ARTICLE
INTRODUCTION
The cytokine IL-15 is a member of the "four-helix-bundle"
family of cytokines [1, 2] which is functionally similar to IL-2 in in
vitro and in vivo systems although it has no sequence similarities
with IL-2. IL-15 has been shown to act on T cells, B cells, NK cells and
others [for review see 3, 4]. IL-15-mediated signal transduction involves
the gammac-chain of the IL-2 receptor; in addition the beta-chain
of the IL-2 receptor and a particular alpha-chain are part of the IL-15
receptor complex [5-7]. IL-15 mRNA has been detected in monocytes/macrophages,
dendritic and Langerhans cells, muscle cells, keratinocytes, epithelial
and fibroblast cell lines [1, 8-13] and can be upregulated by pro-inflammatory
stimuli [8, 11-13]. In vivo IL-15 mRNA is upregulated in inflammatory
situations [14-16], often without concomitant upregulation of IL-2 mRNA
[17]. The presence of extracellular IL-15 protein has been demonstrated
in vivo in chronic inflammatory disease [18-21]. In contrast IL-15
protein cannot be easily detected in supernatants of "normal" cells in
vitro which suggests that normally the secretion of IL-15 is strictly
controlled [4]. In vivo interference with the activity of IL-15
has led to a decrease of experimentally-induced inflammation [22, 23].
The expression pattern of IL-15 and its pleiotropic activities point to
an important role for this cytokine in the initiation and propagation
of chronic inflammatory disease. We have recently shown that the human
lung carcinoma cell line A549 constitutively expressed IL-15 mRNA and
IL-15 protein, both of which could be upregulated by stimulation with
TNF-alpha or IL-1beta [13]. As the use of glucocorticoids is a mainstay
of antiinflammatory therapy, we investigated the effects of dexamethasone
and cyclosporin A on the induction of IL-15 mRNA and protein in this cell
line.
METHODS
Reagents and materials: reagents were obtained as
follows: human recombinant tumor necrosis factor-alpha (TNF-alpha; Peprotech,
London, UK); human recombinant interleukin-1beta (IL-1beta; Genzyme Virotech,
Rüssels-heim, Germany); dexamethasone, cyclosporin A (CyA), benzamidine,
aprotinin and pepstatin (Sigma-Aldrich Chemie, Deisenhofen, Germany),
Pefablock (Boehringer Mannheim, Germany). Dexamethasone was dissolved
in DMSO at 1 mM, from which a series of different concentrations was obtained
by further dilution in DMSO. PBS, DMEM, media supplements and FCS were
obtained from Life Technologies (Eggenstein, Germany). DMEM was supplemented
with 100 U/ml:100 mug/ml penicillin/streptomycin, 2 mM glutamine, 50 muM
2-mercaptoethanol, 1 mM pyruvate and 1% non-essential amino acids and
will be referrred to as complete medium. Tissue culture plastic was from
Costar (Technomara, Fernwald, Germany).
Cell culture: the human lung carcinoma cell line
A549 (ATCC# CCL185), which is of epithelial-like morphology, was grown
in complete medium in the presence of 5% FCS. For induction of IL-15 mRNA,
cells were cultured in 6 cm Ø Petri dishes, and cultures which
had reached 90-95% confluency were stimulated as indicated. Stimulation
was performed in presence of 0.1% FCS. Dexamethasone solutions in 100%
DMSO were added 1:1000 to the culture dishes, so that the final concentration
of DMSO was 0.1% at all concentrations of dexamethasone. Control cultures
received equivalent amounts of DMSO. Total cellular RNA was harvested
after 4 hours. For the production of cellular lysates, cells were stimulated
in complete medium with 0.1% FCS in 75 cm2 tissue culture flasks
for 42 hours or for 22 hours.
Complementary DNA-PCR (cDNA-PCR):
total cellular RNA was obtained using the InViSorb™ RNA Kit II (InViTek,
Berlin, Germany). cDNA synthesis and subsequent PCR were performed as
described [24]. DNA was synthesized using random hexanucleotide primers
(Boehringer Mannheim, Germany) and RAV2 reverse transcriptase (Amersham,
Braunschweig, Germany). Two to three independent cDNA were synthesized
from every RNA sample. PCR was performed using cDNA equivalent to 200
ng RNA. The IL-15 primers were deduced from the sequence of the human
IL-15 cDNA deposited at EMBL (accession no. U 14407). Two different sets
of IL-15 primers were used: most experiments were performed using primer
set "T2": IL-15 sense: 5'-GGA TTT ACC GTG GCT TTG AG-3', IL-15 antisense
5'-TCT ACT GTA TCA TGA ATAC-3. The primer set "T2" is able to distinguish
between the two splice variants described by Meazza et al. [25],
the expected size of amplified material being 358 bp (position 295-652)
for the conventionally spliced mRNA, and 476 bp for mRNA containing an
extra exon spliced in between exon 4 and exon 5. Our specimen of the A549
cell line did not contain significant amounts of alternatively spliced
IL-15 mRNA, thus in most experiments, only signals corresponding to the
358 bp fragment were detectable. It should be noted that the material
amplified by "T2" could clearly be separated from the material amplified
by the GAPDH amplimers, most likely due to unusual separation behaviour
under the conditions employed. The material was fully sequenced and found
to correspond to the published sequence of the expected 358 bp fragment
of IL-15 cDNA (sequencing was performed by GATC, Konstanz, Germany). In
some experiments the primer set "R" was used which does not distinguish
between the two IL-15 splice variants mentioned above: IL-15 sense: 5'-TAA
AAC AGA AGC CAA CTG-3', IL-15 antisense: the same primer as in primer
set "T2"; expected size of amplified material: 205 bp (position 448 to
652). The primers for the detection of IL-8 have been described by Jung
et al. [26]: IL-8 sense: 5'-ATG ACT TCC AAG CTG GCC GTG GCT-3',
IL-8 antisense: 5'-TCT CAG CCC TCT TCA AAA ACT TCT C-3', yielding a 289
bp fragment. GAPDH was used as control gene, primers were: GAPDH sense
5'-CGG GAA GCT TGT GAT CAA TGG-3', GAPDH antisense: 5'-GGC AGT GAT GGC
ATG GAC TG-3', the expected size of amplified material 358 bp. PCR-conditions:
15 sec 95° C/30 sec 55° C/90 sec 72° C; IL-15: 32 cycles
(primer set "T2"); 28 cycles (primer set "R"), IL-8: 22 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). Since the amplimers
were deduced from different exons of the IL-15 gene according to Krause
et al. [27], eventual traces of DNA in the RNA preparation would
not generate a fragment of 358 bp resp. 205 bp 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.
Production of intracellular IL-15: after stimulation
of cells with different cytokines for the time periods indicated, the
supernatants were removed, the adherent A549 cells were washed with PBS
and detached with 10 mM EDTA. Cells were sedimented by centrifugation
and washed, and the pellets were resuspended in 0.5 mL of lysis buffer
(PBS with 1 mM EDTA, 1 mg/mL Pefablock, 2 mM benzamidine, 0.4 Units/mL
aprotinin, 0.7 mug/mL pepstatin). Cells were lysed by repeated shock freezing
in liquid N2. Lysates were centrifuged at 14 000 g and the
content of IL-15 in the supernatant was measured with a commercial IL-15
ELISA (R&D Systems, Wiesbaden-Nordenstadt, Germany). 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 AND DISCUSSION
We have recently shown that the A549 cell line constitutively
expressed IL-15 mRNA as well as intracellular IL-15 protein. The abundance
of IL-15 mRNA and of intracellular protein could be increased by stimulation
with the pro-inflammatory cytokines TNF-alpha and IL-1beta. In contrast,
significant amounts of secreted IL-15 were not detectable in the supernatant
of A549 cells [13]. Corresponding to our earlier findings, Figures
1 and 2 illustrate that 10 ng/ml TNF-alpha or 1 ng/ml IL-1beta increased
the amount of detectable IL-15 mRNA in A549 cells, on average 3.2 ±
0.9 fold (SD, n = 9), and 2.7 ± 0.3 fold (SD, n = 3) respectively.
The same stimuli increased IL-8 mRNA 39 ± 12 fold (SD, n = 3), and
78 fold (n = 2) respectively. Figure
1 also shows that dexamethasone at concentrations of 10
6 M or lower did not diminish the TNF-alpha-induced increase of
IL-15 mRNA. In addition, the amount of IL-15 mRNA in unstimulated cells
was not decreased by dexamethasone at 10 6 M. Figure
2 shows that also the IL-1beta-mediated increase of IL-15 mRNA was
not diminished by dexamethasone. In contrast, dexamethasone dose-dependently
suppressed the TNF-alpha- or IL-1beta-induced increase of IL-8 mRNA in
the same experiment, which demonstrates that the glucocorticoid was active.
In two independent experiments cyclosporin A at 250 ng/ml did not suppress
the TNF-alpha-induced increase of IL-15 mRNA or IL-8 mRNA, although control
experiments showed that the same batch of CyA suppressed CD3-induced 3H-thymidine
incorporation of human mononuclear cells by 97.5% (data not shown). Furthermore,
controls showed that at 32 cycles the cDNA-PCR for IL-15, using the primer
pair "T2", was still in the exponential range of amplification. The non-effectiveness
of dexamethasone in decreasing the TNF-alpha- or IL-1beta-mediated induction
of IL-15 mRNA could be reproducibly detected. A synopsis of the experiments
performed is given in Figure 3,
which also contains data from two experiments which were obtained using
a different pair of primers ("R") for IL-15. It can be concluded from
Figure 3 that there was no difference
between dexamethasone-treated und untreated cells concerning the cytokine-mediated
increase of IL-15 mRNA in A549 cells (p = 0.26, paired t test), in contrast,
the cytokine-mediated increase of IL-8 mRNA was diminished by 10
6 M dexamethasone (p = 0.015, paired t test). Analysis of the subgroups
(stimulation by TNF-alpha or IL-1beta) yielded similar results.
In addition, we investigated whether dexamethasone at 10
6 M inhibited the TNF-alpha- or IL-1beta-induced increase of intracellular
IL-15 protein. Table 1 depicts
that dexamethasone did not suppress the TNF-alpha- or IL-1beta-induced
upregulation of intracellular IL-15 protein in A549 cells. Cyclosporin
A did not show an inhibitory effect here either. Control experiments revealed
that in A549 cells, dexamethasone at 10 6 M inhibited
the TNF-alpha-induced secretion of IL-8 into the supernatant by 80% (detection
by ELISA, data not shown).
Taken together, our data demonstrate
that in the A549 cell line, the induction of IL-15 mRNA and IL-15 protein
by pro-inflammatory cytokines, e.g. TNF-alpha and IL-1beta, is
not affected by dexamethasone in concentrations (10 6
M) which are sufficient to produce a maximal effect in mitogen-stimulated
lymphocytes [28]. To the best of our knowledge, this is the first report
on the non-effectiveness of glucocortidoids, dexamethasone taken as an
example, upon the expression of IL-15. Our findings are supported by a
recent abstract by Weiler et al. [29]. These authors report that
dexamethasone, rapamycin and cyclosporin A have no effect on the basal
and interferon-gamma-induced expression of IL-15 in cultures of human
cortical epithelial cells of the kidney. As the expression of IL-15 seems
to correlate with kidney graft rejection [17], and as IL-2/IL-4 double-knockout
mice still show graft rejection (30), IL-15 might play an important role
in kidney graft rejection. IL-15 protein has also been detected in other
inflammatory conditions such as rheumatoid arthritis [18] and inflammatory
bowel disease [19].The effect of pharmacological medication on expression
levels of IL-15 has not been investigated in all these diseases. Our data
and the abstract by Weiler support the idea that IL-15 might be a valuable
target for a type of anti-inflammatory pharmacological intervention different
from classical glucocorticoids, especially in the case of steroid-resistant
inflammation. In contrast to the findings described above, Gang-Wen Han
et al. [31] reported that dexamethasone inhibited the expression
of IL-15 mRNA in the squamous cell carcinoma cell line HSC-5.
The complete IL-15 receptor is composed of a proprietary
alpha-chain and the beta-chain (CD122) and gammac-chain of
the IL-2 receptor. Data by Dong-Wan Chae et al. [32] suggest that
the PHA-mediated induction of the alpha-chain of the IL-15 receptor is
sensitive to dexamethasone but is not affected by cyclosporin A. Whether
this mechanism is operational in vivo remains to be demonstrated,
as IL-15 can also bind to the CD122/gammac-dimer [33]. Another
level for possible pharmacological interference with IL-15 is the inhibition
of IL-15-mediated events such as proliferation of lymphocytes. In this
respect Degiannis et al. show that rapamycin inhibits the IL-15-mediated
proliferation of OKT-3-preactivated lymphocytes [34].
CONCLUSION
At present, the knowledge concerning the pharmacological
regulation of IL-15 expression is far from complete. As the contribution
of different inflammatory cell types to the overall level of IL-15 may
be different, future work is warranted and should deal with the effect
of steroids on the expression of IL-15 in non-epithelial cells.
Acknowledgements. The authors gratefully acknowledge
the expert technical assistance of Astrid Glöckner and Katharina
Helmerichs.
REFERENCES
REFERENCES
1. Grabstein K H, Eisenman J, Shanebeck K, Rauch C, Srinivasan
S, Fung V, Beers C, Richardson J, Schoenborn M A, Ahdieh M, Johnson L,
Alderson M R, Watson J D, Anderson D M, Giri J G. 1994. Cloning of a T
cell growth factor that interacts with the beta chain of the interleukin-2
receptor. Science 264: 965.
2. Mott H R, Campbell I D. 1995. Four-helix bundle growth
factors and their receptors: protein-protein interactions. (Review) Curr.
Opin. Struct. Biol. 5: 114.
3. Kennedy M K, Park L S. 1996. Characterization of interleukin-15
(IL15) receptor complex. (Review) J. Clin. Immunol. 16: 134.
4. Waldmann T A, Tagaya, Y. 1999. The multifaceted regulation
of interleukin-15 expression and the role of this cytokine in NK cell
differentiation and host response to intracellular pathogens. (Review)
Annu. Rev. Immunol. 17: 19.
5. Giri J G, Ahdieh M, Eisenman J, Shanebeck K, Grabstein
K, Kumaki S, Namen A, Park L S, Cosman D, Anderson D. 1994. Utilization
of the beta and gamma chains of the IL-2 receptor by the novel cytokine
IL-15. EMBO J. 13: 2822.
6. Giri J G, Kumaki S, Ahdieh M, Friend D J, Loomis A,
Shanebeck K, DuBose R, Cosman D, Park L S, Anderson D M. 1995. Identification
and cloning of a novel IL-15 binding protein that is structurally related
to the alpha chain of the IL-2-receptor. EMBO J. 14: 3654.
7. Lin J X, Migone T S, Tsang M, Friedmann M, Weatherbee
J A, Zhou L, Yamauchi A, Bloom E T, Mietz J, John S, Leonard W J. 1995.
The role of shared receptor motifs and common Stat proteins in the generation
of cytokine pleiotropy and redundancy by IL-2, IL-4, IL-7, IL-13 and IL-15.
Immunity. 2: 331.
8. Carson W E, Ross M E, Baiocchi R A, Marien M J, Boiani
N, Grabstein K, Caligiuri M A. 1995. 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.
9. Blauvelt A, Asada H, Klaus-Kovtun V, Altman D J, Lucey
D R, Katz S I. 1996. Interleukin-15 mRNA is expressed by human keratinocytes
Langerhans cells, and blood-derived dendritic cells and is downregulated
by ultraviolet B radiation. J. Invest. Dermatol. 106: 1047.
10. Reinecker H C, MacDermott R P, Mirau S, Dignass A,
Podolsky D K. 1996. Intestinal epithelial cells both express and respond
to interleukin-15. Gastroenterology. 111: 1706.
11. Kumaki N, Anderson D M, Cosman D, Kumaki S. 1996. Expression
of interleukin-15 and its receptor by human fetal retinal pigment epithelial
cells. Curr. Eye Res. 15: 876.
12. Lee Y B, Satoh J, Walker D G, Kim S U. 1996. Interleukin-15
gene expression in human astrocytes and microglia in culture. Neuroreport.
7: 1062.
13. Stoeck M, Kromer W, Gekeler V. 1998. Induction of IL-15
mRNA and protein in A549 cells by pro-inflammatory cytokines. Immunobiology.
199: 14.
14. Kotake S, Schumacher H R Jr, Yarboro C H, Arayssi T
K, Pando J A, Kanik K S, Gourley M F, Klippel J H, Wilder R.L. 1997. In
vivo gene expression of type 1 and type 2 cytokines in synovial tissues
from patients in early stages of rheumatoid, reactive, and undifferentiated
arthritis. Proc. Assoc. Am. Physicians. 109: 286.
15. Agostini C, Trentin L, Facco M, Sancetta R, Cerutti
A, Tassinari C, Cimarosto L, Adami F, Cipriani A, Zambello R, Semenzato
G. 1996. Role of IL-15, IL-2, and their receptors in the development of
T cell alveolitis in pulmanory sarcoidosis. J. Immunol. 157: 910.
16. Kivisäkk P, Matusevicius D, He B, Söderström
M, Fredrikson S, Link H. 1998. IL-15 mRNA expression is up-regulated in
blood and cerebrospinal fluid mononuclear cells in multiple sclerosis
(MS). Clin. Exp. Immunol. 111: 193.
17. Strehlau J, Pavlakis M, Lipman M, Shapiro M, Vasconcellos
L, Harmon W, Strom T B. 1997. Quantitative detection of immune activation
transcripts as a diagnostic tool in kidney transplantation. Proc. Natl.
Acad. Sci. USA 94: 695.
18. McInnes I B, al-Mughales J, Field M, Leung B P, Huang
F P, Dixon R, Sturrock R D, Wilkinson P C, Liew F Y. 1996. The role of
interleukin-15 in T-cell migration and activation in rheumatoid arthritis.
Nature Medicine 2: 175.
19. Kirman I, Nielsen O H. 1996. Increased numbers of interleukin-15
expressing cells in active ulcerative colitis. Am. J. Gastroenterol.
91: 1789.
20. Sakai T, Kusugami K, Nishimura H, Ando T, Yamaguchi
T, Ohsuga M, Ina K, Enomoto A, Kimura Y, Yoshikai Y. 1998. Interleukin-15
activity in the rectal mucosa of inflammatory bowel disease. Gastroenterology
114: 1237.
21. Kumaki S, Minegishi M, Fujie H, Sasahara Y, Ohashi
Y, Tsuchiya S, Konno T. 1998. Prolonged secretion of IL-15 in patients
with severe forms of acute graft-versus-host disease after allogeneic
bone marrow transplantation in children. Int. J. Hematol. 67: 307.
22. Ruchatz H, Leung B P, Wei X Q, McInnes I B, Liew F
Y. 1998. Soluble IL-15 receptor alpha-chain administration prevents murine
collagen-induced arthritis: a role for IL-15 in development of antigen-induced
immunopathology. J. Immunol. 160: 5654.
23. Kim Y S, Maslinski W, Zheng X X, Stevens A C, Li X
C, Tesch G H, Kelley V R, Strom T B. 1998. Targeting the IL-15 receptor
with an antagonist IL-15 mutant/Fc gamma2a protein blocks delayed-type
hypersensitivity. J. Immunol. 160: 5742.
24. Beck J, Handgretinger R, Dopfer R, Klingebiel T, Niethammer
D, Gekeler V. 1995. Expression of mdr1, mrp, topoisomerase II alpha/beta,
and cyclin A in primary or relapsed states of acute lymphoblastic leukaemias.
Br. J. Haematology 89: 356.
25. Meazza R, Verdiani S, Biassoni R, Coppolecchia M, Gaggero
A, Orengo A M, Colombo M P, Azzarone B, Ferrini S. 1996. Identification
of a novel interleukin-15 (IL-15) transcript isoform generated by alternative
splicing in human small cell lung cancer cell lines. Oncogene 12:
2187.
26. Jung, H C, Eckmann L, Yang S K, Panja A, Fierer J,
Morzycka-Wroblewska E , Kagnoff M F. 1995. A distinct array of proinflammatory
cytokines is expressed in human colon epithelial cells in response to
bacterial invasion. J. Clin. Invest. 95: 55.
27. Krause H, Jandrig B, Wernicke C, Bulfone-Paus S, Pohl
T, Diamantstein T. 1996. Genomic structure and chromosomal localization
of the human interleukin 15 gene (IL-15). Cytokine 8: 667.
28. Gillis S, Crabtree G R, Smith K A. 1979. Glucocorticoid-induced
inhibition of T-cell growth factor production. I. The effect on mitogen-induced
lymphocyte proliferation. J. Immunol. 123: 1624.
29. Weiler M, Chaimovitz C, Douvdevani A. 1998. Eur.
Cytokine Netw. 9: 465 (abstract).
30. Li X C, Roy-Chaudhury P, Hancock W W, Manfro R, Zand
M S, Li Y, Zheng X X, Nickerson P W, Steiger J, Malek T R, Strom T B.
1998. IL-2 and IL-4 double knockout mice reject islet allografts: a role
for novel T cell growth factors in allograft rejection. J. Immunol.
161: 890.
31. Han G W, Iwatsuki K, Inoue M, Matsui T, Nishibu A,
Akiba H, Kaneko F. 1999. Interleukin-15 is not a constitutive cytokine
in the epidermis, but is inducible in culture or inflammatory conditions.
Acta Derm. Venereol. 79: 37.
32. Chae D W, Nosaka Y, Strom T B, Maslinski W. 1996. Distribution
of IL-15 receptor alpha-chains on human peripheral blood mononuclear cells
and effect of immunosuppressive drugs on receptor expression. Immunol.
157: 2813.
33. Balasubramanian S, Chernov-Rogan T, Davis A M, Whitehorn
E, Tate E, Bell M P, Zurawski G, Barrett R W. 1995. Ligand binding kinetics
of IL-2 and IL-15 to heteromers formed by extracellular domains of the
three IL-2 receptor subunits. Int. Immunol. 7: 1839.
34. Degiannis D, Kalteziotis V, Thalassinos A, Kapsalis
A, Kroupis C, Koniavitou K. 1998. Effect of cyclosporin and Sirolimus
on interleukin-15-driven proliferation of OKT3-preactivated human lymphocytes.
Transplant. Proc. 30: 948.
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