Published online before print July 6, 2005
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* The University of Texas M.D. Anderson Cancer Center, Houston; and
Introgen Therapeutics, Inc., Houston, Texas
1Correspondence: The University of Texas M.D. Anderson Cancer Center, Experimental Therapeutics, Box 362, 1515 Holcombe Blvd., Houston, TX 77030. E-mail: npoindex{at}mdanderson.org
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, granulocyte macrophage-colony stimulating factor, and IL-1ß stimulate the expression of IL-24 mRNA and protein, whereas interferons and T helper cell type 2 cytokines fail to induce substantial IL-24. When LPS- or PHA-stimulated cells were treated with Actinomycin D, IL-24 mRNA persisted at high levels over the 4-h course of treatment. These data strongly suggest that the expression of IL-24 in human PBMC results from cytokine stimulation and is regulated at the post-transcriptional level through stabilization of IL-24 mRNA.
Key Words: T lymphocytes • monocytes/macrophage • cytokine receptors
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(TNF-) [6
]. Similarly, IL-24 stimulation of human PBMC leads to the production of IL-6 and TNF- [7
]. Currently available data indicate that this family of cytokines is involved in regulation of inflammatory and immune responses [8
, 9
]. IL-24, first called melanoma differentiation-associated gene 7 (MDA-7), was initially identified because of its tumor suppressor properties [10 ]. Early reports indicated that MDA-7 was a nuclear protein [11 ]; however, subsequent studies using higher quality antibodies demonstrated that MDA-7/IL-24 was a secreted protein [8 , 9 ]. Overexpression of IL-24 via adenoviral gene transfer caused growth inhibition in various tumor cells including melanoma [12 , 13 ], and normal cells were not affected by overexpression of this protein [14 ]. Because of its sequence homology to IL-10, its chromosomal location, and its selective expression in tissues related to the immune system, MDA-7 was named IL-24 by the Human Genome Organization gene nomenclature system.
Now established as a cytokine, many laboratories have examined the expression and functional properties of IL-24. We have reported that IL-24 protein will stimulate secondary cytokine release from normal PBMC including TNF-, IL-6, and interferon-
(IFN-) [9
]. Our initial report showed that IL-24 was expressed in CD19- and CD56-expressing cells late during a mitogen-driven response [7
]. Others have reported that IL-24 mRNA is expressed in T cells after stimulation with anti-CD3 and in macrophages after stimulation with LPS [15
, 16
]. Because of the discrepancies in these findings, we examined the kinetics of IL-24 protein and mRNA expression in the total PBMC population and its subsets. As reports in the literature have focused on expression of the IL-24 message rather than protein, we have developed a more-sensitive method, intracellular flow cytometry, to measure IL-24 protein in conjunction with real-time, quantitative reverse transcriptase-polymerase chain reaction (QPCR) to measure IL-24 mRNA. We present evidence showing that IL-24 is expressed in T cells and macrophages as a result of stimulation with polyclonal activators of these cell populations. Furthermore, our results show that the expression of IL-24 in human PBMC results from proinflammatory cytokine stimulation and is regulated at the post-transcriptional level through stabilization of IL-24 mRNA.
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Real-time QPCR analysis
Total RNA was extracted from PBMC after culture with mitogens or cytokines using TRI Reagent (Molecular Research Center, Inc., Cincinnati, OH) following the manufacturer’s instructions. Real-time QPCR was performed using TaqMan One Step procedure, according to the manufacturer’s protocol (Applied Biosystems, Foster City, CA). Gene-specific primer/probes for IL-24, IL-2, IL-10, IL-22 receptor 1 (IL-22R1), and 18S were purchased from Applied Biosystems. The reactions were carried out according to the manufacturer’s protocol using an ABI Prism 7900 HT sequence detection system, and the analysis was conducted using Sequence Detection software, Version 2.1. Amplification of the endogenous control, 18S mRNA, was performed to standardize the amount of sample RNA added to each reaction. This reference gene was chosen, as it demonstrated the least variation during mitogens or cytokine stimulation of PBMC [17
]. The 
threshold cycle (CT) method for relative quantitation was used as described by Applied Biosystems. The IL-24 mRNA level in the absence of stimulation was used as the calibrator. Results are reported as the fold increase in IL-24 mRNA of stimulated PBMC over the unstimulated PBMC.
Flow cytometry and intracellular cytokine analysis
IL-24 protein was detected using a fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody (mAb), 7G11.F2.10, directed against the N terminus of the IL-24 molecule (Introgen, Houston, TX). The specificity of this monoclonal was established based on its ability to stain, by intracellular cytokine analysis, IL-24, produced by the IL-24-transfected human embryonic kidney-293 cell line and not the parental 293 cell line [7
]. This monoclonal was produced, purified, and conjugated by Rockland Immunochemicals (Gilbertsville, PA). The IL-24 protein was detected in cells by intracellular flow cytometry. Briefly, cells were treated with brefeldin A (Sigma-Aldrich) at 10 µg/ml, 4 h prior to harvest, to prohibit secretion of the cytokine. Where applicable, cells were first surface-stained using phycoerythrin (PE)-conjugated antibodies (BD Biosciences, San Jose, CA), and then to detect intracellular cytokines, cells were fixed by treatment with 4% paraformaldehyde, permeabilized with the detergent n-octyl glucopyranoside at 7 mg/ml, and then stained with FITC-anti-IL-24. As the control for permeabilization, cells were also stained with a mAb directed against the intracellular protein vimentin (BioGenex, San Ramon, CA). Standard protocols were followed for immunofluorescence staining. Immunofluorescence was analyzed on a FACSCalibur with CellQuest software (BD Biosciences).
Stabilization of IL-24 mRNA
PBMC were stimulated with PHA or LPS, as described above, for 10–12 h until maximum levels of IL-24 mRNA expression were reached. Actinomycin D (Act D), purchased from Sigma-Aldrich, was added at 5 µg/ml to these cultures, and the incubation continued for 4 h. Cells were harvested at 1-h intervals, and RNA was isolated as described above. mRNA levels were determined by QPCR, and results were expressed as fold increase over unstimulated control cultures of PBMC.
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Table 1. Kinetics of IL-24 mRNA Induction in PHA-Stimulated PBMC
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Figure 1. Kinetic analysis of IL-24 protein expression in PHA-, LPS-, and MLR-stimulated PBMC. Normal PBMC were stimulated with PHA ( ) or LPS ( ) for 72 h. PBMC were stimulated in a MLR ( ) for 7 days (168 h). IL-24 was quantified at specified time-points by intracellular immunocytometry. Results are reported as the percent of IL-24-expressing cells in the total, viable PBMC population. These experiments were repeated at least twice with PBMC from different donors. Shown are representative results from one donor.
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Figure 2. Subset analysis of IL-24 expression in PHA- and LPS-stimulated PBMC, which were stimulated with PHA (5 µg/ml) or LPS (5 µg/ml) for 24 h. Cells were surface-stained with PE-conjugated anti-CD3, -CD19, -CD56, or -CD14, fixed, permeabilized, and stained with FITC-conjugated anti-IL-24. Results are reported as the percent of IL-24-expressing cells in the total PBMC population. Shown in the upper right quadrant are the percent of total PBMC staining positive for IL-24 and the CD marker. Quadrant gates were set to include >99% of control immunoglobulin G (IgG)-stained cells in the lower left quadrant (not shown). These experiments were repeated at least twice with PBMC from different donors. Shown are representative results from one donor.
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Table 2. Kinetics of IL-24 Expression in PBMC Subsets
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Cytokine stimulation of PBMC leads to IL-24 expression
IL-24 was expressed by day 2 of the MLR response and persisted through day 7. This suggested that IL-24 expression could be dependent on secretion of cytokines from activated PBMC, thus explaining the induction of IL-24 by PHA and LPS, mitogens that cause the induction and secretion of high levels of cytokines. To determine if individual cytokines may be driving the expression of IL-24, we began by stimulating PBMC with IL-2, a major cytokine produced during PHA activation and also in a MLR. We measured the increase in IL-24 mRNA and protein. IL-2 stimulation caused a rapid increase in IL-24 mRNA as early as 1 h after stimulation (200-fold over baseline), and maximum levels (3000-fold increase) were achieved by 24 h (Fig. 3
). IL-24 protein in these cells peaked at 24 h and then began to decrease (data not shown). The expression of IL-24 mRNA was only partially blocked by pretreatment of PBMC with anti-IL-2 mAb prior to PHA stimulation, suggesting that other cytokines induce IL-24 expression (data not shown). Therefore, PBMC were analyzed for IL-24 expression after stimulation with 100 U/ml individual cytokines including IL-4, -7, and -15 and IFN- (Fig. 4A
), as well as IL-1ß, IFN-, IFN-ß, TNF-, and granulocyte macrophage-colony stimulating factor (GM-CSF; Fig. 4B
). Shown are two time-points for each cytokine as well as the levels of IL-24 mRNA stimulated by PHA or LPS. Of the cytokines tested, IL-2, IL-7, IL-15, IL-1ß, TNF-, and GM-CSF consistently induced IL-24 mRNA in PBMC from all individuals tested. The T helper cell type 2 cytokine IL-4 failed to induce IL-24 mRNA. It is surprising that IFN-, IFN-ß, and IFN- also failed to induce substantial levels of IL-24. Maximum mRNA levels were seen at 24 h when PBMC were stimulated with IL-2, IL-7, or IL-15, reaching levels similar to PHA stimulation (Fig. 3B)
. Cytokine stimulation of PBMC with these same cytokines also caused the elaboration of IL-24 protein as measured by intracellular flow cytometry. Subset analysis, shown in Figure 4C , demonstrated that in all cases, the major cell types expressing IL-24 were CD3+ T cells and CD14+ macrophages.
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Figure 3. Kinetics of IL-24 mRNA expression in cytokine-stimulated PBMC, which were stimulated with IL-2, IL-7, and IL-15 at 100 U/ml. IL-24 mRNA, measured by real-time QPCR, was expressed early in response to IL-2 (A) and then followed the same kinetics as seen for IL-7- and IL-15-induced expression (B). Results are reported as the fold increase in IL-24 mRNA in cytokine-stimulated PBMC compared with unstimulated PBMC. These experiments were repeated at least twice with PBMC from different donors. Shown are representative results from one donor.
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Figure 4. Individual cytokines induce expression of IL-24. PBMC were stimulated with PHA, IL-2, IL-4, IL-7, IL-15, and IFN-, each at 100 U/ml for 12 and 18 h (A), as well as LPS, IL-1ß, TNF-, GM-CSF, IFN-, and IFN-ß at 100 U/ml for 8 and 12 h (B). Levels of IL-24 mRNA, measured by real-time QPCR, are reported as the fold increase in IL-24 mRNA in stimulated PBMC compared with unstimulated PBMC (A and B). IL-24 protein expression in PBMC subtypes was measured by surface staining and intracellular flow cytometry in cells stimulated for 24 h with cytokines at 200 U/ml (C). These experiments were repeated at least twice with PBMC from different donors. Shown are representative results from one donor.
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-chain (c) [19
]. The IL-7R is composed of a unique -chain and c [20
], and the IL-2R and IL-15R are composed of three subunits: IL-2/IL-15Rß, c, and a unique -chain [21
].
Anti-IL2R antibodies block the expression of IL-24 in PHA-stimulated PBMC
To establish whether signaling through the IL-2R affects IL-24 expression, we attempted to inhibit its expression by the addition of blocking antibodies directed against the three subunits of the IL-2R, IL-2R, IL-2/IL-15Rß, and c (IL-2R). Antibodies were preincubated with PBMC 1 h prior to the initiation of the 18-h culture with PHA. These experiments were repeated three times, each with different donors. In each case, the addition of antireceptor antibodies caused significant but only partial inhibition of the PHA-induced expression of IL-24. Shown in Figure 5
is a representative result from one donor, where each of these antibodies demonstrated partial inhibition of IL-24, at the protein and mRNA level, suggesting that IL-24 expression in T cells occurs, in part, as a result of signaling through IL-2R and its components.
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Figure 5. Blocking of PHA-induced expression of IL-24 mRNA and protein. Antibodies against the IL-2R, IL-2Rß, and IL-2R were added to PBMC 1 h prior to the initiation of the 18-h PHA stimulation. Protein levels were measured by intracellular flow cytometry, and mRNA was measured by real-time QPCR. Results are shown as the percent of the IL-24 expression compared with the control PHA-stimulated PBMC without antibody. These experiments were repeated three times with PBMC from different donors. Shown are representative results from one donor.
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Figure 6. IL-24 mRNA is stabilized during PHA and LPS stimulation. PBMC were stimulated with nothing ( ), PHA (), or LPS () for 12 h before the addition of media or Act D to block transcription. Individual cultures were harvested at the indicated times after the addition of Act D, and total RNA was isolated for determination of IL-24 mRNA (A) or IL-10 mRNA (B) by QPCR analysis. Plotted is the percent of remaining cytokine mRNA after Act D treatment compared with media controls. Similar results were obtained in three separate experiments.
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, and GM-CSF induce IL-24 mRNA and protein expression. Since its identification as a cytokine, there have been numerous reports examining the expression of IL-24 in normal human peripheral blood cells [7 , 15 , 16 , 23 ]. The majority have reported the expression of IL-24 mRNA in activated PBMC, and protein expression was not examined. In this report, we describe the kinetics of IL-24 mRNA and protein expression in normal PBMC populations. Here, we report that robust increases in IL-24 mRNA can be measured as early as 1 h after stimulation, and protein expression can be induced as early as 6 h after polyclonal activation. T cells and macrophages are the major cells expressing IL-24 at early time-points (Table 2) . However, by 72 h after stimulation, IL-24 mRNA and protein in these cells have diminished to levels that are barely detectable by flow cytometry and would not be detected by immunohistochemistry (IHC). In our current analysis, by 48 h, IL-24 protein was detected in natural killer (NK) and B cells, 1.5% of total PBMC coexpresses CD56 and IL-24, and 2.5% of PBMC coexpresses CD19 and IL-24. These numbers represent roughly 15% of the total NK or B cell population in peripheral blood, levels that are detectable by IHC in positively selected populations [7 ]. The function of IL-24 in these populations of lymphocytes is under further investigation in our laboratory.
Our analysis of mRNA and protein expression in activated PBMC corroborates and expands on the work of Wolk and colleagues [15 ] who reported low levels of IL-24 mRNA as early as 2 h in LPS-stimulated human macrophage and by 6 h in anti-CD3-stimulated T cells. Early expression of IL-24 protein was also reported by Wang and colleagues [23 ], who were able to identify by Western analysis IL-24 protein in the supernatant of PBMC after just 2 h stimulation with concanavalin A.
IL-2, IL-7, or IL-15 stimulated the expression of IL-24 in PBMC. These three cytokines share the common cytokine receptor c. The IL-7R is composed of a unique -chain and c [20
], although the IL-2R and IL-15R are composed of three subunits: the IL-2/IL-15Rß, the c, and each with a unique -chain [21
]. We have shown here that blocking these receptors decreases expression of IL-24 in PBMC. These data suggest that signaling through the IL-2R, in particular, c, may regulate expression of IL-24. There is considerable evidence that cytokines that bind to receptors containing c are involved in T cell maintenance and homeostasis [19
, 24
, 25
]. The fact that these cytokines stimulate expression of IL-24 in PBMC, specifically T cells, suggests that IL-24 may also be involved in T cell homeostasis and survival.
The kinetics of IL-24 mRNA induction is rapid: 
2000-fold observed as early as 2 h after PHA stimulation, suggesting that transcriptional activation is not the primary mode of IL-24 gene regulation. Cytokines may function to stabilize IL-24 mRNA via post-transcriptional mechanisms, explaining the large increases we observed in IL-24 mRNA. The results of our experiments using Act D to block new transcription of IL-24 mRNA suggest that stimulation with PHA or LPS blocks the decay of IL-24 mRNA, causing accumulation of IL-24 transcripts in PBMC (Fig. 6)
. Jiang and colleagues [10
] have reported that melanoma cell lines induced to differentiate with mezerein and IFN-ß expressed detectable levels of IL-24 mRNA. In mezerein-treated tumor cells, IFN-ß probably functioned to stabilize IL-24 mRNA, which has been shown to posses adenosine uridine-rich elements (AREs) in its 3'-untranslated region [26
]. These AREs have been identified in other cytokines and function to promote instability through rapid mRNA decay [27
]. The ARE signals in IL-24 were shown to be functional at the post-transcriptional level and decrease the accumulation of MDA-7 transcripts [26
]. Our data suggest that cytokines produced as a result of mitogen stimulation of PBMC or the mitogens themselves promote the stabilization of IL-24 message. The functional significance of this stabilization remains to be determined. LPS treatment results in a half-life of IL-24 mRNA of 5 h, and in PHA-treated cells, the half-life of IL-24 mRNA is considerably longer compared with unstimulated PBMC, where the half-life is approximately 1 h (Fig. 6)
. In contrast, the half-life of IL-10 mRNA is 40 min to 1 h after PHA and LPS treatment, respectively. These data are in good agreement with previous studies, which demonstrate a 1-h half-life for IL-10 mRNA [28
, 29
].
The cytokines that stimulate the production of IL-24 are secreted by cells involved in an inflammatory response. In the skin, the major sources of IL-1ß, GM-CSF, and well as TNF- are macrophages or Langerhans cells. When stimulated through their Toll-like receptors, these cells secrete high levels of these cytokines. We propose that cells of the immune system, including T cells and macrophages, will respond to these cytokines through stabilization of their IL-24 mRNA, leading to the production and secretion of IL-24. In addition, melanocytes, present in the skin, also may serve as a source of IL-24. In this setting, the function of IL-24 remains to be determined. Our data support the hypothesis that IL-24 may play an important role in the inflammatory response in the skin, occurring as a result of a danger signal initiated by viral or bacterial infection or tumor cell growth.
Received February 24, 2005; revised May 20, 2005; accepted June 6, 2005.
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and results in cell apoptosis through TNF- J. Immunol. 169,4288-4297 chain: a functional component of the interleukin-7 receptor Science 262,1877-1880 in human monocyte IL-10 synthesis J. Immunol. 151,6853-6861[Abstract]This article has been cited by other articles:
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