 5% FCS, stimulation was performed at 0.1 % FCS. The stimulation index did not increase when the cells were preincubated before stimulation for 12 h or 36 h at 0.1 % FCS (data not shown). Stimulation of A549 cells with EGF or KGF, or a combination of both did not increase the level of IL-15 mRNA above the value detected in unstimulated cells. Figure 2 shows that stimulation with 1 ng/ml of TNF-a induced a 60-70% increase of IL-15 mRNA. Compared with TNF-a, stimulation with'IFN-y was less effective (Fig. 1, Fig. 2). Only at 100 ng/ml a substantial increase of IL-15 mRNA could be observed. In the range of concentrations tested the combination of TNF-a and IFN-y was not significantly more effective than TNF-a alone. The induction of IL-15 mRNA by TNF-a (or a combination of TNF-a and IFN-y, data not shown) was at maximum after 4 h of stimulation (Fig. 3). Table 1. Increase of IL-15 mRNA in cytokine-stimulated A549 cells compared to unstimulated A549 cells. fold increase of IL-15 mRNA control TNF-a 10 ng/ml IFN-y 10 ng/ml

TNF-a + IFN-y KGF 50 ng/ml EGF 20 ng/ml KGF+EGF IL-l~ 1 ng/ml IL-l~ 10 ng/ml

1.000 2.238 1.298 2.385 1.005 0.995 1.113 1.802 1.837

N = number of independent experiments

SD

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0.315 0.190

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0.313 0.490

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Figure 1. Induction of IL-IS mRNA in AS49 cells. Confluent cultures of AS49 cells were stimulated as follows for 4 h or left unstimulated (control). Experiment A: TNF-a 10 ng/ml, IFN-y 10 ng/ml, IL-l~ 1 ng/ml and 10 ng/ml, experiment B: TNF-a 10 ng/ml, EGF 20 ng/ml, KGF SO ng/ml. Total RNA was transcribed into cDNA and IL-IS mRNA was detected by PCR. Amplification of GAPDH mRNA was used as a control. The values are given as mean of triplicate determinations ± SD (KGF duplicate determination, error bars: range) using one particular mRNA specimen. The results are representative of a series of independent experiments, the summary is given in Table 1. 100

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Figure 2. Induction of IL-IS mRNA in AS49 cells stimulated with different concentrations of TNF-a and IFN-y. Confluent cultures of AS49 cells were stimulated as indicated for 4 h or left unstimulated (control). Total RNA was transcribed into cDNA and IL-IS mRNA was detected by peR. Amplification of GAPDH mRNA was used as a control. Values are given as mean of triplicate determinations ± SD using one particular mRNA specimen. The experiment was repeated with similar results.

18 . M. STOECK, W. KROMER and v. GEKELER

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hours ofstimulation Figure 3. Time kinetics of the induction of li-15 mRNA in A549 cells. Confluent cultures of A549 cells were, stimulated with TNF-a (10 ng/ml) or left unstimulated (control). Total RNA was transribed into eDNA and li-15 mRNA was detected by PCR. Amplification of GAPDH mRNA was used as a control. Values are given as mean of 2 to 5 determinations using one (control: two) particular mRNA specimen. Error bars: range. nd = not done. In a different experiment, stimulation of A549 cells with a combination of 10 ng/ml TNF-a and 10 ng/ml IFN-y yielded similar time kinetics (data not shown).

Induction of Il-15 protein in A549 cells

IL-1S protein could be detected in lysates of unstimulated and stimulated AS49 cells by ELISA (Fig. 4). Stimulation with IL-1~, TNF-a or TNF-a/IFN-y increased the amount of detectable intracellular IL-1S protein, IFN-y(10 ng/ml) was less effective. EGF, KGF and the combination thereof were without effect. These results are consistent with the effects of TNF-a, IFN-y and IL-1~ on the induction of IL-1S mRNA depicted in figures 1-3. For the investigation of possible secretion of IL-1S into cellular supernatants, confluent cultures of AS49 cells in 6 em 0 Petri dishes were stimulated with IL-1~ (1 ng/ml) or IFN-y (10 ng/ml) for up to 72 h or with TNF-a for up to 168 h. Supernatants were harvested every other day and tested for IL-1S in an ELISA. The concentrations of IL-1S secreted into the supernatants were around or below the limit of detection of the ELISA (5-7 pg/ml, data not shown).

Discussion Our data show that the AS49 cell line constitutively expressed IL-1S mRNA as well as intracellular IL-1S protein. The abundance of IL-1S mRNA and protein was increased by stimulation with the pro-inflammatory cytokines TNF-a and IL-1~. In contrast significant amounts of secreted IL-1S could not be detected in

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Figure 4. Induction of intracellular IL-15 protein in A549 cells. Confluent cultures of A549 cells were stimulated with TNF-a (10 ng/ml), IFN-y (10 ng/ml), IL-l~ (1 ng/ml), EGF (20 ng/ml), KGF (50 ng/ml) or left unstimulated. After 6-7 h cellular lysates were prepared and analysed for IL-15 in a commercial IL-15 ELISA. The amount of detectable IL-15 was normalized to soluble protein in the lysates. Values are mean of duplicate determination of a single lysate. Errors bars: range. Two independent experiments (A, B) are shown. Control experiments with and without detection antibody revealed that the cytosolic lysates themselves did not induce the development of a color reaction in the ELISA. (O.D. in ELISA with detection antibody: PBS 0.12, cytosol of unstimulated cells 0.26; O.D. in ELISA without detection antibody: cytosol of unstimulated cells 0.10).

the supernatant of A549 cells. IL-15 mRNA and intracellular protein have also been detected in the human intestinal epithelial tumor-derived cell lines Caco-2 and HT-29 (14). In this publication IL-15 mRNA could be up-regulated in Caco2 cells by IFN-y. Recently constitutive expression of IL-15 mRNA has also been described in human fetal retinal epithelial cell lines, where it could be up-regulated by stimulation with TNF-a or IFN-y (15). These authors also reported the presence of biological IL-15 activity in the supernatant, detected by the short term survival of IL-15 sensitive cells. In addition, LEE et al. (16) observed the induction of IL-15 mRNA and secreted protein in human fetal astrocytes stimulated with IL-1~, IFN-y or TNF-a and in microglia cells stimulated with LPS or IFN-y. These two latter findings might suggest that the mechanisms of secretion of IL-15

20 . M. STOECK, W. KROMER and V. GEKELER in fetal cells differ from non-fetal cells and established cell lines. Our data suggest that in the AS49 cell line the control mechanism regarding the secretion of IL-1S is relatively intact compared to cells from an inflammatory situation (see below). Future work will deal with the modalities of IL-1S release in this cell line. In general, the secretion of IL-1S seems to be strictly controlled as IL-1S is not easily detected in cellular supernatants. Data by BAMFORD et al. (17) highlighted the potential importance of sequences in the S'-UTR of the IL-1S mRNA for the attenuation of IL-1S mRNA translation. ONU et al. (18) have shown that normal human T lymphoblasts transcribed the IL-1S gene but did not secrete the protein and suggested that the IL-1S leader sequence controls the secretion of IL-1S. As many different cell types can express IL-1S, a tight control of the expression of this cytokine seems to be necessary in order to prevent non-appropriate lymphocyte activation in the tissue. It is therefore interesting to note that expression and secretion of IL-1S protein have been observed in pathological situations such as inflammation or viral infection. McINNES et al. (19) reported very high concentrations of IL-1S protein in rheumatoid arthritis synovial fluid. In addition, IL-1S was detected in the serum of patients with ulcerative colitis (20) or in the serum of patients with type C chronic liver disease (21). Also infection of PBMC with human herpesvirus-6 resulted in the induction of IL-1S protein secretion (22). Furthermore, the HTLV-1-associated adult T cell leukemia line HuT102 secreted IL-1S (17). In addition, stimulation of monocytes with bacterial products like LPS (23) or bacterial infection (24) increased the amount of detectable IL-1S protein, and dendritic cells could be triggered by phagocytic stimuli and bacterial products to release IL-1S protein (25). All these data suggest that chronic inflammation or infective stimuli might induce IL-1S production which then could lead to further disturbances of (local) immunological homeostasis. This notion is also supported by the increased expression of IL-1S mRNA in human renal allograft rejection in the absence of significant amounts of IL-2 mRNA (26, 27). Taken together the characteristics of the production of IL-15 and the available clinical data suggest that this cytokine may be of importance for the induction and propagation of autoimmune or chronic inflammatory diseases. The importance of the pulmonary epithelium for the induction and maintenance of local immunological responses has been established (28). Cellular models as described in this communication will be useful for the investigation of possible immunopharmacological modulation of IL-1S expression and for elucidating the modalities of IL-1S protein release. Acknowledgments The authors gratefully acknowledge the expert technical assistance of MONIKA EICKHOFF and MONIKA SCHWARZ.

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A549 and IL-15 . 21

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