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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 10 (May 15), 1999:
pp. 3531-3539
Human Monocytes Constitutively Express Membrane-Bound, Biologically
Active, and Interferon- -Upregulated Interleukin-15
By
Tiziana Musso,
Liliana Calosso,
Mario Zucca,
Maura Millesimo,
Daniela Ravarino,
Mirella Giovarelli,
Fabio Malavasi,
Alessandro Negro Ponzi,
Ralf Paus, and
Silvia Bulfone-Paus
From the Department of Public Health and Microbiology, Postgraduate
School of Clinical Pathology, Department of Genetics, Biology and
Medical Chemistry, and Department of Clinical and Biological Sciences,
University of Turin, Turin, Italy; the Institute of Biology and
Genetics, University of Ancona, Ancona, Italy; the Department of
Dermatology, Charité, Humboldt University, Berlin, Germany; and
the Institute for Immunology, University Hospital Benjamin Franklin,
Free University Berlin, Berlin, Germany.
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ABSTRACT |
Interleukin-15 (IL-15) is a potent regulator of T-, B-, and natural
killer cell proliferation and displays unusually tight controls of
secretion. Even though IL-15 mRNA is constitutively expressed in
monocytes/macrophages and is upregulated by a variety of stimuli,
evidence for IL-15 cytokine secretion is only found exceptionally, eg,
conditions of pathological, chronic inflammation. This raises the
possibility that monocytes express membrane-bound IL-15 rather than
secrete it. The current study explores this hypothesis. We demonstrate
here that biologically active IL-15 is indeed detectable in a
constitutively expressed, membrane-bound form on normal human
monocytes, as well as on monocytic cell lines (MONO-MAC-6, THP-1, and
U937), but not on human T or B cells (MT4, M9, C5966, JURKAT, DAUDI,
RAJI, and Epstein-Barr virus-immortalized B-cell clones). Furthermore,
cell surface-bound IL-15 is upregulated upon interferon-
stimulation. Interestingly, monocyte/macrophage inhibitory cytokines
such as IL-4 and IL-13 fail to downregulate both constitutive and
induced cell-surface expression of IL-15. Membrane-bound IL-15 does not
elute with acetate buffer or trypsin treatment, suggesting that it is
an integral membrane protein and that it is not associated with the
IL-15 receptor complex. Finally, membrane-bound IL-15 stimulates T
lymphocytes to proliferate in vitro, indicating that it is biologically
active. These findings enlist IL-15 in the fairly small family of
cytokines for which the presence of a biologically active
membrane-bound form has been demonstrated (eg, IL-1, tumor necrosis
factor- , and IL-10) and invites the speculation that most of the
biological effects of IL-15 under physiological conditions are exerted
by the cell surface-bound form.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
INTERLEUKIN-15 (IL-15) was originally
discovered as a T-cell stimulatory activity that mimics several
biological effects of IL-2.1,2 For example, like IL-2,
IL-15 induces T-cell proliferation and chemotaxis, stimulates natural
killer (NK) cell growth and interferon- (IFN-) production,
generates cytotoxic effector cells, and costimulates B-cell growth and
Ig production.2,3-6 In addition, IL-15 induces cytokine
production by monocytes and polymorphonuclear cells and stimulates the
antimicrobial activity of phagocytes.7-11 For binding and
signaling, IL-15 uses the IL-2R and chain12-14 as
well as the specific IL-15R chain.15,16
Although IL-2 is mainly produced by T cells, IL-15 mRNA is
constitutively expressed by a large variety of cell types and tissues, including monocytes/macrophages, fibroblasts, keratinocytes, and skeletal muscle.1,17 In monocytes/macrophages, IL-15 mRNA expression is upregulated by exogenous stimuli such as IFN- and lipopolysaccharide (LPS) or by bacteria, protozoa, and virus
infection.5,17,18-21 However, despite the widespread
expression of IL-15 mRNA, it has been very difficult to show IL-15
activity in cell culture supernatants using either bioassay or
enzyme-linked immunosorbent assay (ELISA).
Two IL-15 isoforms have been identified that differ in the length of
the signal peptide.22-25 IL-15 associated with the short signal peptide (SSP-IL-15) is not secreted, but rather stored intracellularly, where it appears in the nucleus and cytoplasm. The
alternative isoform, characterized by the longer signal peptide (LSP-IL-15), is located in the endoplasmic reticulum and is supposed to follow a pathway ending in its
secretion.22,23
This discrepancy between abundant IL-15 message, intracellularly
detectable IL-15 protein, yet inefficient or absent cytokine secretion
prompted us to speculate that, under physiological conditions, IL-15
may mainly be present in a membrane-bound, nonsecreted form. In this
study, we have explored this hypothesis, studying normal human
monocytes and monocytic cell lines as well as T and B cells. Biological
activity was tested by a T-cell proliferation assay, and association of
surface-bound IL-15 with plasma membrane or IL-15 receptor was
investigated using acetate buffer and trypsin treatment. Collectively,
our experiments suggest that human monocytes constitutively express
membrane-bound, biologically active, and IFN--inducible IL-15.
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MATERIALS AND METHODS |
Monocytes preparation and cell lines.
Peripheral blood mononuclear cells (PBMC) were obtained by
Ficoll-Hypaque gradient centrifugation from normal volunteers after informed consent was received. Monocytes were purified by Percoll gradient. The purity of the monocytes preparations used in this study
was 90% ± 4%, as assessed by morphology on Giemsa-stained cytocentrifuge preparations and by flow cytometry, using the
monocyte-specific monoclonal antibody (MoAb) LeuM3.7 Cells
were cultured in RPMI 1640 (GIBCO, Grand Island, NY) containing 100 U/mL penicillin, 100 µg/mL streptomycin, 2 mmol L-glutamine, 20 mmol
HEPES (GIBCO), and 10% heat-inactivated fetal bovine serum (HyClone
Lab, Logan, UT). This will be referred to as complete medium. Human
THP-1, U937, RAJI, DAUDI, JURKAT, SUPT-1, MT4, M9, and C5966 (American Type Culture Collection, Manassas, VA) were cultured in complete medium. The MONO-MAC-6 cell line, an acute leukemia monocytic cell line
originally established by H.W. Ziegler-Heitbrok, was obtained from the
German Collection of Microorganisms and Cell Cultures (Braunschweig,
Germany).26 These cells were maintained in complete medium
supplemented with 1 mmol/L sodium pyruvate and 9 µg bovine insulin
(Sigma Chemical Co, St Louis, MO) per milliliter.
Cytokines, antibodies, and reagents.
Human recombinant IFN-, IL-4, IL-13, and IL-10 were purchased from
Peprotech (Rocky Hill, NJ). Recombinant IL-15 was a kind gift of Dr A. Troutt (Immunex, Seattle, WA). Escherichia coli 0111:B4 LPS was
purchased from Sigma. Anti-IL-15 M111 and anti-IL-15 M112 (IgG2b)
MoAbs were purchased from Genzyme (Boston, MA). Anti-IL-2 was
purchased from Pharma Biotecnologie (Hannover, Germany). Anti-IL-2R A41 was prepared as described.27 Control IgG2b was
purchased from Becton Dickinson (San Jose, CA).
Fluorescein isothiocyanate (FITC)-labeled goat antimouse Ab used for
fluorescent staining was purchased from Southern Biotechnology
Associates, Inc (Birmingham, AL).
To obtain human IL-15 mouse Ig G2b (IgG2b) fusion protein, cDNA
encoding IL-15 was fused to genomic DNA encoding for the Fc portion of
mouse IgG2b as described.28 Biotinylation of human IL-15
mouse IgG2b fusion protein resulted in high stability of the cytokine
without reduction of its biological activity, as determined by
CTLL proliferation assay.29
Flow cytometry analysis.
For indirect immunofluorescence analysis, cells were preincubated for
30 minutes at 4°C in phosphate-buffered saline (PBS) containing 2%
goat serum plus 0.2% sodium azide (NaN3), washed with 1%
bovine serum albumine (BSA), and incubated with mouse MoAbs for 30 minutes at 4°C or with an isotype-matched MoAb. The cells were then
washed twice with 1% BSA in PBS and incubated with goat antimouse
Ig-FITC for a further 30 minutes at 4°C. Cytoplasmic IL-15 was
evaluated after permeabilization of the cell membranes using PermeaFix
(1:2 dilution; Ortho, Raritan, NJ). To determine the binding of
IL-15-IgG2b fusion protein, cells were incubated for 1 hour at 4°C
with the biotinylated IL-15-IgG2b fusion protein or with the
isotype-matched biotin-conjugated IgG (Pharmingen, San Diego, CA). The
cells were then washed twice with 1% BSA in PBS and further incubated
with FITC-conjugated avidin (Sigma) for 20 minutes at 4°C. IL-15
binding was assessed by flow cytometry. Labeled cells were analyzed by
flow cytometry analysis with a FACSort (Becton Dickinson
Immunocytometry Systems, San Jose, CA). Lysis II software was used to
analyze FACS data and significance was analyzed by comparison between
treated and untreated cells with z-test (*P < .001).
Reverse transcription-polymerase chain reaction (RT-PCR) analysis.
RNA extraction and RT-PCR analysis were performed as
described.10 Briefly, total RNA was purified with TRIzol
(Life Technologies, Gaithersburg, MD) as specified by the
manufacturer's instructions. cDNA synthesis was performed with 2 µg
of RNA in a total volume of 20 µL, containing 50 mmol/L Tris-HCl, pH
8.3, 75 mmol/L KCl, 3 mmol/L MgCl2, 0.1 mol/L
dithiothreitol, 40 U of RNase inhibitor (RNase OUT; Life Technologies),
0.5 µg oligo (dT), and 200 U Superscript II reverse transcriptase
(Life Technologies). The reaction mixture was incubated at 42°C for
50 minutes and stopped at 90°C for 5 minutes. A 2-µL aliquot of
the obtained cDNA was amplified in a 50 µL reaction containing 500 mmol/L KCl, 100 mmol/L Tris-HCl, pH 8.8, 25 mmol/L MgCl2, 2 mmol/L of each dNTP, 2 mg/mL BSA (Pharmacia, Uppsala, Sweden), 200 nmol/L of each primer, and 5 U Taq DNA polymerase (Life Technologies).
The mixture was capped with 50 µL of sterile mineral oil. To ensure
that equivalent amounts of cDNA were used in each reaction, PCR was
also performed for -actin from each sample, and the cDNA was
adjusted to equivalent levels. The following oligonucleotides were used
in the PCR reactions: IL-15 sense, 5'-GGATTTACCGTGGCTTTGAGTAATGAG-3', and IL-15 antisense,
5'-GAATCAATTGCAATCAAGAAGTG-3' (cycling conditions: 1 minute
at 94°C, 1 minute at 80°C, and 2 minutes at 72°C, for 35 cycles25); and -actin sense,
5'-GAGCGGGAAATCGTGCGTGACATT-3', and -actin antisense,
5'-GAAGGTAGTTTCGTGGATGCC-3' (cycling conditions: 1 minute
at 94°C, 1 minute and 30 seconds at 62°C, and 2 minutes at
72°C for 27 cycles).30 A sample (15 µL) of each PCR
reaction was electrophoresed through a 2% agarose gel and visualized
with ethidium bromide.
Acidic elution.
Acidic elution was performed as described.31 The cells were
pelleted by centrifugation and resuspended in acetate buffer, pH 4.4, containing 0.05 mol/L sodium acetate, 0.09 mol/L NaCl, 0.005 mol/L KCl,
and 0.05% fetal calf serum (FCS). The suspension was neutralized after
1 to 15 minutes by adding PBS containing 0.05% FCS. The suspended
cells were immediately underlayed with 1 mL FCS and centrifuged to
pellet the cells. The cells were then washed three times and stained as
indicated above. Up to 15 minutes of acid treatment did not
significantly change the cell viability as determined by forward and
right angle scatter and trypan blue exclusion.
Trypsin treatment.
Cells were washed with PBS and treated with 10 µg/mL
L-p-tosylamino-2-phenylethyl chloromethyl ketone (TPCK)-trypsin (Sigma) for 10 minutes at 37°C. The trypsin activity was neutralized by adding 100 µg/mL soybean trypsin inhibitor (Sigma).32
CD3+ T-cell proliferation assays.
CD3+ T cells were purified from peripheral blood
lymphocytes (PBL) cell suspensions by using
CD3+ T-cell subset enrichment columns (R&D Systems,
Minneapolis, MN). For mitogen assays, CD3+ were cultured
alone or with 12.5 × 103 monocytes in the presence of
10 µg/mL ConA (Sigma). Before the proliferation assay, monocytes were
treated with IFN- for 72 hours and with mitomycin-C (Sigma) at 25 µg/107 cells/mL for 30 minutes at 37°C. Each
experiment was performed in triplicate. Cultures were incubated for 72 hours at 37°C. Sixteen hours before the termination of the
cultures, the plates were pulsed with 0.5 µCi/well 3H
thymidine (Dupont, NEN, Boston, MA). The cells were harvested on paper
filters and 3H thymidine uptake was measured in a liquid
scintillation counter and expressed as total cpm.
Statistical analysis.
All in vitro experiments were performed independently at least three
times. Comparison among treatments was performed by analysis of
variance. When a difference among multiple treatments was found, the
Fisher's least significant difference test was used to identify which
of the means were significantly different from the others at the .05 significance level.
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RESULTS |
Monocytes display membrane-bound IL-15.
Constitutive expression of IL-15 mRNA has previously been demonstrated
in monocytes/macrophages.1,17 However, the IL-15 cytokine
could be hardly detected in the supernatant by standard ELISA and
CTL proliferation assays. Because of this apparent lack in
cytokine secretion, we have tested the hypothesis that IL-15, rather
than being released, is constitutively retained in functional form on
the cell surface of monocytes.
Using the anti-IL-15 antibody M112 in an indirect fluorescence assay,
purified peripheral blood monocytes as well as the monocytic cell lines
MONO-MAC-6, THP-1, and U937 demonstrated specific immunoreactivity on
the surface of tested cells. Immunoreactivity was not seen with
purified T lymphocytes (neither resting nor phytohemagglutinin [PHA]-activated T cells) or the JURKAT T-cell line
(Fig 1). In addition, the screened panel of
T- and B-cell lines (MT4, M9, C5966, SUPT-1; RAJI; DAUDI; and
Epstein-Barr virus [EBV]-immortalized B-cell clones) gave negative
immunostaining results. These data were fully reproduced using a
different MoAb against IL-15 (M111; data not shown).
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| Fig 1.
Flow cytometric analysis with anti-IL-15 M112 antibody.
Human peripheral blood purified monocytes (MONOCYTES) and T lymphocytes
(LYMPHOCYTES), human monocytic cell lines (MONO-MAC-6, THP-1, U937),
and a human T-cell leukemia line (JURKAT) were stained with M112 MoAb
followed by goat antimouse FITC-conjugated antibody. Fluorescence
intensity is represented by white histograms; black histograms refer to
the background staining of isotype-matched control MoAb. Data show one
representative of five independent experiments.
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To show the specificity of the binding, we tested whether the addition
of exogenous IL-15 competed for the binding of the M112 antibody to
MONO-MAC-6 cells. This was the case, because a dose-dependent decrease
in antibody staining was observed by preincubating the cells with IL-15
in a dosage range from 10 µg to ng dilution
(Fig 2).
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| Fig 2.
Inhibition of M112 MoAb binding by IL-15. MONO-MAC-6
cells were preincubated for 30 minutes at 4°C with medium (A), 0.01 µg (B), 0.1 µg (C), 1 µg (D), or 10 µg (E) of human recombinant
IL-15 and then stained with M112 MoAb as described in Fig 1. Open
histograms with a bold line represent M112 staining; open histograms
with a gray line represent the residual staining after preincubation
with IL-15; and solid histograms refer to the background
fluorescence.
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Thus, IL-15 is constitutively present in membrane-bound form on
peripheral blood monocytes as well as on the tested monocytic cell
lines and can be specifically recognized by antibodies normally used
against the soluble cytokine.
IFN- significantly upregulates expression of the
membrane-associated form of IL-15.
Various monocyte activators have been shown to upregulate IL-15 mRNA
expression and to augment the levels of cytoplasmic
IL-15.5,17,18 Monocyte activators may also give rise to
detectable IL-15 activity in the supernatant of treated cells, although
this remains controversial.17 Therefore, we next determined
if IFN- and/or LPS, two standard monocyte/macrophage activating
agents, are able to modulate IL-15 membrane expression as well as the
cytoplasmic and mRNA levels of the cytokine.
Purified peripheral blood monocytes
(Fig 3A) and MONO-MAC-6 cells
(Fig 3B) were stimulated for 24 hours with IFN- (500 U/mL), LPS (5 µg/mL), or LPS plus IFN- or left untreated and stained with M112
MoAb. As shown in Fig 3A and B, IFN- significantly increased IL-15
membrane levels, whereas LPS did not affect membrane IL-15 expression
and did not potentiate the effect induced by IFN-.
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| Fig 3.
IFN- upregulates IL-15 expression. The
membrane and cytoplasmic forms of IL-15 were evaluated by FACS
analysis. Human peripheral blood monocytes (A) and MONO-MAC-6 cells (B)
were treated with IFN-, LPS, or LPS + IFN- or left untreated
and were stained with the M112 MoAb as described in Materials and
Methods. The mean ± SD from three independent experiments is shown.
Significance was analyzed by comparison between treated and untreated
cells with z-test (*P < .001). (C) cDNA derived from
MONO-MAC-6 cells incubated for 6 hours with medium alone or
supplemented with IFN-, LPS, or LPS plus IFN- were amplified with
primers specific for IL-15. -actin served as the control.
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The expression of cytoplasmic IL-15 was evaluated by permeabilizing
cell membranes before M112 staining. Untreated monocytes and MONO-MAC-6
cells both showed a basal level of intracellular staining that
significantly increased after treatment with IFN-. However, LPS did
not significantly affect intracytoplasmic IL-15 (Fig 3A and B). This
pattern of response was confirmed at the gene level by RT-PCR analysis
on MONO-MAC-6 cells. As shown in Fig 3C by a representative example, a
low constitutive level of IL-15 mRNA expression was detectable in
untreated MONO-MAC-6 but was strongly upregulated by incubation with
IFN- for 6 hours. Stimulation with LPS alone did not significantly
affect IL-15 mRNA and costimulation with LPS plus IFN- did not
further upregulate the IL-15 mRNA level over the expression level
stimulated by IFN- alone. The same pattern of response at the mRNA
level was observed with peripheral blood monocytes (not shown).
When supernatants of LPS-treated, IFN--treated, or LPS plus
IFN--treated monocytes and MONO-MAC-6 cells were tested for the
presence of secreted, soluble IL-15 protein by ELISA and for IL-15
activity by CTLL proliferation assay, no detectable IL-15 could be
demonstrated (not shown).
To determine the effect of the IFN- concentration on IL-15 membrane
expression, MONO-MAC-6 were cultured for 24 hours with increasing
concentrations of IFN- and were subsequently stained with M112 MoAb
as described above. IFN- at 20 U/mL significantly increased IL-15
membrane expression, which reached a maximum at 100 U/mL
(Fig 4A).
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| Fig 4.
Dose- and time-dependent upregulation of membrane
IL-15 after IFN- treatment. MONO-MAC-6 were treated with the
indicated amounts of IFN- for 24 hours (A). In (B), MONO-MAC-6 were
treated with medium alone or with IFN- (500 U/mL) for the indicated
time. Cells were then stained with M112 MoAb as described in Materials
and Methods and were analyzed by FACS. Results represent mean ± SD of
three independent experiments. Significance was analyzed by comparison
between treated and untreated cells with the z-test (*P < .001).
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To determine the kinetic of the induction, membrane IL-15 was assessed
at different time points after exposure to IFN-. As shown in Fig 4B,
an increase in the IL-15-associated fluorescence intensity was
observed 6 hours after IFN- stimulation and reached a plateau at 24 hours. Similar kinetics and dose-response were obtained with purified
peripheral blood monocytes (not shown).
Thus, the constitutive expression of membrane-bound IL-15 is strongly
and significantly upregulated upon monocyte activation in a
dose-dependent manner by IFN- treatment. In our experimental setting, LPS failed to significantly induce IL-15 expression.
The membrane form of IL-15 is not associated with the IL-15R complex.
From the data presented above, it cannot be excluded that IFN-
induces IL-15 to be secreted and that, after release, the cytokine
binds to its own receptor complex. In fact, monocytes constitutively
express the 33 and 34 chains of the
IL-2R, which are shared by the IL-15R complex. In addition, IFN-
treatment is known to upregulate IL-2R and to induce IL-15R
expression.16,34 To investigate this possibility,
IFN--treated MONO-MAC-6 were incubated with acetate buffer, pH 4.4, which solubilizes cytokines associated with their cognate
receptors.31,35
Figure 5A shows that there was no change in
the mean fluorescence intensity of MONO-MAC-6 stained with anti-IL-15
MoAb after 10 minutes of treatment with acid buffer compared with cells
incubated in normal medium. This suggests that membrane-bound IL-15 is
not attached to its receptor but is an integral protein. As a control, we tested the effect of the acetate buffer treatment on the binding of
an IL-15-IgG2b fusion protein to MONO-MAC-6 cells. As shown in Fig 5B
(center panel), the binding of the IL-15 component to the IL-15R was
totally displaced by such treatment.
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| Fig 5.
Cell surface IL-15 is not associated with its own
receptor. (A) MONO-MAC-6 were treated with IFN- (500 U/mL) for 24 hours. Cells were then washed and incubated in PBS, acetate buffer (pH
4.4), or trypsin as described in Materials and Methods. All groups were
stained with M112 MoAb and analyzed on FACSort. Fluorescence intensity
is represented by open histograms; solid histograms refer to the
background staining of isotype-matched control MoAb. (B) MONO-MAC-6
treated with IFN- as described above were incubated with PBS (left
panel) or trypsin (right panel). Cells were then incubated with
biotin-conjugated IL-15 IgG2b fusion protein (open histogram) or with
equal amount of isotype-matched biotinylated IgG (solid histogram). In
the central panel, cells were incubated with the IL-15 IgG2b fusion
protein and then with acetate buffer, pH 4.4. The x axis represents the
intensity of green fluorescence expressed in a log scale as mean
channel, and the y axis represents the number of cells per channel.
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This concept was further probed by an additional experimental approach.
We have observed that IL-15R can be cleaved by trypsin treatment so
that the cells lose their ability to bind the IL-15-IgG2b fusion
protein used as receptor detector. This made it reasonable to expect
that, if IL-15 detected on the membrane was indeed bound to its
receptor, the whole ligand-receptor complex should be cleaved by
trypsin, and trypsinized cells should not bind either M112 or
IL-15-IgG2b. Therefore, IFN--induced MONO-MAC-6 were treated with
10 µg/mL trypsin for 10 minutes at 37 °C and stained with M112
antibody or IL-15-IgG2b. As shown in Fig 5A, the fluorescence intensity due to M112 staining was unchanged, whereas IL-15-IgG2b did
not bind any longer to the cell surface, indicating that IL-15R had
successfully been cleaved from the cell membrane (Fig 5B).
These results indicate that the cell surface IL-15 immunoreactivity is
not due to IL-15 bound to its cognate receptor.
Monocyte-inhibitory cytokines do not affect IL-15 membrane
expression.
Many of the functionally important monocyte/macrophage activities
induced by IFN- and other monocyte activators can be downregulated by IL-4, IL-13, and/or IL-10.36-41 However, it has recently
been reported that IL-4 and IL-13 fail to significantly inhibit IL-15 gene expression, whereas IL-10 was even able to upregulate the message
for IL-15 in murine macrophages.17 Therefore, it was of
interest to establish whether these cytokines could modulate the
expression of membrane-bound IL-15. To test this, anti-IL-15 staining
of MONO-MAC-6 treated for 24 hours with medium alone, IFN- (500 U/mL), IL-4 (100 U/mL), IL-13 (50 ng/mL), IL-10 (100 U/mL), or IFN-
plus IL-4, IL-13, or IL-10 was performed.
Figure 6 shows that, among the tested
cytokines, only IFN- significantly upregulated membrane IL-15,
whereas IL-4, IL-13, and IL-10 had no modulatory effects when added to
the cultures singly. Furthermore, these cytokines were unable to
counteract the stimulatory effect of IFN-. Conversely, IL-4, IL-13,
and IL-10 treatment of MONO-MAC-6 inhibited monocyte chemotactic
protein-1 (MCP-1) production induced by IFN- (not shown)
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| Fig 6.
Inhibitory cytokines do not affect IL-15 membrane
expression. MONO-MAC-6 were treated as indicated for 24 hours and
stained with M112 MoAb as described in Materials and Methods. One
representative experiment of three independent experiments, which gave
similar results, is shown.
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Membrane-bound IL-15 is biologically active.
IL-15 is a potent growth factor for T-cell
proliferation.1,42 To investigate the ability of
membrane-bound IL-15 to support T-cell proliferation, ConA-stimulated
human purified CD3+ T cells were cocultured with
mitomycin-treated monocytes that had previously been stimulated with
IFN- for 72 hours to assure an optimal membrane level of IL-15.
T-cell proliferation was assessed by a [3H] thymidine
uptake test.
This assay showed that proliferation of CD3+ T cells was
significantly enhanced by the presence of IFN--treated monocytes as
compared with T cells alone (Fig 7).
Monocytes from 8 subjects were treated with IFN-, which increased
their ability to stimulate T-cell proliferation up to 33% as compared
with untreated monocytes (181.140 ± 22.700 v 136.630 ± 33.370, P < .01). The addition of neutralizing
anti-IL-1543 and blocking anti-IL-2R12,13
antibodies to the culture significantly inhibited the CD3+
T-cell proliferation induced by coincubation with IFN--activated, mitomycin-treated monocytes (Fig 7). Furthermore, using ELISA it was
determined that the anti-IL-15 treatment of the coculture inhibited
the IFN- release from activated T cells in the supernatants by 50%
(not shown). The addition of anti-IL-2 antibodies also had an
inhibitory effect on proliferation by blocking the IL-2 released by
stimulated T cells. The combination of neutralizing anti-IL-15 and
anti-IL-2 antibodies showed an additive inhibitory effect on
proliferation. The isotype-matched antibody showed no effect.
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| Fig 7.
Membrane-bound IL-15 is biologically active. Human
purified CD3+ T cells were incubated with medium, IL-15
(10 ng/mL), IFN--treated and mitomycin C-treated monocytes, or the
latter plus anti-IL-15, anti-IL-2, or anti-IL-2R antibodies (10 µg/mL), all in presence of ConA (10 µg/mL). Proliferative activity
was assessed by a (3H) thymidine incorporation assay. One
representative experiment of three that produced similar results is
shown. Significance was analyzed by comparison between antibody-treated
and untreated cells with Fisher's Least-test. *Significant induction
by treatment (P < .05). §Significant inhibition
by treatment (P < .05).
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To exclude the possibility that the functional effects described above
simply resulted from the shedding of previously membrane-bound IL-15
into the medium, the supernatants were also tested by ELISA for the
presence of soluble IL-15. However, no IL-15 protein could be detected
(data not shown), suggesting that the CD3+ proliferative
effect observed after coculture with monocytes reflects genuine
bioactivity of membrane-bound IL-15.
Taken together, these experiments suggest that IL-15 expressed on the
cell surface of IFN--stimulated monocytes is functionally active.
| |
DISCUSSION |
In this study, we show that IL-15 exists as a biologically active, cell
surface-bound molecule on human monocytes and monocytic cell lines. Its
expression is upregulated by IFN- treatment, but unaffected by IL-4,
IL-13, and IL-10, and it is not associated with the IL-15R complex.
We have shown that, although IFN- is a potent inducer of IL-15 mRNA
and of intracytoplasmic and membrane-bound IL-15 in both monocytes and
MONO-MAC-6 cells, LPS does not significantly affect IL-15 expression at
any level, although, in other respects, MONO-MAC are responsive to
LPS.44 These results differ from those of Carson et
al,5 who observed induction of intracytoplasmatic IL-15 in
monocytes treated with LPS. However, these investigators used monocytes
purified by adherence, a procedure known to provide additional stimuli
for cytokine induction.45 Bamford et al19 have
shown that high levels of IL-15 mRNA are expressed in monocytes treated
with LPS plus IFN-. However, the effect of LPS or IFN- alone is
not mentioned in that study,19 so the reported effect might
well reflect the same IL-15 upregulation by IFN- reported here.
In murine systems, IL-15 mRNA is upregulated by several microbiological
agents, including mycobacteria, Toxoplasma gondii, and
LPS.17,19 Pretreatment of mouse macrophages with IFN- synergizes with BCG to increase IL-15 mRNA expression, but IFN- has
little effect if added alone or after microbial stimulation. Our
current data suggest that, in human monocytes, IFN- alone is a
potent stimulus for IL-15 mRNA expression. This discrepancy could be
explained by the differences between the two species in
question as well as by the sources of cells (bone marrow or peritoneal
macrophages v monocytes from peripheral blood). During the
activation of murine macrophages, IFN- usually acts as a priming
stimulus, and to achieve activation a second signal either physiologic
(eg, IL-2) or environmental (eg, LPS) is required. In contrast, human
monocytes can be activated by IFN- alone without the need for
costimulatory signals.46
IL-4, IL-13, and IL-10 downregulate the activation of mononuclear
phagocytes and antagonize IFN--induced responses, including the
production of several cytokines. Whereas IL-4, IL-13, and IL-10
inhibited IFN--induced expression of MCP-1 (data not shown), the
same cytokines failed to inhibit the constitutive expression of
membrane IL-15 as well as the upregulation induced by IFN- treatment. Our results are in agreement with the study by Doherty et
al17 in which the same cytokines did not influence IL-15 message expression in the murine system. Thus, IL-15 cytokine expression levels appear to be much more stable and less easily influenced than those of other proinflammatory cytokines.
Despite the very widespread distribution of IL-15 mRNA, it has been
difficult to demonstrate IL-15 in the supernatants of many cells that
express its message. In fact, only a single report demonstrates that
murine macrophages activated in vitro with live mycobacteria produce
detectable levels of IL-15 bioactivity as assessed with the CTLL-2
bioassay.17 We were unable to detect any IL-15 activity or
IL-15 protein in the culture of monocytic cells, even upon a strong
upregulation of IL-15 at the level of mRNA, membrane, and cytoplasmic
form after IFN- stimulation. However, addition of neutralizing
anti-IL-15 antibody to different in vitro models specifically
abrogated IL-15 biological functions.5,18,43 In the current
study, we show that, in the absence of detectable IL-15 in the
supernatants and in the presence of a membrane-bound IL-15 form,
IFN--treated and mitomycin-treated monocytes can stimulate T-cell
proliferation as well as IFN- release, which can be blocked by IL-15
neutralizing antibodies.
Thus, we can postulate that the membrane-bound form of IL-15 is likely
to be the biologically active cytokine that directs the activities
mentioned above. We are currently investigating if the IL-15
membrane-associated form also exerts other biological activities
attributed to IL-15 such as preventing apoptosis in multiple
systems.28,47
IL-15 production seems to be regulated at multiple levels. It has been
proposed that the control is mainly posttranscriptional, ie, at the
level of protein translation and intracellular trafficking rather than
transcription.19,22,23 Two isoforms differing in the leader
peptide sequences have been identified. Transfection of COS cells with
the different isoforms showed that IL-15 associated with the short
signal peptide is not secreted but is stored intracellularly, appearing
in the nucleus and cytoplasmic components; on the contrary, the long
signal peptide isoform is observed in the ER but its secretion is
inefficient.22,23 Preliminary transfection experiments, with constructs carrying the two different isoforms, are being performed to clarify if the long signal peptide isoform is the form
presented on the membrane. The results so far suggest that the
expression of the membrane form of IL-15 does not depend on the signal
peptide sequence.
The concept of the existence of a membrane-bound form of IL-15 is
intriguing, because it enlists IL-15 into a small family of cytokines
for which such cell surface-associated isoforms have also been
detected. Like IL-15, other cytokines playing a pivotal role in the
modulation of immune responses such as tumor necrosis factor-
(TNF-), IL-1, IL-10, and transforming growth factor (TGF) have been reported to be secreted and to have a
membrane form that is biologically active.31,35,48-51 It
now needs to be clarified whether the secreted and membrane-bound IL-15
isoforms act differently and whether the membrane-bound IL-15 can be
cleaved and released. We are currently testing if the activity of
membrane bound IL-15 in juxtacrine signaling may be controlled by
protease/antiprotease systems.52 It is tempting to
speculate that membrane-associated IL-15 modulates constitutive,
physiologic immune response, whereas the intracytoplasmic IL-15 pool is
kept in store to be secreted only upon special demands and under
pathological conditions.
| |
ACKNOWLEDGMENT |
The authors thank Dr Raffaele Badolato and Dr Thomas Pohl for helpful discussions.
| |
FOOTNOTES |
Submitted August 26, 1998; accepted January 17, 1999.
Supported in part by grants from the 2nd National Project
on Tuberculosis (ISS Rome, Italy), from AIRC (Milan, Italy), and from the Deutsch Forschungsgemainschaft (SFB 506/C5).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Tiziana Musso, PhD, Institute
of Microbiology, Via Santena 9, 10126 Torino, Italy; e-mail:
musso{at}molinette.unito.it.
| |
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PNAS,
June 14, 2005;
102(24):
8662 - 8667.
[Abstract]
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R. Ing, P. Gros, and M. M. Stevenson
Interleukin-15 Enhances Innate and Adaptive Immune Responses to Blood-Stage Malaria Infection in Mice
Infect. Immun.,
May 1, 2005;
73(5):
3172 - 3177.
[Abstract]
[Full Text]
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L. Wenxin, F. Jinxiang, W. Yong, L. Wenxiang, S. Wenbiao, and Z. Xueguang
Expression of membrane-bound IL-15 by bone marrow fibroblast-like stromal cells in aplastic anemia
Int. Immunol.,
April 1, 2005;
17(4):
429 - 437.
[Abstract]
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B. W. Blaser, S. Roychowdhury, D. J. Kim, N. R. Schwind, D. Bhatt, W. Yuan, D. F. Kusewitt, A. K. Ferketich, M. A. Caligiuri, and M. Guimond
Donor-derived IL-15 is critical for acute allogeneic graft-versus-host disease
Blood,
January 15, 2005;
105(2):
894 - 901.
[Abstract]
[Full Text]
[PDF]
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R. H. McIntire, P. J. Morales, M. G. Petroff, M. Colonna, and J. S. Hunt
Recombinant HLA-G5 and -G6 drive U937 myelomonocytic cell production of TGF-{beta}1
J. Leukoc. Biol.,
December 1, 2004;
76(6):
1220 - 1228.
[Abstract]
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C.-S. Park, S.-O. Yoon, R. J. Armitage, and Y. S. Choi
Follicular Dendritic Cells Produce IL-15 That Enhances Germinal Center B Cell Proliferation in Membrane-Bound Form
J. Immunol.,
December 1, 2004;
173(11):
6676 - 6683.
[Abstract]
[Full Text]
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A. HAILU, T. VAN DER POLL, N. BERHE, and P. A. KAGER
ELEVATED PLASMA LEVELS OF INTERFERON (IFN)-{gamma}, IFN-{gamma} INDUCING CYTOKINES, AND IFN-{gamma} INDUCIBLE CXC CHEMOKINES IN VISCERAL LEISHMANIASIS
Am J Trop Med Hyg,
November 1, 2004;
71(5):
561 - 567.
[Abstract]
[Full Text]
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A. Ozawa, H. Tada, Y. Sugawara, A. Uehara, T. Sasano, H. Shimauchi, H. Takada, and S. Sugawara
Endogenous IL-15 Sustains Recruitment of IL-2R{beta} and Common {gamma} and IL-2-Mediated Chemokine Production in Normal and Inflamed Human Gingival Fibroblasts
J. Immunol.,
October 15, 2004;
173(8):
5180 - 5188.
[Abstract]
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V. Budagian, E. Bulanova, Z. Orinska, T. Pohl, E. C. Borden, R. Silverman, and S. Bulfone-Paus
Reverse Signaling through Membrane-bound Interleukin-15
J. Biol. Chem.,
October 1, 2004;
279(40):
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[Abstract]
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[PDF]
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V. Budagian, E. Bulanova, Z. Orinska, A. Ludwig, S. Rose-John, P. Saftig, E. C. Borden, and S. Bulfone-Paus
Natural Soluble Interleukin-15R{alpha} Is Generated by Cleavage That Involves the Tumor Necrosis Factor-{alpha}-converting Enzyme (TACE/ADAM17)
J. Biol. Chem.,
September 24, 2004;
279(39):
40368 - 40375.
[Abstract]
[Full Text]
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G. G. Neely, S. Epelman, L. L. Ma, P. Colarusso, C. J. Howlett, E. K. Amankwah, A. C. McIntyre, S. M. Robbins, and C. H. Mody
Monocyte Surface-Bound IL-15 Can Function as an Activating Receptor and Participate in Reverse Signaling
J. Immunol.,
April 1, 2004;
172(7):
4225 - 4234.
[Abstract]
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R. Vankayalapati, P. Klucar, B. Wizel, S. E. Weis, B. Samten, H. Safi, H. Shams, and P. F. Barnes
NK Cells Regulate CD8+ T Cell Effector Function in Response to an Intracellular Pathogen
J. Immunol.,
January 1, 2004;
172(1):
130 - 137.
[Abstract]
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[PDF]
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J. Giron-Michel, A. Caignard, M. Fogli, D. Brouty-Boye, D. Briard, M. van Dijk, R. Meazza, S. Ferrini, C. Lebousse-Kerdiles, D. Clay, et al.
Differential STAT3, STAT5, and NF-{kappa}B activation in human hematopoietic progenitors by endogenous interleukin-15: implications in the expression of functional molecules
Blood,
July 1, 2003;
102(1):
109 - 117.
[Abstract]
[Full Text]
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E. Bulanova, V. Budagian, Z. Orinska, H. Krause, R. Paus, and S. Bulfone-Paus
Mast Cells Express Novel Functional IL-15 Receptor {alpha} Isoforms
J. Immunol.,
May 15, 2003;
170(10):
5045 - 5055.
[Abstract]
[Full Text]
[PDF]
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J. H. Von der Thusen, J. Kuiper, T. J. C. Van Berkel, and E. A. L. Biessen
Interleukins in Atherosclerosis: Molecular Pathways and Therapeutic Potential
Pharmacol. Rev.,
March 1, 2003;
55(1):
133 - 166.
[Abstract]
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M. Kurowska, W. Rudnicka, E. Kontny, I. Janicka, M. Chorazy, J. Kowalczewski, M. Ziolkowska, S. Ferrari-Lacraz, T. B. Strom, and W. Maslinski
Fibroblast-Like Synoviocytes from Rheumatoid Arthritis Patients Express Functional IL-15 Receptor Complex: Endogenous IL-15 in Autocrine Fashion Enhances Cell Proliferation and Expression of Bcl-xL and Bcl-2
J. Immunol.,
August 15, 2002;
169(4):
1760 - 1767.
[Abstract]
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D. Briard, D. Brouty-Boye, B. Azzarone, and C. Jasmin
Fibroblasts from Human Spleen Regulate NK Cell Differentiation from Blood CD34+ Progenitors Via Cell Surface IL-15
J. Immunol.,
May 1, 2002;
168(9):
4326 - 4332.
[Abstract]
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[PDF]
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P. Y. Ong, Q. A. Hamid, J. B. Travers, I. Strickland, M. A. Kerithy, M. Boguniewicz, and D. Y. M. Leung
Decreased IL-15 May Contribute to Elevated IgE and Acute Inflammation in Atopic Dermatitis
J. Immunol.,
January 1, 2002;
168(1):
505 - 510.
[Abstract]
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[PDF]
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U. Schonbeck and P. Libby
CD40 Signaling and Plaque Instability
Circ. Res.,
December 7, 2001;
89(12):
1092 - 1103.
[Abstract]
[Full Text]
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E. Bulanova, V. Budagian, T. Pohl, H. Krause, H. Durkop, R. Paus, and S. Bulfone-Paus
The IL-15R{alpha} Chain Signals Through Association with Syk in Human B Cells
J. Immunol.,
December 1, 2001;
167(11):
6292 - 6302.
[Abstract]
[Full Text]
[PDF]
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G. G. Neely, S. M. Robbins, E. K. Amankwah, S. Epelman, H. Wong, J. C. L. Spurrell, K. K. Jandu, W. Zhu, D. K. Fogg, C. B. Brown, et al.
Lipopolysaccharide-Stimulated or Granulocyte-Macrophage Colony-Stimulating Factor-Stimulated Monocytes Rapidly Express Biologically Active IL-15 on Their Cell Surface Independent of New Protein Synthesis
J. Immunol.,
November 1, 2001;
167(9):
5011 - 5017.
[Abstract]
[Full Text]
[PDF]
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F. Mattei, G. Schiavoni, F. Belardelli, and D. F. Tough
IL-15 Is Expressed by Dendritic Cells in Response to Type I IFN, Double-Stranded RNA, or Lipopolysaccharide and Promotes Dendritic Cell Activation
J. Immunol.,
August 1, 2001;
167(3):
1179 - 1187.
[Abstract]
[Full Text]
[PDF]
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T. A. Fehniger and M. A. Caligiuri
Interleukin 15: biology and relevance to human disease
Blood,
January 1, 2001;
97(1):
14 - 32.
[Full Text]
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C. AGOSTINI, M. FACCO, M. SIVIERO, D. CAROLLO, S. GALVAN, A. M. CATTELAN, R. ZAMBELLO, L. TRENTIN, and G. SEMENZATO
CXC Chemokines IP-10 and Mig Expression and Direct Migration of Pulmonary CD8+/CXCR3+ T Cells in the Lungs of Patients with HIV Infection and T-Cell Alveolitis
Am. J. Respir. Crit. Care Med.,
October 1, 2000;
162(4):
1466 - 1473.
[Abstract]
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[PDF]
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K. Kitaya, J. Yasuda, I. Yagi, Y. Tada, S. Fushiki, and H. Honjo
IL-15 Expression at Human Endometrium and Decidua
Biol Reprod,
September 1, 2000;
63(3):
683 - 687.
[Abstract]
[Full Text]
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R. Ruckert, K. Asadullah, M. Seifert, V. M. Budagian, R. Arnold, C. Trombotto, R. Paus, and S. Bulfone-Paus
Inhibition of Keratinocyte Apoptosis by IL-15: A New Parameter in the Pathogenesis of Psoriasis?
J. Immunol.,
August 15, 2000;
165(4):
2240 - 2250.
[Abstract]
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[PDF]
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P. Jourdan, J.-P. Vendrell, M.-F. Huguet, M. Segondy, J. Bousquet, J. Pene, and H. Yssel
Cytokines and Cell Surface Molecules Independently Induce CXCR4 Expression on CD4+ CCR7+ Human Memory T Cells
J. Immunol.,
July 15, 2000;
165(2):
716 - 724.
[Abstract]
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J. A. Gollob, J. W. Mier, K. Veenstra, D. F. McDermott, D. Clancy, M. Clancy, and M. B. Atkins
Phase I Trial of Twice-Weekly Intravenous Interleukin 12 in Patients with Metastatic Renal Cell Cancer or Malignant Melanoma: Ability to Maintain IFN-{{gamma}} Induction Is Associated with Clinical Response
Clin. Cancer Res.,
May 1, 2000;
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1678 - 1692.
[Abstract]
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[PDF]
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TOP
ABSTRACT
INTRODUCTION