REVERSE SIGNALING THROUGH MEMBRANE-BOUND INTERLEUKIN-15

This study demonstrates that membrane-anchored interleukin-15 (IL-15) constitutively expressed on the cell surface of PC-3 human prostate carcinoma cells and IFN (cid:74) -activated human monocytes is implicated in reverse signaling upon stimulation with sIL-15R (cid:68) or anti-IL-15 antibodies, mediating the outside-to-inside signal transduction that involves the activation of members of MAP kinase family ERK and p38, and focal adhesion kinase. The presence of membrane-bound IL-15 is not dependent on the expression of the trimeric IL-15 receptor complex by these cells and resists the treatment with acidic buffer or trypsin. The reverse signaling through membrane-bound IL-15 considerably increases the production of several pro-inflamatory cytokines by monocytes such as IL-6, IL-8, and TNF (cid:68) , indicating thereby the relevance of this process to the complex immunomodulatory function of these cells. Furthermore, the stimulation of transmembrane IL-15 also enhances the transcription of IL-6 and IL-8 in PC-3 cell line, and promotes migration of PC-3 cells as well as LnCap human prostate carcinoma cells stably expressing IL-15 upon the cell surface. Thus, IL-15 can exist as a biologically active transmembrane molecule that possesses dual ligand/receptor qualities with potentiality to induce bi-directional signaling. This fact highlights a new level of complexity in the biology of IL-15 and offers novel important insights to our understanding of the cellular responses modulated by this pleiotropic cytokine. Nuclei Data analysis – All experiments were performed in at least three independent assays, which yielded highly comparable results. Protein sequences were analysed using ProteinPredict software for sequence analysis and prediction of protein function and structure (http://www.embl-heidelberg.de/predictprotein/predictprotein.html). Blots were quantitated using ImageQuant TL software (Amersham Pharmacia). Data are summarized as mean + SD. Statistical analysis of the results was performed by Student’s t test for unpaired samples. A p value of <0.05 was considered as statistically significant.


INTRODUCTION
Interleukin-15 (IL-15) 1 is a pleiotropic cytokine which belongs to the four-helix bundle cytokine family and was first identified because of its ability to substitute IL-2 in supporting the growth of murine IL-2-dependent CTLL cell line (1). IL-15 shares with IL-2 the IL-2 receptor beta (IL-2R ) and IL-2 receptor gamma (IL-2R / c) chains (2), but has a unique high affinity chain (IL-15R ) responsible for the differential functional effects of IL-15 versus IL-2 on cells of the same type (3). IL-15 can replace IL-2 in most of its activities in the immune system, including induction of T-cell proliferation and chemotaxis, stimulation of natural killer (NK) cell growth and interferon (IFN ) production, generation of cytotoxic effector cells, and co-stimulation of B-cell growth and immunoglobulin synthesis (4)(5)(6). IL-15 and IL-15R knock-out mice display a marked reduction in numbers of NK cells, memory phenotype CD8+ T lymphocytes, and a distinct population of intestine intraepithelial lymphocytes, suggesting an important role for IL-15 in the development and/or survival of these cells (7,8). In addition, IL-15 has a potent anti-apoptotic function, inhibiting apoptosis of activated T and B cells, keratinocytes and melanoma cells in vitro, and protecting mice from Fas-induced hepatic failure and multi-system apoptosis in vivo (9)(10)(11).
IL-15 mRNA is constitutively expressed by a large variety of cell types and tissues, including monocytes/macrophages, fibroblasts, keratinocytes, kidney epithelial cells, nerve cells, placenta, heart, and skeletal muscle (4,7,(12)(13)(14)(15). Interestingly, most primary cells and cell lines which express IL-15 mRNA do not release detectable amounts of this cytokine into the culture medium. This discrepancy is explained by the fact that IL-15 has a complex, multifaceted control of expression with regulation at the Reverse signaling through membrane-bound IL- 15 5 levels of transcription, translation, and intracellular trafficking. The existence of two IL-15 isoforms has been reported which differ in the length of the signal peptide (16). These isoforms exhibit a differential intracellular trafficking, secretion, and endosomal localization, indicating an important role for signal peptide in multiple mechanisms controlling IL-15 production (17,18). IL-15 associated with the short signal peptide (IL-15SSP) is not secreted but rather stored intracellularly in the nucleus and cytoplasm, whereas the alternative isoform characterized by the longer signal peptide (IL-15LSP) is located in the Golgi, early endosomes, and the endoplasmic reticulum (ER), and has been suggested to follow a pathway that may result in the cytokine secretion (17,18).
In monocytes/macrophages, IL-15 is expressed in a biologically active membrane-bound form, and its mRNA expression can be upregulated by exogenous stimuli, such as IFN and lipopolysaccharide (LPS) (12). Furthermore, TNF -stimulated dermal fibroblasts were able to sustain proliferation of activated T cells through expression of membrane-bound IL-15 (13). The presence of biologically active membrane-anchored IL-15 upon the cell surface of normal human monocytes, several monocytic cell lines, and TNF -stimulated dermal fibroblasts suggests that, under physiological conditions, IL-15 may mainly be present in a membrane-bound rather than secreted form. This report highlights the ability of membrane-anchored IL-15 to mediate outside-to-inside (reverse) signaling that activates focal adhesion kinase (FAK) and MAP For immunoprecipitation studies, lysates containing 500 g of proteins were immunoprecipitated overnight at 4°C by incubation in 0.5% NP-40 buffer with 2 g/ml of Abs. Immunocomplexes were captured on protein A/G-agarose. After washing, pellets were re-suspended in SDS-PAGE sample buffer, boiled for 5 min and analysed in 10% SDS-PAGE. The resolved proteins were transferred onto nitrocellulose (Bio-Rad). Blots were blocked for 1 h in PBS with 0.05% Tween-20 and 3% BSA (Sigma-Aldrich). After incubations with first and second Abs and washing with PBS-T, visualization of specific proteins was carried out by an enhanced chemiluminescence (ECL) method using ECL Western blotting detection reagents (Amersham Pharmacia) according to the manufacturer's instructions.
RT-PCR -Total RNA was extracted from cells using TRIZOL reagent (Invitrogen). cDNA was synthesized from 5 g of total RNA using random oligonucleotides as primers and SuperScriptII TM kit (Invitrogen). cDNA was amplified by Samples were amplified in a DNA Thermocycler (Eppendorf, Hamburg, Germany) for 30 cycles. Each cycle consisted of denaturation at 94°C for 15 sec, annealing at 60°C for 30 sec and elongation at 72°C for 30 sec, preceded by initial denaturation at 94°C for 5 min and followed by a final extension step at 72°C for 5 min. Reverse signaling through membrane-bound IL- 15 11 To evaluate mRNA expression semi-quantitatively, in addition to the PCR product from 30 cycles, 15 l of the PCR product from the 26, 28 cycles and the 32 cycles were run simultaneously. Aliquots of PCR products were electrophoresed on 1.5% agarose gel and visualized by ethidium bromide staining. -actin message was used to normalize the cDNA amount to be used. A mock PCR (without cDNA) was included to exclude contamination in all experiments.
Wound healing assay -Exponentially growing cells (2 x 10 6 ) were plated onto rat tail collagen (Roche, Heidelberg, Germany) coated (10 g/ml) cell culture plates in complete growth media. After 8 hours, the monolayers of cells were wounded by manual scratching with a pipet tip, washed with PBS, placed into complete growth medium and photographed in phase-contrast with a Nikon microscope (Nikon Diaphot 300, Tokyo, Japan). Matched pair marked wound regions were photographed again after 18 (PC-3

cells) or 8 (LnCap cells) hours.
Flow cytometric analysis -Cells were stained with monoclonal Abs or IL-15-IgG 2b fusion protein as previously described (21), and analyzed on a FACScalibur (Becton Dickinson, San Jose, CA) using CELL Quest software. Negative controls consisted of isotype-matched, directly conjugated, nonspecific Abs (BD PharMingen).
Confocal Microscopy -Cells were seeded in concentration of 5 x 10 4 cells/well in 12-well plates containing 18 mm glass coverslips. Next day coverslips were fixed with 2% paraformaldehyde for 10 min at room temperature. To stain cell membranes, fixed Data analysis -All experiments were performed in at least three independent assays, which yielded highly comparable results. Protein sequences were analysed using ProteinPredict software for sequence analysis and prediction of protein function and structure (http://www.embl-heidelberg.de/predictprotein/predictprotein.html). Blots were quantitated using ImageQuant TL software (Amersham Pharmacia). Data are summarized as mean + SD. Statistical analysis of the results was performed by Student's t test for unpaired samples. A p value of <0.05 was considered as statistically significant.

PC-3 cells and IFN -activated monocytes express membrane-bound IL-15 -
Biologically active form of membrane-bound IL-15 is constitutively expressed on the cell surface of human monocytes and several monocytic cell lines (12,22), whereas IFN stimulation further upregulates its surface expression (12). Therefore, monocytes were stimulated with IFN for 24 h in all experiments described thereafter. Furthermore, we found that PC-3 prostate carcinoma cell line expresses only membrane-bound form of IL-15, whereas the IL-15R , IL-2R , IL-2R / c chains were absent in these cells. To assess the expression of IL-15 in PC-3 cells and monocytes in more detail, we first analyzed its mRNA content by RT-PCR using a pair of primers that recognize two distinct IL-15 isoforms corresponding to the cytokine with the short (21 aa) and long (48 aa) signal peptide (16). As shown at Reverse signaling through membrane-bound IL-15 14 microscopy (Fig. 1D). IL-15LSP associated with the cell membrane and was also found in the nuclei, which is in agreement with recent findings (11). Thus, only IL-15LSP isoform is present in PC-3 cells, whereas both IL-15LSP and IL-15SSP are found in IFN -activated monocytes.  The phenomenon of reverse signaling or the ability of a membrane-bound ligand to induce activation of intracellular mediators has surfaced recently as an important mechanism to regulate qualitatively distinct cellular responses to specific stimuli (23).

Membrane-anchored IL-15 mediates reverse signaling that involves protein phosphorylation, and activation of MAP kinases and FAK in PC-3 cells and monocytes -
Many ligand-receptor pairs have been shown capable of bi-directional signal transduction (24)(25)(26)(27)(28)(29). The presence of membrane-bound IL-15 on the cell surface of PC-3 cells and monocytes strongly invited to investigate its biological relevance for host cells. To test whether membrane IL-15 may mediate reverse signaling events, PC-3 and monocytes were treated with recombinant sIL-15R for different time intervals, and the pattern of phosphorylation of cellular proteins was assessed. Notably, the concentration of endotoxin in all sIL-15R preparations was extremely low and LPS in such concentration was not able to induce any signaling in PC-3 cells and monocytes (data not shown).
Nevertheless, in order to verify that the observed effects of membrane IL-15 stimulation were genuine and not due to nonspecific activation through contamination with endotoxin associated with sIL-15R preparations, we also stimulated cells with anti-IL-15 Abs, whereas treatment with isotype-matched Abs was used as a control. Next, we sought to establish the identity of the phosphorylated molecules. To this end, the membranes were reprobed with Abs directed against several cellular proteins which reportedly play important roles in mediating downstream signaling from a wide range of the membrane-expressed receptor molecules. These experiments revealed that the stimulation with sIL-15R induced the phosphorylation of ERK and p38 (Fig. 3C).
Interestingly, the predominant tyrosine phosphorylation of ERK2 (p42) was observed in human monocytes, and to a lesser extent, in PC-3 cells (Fig. 3C). Moreover, the phosphorylation of FAK was detected. However, the phosphorylation status of JNK (Fig.   3C) and the FAK-related kinase Pyk2 (data not shown) was not altered. Similar results were obtained after the stimulation with anti-IL-15 Abs, whereas treatment with the acidic buffer or trypsin was not able to abolish these effects (data not shown). were unresponsive (Fig. 4B). However, when cells were stably transfected with IL-15SSP or IL-15LSP, tyrosine-phosphorylated proteins were observed in cells expressing both constructs, although the phosphorylation was significantly stronger in the IL-15LSPtransfected cells (Fig. 4C). In accordance with these findings, reprobing of the membrane with specific Abs allowed us to identify ERK as one of the phosphorylated substrates ( Fig. 4B and C). We did not observe the phosphorylation of JNK or p38 in these cells Reverse signaling through membrane-anchored IL-15 triggers cytokine production -The MAP kinase cascade represents a key signaling pathway critical for the linking membrane receptors to cytoplasmic and nuclear effectors, and regulates a wide range of cellular functions. Given that treatment with sIL-15R clearly induced dramatic changes in the level of phosphorylated proteins in PC-3 cells and monocytes, including ERK and p38, we next tested the production of pro-inflamatory cytokines by these cells.
The stimulation with sIL-15R or anti-IL-15 Abs for 24 h considerably enhanced the expression of TNF , IL-6, and IL-8 by monocytes, as detected by RT-PCR (Fig. 5A) and ELISA (Fig. 5B), whereas treatment with the acidic buffer was not able to abolish these responses (Fig. 5B). Isotype-matched control Abs were without effect.  (31,32). It has been shown that FAK is required for both integrin-and growth factor-stimulated cell motility (32,33). FAK is highly tyrosine-phosphorylated at a number of different sites in either growing, intregrin-stimulated, or in migrating cells (31). Our results suggest that IL-15 on activated monocytes is directly anchored to the cell membrane rather than binds to the IL-15R chain (12,20). This suggestion is in agreement with two recent studies which also reported the presence of membrane-bound, biologically active IL-15 on monocytes (12,22). The bioactivity of IL-15 was convincingly demonstrated by the ability of mitomycin-treated or fixed monocytes to support proliferation of concanavalin A-stimulated human T cells or an IL-15-dependent CTLL cell line, respectively (12,22). The presence of biologically active membrane IL- Reverse signaling through membrane-bound IL- 15 26 to have several consensus sequences, including potential phosphorylation sites for casein kinase II and protein kinase C (data not shown). Importantly, we observed the phosphorylation of transmembrane IL-15 at serine but not tyrosine residues, although no physical association between IL-15 and MAP kinases or FAK was detected in the immunoprecipitation experiments upon the stimulation with sIL-15R or anti-IL-15 Abs.
Noteworthy, the predicted cytoplasmic portion of IL-15LSP contains several serine residues (Fig. 7). Current studies in our laboratory are focused on the mutational analysis Reverse signaling through membrane-bound IL- 15 27 intracellular trafficking rather than transcription. Two distinct isoforms of IL-15 exhibit different patterns of intracellular distribution and trafficking (16)(17)(18). Whereas IL-15LSP is found in the Golgi and early endosomes (17), and in the ER (18), IL-15SSP is present in the cytosol but does not co-locolize either with the Golgi, early endosomes, or the ER (17,18). However, both IL-15LSP and IL-15SSP may exhibit nuclear localization (11).
Although and TNF showed striking similarities in the protein structure organization (Fig. 7).
Moreover, as mentioned above, IL-15 has been predicted to contain several consensus sequences, including potential phosphorylation sites for casein kinase II and protein kinase C, sharing thereby not only structural similarities with TNF (data not shown).