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Departments of
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Medical Sciences,
Oncology,
Biochemistry and Molecular Biology,
Microbiology and Infectious Diseases,
¶ Molecular Pathology,
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Medicine, and
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Internal Medicine, University of Calgary, Calgary, Alberta, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cell growth factors, which include the type I cytokines IL-2 and
IL-15, are essential during the initial development of lymphocyte
responses. Through the 
and 
common chains and Janus
kinase/STAT signaling apparatus (1, 2, 3, 4, 5), IL-2 and
IL-15 provide signals for activation, differentiation, and homeostatic
control of T cells and NK cells (6, 7, 8, 9, 10).
The tissue source and factors controlling IL-2 are well established and appear to be quite different from those controlling IL-15. Resting T cells do not express IL-2, but transcript is expressed upon Ag-specific T cell activation (11, 12, 13). This transcript codes for IL-2 containing a signal peptide that directs IL-2 protein through the endoplasmic reticulum and promotes efficient secretion (14). By contrast, there are two isoforms of the IL-15 transcript. One, encoding a 48-aa long signal peptide, IL-15-LSP, results in inefficient secretion (1, 15, 16), while the other, with a 21-aa short signal peptide, IL-15-SSP (17), does not direct secretion (16). The role of soluble IL-15 has been determined in a variety of studies (18). However, a recent report indicates that IL-15-LSP, which results in inefficient secretion, is more effective in viral host defense than IL-15 that has been engineered to express an efficient signal sequence (19). Although these studies suggest that IL-15 may play an important role in host defense by a mechanism other than secretion, the role of unsecreted IL-15, potentially the predominant fate of IL-15, is not clear.
In the current study, we address the regulatory stages limiting IL-15 expression in human monocytes. Using semiquantitative RT-PCR, immunoblotting, intracellular cytokine labeling and flow cytometric analysis, and a cell-surface ELISA, we examined IL-15 mRNA and protein expression in resting human PBMC. Expression of IL-15 protein in PBMC subpopulations was addressed using two-color flow cytometry. Using cell-surface flow cytometry, we define the role of transcription and translation in inducible IL-15 surface expression. Finally, using a modified CTLL-2 assay, we explore the function of surface-bound IL-15 on activated monocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peripheral blood was obtained by venipuncture from healthy adults. Blood was anticoagulated by adding 10 U/ml heparin (Organon Teknika-Cappel, Scarborough, Ontario, Canada). PBMC were purified by centrifugation (800 x g for 20 min) on a Ficoll-Hypaque density gradient (C-SIX Diagnostics, Mequon, WI). The cells were washed three times in HBSS (Life Technologies, Burlington, Ontario, Canada), counted in a hemocytometer, and resuspended in complete medium containing RPMI 1640 (Life Technologies), 5% heat-inactivated human AB serum (BioWhittaker, Walkersville, MD), 2 mM L-glutamine (Life Technologies), 100 U/ml penicillin, 100 µg/ml streptomycin, 0.2 µg/ml amphotericin B (Life Technologies), 1 mM sodium pyruvate (Life Technologies), and 0.1 mM nonessential amino acids (Life Technologies).
In some cases, PBMC (1.5 x 106/well) were allowed to adhere to plastic in 24-well flat-bottom plates (Falcon, Franklin Lakes, NJ) for 2 h at 37°C and 5% CO2 in complete medium. Medium and nonadherent cells were then gently aspirated, adherent cells were washed three times with RPMI 1640, and fresh complete medium was added to the wells.
RNA extraction
PBMC (1.5 x 106/ml) were cultured in 24-well plates in the presence and absence of 1 µg of Escherichia coli LPS (Sigma, Oakville, Ontario, Canada). At various intervals (see individual experiment), the cells were lysed with a guanidinium-isothiocyanate solution and the total RNA was extracted using the MicroRNA isolation kit (Stratagene, La Jolla, CA) following the manufacturer’s protocol. RNA was stored at -70°C. The amount and quantity of the RNA was determined using the A280:A260 ratio.
cDNA synthesis and PCR amplification
RNA (2 µg) was reverse transcribed into cDNA and stored at -20°C. PCR amplification was performed using primers specific for IL-15 (5' primer: 5'-CTGAAGCTGGCATTCATGTCTTCA-3'; and 3' primer: 5'-GAGTTCATCTGATCCAAGGTCT-3') and GAPDH (5' primer: 5'-CGGAGTCAACGGATTTGGTCGTAT-3'; and 3' primer: 5'-AGCCTTCTCCATGGTGGTGAAGAC-3'). PCR products were separated by electrophoresis in ethidium bromide-stained 1.4% agarose gels and visualized with UV illumination.
Flow cytometry
Resting PBMC (1.5 x 106/ml) were
cultured in complete RPMI 1640 (Life Technologies) or medium containing
10 ng/ml recombinant human
(rh)4IFN- (R&D
Systems, Minneapolis, MN), 30 ng/ml rhGM-CSF (R&D Systems), or 1 µg
of E. coli LPS (Sigma) with or without 20 µg of
cycloheximide (CHX) (Sigma). For cell-surface labeling, 1.5 x
106 PBMC were washed three times with PBS (1%
FCS, 0.1% NaN3) and incubated in the dark at
4°C with anti-IL-15 Ab or isotype-matched control Ab. Anti-IL-15
Abs were mAb 247, mAb 647 (both from R&D Systems), and 16E3-PE
(BioSource International, Camarillo, CA). The control Abs were an
unlabeled isotype-matched control Ab (R&D Systems) or a directly
labeled isotype-matched Ab (BD Biosciences, Mountain View, CA). Cells
were washed twice and incubated with goat anti-mouse IgG1-PE
(Molecular Probes, Eugene, OR). Cells were washed three times and
incubated with anti-CD3 PerCP, anti-CD14 FITC, anti-CD19
FITC, or anti-CD56 FITC (BD Biosciences) for 30 min. Cells were
fixed with 1% paraformaldehyde for 20 min before fluorescent analysis
using Lysis II software on a FACScan fluorocytometer (BD
Biosciences).
For intracellular staining of resting PBMC, 1.5 x 106 cells were washed three times with PBS (1% FCS, 0.1% NaN3) and permeabilized using the Cytofix/Cytoperm reagent (BD PharMingen, San Diego, CA) according to the manufacturer’s instructions. Cells were washed twice in "perm wash" (BD PharMingen) and incubated for 30 min at 4°C with mouse monoclonal anti-human IL-15 Ab (R&D Systems or BioSource International) or mouse isotype-matched control (R&D Systems or BD Biosciences). Cells were washed twice and incubated with goat anti-mouse IgG1-PE (Molecular Probes). For identification of cell populations expressing IL-15, cells were then washed three times with PBS (1% FCS, 0.1% NaN3) and incubated with anti-CD3 PerCP, anti-CD14 FITC, anti-CD19 FITC, or anti-CD56 FITC (BD Biosciences).
Cell-surface ELISA
Adherent monocytes were fixed in 1% paraformaldehyde, washed, and suspended in 1% human AB serum, 1% BSA, and 0.01% NaN3 in PBS. Cells were labeled with the IL-15-specific mAb 247 or isotype-matched control Ab (R&D Systems) followed by goat anti-mouse HRP (Amersham, Buckinghamshire, U.K.). The ELISA was developed using 200 µl of a 1/1 mixture of H202 and tetramethylbenzidine solutions (Genzyme Diagnostics, San Carlos, CA). Reaction was stopped with 200 µl of 1 M H2SO4 and read spectrophotometrically at 450 nm. The values are expressed as the fold increase in absorption over control cells (absorption from cells labeled with anti-IL-15 Ab / absorption from cells labeled with isotype-matched Ab). For comparisons between different culture conditions, net IL-15 A450 values were obtained and expressed relative to control cells.
Immunoblotting
Freshly isolated cells were lysed in hypotonic buffer (10 mM Tris-hydrochloric acid (pH 8.0), 10 mM KCl) with fresh protease inhibitors (1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin (Sigma)), followed by Dounce homogenization. The cellular lysate was isolated from the homogenate by centrifugation (1000 x g for 10 min) and resuspended in 1x Laemmli SDS sample buffer. The lysate was applied to 15% SDS-PAGE and transferred onto nitrocellulose paper (Bio-Rad, Hercules, CA). The membrane was blocked with 5% BSA in PBS/0.5% Tween 20 and probed with polyclonal rabbit anti-human IL-15 (P2; Immunex, Seattle, WA), followed by HRP-conjugated goat anti-rabbit IgG (Bio-Rad). Immunoreactive protein bands were visualized using ECL (Amersham).
Surface IL-15 bioassay
Freshly isolated PBMC (2 x 105) were cultured in complete RPMI 1640 overnight and monocytes were obtained by adherence to plastic. Monocytes were stimulated for 1 h with 10 ng/ml GM-CSF, washed, and fixed with 0.05% gluteraldehyde. Cells were washed twice with cold PBS and then incubated in complete RPMI 1640 for 1 h. CTLL-2 cells (2 x 104) were added to fixed adherent monocytes in the presence or absence of mAb 247 or an isotype-matched control Ab. For optimal CTLL-2 response, 0.4 ng of IL-2 was added to each group. After 48 h, 10 µl of a 5 mg/ml MTT solution was added to each well and cells were incubated for an additional 4 h at which time 100 µl of isopropanol/0.04 N hydrochloric acid solution was added to each well and read spectrophotometrically at 570 nm.
Statistics
When applicable, data are presented as mean ± SE. Different experiments were performed on different subjects on different days. Statistical analysis was performed by use of the Scheffe test for analysis of variance, when allowed by the F test. For these tests, p < 0.05 was considered significant. For CTLL-2 proliferation, a Student t test was performed using the Bonferroni correction for the comparison of multiple groups.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To characterize the regulatory stages controlling IL-15 protein
expression, we examined IL-15 mRNA and protein expression in resting
human PBMC. Semiquantitative RT-PCR was performed on freshly isolated
cells that were left in culture for various lengths of time. IL-15 mRNA
was found constitutively expressed in PBMC, and there was no change in
IL-15 mRNA expression with up to 72 h in culture (Fig. 1
a). Immunoblotting revealed
that freshly isolated PBMC also constitutively express cell-associated
IL-15 protein, showing one strong immunoreactive band at 
19 kDa, and
one weak band at 15 kDa (Fig. 1b). These correspond to
the previously described molecular mass of mature IL-15 and the variant
with the short signal peptide (20).
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Because both IL-15 mRNA and protein are constitutively expressed
in resting PBMC, experiments were performed to determine whether PBMC
display surface-bound or intracellular IL-15 protein. PBMC labeled with
a mAb specific for IL-15 showed no surface-bound IL-15 protein (Fig. 2a). However, when PBMC were
permeabilized before labeling, IL-15 protein was detected
intracellularly (Fig. 2b). The specificity of IL-15 staining
was demonstrated by adding excess rIL-15 to cells before addition of
the anti-IL-15 Ab, which reduced the intensity of fluorescence to
that of the cells labeled with an isotype-matched control Ab (Fig. 2c).
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Having determined that PBMC express intracellular IL-15,
experiments were performed to determine which subsets of PBMC express
IL-15. Two-color flow cytometry revealed that IL-15 protein is
expressed largely in CD14+ monocytes, but
labeling also occurred in CD3+ T cells, and to a
lesser extent in CD19+ B cells and
CD56+ NK cells (Fig. 3a). Surface expression of
IL-15 on the major populations of resting PBMC was not detected by
two-color flow cytometry (Fig. 3b). Similar results were
obtained using the mouse mAbs 247, 647, and 16E3. Monocyte IL-15
surface labeling was detected with the M112 Ab, but, consistent with
previous reports (21), when rIL-15 was added before
labeling, we were unable to reduce the level of binding by M112,
suggesting that this IL-15 signal detected using M112 was nonspecific.
Thus, subsets of PBMC do not constitutively express IL-15 on their
surface but do express intracellular IL-15.
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Because our inability to detect constitutive IL-15 surface
expression on monocytes by flow cytometric analysis may have been due
to a lack of sensitivity of the assay, we developed a highly sensitive
ELISA to detect IL-15 on the surface of monocytes. This revealed that
only one donor of four expressed trace amounts of IL-15 on the surface
of resting adherent monocytes (Fig. 4a). Because previous studies
had demonstrated constitutive surface expression of IL-15, studies were
performed to determine whether the surface expression might be
influenced by the PBMC purification process. Surface labeling of IL-15
was measured at various stages of cellular purification. Monocytes
obtained from whole blood did not express IL-15 on their cell surface
(Fig. 4b). However, monocytes tested immediately after
Ficoll-Hypaque gradient centrifugation displayed membrane-bound IL-15
(Fig. 4c) that was quickly down-regulated when cells were
allowed to rest in culture (Fig. 4, d and e).
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To determine whether mediators of inflammation induce IL-15
surface expression, resting PBMC were stimulated with LPS, which is
known to induce modest levels of IL-15 secretion (22).
Resting PBMC cultured in medium alone showed no IL-15 surface
expression on CD14+ monocytes (Fig. 5, a and b). In
contrast, CD14+ monocytes stimulated with 1 µg
of LPS displayed IL-15 surface expression at both 30 and 60 min after
stimulation (Fig. 5, c and d). The macrophage
stimulus GM-CSF (Fig. 5e), but not IFN- (Fig. 5f), also induced IL-15 surface expression on monocytes.
Although we detected surface expression of IL-15, ELISA did not detect
IL-15 in the culture supernatants at these early time points (data not
shown). Thus, both LPS and GM-CSF induced surface expression of IL-15,
while IFN- did not, and surface expression was not accompanied by
secretion of IL-15.
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Because IL-15 surface expression occurred rapidly following LPS
stimulation, we considered the possibility that surface expression was
from presynthesized IL-15 rather than the result of transcriptional or
translational up-regulation. To determine whether the transcript
increased, IL-15 mRNA was assessed following LPS stimulation. PBMC
stimulated with LPS did not up-regulate IL-15 mRNA (Fig. 6a). To determine whether
translation was required, PBMC were stimulated with LPS in the presence
of the protein synthesis inhibitor CHX. Both flow cytometry (Fig. 6, b–d) and cell-surface ELISA (Fig. 6e)
demonstrated that monocytes stimulated with LPS in the presence of CHX
expressed IL-15 on their cell surface similar to cells stimulated with
LPS in the absence of CHX. There was no reduction in viability of cells
due to CHX exposure as assessed by trypan blue exclusion or forward and
side light scatter (data not shown), and the concentration of CHX used
was capable of inhibiting protein synthesis, because CHX treatment
abrogated the secretion of IL-10 by PBMC stimulated with Con A (data
not shown). These results suggest that early IL-15 surface expression
is not the result of de novo protein synthesis, but, instead, IL-15 is
translocated to the cell surface from preformed intracellular
stores.
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Because monocytes express IL-15 on their surface following
activation, experiments were performed to determine whether the
surface-expressed IL-15 was biologically active. To avoid the
possibility that IL-15 was secreted into the medium, monocytes were
stimulated, fixed, and washed before assessing the activity of surface
IL-15. Fixed, stimulated monocytes were incubated with the
IL-15-responsive T cell line CTLL-2. Unstimulated monocytes did not
support CTLL-2 proliferation, supporting our previous observation that
monocytes do not constitutively express surface IL-15 (Fig. 7). After stimulation with GM-CSF and
fixation, monocytes induced proliferation of CTLL-2 cells, which was
inhibited by an anti-IL-15 neutralizing Ab. By correlating the
CTLL-2 proliferation with the activity in response to recombinant
standards, we estimated that activated monocytes had 4.4 ng of IL-15
biological activity per 100,000 cells. Thus, surface-expressed IL-15 is
biologically active.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Many cell types show constitutive expression of IL-15 mRNA (1, 23, 24, 25, 26, 27, 28, 29), suggesting that IL-15 is regulated through
posttranscriptional mechanisms. Nevertheless, the purpose of this mRNA
was not clear, because to date only a limited number of studies have
demonstrated constitutive IL-15 protein expression. IL-15 protein is
present in umbilical vein endothelial cells, renal cortical tubular
epithelial cells, and human cancer cell lines (29, 30, 31, 32).
Using intracellular cytokine analysis, the current studies indicate
that the constitutively expressed IL-15 mRNA is translated to protein
in the major subsets of PBMC, which possess IL-15 protein stored
intracellularly. Previously pulse/chase experiments have shown that
cell-associated IL-15 protein has a slow turnover rate
(20). Taken in conjunction with the present study, we
conclude that monocytes store IL-15 protein in relatively long-lived
intracellular stores that become rapidly exported to the plasma
membrane following stimulation. Although we have demonstrated that some
biologically relevant stimuli (LPS or GM-CSF) are sufficient to signal
CD14+ monocytes to express IL-15 on their
surface, while others (IFN-) are not, the downstream pathways
required and their specificity are unknown.
Other type I T cell growth factors, including IL-2
(11, 12, 13) and IL-4 (12), are primarily under
transcriptional regulation. Although this tight regulation prevents
inappropriate growth factor signaling, it imposes a number of
limitations on the expression of these cytokines, including the time
and physiologic mechanisms required for transcription and translation
before expression. Preformed cytokines circumvent these limitations
and, in the case of IL-15, might permit immediate support for T cell
responses and other T cell growth factor-dependent responses until
additional growth factors can be synthesized. Although
posttranslational regulation is atypical for other T cell growth
factors, some monokines are regulated in this manner. For example,
monocytes store TGF-
in secretory granules, and these stores are
liberated following treatment with IL-10 and IFN- (33).
The current studies indicate that IL-15 can bypass the requirement for
transcriptional and translational control by existing as a mobile
preformed cytokine in peripheral blood monocytes.
Although many cytokines are secreted proteins that allow action at
sites distant from their elaboration, some other monokines have
membrane-associated forms. Membrane-bound IL-1 plays an important role
in accessory cell support of anti-CD3-induced T cell proliferation
(34). Surface TNF- mediates macrophage
contact-dependent cytotoxicity (35, 36, 37, 38) and IL-10
induction (39). Macrophage-expressed surface IL-10
regulates both phagocytic function (40) and bactericidal
activity (41), and membrane lymphotoxin-
contributes to lymphokine-activated killer cell cytotoxicity
(42). Membrane retention provides an important level of
control over immune responses, because cells must be in direct contact
for these membrane-bound cytokines to exert their effects.
Monocytes have the capacity to support lymphocyte activity by a number
of mechanisms that include accessory receptor-ligand interactions and
the release of cytokines. Although soluble IL-15 can clearly promote T
cell responses (6, 21), we have shown a potent capacity
for membrane-bound IL-15 to promote T cell proliferation. Recent
investigations suggest that a form of IL-15 that is not secreted
confers a more protective effect than secreted IL-15 in in vivo
anti-vaccinia virus responses (19). In the current
study we observed 4.4 ng of IL-15 activity on the surface of 100,000
monocytes. This concentration is higher than what we have estimated
using other techniques (our unpublished observations). A
potential explanation for the observation that a lower level of IL-15
protein was found on the surface of activated monocytes (<1 ng per
105 monocytes) despite a higher level of IL-15
activity detected (4.4 ng per 105 monocytes, Fig. 7
) may depend on the fact that membrane-localized IL-15 activity may be
more potent than its soluble isoform. That is, an additional feature of
membrane-bound IL-15 may be its ability to promote clustering of its
cognate receptor, thereby amplifying the effective dose of membrane
IL-15, when compared with its soluble counterpart. This provides a
potential mechanism for the protective effect of cell-associated IL-15.
Additionally, because IL-15 is a potent T cell growth factor, cognate
interactions between IL-15-expressing monocytes and Ag-specific T cells
would limit the extent of bystander activation in the initiation of an
Ag-specific immune response. Cognate roles for IL-15 may also be
important in the developmental education of intraepithelial lymphocytes
and NK cells, processes both known to be dependent on the IL-15
receptor (9).
We have shown that PBMC store IL-15 intracellularly. A recent study by Musso et al. (21) reported that monocytes and monocytic cell lines constitutively express IL-15 on their surface. We have confirmed that some cell lines, such as THP-1, constitutively express IL-15 on their cell surface (data not shown), but, using multiple different IL-15 mAbs, we were unable to detect IL-15 on monocytes from the majority of healthy adults. We believe that differences in techniques are the explanation for these discrepant results. First, we have used four different mAbs. We have found that three commercially available mAbs consistently show IL-15 only on activated monocytes, while M112 used by Musso et al. (21) appears to have a level of nonspecific binding, which gives the appearance of constitutive surface expression (after stimulation, M112 does not reveal any rapid increase in surface labeling). Secondly, Musso et al. (21) purified monocytes from total PBMC by Ficoll-Hypaque followed by Percoll gradient centrifugation. We made similar observations concerning surface expression of IL-15 after Ficoll-Hypaque separation of PBMC. The transient surface expression may be due to the activation of cells during the purification process. Specifically, Percoll, which is a modified mixture of colloidal silica (43), has been shown to cause membrane disruption (44), decrease plasma membrane phospholipid levels (45), induce histamine release from human basophils (46), and induce random migration and elevated H202 production from neutrophils (47). In addition, we have shown that, while resting monocytes do not have active IL-15 on their cell surface, activated monocytes can readily support T cell proliferation, which is blocked by an anti-IL-15 Ab. Although Musso et al. (21) reported constitutive IL-15 membrane localization on resting monocytes, they failed to attribute biological function to this observation. Finally, our data corroborate the observation that healthy alveolar macrophages do not express surface IL-15 (48). Thus, our results suggest that IL-15 is not constitutively expressed on the membrane, but becomes rapidly expressed on the cell surface following monocyte activation.
Although IL-2 is produced by Ag-specific T cells hours to days after Ag presentation (12, 49), and is not necessary until after the fifth round of division (50), IL-15 is required for the afferent phase of a T cell response in vivo (50) and surface expression is rapidly induced by monocyte activators like the cytokine GM-CSF or the innate T-independent immunostimulant LPS. This provides monocytes with the capacity to use LPS (and perhaps other signals) as an initial bridge between innate and acquired immunity, resulting in immediate IL-2-independent support for the initiation of an Ag-specific T cell response.
In summary, IL-15 is constitutively expressed in subsets of resting PBMC at the level of both message and protein. Therefore, mechanisms that regulate the cellular export of IL-15 protein are likely the limiting factor in IL-15 expression. Furthermore, monocytes rapidly express IL-15 on their surface following activation. The broad expression of preformed IL-15 stores, and the ability of the stores to be mobilized in a T-independent manner to a biologically active form, suggest that IL-15 is operationally ready to provide an initial bridge between innate and acquired immunity.
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Acknowledgments |
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Footnotes |
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2 This work conforms to the guidelines established by the Conjoint Committee on Medical Ethics at the University of Calgary, Calgary, Alberta, Canada.
3 Address correspondence and reprint requests to Dr. Christopher H. Mody, University of Calgary, Room 273 Heritage Medical Research Building, Calgary T2N 4N1, Alberta, Canada. E-mail address: cmody{at}ucalgary.ca
4 Abbreviations used in this paper: rh, recombinant human; CHX, cycloheximide.
Received for publication November 21, 2000. Accepted for publication September 4, 2001.
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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] [Full Text] [PDF] |
