Inhibition of lymphocyte apoptosis by pancreatic stellate cells: impact of interleukin-15

Gisela Sparmann,1 Änne Glass,2 Peter Brock,1 Robert Jaster,1 Dirk Koczan,3 Hans-Jürgen Thiesen,3 Stefan Liebe,1 and Jörg Emmrich1

1Division of Gastroenterology, Department of Medicine, 2Institute for Medical Informatics and Biometry, and 3Institute of Immunology, University of Rostock, Rostock, Germany

Submitted 25 October 2004 ; accepted in final form 3 July 2005


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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There is growing evidence that pancreatic stellate cells (PSCs) produce cytokines and take part in the regulation of inflammatory processes in the pancreas. IL-15 inhibits apoptosis of various cell populations. This study was performed to investigate whether PSCs produce IL-15 and thereby can affect lymphocytes. Primary PSCs were isolated from the rat pancreas using density gradient centrifugation. mRNA expression of IL-15 was demonstrated by RT-PCR, and IL-15 protein was analyzed by immunoblotting. Lymphocytes obtained from rat mesenterial lymph nodes were cocultured with in vitro activated PSCs. Apoptosis has been quantified by the binding of annexin V-FITC with a flow cytometer. Proliferation was monitored using [3H]thymidine incorporation. PSCs express two splice variants of IL-15. The protein was detectable only in cell lysates but not in the cell culture supernatant. Cocultivation of lymphocytes with PSCs and IL-15 inhibited spontaneous lymphocyte apoptosis, and this effect was reduced by an anti-IL-15 antibody. Lymphocytes induced vice versa the proliferation and collagen production of PSCs. The inhibition of spontaneous lymphocyte apoptosis in cocultures with PSCs was at least partially mediated by cell-bound IL-15. This effect and the stimulation of PSCs by lymphocytes may lead to a circulus vitiosus, resulting in the persistence of inflammatory processes and the development of fibrosis during chronic pancreatitis.

rat; cytokine; chronic inflammation


CHRONIC PANCREATITIS is histomorphologically characterized by an inflammatory cell infiltration and a marked organ fibrosis (20). The molecular mechanisms underlying the development of the persistent inflammation with deposition of connective tissue are poorly understood. The key event in the cellular pathogenesis of pancreas fibrosis is the activation of retinoid-storing pancreatic stellate cells (PSCs; see Refs. 3, 4, 15, and 18). After isolation, PSCs in culture undergo a similar process and differentiate to a myofibroblast-like, {alpha}-smooth muscle actin-expressing phenotype. The morphological transformation of PSCs is characterized by reduction of vitamin A-containing fat droplets, increased proliferation, and enhanced expression of various types of extracellular matrix components (ECM; see Refs. 3, 4, 19, and 34).

In addition to an extended fibrosis, chronic pancreatitis was characterized by massive cell infiltrations (10, 20). Studies on infiltrating leukocytes suggested that T cell-mediated immune reactions may be involved in the pathogenesis of the disease (9, 10, 17).

It is well accepted that the pivotal role of leukocytes in inflammation is mainly mediated by the secretion of cytokines. PSCs were shown to respond to different cytokines in terms of variations in the proliferation rate and in the expression of ECM components (2, 3, 24, 31). In addition, stellate cells themselves are able to synthesize various cytokines and chemokines (2, 21, 34).

Taken as a whole, these results have given rise to the concept of interactions between lymphocytes and stellate cells supporting the inflammatory process in the pancreas.

IL-15 has been described as a cytokine produced also by nonimmune cells influencing proliferation and apoptosis of lymphocytes (11, 25). The cytokine was initially identified through its ability to mimic IL-2-induced T cell proliferation (13). However, numerous reports have shown distinct and nonredundant functions of IL-2 and IL-15 (reviewed in Ref. 11). Furthermore, unlike IL-2, IL-15 mRNA has been identified in a wide range of different cell types, including monocytes/macrophages, epithelial cells, and fibroblasts (8, 13, 26, 28, 29). In addition to the stimulation of immune cells, IL-15 was shown to be a potent inhibitor of several apoptosis pathways in various cell populations (6, 25, 28, 32).

The purpose of the current study was to address the following two questions: 1) determine whether PSCs express IL-15 and 2) test the hypothesis that stellate cells thus affect immune- competent cells.


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
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Reagents were purchased from the following sources: Histopaque 1083, protease IX, monoclonal mouse anti-vimentin antibody, and standard laboratory chemicals from Sigma (Deisenhofen, Germany); RPMI 1640, nonessential amino acids, penicillin, streptomycin, and trypsin, all from Invitrogen (Karslruhe, Germany); collagenase P from Roche (Mannheim, Germany); Iscove's modified Dulbecco's medium (IMDM) and lymphocyte medium Quantum007 from PAA (Cölbe, Germany); FCS from Biochrom (Berlin, Germany); 70-µm mesh cell streamer and cell culture inserts containing a 0.4-µm pore size membrane from Becton-Dickinson Labware; annexin V-FITC Apoptosis Detection Kit from BD Pharmingen (Heidelberg, Germany); [3H]thymidine, [2,3-3H]proline, enhanced chemiluminescence (ECL) plus kit, and horseradish peroxidase-labeled anti-rabbit or anti-mouse Ig antibody from Amersham (Freiburg, Germany); rabbit anti-phospho-signal transducer and activator of transcription (STAT)-3 (705Tyr) from Cell Signaling Technology (Beverly, MA); rabbit anti-STAT-3 from Santa Cruz Biotechnologies (Santa Cruz, CA); recombinant mouse IL-15 from Chemicon (Temecula, CA); IL-2 and interferon (IFN)-{gamma} from R&D Systems (Wiesbaden, Germany); RNeasy Mini RNA extraction kit and Taq Polymerase from Qiagen (Hilden, Germany); all reagents for reverse transcription [Superscript II RT, oligo(dT)12–18, T7-(dT)24 primer, dNTP] and Superscript Choice system from Invitrogen (Karlsruhe, Germany); primers for PCR were generated using the National Center for Biotechnology Information (NCBI) gene bank as a source for any sequences and synthesized by BioTez (Berlin, Germany); and microarray Rat 230_2 from Affymetrix (Santa Clara, CA). Male Lewis inbred rats were obtained from Charles River (Sulzfeld, Germany).

Cell Isolation and Cultivation

PSCs were isolated from the pancreas of male Lewis rats by collagenase P/protease IX digestion and purified by isopycnic density centrifugation as previously described (34). PSCs were cultured in IMDM supplemented with 10% FCS, 1% nonessential amino acids (dilution of a 100x stock solution), 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in a 5% CO2 humidified atmosphere.

To investigate the effect of IFN-{gamma} on RNA expression, the respective cytokine concentrations were added to PSCs cultured in six-well plates followed by the extraction of RNA after the indicated incubation time.

The lymph node cells (LnLy) were obtained by mincing mesenteric lymph nodes and then gently pressing them through a 70-µm mesh cell streamer. The cell suspension was further purified by density gradient centrifugation on Histopaque 1083. The obtained cells consisted of ≥95% lymphocytes, as assessed by fluorescence-activated cell sorter (FACS) analysis.

Coculture experiments were performed using subconfluent PSCs activated by in vitro cultivation 7–10 days after isolation. Culture medium was removed, and 1 x 106 freshly isolated LnLy diluted in lymphocyte medium Quantum007 were added. To prevent cell-cell contact, PSCs and lymphocytes were separated by a transwell system with cell culture inserts. LnLy cultured without PSCs were used as controls.

Immunofluorescence

PSCs were cultivated on glass coverslips. For immunofluorescence staining, cells were fixed with 0.5% paraformaldehyde followed by incubation with a rabbit polyclonal antibody directed against rat IL-15 (AB15). Binding of AB15 was determined by a fluorescein-labeled goat anti-rabbit IgG and visualized using a fluorescence microscope.

RNA Isolation and RT-PCR

Analysis of RNA expression has been performed as described in detail previously (33). Total cell RNA was isolated using the RNeasy kit according to the manufacturer's instructions. Total RNA (1 µg) was reverse transcribed into cDNA using oligo(dT)12–18 primer and Superscript II. To correct for variations in different preparations, the cDNA samples were adjusted to equal the input cDNA concentrations, based on their hypoxanthine phosphoribosyl transferase or {beta}-actin content as housekeeping genes. For this purpose, each cDNA sample was subjected to a competitive PCR using a synthetic DNA control fragment as an internal standard. The PCR products were separated electrophoretically in an agarose gel containing 0.3 µg/ml ethidium bromide. The intensity of the ethidium bromide fluorescence, reflecting the amount of cDNA, was measured with an electronic camera analyzing the data with the EASY program (Herolab, Wiesloch, Germany).

IL-15 primers (forward 5'-CTT CTT AAC TGA GGC TGC ATC-3' and reverse 5'-GTG AAG TTT CTC TCC TCC AGC T-3') were designed based on the rat-specific cDNA sequences for rat IL-15 (U-69272).

Microarray Analysis

The cRNA preparation and hybridization on Rat 230_2 Arrays (Affymetrix) was conducted according to Affymetrix standard protocols (34). For each of the three treatment groups (controls, cocultured lymphocytes with PSCs, or IL-15), mRNA from three replicated samples was hybridized. We used MAS 5.0 to process raw microarray probe set data and to generate 31,099 gene expression values ("signals") per array with a corresponding validity value ("detection call") each. Data were scaled based on total intensity (for details, see www.affymetrix.com, Statistical Algorithms Reference Guide, Part Number 701137 Rev 3). To perform a quality control for replicated microarrays, the coefficient of determination r2 was calculated between pairs of arrays within each treatment group (e.g., 3 array pairs for triplicates of the control group: 1–2, 2–3, and 1–3). Thus the array similarity of replicated arrays was determined in relation to the signal parameter. A gene sequence was included into the differential gene expression analysis if both of the following conditions were complied with: all three replicated signal values of the gene sequence were similar to their median of at least 51% (reproducibility of signals). At least two of the three corresponding detection call values were "present" (validity of signals). The first step of differential gene expression analysis was the aggregation of reproducibility and valid replicated gene signals by their median. The three resulting median arrays (one for each group) were analyzed for differential gene expression of lymphocytes with PSCs vs. control and lymphocytes with IL-15 vs. control. A gene with an at least +2/–2-fold change in its expression was defined to be upregulated/downregulated. Intersections of the four resulting sets of regulated genes (i.e., upregulated/downregulated in PSC cocultures and upregulated/downregulated in IL-15 cocultured lymphocytes) were analyzed.

Sequencing of IL-15 cDNA Variants

Electrophoretically separated RT-PCR products were sequenced from both primer sites using dye-terminator technology. The sequence reactions were analyzed by capillary electrophoresis (ABI PRISM 310 Genetic Analyzer; Applied Biosystems).

Detection of Lymphocyte Apoptosis by Flow Cytometry

Apoptotic lymphocytes were evaluated using the annexin V-FITC Apoptosis Detection Kit according to the manufacturer's instructions. Briefly, 1 x 105 LnLy were harvested from the culture plates, washed two times with PBS (pH 7.4), and incubated in the annexin V-binding buffer. Annexin V-FITC and propidium iodide (PI) were added followed by the measurement in the flow cytometer FACSort using Cellquest software (Becton-Dickinson).

[3H]thymidine Incorporation

LnLy (1 x 106/ml) suspended in serum-free RPMI 1640 were seeded in 12-well plates and incubated for 24 h with 1.0 µCi [3H]thymidine/ml in the presence of PSCs, IL-15, and IL-15 plus the anti-IL-15 antibody. LnLy cultured in the [3H]thymidine-containing medium were used as controls. Afterward, LnLy were harvested and washed, and [3H]thymidine incorporation was evaluated using a liquid scintillation counter. Data were calculated from sixfold determinations and expressed as counts per minute (cpm).

Cell Counting

Adherent growing PSCs were detached from the culture plates by trypsin-EDTA using standard protocols. Cell numbers were determined by counting them microscopically in a Fuchs-Rosenthal hemocytometer. Cell viability was detected through the trypan blue dye exclusion test.

Measurement of Collagen Production

The collagen production by PSCs was quantified by assessing the incorporation of [2,3-3H]proline, as previously described (34). Briefly, PSCs maintained in 12-well culture plates (1 x 104 cells/ml) were incubated for 24 h in the presence of 2.5 µCi/ml [3,4-3H]proline without or cocultured with 1 x 106 LnLy/ml RPMI medium supplemented with 10% FCS. Lymphocytes were removed from the cell culture medium by centrifugation. The [3H]proline-containing proteins of the cell supernatant were subjected to precipitation/solubilization procedures followed by evaluation using liquid scintillation counting. Results were calculated from sixfold determinations and expressed as cpm per milliliter.

Generation of a Rat Specific anti-AB-IL-15

Based on the rat IL-15 cDNA sequence (U-69272, GenBank provided by NCBI), two appropriate peptides comprising 12 and 16 amino acids have been synthesized (Eurogentec, Herstal, Belgium). To obtain a rat-specific anti-IL-15 antibody, two rabbits have been immunized using a mixture of both peptides (Eurogentec). In a second step, the immune serum was purified using the affinity chromatography technique (Biogenes, Berlin, Germany). The procedure resulted in a monospecific IgG fraction recognizing the rat IL-15 protein (AB15).

Immunoblotting

Protein extracts of PSCs were prepared as previously described (34). Proteins were separated by SDS-PAGE and blotted on nitrocellulose filters. Blots were incubated with the respective primary antibody overnight at 4°C. For visualization of the antibody binding, filters were exposed to a horseradish peroxidase-labeled anti-rabbit or anti-mouse Ig antibody and developed using the ECL Plus kit.

The PSC culture supernatant was concentrated by centrifugal filter devices (Amicon Ultra-15; Millipore) and subjected to the immunoblotting procedure described above.

All results shown are representative of at least three independent experiments.

Statistical Analysis

Results are expressed as means ± SE. Data were analyzed using Wilcoxon's rank sum test, and P < 0.05 was considered to be statistically significant.


    RESULTS
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IL-15 Expression in PSCs

Figure 1A shows the expression of mRNA encoding IL-15 in PSCs at different time points after cell isolation detected by RT-PCR. The activation of PSCs induced through in vitro cultivation was accompanied by an increase of the IL-15 transcript levels. Surprisingly, in addition to the expected amplicon of 363 bp in the gel electrophoresis of RT-PCR products, there was observed a second smaller fragment (Fig. 1A). Sequencing of both amplicons revealed an mRNA splice variant different from the known sequence by an in- frame deletion of 48 nucleotides. The ratio of the fluorescence intensity of the PCR products 363 bp/315 bp, reflecting the respective transcript levels, was ~3.7. This relation of the two splice variants was found to be constant in different rat cell populations (data not shown).



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Fig. 1. Expression of IL-15 in rat pancreatic stellate cells (PSCs). A: time course of IL-15 mRNA expression in cultivated PSCs determined by RT-PCR. Total cellular RNA was isolated from PSCs after 1 (lane 2), 2 (lane 3), 3 (lane 4), 5 (lane 5), 7 (lane 6), and 14 (lane 7) days in culture and subsequently reverse transcribed into cDNA using an oligo(dT)12–18 primer. The cDNA probes were subjected to amplification with the IL-15-specific primers, giving rise to 2 fragments of 363 and 315 bp, respectively. To adjust the various cDNA samples to equal input concentrations, PCR was performed for the housekeeping gene hypoxanthine phosphoribosyl transferase (HPRT) in the presence of a synthetic DNA fragment used as an internal standard (control fragment, CF), resulting in the 608-bp (cDNA) and the 499-bp (CF) fragments. PCR products were electrophoretically separated in an agarose gel containing 0.3 µg/ml ethidium bromide. Results shown are representative of 3 independent experiments. M, 100-bp molecular weight marker (lane 1). B: detection of IL-15 protein in PSCs using immunoblot techniques. Protein extracts of PSCs after 5 (lane 1), 7 (lane 2), and 14 (lane 3) days in culture were harvested and electrophoretically separated on a 10% SDS polyacrylamide gel. After protein transfer on a nitrocellulose membrane, blots were incubated with a rabbit anti-IL-15 antibody. Detection of primary antibody binding was performed by using a horseradish peroxidase-labeled anti-rabbit IgG, and horseradish peroxidase activity was visualized with the ECL Plus kit. To demonstrate equal protein loading, the blot was probed with an anti-vimentin antibody. Results shown are representative of 3 independent experiments. C: immunocytochemical localization of IL-15 expression in PSCs. Cells grown on glass coverslips were fixed and incubated with an antibody against rat IL-15 generated in rabbits. Binding of the primary antibody was determined with a fluorescein-labeled goat anti-rabbit IgG, resulting in fluorescence (a). To demonstrate specificity of the primary antibody, PSCs were incubated only with the fluorescein-labeled IgG as a control (b). Original magnification: x630.

 
To determine if PSCs are able to produce IL-15 protein, Western blot analysis was performed on total lysates of PSCs after 4, 7, and 14 days in culture. As shown in Fig. 1B, PSCs contained two proteins of ~16 and 14 kDa representing probably both splice variants. According to the IL-15 mRNA expression, there was an increase of the protein level during the cultivation time after PSC isolation.

In the culture medium supernatants, IL-15 protein was not detectable, suggesting a low secretion capacity (data not shown).

Immunofluorescence staining of PSCs using the anti-IL-15 antibody revealed small spots evenly spread on the cell surface (Fig. 1C) indicating a membrane-associated localization of IL-15 protein.

In the context of inflammatory processes, the responsiveness of PSCs to respective mediators is of special interest. Figure 2 shows that the expression of IL-15 in PSCs could be induced by the lymphocyte-specific T helper 1 (Th1) cytokine IFN-{gamma} in a dose-dependent manner. Both splice variants increased in parallel, and the induction of IL-15 by the proinflammatory cytokine did not affect the ratio of the two transcript levels.



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Fig. 2. Induction of IL-15 expression in PSCs by interferon (IFN)-{gamma}. RT-PCR was performed as described in Fig. 1A. A: PSCs were cultured for 4 h without (lane 1) and in the presence of 20 ng/ml (lane 2) and 100 ng/ml (lane 3) IFN-{gamma}. To adjust the various cDNA samples to equal input concentrations, PCR was performed for the housekeeping gene HPRT in the presence of a synthetic DNA fragment used as an internal standard (CF). B: densitometric evaluation of the ethidium bromide fluorescence of the PCR products reflecting the IL-15 transcript levels. Results were expressed as the degree of change with respect to untreated control cells. Data are given as means ± SE (n = 4 experiments). *P < 0.05.

 
Cocultures of Lymphocytes with PSCs

Spontaneous apoptosis of lymphocytes. Rat lymphocytes obtained from mesenterial lymph nodes (LnLy) were cultivated in the absence or presence of a subconfluent monolayer of primary PSCs. The coculture experiments were performed with PSCs that were activated in vitro by growing on plastic for 7–10 days. After 24, 48, and 72 h, an aliquot of the LnLy was prepared for FACS analysis, measuring the binding of annexin V-FITC indicating cell apoptosis and the intake of PI that can penetrate only the damaged cell membrane. Lymphocytes positive for both annexin V and PI were defined as late apoptotic.

Figure 3 shows representative dot plots obtained by FACS analysis of in vitro cultured LnLy for 24 h (A and B), 48 h (C and D), and 72 h (E and F) in the absence (A, C, and E) or presence (B, D, and F) of stellate cells.



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Fig. 3. Representative dot plots of lymphocytes incubated with annexin V-FITC and propidium iodide (PI) followed by fluorescence-activated cell sorter (FACS) analysis. Lymphocytes isolated from rat mesenterial lymph nodes (LnLy) were maintained for 24 h (A and B), 48 h (C and D), and 72 h without (A, C, and E) or in coculture with (B, D, and F) PSCs. Vital lymphocytes were defined to be negative for both annexin V and PI (bottom left quadrant). Apoptotic cells bind annexin V (bottom right quadrant) followed by the loss of membrane integrity, affording the penetration of PI (top right quadrant). The percentages of cells in relation to the whole lymphocyte compartment are indicated.

 
Interestingly, the portion of early apoptotic cells defined by annexin V-positive but PI-negative fluorescence did not increase continuously to the same extent compared with the late-apoptotic lymphocytes. Consequently, the percentage of exclusively annexin V-positive lymphocytes had a maximum after 48 h followed by a decline during further cultivation. In contrast, the number of lymphocytes positive for both annexin V and PI increased proportionally to the reduction of surviving cells. These results suggest that the apoptotic process, once induced in lymphocytes, was succeeded immediately by the following reactions of the death machinery, resulting in the damage and leakage of the cell membrane. Therefore, we have defined apoptotic cells as the sum of annexin V-positive and PI-positive lymphocytes.

Figure 4A summarizes the time course of the lymphocyte viability under different culture conditions. The number of annexin V-negative vital lymphocytes was decreased after 3 days to ~45%, whereas the life span of LnLy could be increased up to 90% by cocultivation with PSCs. The protecting effect of PSCs on LnLy could be attenuated by cocultivation in the presence of an anti-IL-15 antibody. Furthermore, there was only a modest increase of surviving cells cocultured with PSCs in a Transwell system preventing cell-cell contact between PSCs and lymphocytes.



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Fig. 4. A: vital lymphocytes after 24, 48, and 72 h in culture analyzed by flow cytometry as described in Fig. 3. Cells were cultivated in medium (LnLy) or cocultured with PSCs (CoCu) in the presence of PSCs with the addition of anti-IL-15 antibody (CoCu + AB15). To prevent the cell-cell contact between PSCs and lymphocytes, cocultures were performed in a Transwell system (TW) with inserts containing a 0.4-µm pore size membrane. B: influence of recombinant (r)IL-15 on lymphocyte survival. LnLy were cultivated for 24 h without (C) and with rIL-15 at the indicated concentrations. An aliquot of lymphocytes was harvested and incubated with annexin V-FITC and PI followed by FACS analysis. Vital lymphocytes were defined as negative for both annexin V and PI. Results are expressed as the mean percentages of vital cells ± SE (n = 6). *P < 0.05 with respect to control cells.

 
The addition of recombinant (r)IL-15 also significantly inhibited the spontaneous lymphocyte apoptosis in a dose-dependent manner (Fig. 4B). However, the prolonging effect of rIL-15 on the life span of LnLy was lower compared with the influence of the PSC cocultures.

Figure 5 shows the effect of rIL-15 and the coculture procedure on proliferation of lymphocytes by means of [3H]thymidine uptake. Compared with untreated controls, the [3H]thymidine incorporation was heightened significantly by rIL-15 and by cocultivation with stellate cells. Addition of the anti-IL-15-AB attenuated the stimulation exerted by PSC cocultures, although it did not reverse the values reaching normal values.



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Fig. 5. Lymphocyte poliferation evaluated by means of [3H]thymidine incorporation. Cells (1 x 106/ml) were grown for 24 h in the presence of 1.0 µCi [3H]thymidine/ml under the following different conditions: without (C) or with rIL-15 at the indicated concentrations, cocultered with PSCs (CoCu), and after the addition of an anti-IL-15 antibody to the cocultures (CoCu + AB). Results are expressed as mean counts/min (cpm) ± SE (n = 6). *P < 0.05.

 
The proliferation-stimulating effects of the "classic" lymphocyte-activating cytokine IL-2 and the unspecific mitogen concanavalin A (ConA) have been to an extremely high degree stronger compared with IL-15 (Fig. 6A). However, in contrast to rIL-15 and the cocultivation with PSCs, both IL-2 and ConA markedly accelerated the spontaneous apoptosis of LnLy (Fig. 6B). Furthermore, rIL-15 and PSC cocultivation was able to reverse the ConA-mediated proapoptotic effect. Similar but less pronounced results were found regarding the interactions between rIL-15 and rIL-2 (data not shown).



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Fig. 6. Impact of concanavalin A (ConA) and IL-2 on proliferation and survival of lymphocytes. A: lymphocytes were cultured for 24 h in presence of 1.0 µCi [3H]thymidine/ml with ConA (1 µg/ml) and with rIL-2 (20 and 100 ng/ml). Untreated cells were used as control (C). [3H]thymidine uptake was evaluated using a liquid scintillation counter. Results are expressed as mean cpm ± SE (n = 6). *P < 0.05. B: lymphocytes were cultivated for 24 h in presence of 1 µg/ml ConA, 100 ng/ml rIL-2, and ConA plus 100 ng/ml rIL-15 (ConA + IL-15). Furthermore, 1 µg/ml ConA was added to lymphocytes cocultured with PSCs. At the end of incubation time, an aliquot of lymphocytes was subjected to FACS analysis, measuring fluorescence intensity of annexin V-FITC and PI. Vital lymphocytes were defined as negative for both annexin V and PI. Results are expressed as mean percentages of vital cells ± SE (n = 6). *P < 0.05 with respect to control cells.

 
Regarding the intracellular signal transfer in lymphocytes, we have shown that rIL-15 and PSC cocultivation was associated with an activation of the STAT-3 (Fig. 7). Addition of the anti-IL-15 antibody to the PSC-lymphocyte cocultures resulted in an attenuation of the STAT-3 phosphorylation.



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Fig. 7. Detection of phosphorylated signal transducer and activator of transcription (STAT)-3 in isolated lymphocytes. Cells were cultured for 60 min with 100 ng/ml rIL-15 (lane 2), in the presence of PSCs (lane 3), and with PSCs and the anti-IL-15 antibody (lane 4). Untreated lymphocytes were used as control (lane 1). At the end of incubation time, lymphocytes were harvested followed by centrifugation and cell lysis. Cell lysates were electrophoretically separated on an 8% SDS-polyacrylamide gel. After protein transfer to a nitrocellulose membrane, blots were incubated with a rabbit anti-phospho (P)-STAT-3 antibody (IL-15 antibody). Detection of primary antibody binding was performed using a horseradish peroxidase-labeled anti-rabbit IgG, and horseradish peroxidase activity was visualized with the ECL Plus kit. To demonstrate equal protein loading, the blot was probed with an anti-STAT-3 antibody. Results shown are representative of 3 independent experiments.

 
Expression profile analysis. To get insight into the molecular mechanisms underlying the apoptosis inhibition, we generated gene expression profiles of lymphocytes in different cocultures by means of microarray analysis. The replicated arrays showed a high degree of similarity within each coculture in relation to the signal parameter. The high similarity was reflected in the linear scatterplots with the indication of r2 for all groups alike between 0.97 and 0.98 (plots not shown). For comparison, the values of r2 for array pairs between the groups ranged between 0.92 and 0.96. When gene expression analysis was performed, the number of remaining gene sequences in each of the resulting median arrays was nearly 16,000 likewise. According to the stringent criteria of differential expression, genes were upregulated/downregulated with similar expression profiles in lymphocytes whether cocultured with PSCs or rIL-15. In total, 411 genes were upregulated and 245 were downregulated. The intersection calculation resulted in 57 common upregulated and 7 common downregulated genes (Fig. 8). It must be averred that there was no gene detected with an opposite regulation in both cocultures (upregulated in lymphocytes cocultured with PSCs and downregulated with IL-15 and vice versa).



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Fig. 8. Microarray data analysis was performed to screen genes relevant to the molecular mechanisms in apoptosis at the transcriptional level. The degree of changes of gene expression in cocultures vs. control was calculated. For sample description, see MATERIALS AND METHODS. Genes are considered to be significant changed in their gene expression by an at least 2-fold change. Similar gene expression profiles were retrieved in lymphocytes cocultured with both PSCs and IL-15 vs. control. Fifty-seven sequences for 18 different annotated genes and 37 estimated sequence tags (ESTs) were significantly upregulated in both cocultures, and 7 sequences (1 gene and 6 ESTs) were downregulated. The NCBI GenBank Accession Numbers for the Human Genome Organization Gene Nomenclature Committee annotated genes are given on right. There was no gene detected with an opposite regulation in either coculture (upregulated in PSC cocultures, downregulated in cocultures with IL-15, and vice versa). CISH, cytokine-inducible SH2 protein.

 
To control microarray data, we investigated the expression of CISH, a member of the cytokine-inducible SH2 protein family that has been found clearly upregulated in LnLy cocultured with PSCs or rIL-15. Using RT-PCR, we could confirm the rIL-15- and PSC-mediated increase of CISH encoding mRNA (Fig. 9). Furthermore, addition of the anti-IL-15 antibody attenuated the PSC-triggered induction of CISH expression.



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Fig. 9. A: expression of CISH in lymphocytes assessed by RT-PCR. Lymphocytes were cultivated for 4 h with 100 ng/ml rIL-15 (lane 2), in the presence of PSCs (lane 3), and with PSCs and the anti-IL-15 antibody (lane 4). Untreated lymphocytes were used as controls (lane 1). At the end of the incubation time, lymphocytes were harvested followed by the extraction of total cellular RNA. Subsequently, RNA was reverse transcribed into cDNA using an oligo(dT)12–18 primer. The cDNA probes were subjected to amplification with CISH-specific primers, giving rise to a fragment of 321 bp. To adjust the various cDNA samples to equal input concentrations, PCR was performed for the housekeeping gene {beta}-actin in the presence of a synthetic DNA fragment used as an internal standard (CF), resulting in the 762-bp (cDNA) and 601-bp (CF) amplicons. PCR products were electrophoretically separated in an agarose gel containing 0.3 µg/ml ethidium bromide. Results shown are representative of 3 independent experiments. B: results expressed as the degree of change with respect to untreated control cells. Data are given as means ± SE (n = 3).

 
Influence of Lymphocytes on PSCs

To investigate effects of lymphocytes on stellate cell functions, 24 h after starting the coculture system, PSC proliferation and their collagen synthesis was assessed. Figure 10 shows that cell growth (Fig. 10, left) and collagen secretion (Fig. 10, right) by PSCs in the presence of lymphocytes increased significantly compared with controls.



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Fig. 10. Proliferation (left) and collagen secretion (right) of PSCs without and cocultured with lymphocytes (LnLy). Left: after PSCs were detached from the culture flasks, cell number was counted using a hemocytometer. Cell numbers are expressed as means ± SE (n = 8). *P < 0.05. Right: determination of collagen synthesis by means of [3H]proline incorporation. PSCs were grown for 24 h in 12-well culture plates in the presence of 2.5 µCi/ml [3,4-3H]proline without or cocultured with lymphocytes. The content of [3H]proline in the cell culture supernatant was measured using liquid scintillation counting. Results are expressed as cpm/ml, representing means ± SE (n = 6). *P < 0.05.

 

    DISCUSSION
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Leukocytes are considered to play a crucial role in the inflammatory process of chronic pancreatitis (5, 10, 12, 17, 18). On the other hand, PSCs as the main producers of the ECM have been essentially implicated in pancreatic fibrosis, a characteristic pathomorphological symptom of the disease. There is striking evidence that PSCs represent a substantial target cell population for leukocyte-mediated effects (3, 2224, 30, 31).

A wide range of cellular functions are known to be realized by cytokines. Besides activation processes, the modulation of pathways leading to cell apoptosis is suggested to be a critical regulatory mechanism during inflammation (27). Generally, at the end of an inflammatory reaction, immune cells that were recruited and expanded during the active phase of response are normally removed by apoptosis (1, 27). In chronic inflammation, this resolution mechanism becomes disordered, leading to the persistence of the cellular infiltrate, and, ultimately, tissue destruction and fibrosis (1). Consequentially, mechanisms that inhibit apoptotic pathways in lymphocytes may be involved in the defective tissue regeneration during chronic inflammation.

Here, we have shown that coculture of lymphocytes with in vitro activated PSCs resulted in a significant reduction of spontaneous lymphocyte apoptosis. The reversal of the anti-apoptotic effect of PSCs on lymphocytes by addition of an anti-IL-15 antibody to the cocultures suggests that IL-15 is an important factor of the PSC-induced lymphocyte rescue.

IL-15 has been shown to inhibit apoptosis in various cell populations, including lymphocytes (6, 7, 25, 28, 32, 36).

In the present study, we have shown for the first time that rat PSCs express IL-15 on the RNA and protein levels. The activation of PSCs during their in vitro cultivation has been accompanied by an induction of the expression of the cytokine. Furthermore, we have identified a spliced IL-15 mRNA variant. In all cells tested so far, the expression of the larger isoform was approximately fourfold higher than the shorter sequence.

The localization of IL-15 in PSCs using immunofluorescence suggested a tight association of the protein with the plasma membrane. Accordingly, IL-15 protein was detected exclusively in cell lysates but not in the cell culture supernatant. In the consequence of the lack of IL-15 secretion, the antiapoptotic effect of PSCs on lymphocytes was found to be strongly diminished by the prevention of cell-cell contact between both cell populations using a transwell system. These results are in agreement with reports showing the functional activity of membrane-bound IL-15 (26, 28).

Our results are consistent with comprehensive studies on IL-15 expression in different systems that revealed an unusual complex and tight posttranscriptional control of the IL-15 gene product and a low secretion potential of the signal peptide. These specific molecular characteristics resulted in low translation rates and little secretion capacity of IL-15 compared with other cytokines (35). A mechanism by which IL-15 could exert biological effects while being undetectable in culture supernatants has been suggested.

The lower efficiency of rIL-15 in prolonging lymphocyte survival compared with the coculture system might be caused by various reasons. Thus the permanent and stable attendance of the adequate IL-15 splice variants presented by the cells may have a more extending impact on lymphocyte viability than the recombinant protein with a limited half-life. However, our data indicate that, apart from IL-15, additional factors generated by PSCs protect lymphocytes from apoptosis. Consequently, the prevention of cell-cell contact using the Transwell system did not abolish the coculture effect completely.

IL-15-mediated signal transfer has been shown to include the STAT pathway (35). The phosporylation of STAT-3 in lymphocytes provoked by both rIL-15 and PSC cocultivation and the reduction of activated STAT-3 by the anti-IL-5 antibody indicate a correlation of apoptosis and IL-15-mediated STAT signal cascade.

The gene expression analysis of cocultured lymphocytes with PSCs or rIL-15 shows a sufficient set of differential regulated genes with similar expression profiles in both culture systems. Sixty-four genes and estimated sequence tags met the stringent criteria of differential regulation and showed common regulation effects in both coculturing lymphocytes. In contrast, there was no gene with an opposite regulation in either coculture detected, which corroborates the theory of IL-15-mediated apoptosis inhibition in cocultured lymphocytes.

The clear increase of CISH expression induced by both rIL-15 and PSC cocultures strongly indicate an involvement of the cytokine-induced SH2 protein [CIS/suppressor of cytokine signaling (SOCS)] family in IL-15-mediated biological effects. The SOCS proteins have been shown to control cytokine responses (16). Thus we can hypothesize that the IL-15-mediated upregulation of CISH provides a feedback mechanism preventing an aberrant cytokine effect.

The stimulation of lymphocyte proliferation by PSCs or rIL-15 has been modest compared with the growth effects of the classic lymphocyte activator IL-2 or the mitogen ConA. However, in contrast to IL-15, IL-2 and ConA shortened the in vitro life time mediated by the activation-induced cell death mechanism (36). IL-15 or the PSC cocultures could effectively reduce the ConA-induced apoptosis. Therefore, the inhibition of apoptosis appears the most important effect of PSC-bound IL-15 on lymphocytes.

The expression of IL-15 in PSCs could be induced by the lymphocyte-restricted Th1 cytokine IFN-{gamma}. Moreover, it is well known that IL-15 upregulates IFN-{gamma} synthesis in lymphocytes (8, 26). Taken together, the complex cross talk between PSCs and lymphocytes could comprise the mutual paracrine induction of IL-15 and IFN-{gamma} expression. This hypothesis was supported by results that we obtained in a rat model of chronic pancreatitis. In that model, we could show that IFN-{gamma} was substantially involved in the persistence of inflammation and the development of pancreatic fibrosis (33).

In summary, PSCs have been clearly shown to exert a drastic anti-apoptotic effect on immune cells. Our data suggest that IL-15 may be one of the components generated by PSCs that implement this biological function. Further studies are necessary to clarify the underlying molecular mechanisms.

In addition, cocultures with lymphocytes significantly induced PSC growth and their collagen secretion. Our results are in agreement with numerous reports showing the stimulation of stellate cells through different mediators generated by inflammatory cells (3, 14, 18, 2224). Taken together, these data support the concept of a mutual stimulation of PSCs and infiltrating lymphocytes, ultimately resulting in the persistence and progression of pancreatic inflammation accompanied by the development of fibrosis in chronic pancreatitis.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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The study was supported by Grant 01ZZ0108 from the Bundesministerium für Bildung und Forschung and by the Ministry for Education of the German federal state Mecklenburg-Vorpommern with European Regional Development Funds EFRE0400210/2004.


    ACKNOWLEDGMENTS
 
We thank Edith Prestin and Katrin Sievert for excellent technical assistance and for help with animal experiments.


    FOOTNOTES
 

Address for reprint requests and other correspondence: G. Sparmann, Dept. of Gastroenterology, Univ. Hospital of Rostock, Ernst-Heydemann-Strasse 6, D-18057 Rostock, Germany (e-mail: gisela.sparmann{at}med.uni-rostock.de)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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  1. Akbar AN and Salmon M. Cellular environments and apoptosis: tissue microenvironments control activated T-cell death. Immunol Today 18: 72–76, 1997.[CrossRef][ISI][Medline]
  2. Andoh A, Takaya H, Saotome T, Shimada M, Hata K, Araki Y, Nakamura F, Shintani Y, Fujiyama Y, and Bamba T. Cytokine regulation of chemokine (IL-8, MCP-1, and RANTES) gene expression in human pancreatic periacinar myofibroblasts. Gastroenterology 119: 211–219, 2000.[CrossRef][ISI][Medline]
  3. Apte MV, Haber PS, Darby SJ, Rodgers SC, McCaughan GW, Korsten MA, Pirola RC, and Wilson JS. Pancreatic stellate cells are activated by proinflammatory cytokines: implications for pancreatic fibrogenesis. Gut 44: 534–541, 1999.[Abstract/Free Full Text]
  4. Bachem MG, Schneider E, Gross H, Weidenbach H, Schmid RM, Menke A, Siech M, Beger H, Grunert A, and Adler G. Identification, culture, and characterization of pancreatic stellate cells in rats and humans. Gastroenterology 115: 421–432, 1998.[ISI][Medline]
  5. Beger HG, Gansauge F, and Mayer JM. The role of immunocytes in acute and chronic pancreatitis: when friends turn into enemies. Gastroenterology 118: 626–629, 2000.[ISI][Medline]
  6. Bulfone-Paus S, Bulanova E, Pohl T, Budagian V, Durkop H, Ruckert R, Kunzendorf U, Paus R, and Krause H. Death deflected: IL-15 inhibits TNF-{alpha}-mediated apoptosis in fibroblasts by TRAF2 recruitment to the IL-15R{alpha} chain. FASEB J 13: 1575–1585, 1999.[Abstract/Free Full Text]
  7. Bulfone-Paus S, Ungureanu D, Pohl T, Lindner G, Paus R, Ruckert R, Krause H, and Kunzendorf U. Interleukin-15 protects from lethal apoptosis in vivo. Nat Med 3: 1124–1128, 1997.[CrossRef][ISI][Medline]
  8. Carson WE, Ross ME, Baiocchi RA, Marien MJ, Boiani N, Grabstein K, and Caligiuri MA. Endogenous production of interleukin 15 by activated human monocytes is critical for optimal production of interferon-gamma by natural killer cells in vitro. J Clin Invest 96: 2578–2582, 1995.[ISI][Medline]
  9. Ebert MP, Ademmer K, Muller-Ostermeyer F, Friess H, Buchler MW, Schubert W, and Malfertheiner P. CD8+CD103+ T cells analogous to intestinal intraepithelial lymphocytes infiltrate the pancreas in chronic pancreatitis. Am J Gastroenterol 93: 2141–2147, 1998.[CrossRef][ISI][Medline]
  10. Emmrich J, Weber I, Nausch M, Sparmann G, Koch K, Seyfarth M, Lohr M, and Liebe S. Immunohistochemical characterization of the pancreatic cellular infiltrate in normal pancreas, chronic pancreatitis and pancreatic carcinoma. Digestion 59: 192–198, 1998.[CrossRef][ISI][Medline]
  11. Fehniger TA and Caligiuri MA. Interleukin 15: biology and relevance to human disease. Blood 97: 14–32, 2001.[Free Full Text]
  12. Friess H, Buchler MW, Mueller C, and Malfertheiner P. Immunopathogenesis of chronic pancreatitis. Gastroenterology 115: 1018–1022, 1998.[ISI][Medline]
  13. Grabstein KH, Eisenman J, Shanebeck K, Rauch C, Srinivasan S, Fung V, Beers C, Richardson J, Schoenborn MA, Ahdieh and M. Cloning of a T cell growth factor that interacts with the beta chain of the interleukin-2 receptor. Science 264: 965–968, 1994.[ISI][Medline]
  14. Gressner AM. Cytokines and cellular crosstalk involved in the activation of fat-storing cells. J Hepatol 22: 28–36, 1995.[CrossRef][ISI][Medline]
  15. Haber PS, Keogh GW, Apte MV, Moran CS, Stewart NL, Crawford DH, Pirola RC, McCaughan GW, Ramm GA, and Wilson JS. Activation of pancreatic stellate cells in human and experimental pancreatic fibrosis. Am J Pathol 155: 1087–1095, 1999.[Abstract/Free Full Text]
  16. Hanada T, Kinjyo I, Inagaki-Ohara K, and Yoshimura A. Negative regulation of cytokine signaling by CIS/SOCS family proteins and their roles in inflammatory diseases. Rev Physiol Biochem Pharmacol 149: 72–86, 2003.[ISI][Medline]
  17. Hunger RE, Mueller C, Z'graggen K, Friess H, and Buchler MW. Cytotoxic cells are activated in cellular infiltrates of alcoholic chronic pancreatitis. Gastroenterology 112: 1656–1663, 1997.[ISI][Medline]
  18. Jaskiewicz K, Nalecz A, Rzepko R, and Sledzinski Z. Immunocytes and activated stellate cells in pancreatic fibrogenesis. Pancreas 26: 239–242, 2003.[CrossRef][ISI][Medline]
  19. Jaster R, Sparmann G, Emmrich J, and Liebe S. Extracellular signal regulated kinases are key mediators of mitogenic signals in rat pancreatic stellate cells. Gut 51: 579–584, 2002.[Abstract/Free Full Text]
  20. Kloppel G and Maillet B. Pathology of acute and chronic pancreatitis. Pancreas 8: 659–670, 1993.[ISI][Medline]
  21. Kruse ML, Hildebrand PB, Timke C, Folsch UR, and Schmidt WE. TGFbeta1 autocrine growth control in isolated pancreatic fibroblastoid cells/stellate cells in vitro. Regul Pept 90: 47–52, 2000.[CrossRef][ISI][Medline]
  22. Luttenberger T, Schmid-Kotsas A, Menke A, Siech M, Beger H, Adler G, Grunert A, and Bachem MG. Platelet-derived growth factors stimulate proliferation and extracellular matrix synthesis of pancreatic stellate cells: implications in pathogenesis of pancreas fibrosis. Lab Invest 80: 47–55, 2000.[ISI][Medline]
  23. Maher JJ. Leukocytes as modulators of stellate cell activation. Alcohol Clin Exp Res 23: 917–921, 1999.[ISI][Medline]
  24. Mews P, Phillips P, Fahmy R, Korsten M, Pirola R, Wilson J, and Apte M. Pancreatic stellate cells respond to inflammatory cytokines: potential role in chronic pancreatitis. Gut 50: 535–541, 2002.[Abstract/Free Full Text]
  25. Mueller YM, Makar V, Bojczuk PM, Witek J, and Katsikis PD. IL-15 enhances the function and inhibits CD95/Fas-induced apoptosis of human CD4+ and CD8+ effector-memory T cells. Int Immunol 15: 49–58, 2003.[Abstract/Free Full Text]
  26. Musso T, Calosso L, Zucca M, Millesimo M, Ravarino D, Giovarelli M, Malavasi F, Ponzi AN, Paus R, and Bulfone-Paus S. Human monocytes constitutively express membrane-bound, biologically active, and interferon-gamma-upregulated interleukin-15. Blood 93: 3531–3539, 1999.[Abstract/Free Full Text]
  27. Orteu CH, Poulter LW, Rustin MH, Sabin CA, Salmon M, and Akbar AN. The role of apoptosis in the resolution of T cell-mediated cutaneous inflammation. J Immunol 161: 1619–1629, 1998.[Abstract/Free Full Text]
  28. Rappl G, Kapsokefalou A, Heuser C, Rossler M, Ugurel S, Tilgen W, Reinhold U, and Abken H. Dermal fibroblasts sustain proliferation of activated T cells via membrane-bound interleukin-15 upon long-term stimulation with tumor necrosis factor-alpha. J Invest Dermatol 116: 102–109, 2001.[CrossRef][ISI][Medline]
  29. Reinecker HC, MacDermott RP, Mirau S, Dignass A, and Podolsky DK. Intestinal epithelial cells both express and respond to interleukin 15. Gastroenterology 111: 1706–1713, 1996.[ISI][Medline]
  30. Schmid-Kotsas A, Gross HJ, Menke A, Weidenbach H, Adler G, Siech M, Beger H, Grunert A, and Bachem MG. Lipopolysaccharide-activated macrophages stimulate the synthesis of collagen type I and C-fibronectin in cultured pancreatic stellate cells. Am J Pathol 155: 1749–1758, 1999.[Abstract/Free Full Text]
  31. Schneider E, Schmid-Kotsas A, Zhao J, Weidenbach H, Schmid RM, Menke A, Adler G, Waltenberger J, Grunert A, and Bachem MG. Identification of mediators stimulating proliferation and matrix synthesis of rat pancreatic stellate cells. Am J Physiol Cell Physiol 281: C532–C543, 2001.[Abstract/Free Full Text]
  32. Shinozaki M, Hirahashi J, Lebedeva T, Liew FY, Salant DJ, Maron R, and Kelley VR. IL-15, a survival factor for kidney epithelial cells, counteracts apoptosis and inflammation during nephritis. J Clin Invest 109: 951–960, 2002.[Abstract/Free Full Text]
  33. Sparmann G, Behrend S, Merkord J, Kleine HD, Graser E, Ritter T, Liebe S, and Emmrich J. Cytokine mRNA levels and lymphocyte infiltration in pancreatic tissue during experimental chronic pancreatitis induced by dibutyltin dichloride. Dig Dis Sci 46: 1647–1656, 2001.[CrossRef][ISI][Medline]
  34. Sparmann G, Hohenadl C, Tornoe J, Jaster R, Fitzner B, Koczan D, Thiesen HJ, Glass A, Winder D, Liebe S, and Emmrich J. Generation and characterization of immortalized rat pancreatic stellate cells. Am J Physiol Gastrointest Liver Physiol 287: G211–G219, 2004.[Abstract/Free Full Text]
  35. Tagaya Y, Bamford RN, DeFilippis AP, and Waldmann TA. IL-15: a pleiotropic cytokine with diverse receptor/signaling pathways whose expression is controlled at multiple levels. Immunity 4: 329–336, 1996.[CrossRef][ISI][Medline]
  36. Waldmann T. The contrasting roles of IL-2 and IL-15 in the life and death of lymphocytes: implications for the immunotherapy of rheumatological diseases. Arthritis Res 4, Suppl 3: S161–S167, 2002.[CrossRef][Medline]




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