Modulation of Mycoplasma arthritidis-Derived Superantigen-Induced. Cytokine Gene Expression by Dexamethasone and Interleukin-4. AL-DACCAK,1t.
INFECTION AND IMMUNITY, Nov. 1994, p. 4716-4721 0019-9567/94/$04.00+0 Copyright C 1994, American Society for Microbiology
Vol. 62, No. 11
Modulation of Mycoplasma arthritidis-Derived Superantigen-Induced Cytokine Gene Expression by Dexamethasone and Interleukin-4 AL-DACCAK,1t
MECHERI,3 J. HIBERT,1 AND W. MOURADl*
Centre de recherche en Rhumatologie et Immunologie, Departement de Medecine, Universite Laval and Centre de recherche du CHUL, St-Foy, Quebec, Canada'; Institute of Immunology and Transfusion Medicine, University of Lubeck Medical School, Lubeck Germany2; and Institut Pasteur, Paris, France3 Received 2 May 1994/Returned for modification 19 July 1994/Accepted 9 August 1994
Activation of human monocytes or monocytic cell lines with all known stimuli coordinately induces the gene expression of various cytokines, including tumor necrosis factor alpha (TNF-a), interleukin-1I (IL-1"), and the IL-1 receptor antagonist (IL-iRa). In contrast, superantigens induce TNF-a and IL-1i8 but fail to affect IL-iRa gene expression, suggesting that activation of monocytes via major histocompatibility complex class II is distinct from other signal transduction pathways. In the present study, we analyzed the regulation of the Mycoplasma arthritidis-derived superantigen (MAM)-induced IL-1p and TNF-a gene expression by studying the effects of two different anti-inflammatory agents: dexamethasone (DEX) and the T-cell-derived cytokine IL-4. Both agents contributed to the downregulation of MAM-induced IL-1i and TNF-a gene expression. They accelerated the normal decline of the gene expression of both MAM-induced cytokines by decreasing the stability of mRNAs via the induction or enhanced synthesis of one or more regulatory proteins. In addition, IIA, but not DEX, induced a strong and rapid expression of IL-lRa mRNA in MAM-stimulated and unstimulated THP-1 cells in a de novo protein synthesis-independent manner. The capacity of IL-4 to induce IL-lRa gene expression reinforces its anti-inflammatory activity. This study illustrates some of the mechanisms by which MAM-induced proinflammatory monokine gene expression can be downregulated by IL-4 and DEX. 13-acetate, and cytokine stimulation (16, 23, 32, 33). DEX is a glucocorticoid used largely as an anti-inflammatory agent, whereas IL-4, a lymphokine produced primarily by CD4+ T cells, was initially described as a B-cell stimulatory factor (13). The role of IL-4 in the downregulation of gene expression and synthesis of different monocyte-derived inflammatory mediators (11, 19, 31) suggests that it is an anti-inflammatory agent (9, 12, 30, 34, 35). Because activation of human monocytes via MHC class II molecules seems to be distinct from other signal transduction pathways (2), analysis of the effects of DEX and IL-4 on superantigen-induced response is of special interest. In part, it would elucidate some of the regulatory mechanisms governing the superantigen response, in particular that of MAM, which will consequently contribute to the comprehension of MAM-mediated diseases. Our results show that DEX and IL-4 downregulate the level of the MAM-induced IL-13 and tumor necrosis factor alpha (TNF-at) mRNA, mainly by accelerating turnover of the mRNAs. In addition, in contrast to DEX, IL-4 induces IL-1 receptor antagonist (IL-lRa) gene expression, which may have a role in its inhibitory activity.
Microbial superantigens are among the most potent activators of T lymphocytes. Following their interaction with the
major histocompatibility complex (MHC) class II molecules on antigen-presenting cells, the complex so formed interacts with the T-cell receptor of T lymphocytes in a VP-restricted fashion and induces a series of events in both cell types, resulting in a massive cytokine release (1, 3, 5, 20-22, 36). A possible role of superantigens in the pathology of several autoimmune diseases has been proposed (14, 24, 25). Mycoplasma arthritidis-derived superantigen (MAM) (15) is produced by a microorganism associated with an autoimmune-like disorder of rodents that resembles human rheumatoid arthritis (6) and has the capacity to induce different immunological responses in immunocompetent cells (26). Therefore, it has been suggested that MAM or a MAM-like superantigen may contribute to the pathogenesis of autoimmune diseases such as rheumatoid arthritis (1, 7, 10, 17). Unlike lipopolysaccharides (LPS) and phorbol 12myristate 13-acetate, which coordinately induce interleukin-1, (IL-113) and IL-1 receptor antagonist (IL-iRa) gene expression in human monocytes, superantigens (MAM and staphylococcal enterotoxin A) activate only the expression of IL-13 (2). Alterations in the balance of these cytokines mediate inflammatory reactions; therefore, our recent study (2) further supports the involvement of superantigens in the pathology of autoimmune diseases associated with inflammatory reactions and suggests that activation via MHC class II molecules is distinct from other signal transduction pathways such as that via CD14 and that induced by the protein kinase C activator. Various studies have demonstrated that dexamethasone (DEX) and IL-4 downregulate the expression of several genes in different cell types following LPS, phorbol 12-myristate
MATERUILS AND METHODS
Reagents. MAM was prepared as previously described (27) (MAM represented 20% of the protein preparation), and the preparation was demonstrated to be LPS free in a system with C3H/HeJ (MAM-responder and LPS-nonresponder) and C57BL/6 (MAM-nonresponder and LPS-responder) mice. The MAM preparations are defined as reactive for the superantigen and free of LPS when a response is detected in C3H/HeJ culture and absent in C57BL/6 culture. The parameter for responsiveness is proliferation in spleen cell cultures and cytokine induction by murine monocytes. LPS was obtained from Difco Laboratories, Detroit, Mich.; human recombinant gamma interferon (rIFN--y) was obtained from Becton Dick-
* Corresponding author. Mailing address: CRII, Rm. 9800, 2705 Blvd. Laurier, St-Foy, Quebec, Canada G1V 4G2. Phone: (418) 654-2240. Fax: (418) 654-2765. t K. Mehindate and R. Al-Daccak contributed equally to this work.
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inson Labware, Bedford, Mass.; actinomycin D was obtained from Boehringer Mannheim Canada, Laval, Quebec, Canada; cycloheximide (CHX) was obtained from ICN Biochemicals Canada Ltd., Mississauga, Ontario, Canada; IL-4 was obtained from R&D Systems, Minneapolis, Minn.; and DEX was obtained from Sigma Diagnostics Canada, Mississauga, Ontario, Canada. The cDNA probes for IL-113, TNF-oa, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained from the American Type Culture Collection, Rockville, Md.; and the IL-lRa probe was a generous gift from D. E. Tracey, Upjohn Co., Kalamazoo, Mich. Cells. The THP-1 monocytic human cell line was obtained from the American Type Culture Collection and maintained in RPMI 1640 medium containing 10% fetal calf serum and antibiotics. THP-1 cells were stimulated with IFN--y (200 U/ml) for 48 h to express MHC class II molecules. Before use, cells were washed twice with Hanks balanced salt solution and resuspended at a concentration of 107/ml in RPMI 1640 medium containing 10% fetal calf serum. The cell line used in this study was screened systematically for mycoplasma contamination and was always demonstrated to be free. Quantification of the levels of IL-1fB, TNF-a, and IL-1Ra mRNA. Stimulation conditions for each experiment are detailed in the appropriate figure legends. Cytoplasmic RNA was purified by standard methods (28), and 10 ,ug of RNA was loaded onto 1% agarose gels. The RNA was then transferred onto Hybond-N filter paper and hybridized with randomprimer-labeled cDNA probes for IL-113, TNF-a, and IL-lRa. Equal loading of RNA was confirmed by hybridization with GAPDH cDNA probe. All washes were performed under stringent conditions. The mRNA hybridizing with the two cytokine cDNA probes was visualized by autoradiography. RESULTS Effect of DEX on MAM-induced monokine gene expression. We recently reported that treatment of the THP-1 monocytic cell line with IFN-y induces HLA-DR, -DQ, and -DP expression and that this MHC class II expression enables the cells to respond to stimulation with superantigens, resulting in an increase in the level of IL-1, and TNF-a mRNA (1). Different reports showed the capacity of superantigens to induce different cytokines in purified human monocytes (5, 27), with profiles similar to those obtained with IFN-y-treated THP-1 cells, which can confirm that the phenomena we observed are due to superantigenic activities. However, we cannot completely rule out the priming effect of INF-y on superantigeninduced gene expression. Here the effect of DEX on MAMinduced IL-13 and TNF-a gene expression was evaluated by using the same cell line. THP-1 cells were pretreated with different concentrations of DEX for 2 h at 37°C and then stimulated with MAM for a further 1 h. Northern (RNA) blot analysis and mRNA decay curves showed a dose-dependent inhibition of both IL-13 and TNF-ax mRNA (Fig. 1). The observed inhibition of MAM-induced monokine gene expression was not due to altered MHC class II expression, because treatment of THP-1 with DEX for 3 h did not affect the level of MHC class II expression of molecules as determined by fluorescence-activated cell sorter analysis (data not
shown). We examined the possibility that the inhibition of MAMinduced IL-1lB mRNA accumulation following DEX treatment was indirectly mediated by the induction of IL-1Ra mRNA expression. Our results showed that DEX was not able to induce IL-lRa transcripts in THP-1 cells even at high concen-
4717
A DEX Conc (M) MAM DEX
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Concentration of DEX (M)
FIG. 1. Dose-dependent inhibitory effect of DEX on IL-13 and TNF-a mRNA accumulation in MAM-stimulated THP-1 cells. IFN-y-treated THP-1 cells were incubated for 2 h at 370C with DEX at various concentrations and then stimulated with MAM (1:100) for 1 h. LPS (1 ,±g/ml) was us -d as a control. Total RNA was extracted, and Northern blot analysis was performed. (A) Northern blot analysis for IL-11, TNF-cx, and IL-1Ra mRNA expression. The GAPDH probe confirmed equal RNA loading. (B) Decay curves of IL-lp and TNF-a mRNAs following DEX treatment, as determined from densitometric analysis and after corrections depending on RNA loading. Results are representative of a total of three experiments.
trations (Fig. 1). This may indicate that IL-1Ra induction does not participate in the inhibitory effect of DEX in these cells. Effect of CHX on DEX-induced inhibition of MAM-induced monokine gene expression. We tested the effect of CHX, a protein synthesis inhibitor, on DEX-induced inhibition. DEXtreated and untreated THP-1 cells were stimulated with MAM in the presence or absence of CHX. Analysis of the level of IL-1 and TNF-ca mRNA expression showed, as we previously reported (1), that CHX enhanced the MAM-induced mRNA expression of both monokines, suggesting that MAM-induced monokine gene expression does not require protein synthesis. Inhibition of protein synthesis by CHX abrogated the inhibitory effect of DEX (Fig. 2), indicating that the inhibitory activity of DEX requires de novo synthesis of one or more proteins. Effect of IL-4 on MAM-induced monokine gene expression. Previous studies have shown that IL-4 downregulates the production of different monokines in LPS-stimulated human monocytes (9, 30, 34, 35). Because activation of human monocytes via MHC class II molecules is distinct from that induced via CD14 (2), it is very important to study the effect of IL-4 on MAM-induced gene expression of IL-13 and TNF-ac in the THP-1 monocytic cell line. Previous data suggested the necessity of a minimum of 2 to 3 h for IL-4 to exhibit an inhibitory effect on the levels of IL-13 mRNA in LPS-stimulated human monocytes (8); THP-1 cells were treated with various concentrations of IL-4 for 3 h and then stimulated with MAM (1:100) for an additional 1 h. Pretreatment with IL-4, at levels as low as 20 U/ml, decreased the level of MAM-induced IL-13 and
4718
MEHINDATE ET AL.
INFECT. IMMUN.
MAM
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FIG. 2. Effect of CHX on the DEX-mediated suppression of monokine mRNA levels. IFN-y-treated THP-1 cells were treated with medium alone, CHX (1 ,ug/ml), DEX (10-v M), or CHX plus DEX for 2 h at 37°C. Then MAM (1:100) was added, and cells were incubated for another 1 h. At the end of the incubation period, total RNA was prepared, and the Northern blot was hybridized sequentially with IL-1,, TNF-CL, and GAPDH probes. Results are representative of a total of three experiments.
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TNF-ot mRNA (Fig. 3). Higher doses of IL-4 (100 or 500 U/ml) nearly completely inhibited transcription of both genes. As previously reported (9), IL-4 alone, even at high concentrations, did not induce any detectable levels of IL-1 or TNF-a mRNA.
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FIG. 4. Time effect on IL-4 activity in MAM-stimulated THP-1 cells. (A) Lanes: 1 and 2, cells were stimulated with MAM (1:100) or medium alone for 1 h; 3 and 4, cells were pretreated for 3 h with IL-4 (100 U/ml) and then stimulated for an additional 1 h with MAM (1:100) or medium alone for an additional 1 h; 5 and 6, cells were pretreated with IL-4 (100 U/ml) for 2 h and then stimulated with MAM (1:100) or medium alone; 7 and 8, cells were stimulated simultaneously with IL-4 (100 U/ml) and MAM (1:100) or with IL-4 alone for 1 h. All stimulations were done at 37°C. At the end of the incubation, total RNA was prepared. Northern blot analysis for the levels of IL-1f, TNF-ot, and IL-lRa mRNA was carried out. The membrane was also hybridized with the housekeeping GAPDH probe to ensure equal RNA loadings. (B) Inhibition percentages of MAMinduced IL-lp and TNF-ae as a function of the time of pretreatment with IL-4. Values were determined according to densitometric analysis of the above blot. (C) Relative IL-lRa mRNA levels induced as a function of the time of pretreatment with IL-4 in MAM-stimulated (R) and unstimulated (O) cells. Values were obtained from scanning the above blot. All values were corrected depending on RNA loading. Results are representative of a total of three experiments.
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FIG. 3. IL-11, TNF-a, and IL-lRa mRNA levels in MAM-stimulated THP-1 cells are regulated by IL-4 in a dose-dependent manner. IFN-y-treated THP-1 cells were cultured with different concentrations of IL-4 for 3 h at 37°C and then stimulated with MAM (1:100) for another 1 h. At the end of the incubation period the cells were harvested, and total RNA was prepared. (A) Northern blot analysis of the levels of IL-1p, TNF-a, and IL-IRa mRNA. The membrane was also hybridized with the housekeeping GAPDH probe to ensure equal RNA loadings. (B) RNA decay curves of MAM-induced IL-lp and TNF-a mRNA in the presence of different concentrations of IL-4 as determined from the densitometric analysis of data in panel A. C) Relative IL-lRa mRNA levels induced in the presence of IL-4 in MAM-stimulated (-) and unstimulated (l) cells as determined from the densitometric analysis of data in panel A. All values were corrected depending on RNA loading. Results are representative of a total of three experiments.
We then examined the temporal effect of IL-4 treatment on MAM stimulation. THP-1 cells treated simultaneously with MAM and IL-4 (100 U/ml) for 1 h at 37°C expressed lower levels of both IL-13 and TNF-a mRNA than did control cells stimulated with MAM alone (Fig. 4). However, a complete repression of gene expression of both monokines was obtained only when THP-1 cells were pretreated with IL-4 for 2 or 3 h before exposure to MAM (Fig. 4). Use of a higher concentration of IL-4 (500 U/ml) resulted in similar time-dependent inhibition profiles (data not shown). These findings indicate that although a significant suppression of MAM-induced IL-11 and TNF-ot gene expression can be obtained by simultaneous treatment with IL-4, pretreatment with IL-4 appears to be critical for complete inhibition. IL-4 induces IL-1Ra gene expression in MAM-stimulated and unstimulated THP-1 cells. A recent report demonstrated the ability of IL-4 to directly induce IL-lRa gene expression and peptide secretion in human monocytes independently from IL-1lB production (34). On the same line of research, this study shows that IL-4 alone or in the presence of MAM strongly induces IL-1Ra gene expression in THP-1 cells in a dose-dependent manner (Fig. 3), with maximum expression observed when 100 U of IL-4 was used. The kinetics of this
REGULATION OF
VOL. 62, 1994
MAM IL-4 CHX
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FIG. 5. Abrogation of the suppressive effect of IL-4 on MAMinduced IL-1,B and TNF-a mRNA by CHX. IFN-y-treated THP-1 cells were cultured in the presence of medium alone, IL-4 (100 U/ml), CHX (1 p.g/ml), or IL-4 plus CHX for 2 h at 37°C and then stimulated with MAM (1:100) for another 1 h. RNA was extracted, and the levels of IL-1p, TNF-a, and IL-lRa mRNA were analyzed. Hybridization with GAPDH ensured equal RNA loading. Results are representative of a total of three experiments.
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expression was analyzed in MAM-stimulated and unstimulated cells. As shown in Fig. 4, THP-1 cells treated with IL-4 (100 U/ml) in the presence or absence of MAM exhibited a time-dependent increase in the level of IL-lRa mRNA. Densitometric analysis indicated that maximum IL-lRa gene expression occurred when cells were treated with IL-4 for 4 h. The presence of MAM had no significant effect on the level of IL-4-induced IL-lRa mRNA. In fact, the only slight decrease in the level of IL-4-induced IL-lRa mRNA observed in the presence of MAM (Fig. 4, lane 6) did not have any statistical significance. Taken together, these results suggest that induction of IL-lRa gene expression following IL-4 stimulation may have a role in the repression of IL-1B transcription. Effect of CHX on IL-4-mediated reduction of MAM-induced gene expression. The effect of IL-4 on MAM-induced gene expression was further examined in the presence of CHX. The addition of CHX completely blocked the IL-4-mediated reduction of MAM-induced IL-lp and TNF-a mRNA levels (Fig. 5). These data strongly suggest that the inhibitory effect of IL-4 is via the induction or enhancement of certain regulatory proteins. On the other hand, the addition of CHX augmented the level of IL-1Ra mRNA induced by IL-4 (in the presence or absence of MAM) at least twofold. Because MAM can neither induce nor increase IL-lRa gene expression, it can be concluded that IL-4-induced IL-1Ra mRNA does not require de novo protein synthesis. Effect of DEX and IL-4 on the stability of MAM-induced monokine mRNA. We determined whether the downregulation of mRNA levels of MAM-induced monokines by DEX and IL-4 is accompanied by an acceleration in the normal decline of these mRNA levels. IL-13 and TNF-a mRNA levels in THP-1 cells treated with MAM alone for different periods were compared with those of cells stimulated with MAM for 1 h (reaching maximum expression) and then treated with IL-4 or DEX for the same different period. Northern blot analysis and mRNA decay curves demonstrated that DEX and IL-4 accelerated the normal decline of MAM-induced IL-1, and TNF-a mRNA levels (Fig. 6), indicating clearly the effect of both agents on the stability of the mRNAs. To further clarify the mechanism of accelerated decline of MAM-induced IL-1P and TNF-ot mRNA levels in the presence of IL-4 or DEX, we analyzed the effect of these two inhibitors on the turnover of the mRNAs of both monokines by using the
+
A
20
0
1
2 TIME (hr)
3
2 TIME (hr)
FIG. 6. Effect of IL-4 and DEX on the normal decline of MAMinduced IL-1i and TNF-ot mRNA levels. (A) Cells were treated with MAM (1:100) for 1 h at 37°C to reach maximal expression of IL-1,P and TNF-a mRNAs. Then IL-4 (100 U/ml) or DEX (10-9 M) was added for the indicated time intervals. RNA was prepared and analyzed by Northern blotting. (B) MAM-induced IL-1B mRNA decay curves in the presence or absence of IL-4 or DEX as determined from densitometric analysis of the above data. (C) MAM-induced TNF-a mRNA decay curves in the presence or absence of IL-4 or DEX as determined from densitometric analysis of the above data. All values were corrected depending on RNA loading. Results are representative of a total of three experiments.
transcriptional inhibitor actinomycin D. THP-1 cells were incubated with MAM (1:100) alone, MAM plus IL-4 (100 U/ml), or MAM plus DEX (10-9 M) for 1 h. Then actinomycin D (10 ,ug/ml) was added, and mRNA decay was monitored over the next 6 h (Fig. 7A). The approximate half-lives (tj/2) of MAM-induced IL-1B and TNF-a as determined from mRNA decay curves were 2 and 1 h, respectively. The presence of DEX or IL-4 reduced the tj/2 of IL-1B mRNA and of TNF-a mRNA to approximately 45 and 30 min, respectively, indicating that these two inhibitors have a direct effect on the turnover of mRNAs (Fig. 7B and C). The effect of IL-4 and DEX on the turnover of MAM-induced mRNAs was also determined by following another experimental protocol. THP-1 cells were stimulated with MAM for 1 h, actinomycin D (10 ,ug/ml) was added alone or in combination with IL-4 or DEX, and mRNA decay was monitored over the next 2 h. The determined tj/2 of the mRNAs of both monokines (data not shown) were exactly the same as those determined by following the previous protocol. In contrast, no significant difference was observed in the levels of IL-lRa mRNA induced in presence of IL-4 either 2 or 4 h after the addition of actinomycin D (Fig. 7). DISCUSSION An imbalance in the level of IL-lp and IL-lRa may play an important role in the initial step and/or severity of inflammatory reactions associated with some diseases. Recently, we have
4720
MEHINDATE ET AL.
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and TNF-ot mRNA and the approximate half-life of mRNAs. (A) Cells were treated with MAM (1:100) alone, MAM plus IL-4 (100 U/ml), or MAM plus DEX (10-' M) for 1 h at 37°C. Then actinomycin D (AD) (10 ,ug/ml) was added, and the incubation was continued at 37°C for the indicated time intervals. RNA was extracted, and the levels of IL-113, TNF-a, and IL-lRa mRNA were analyzed by Northern blotting. Hybridization with GAPDH ensured equal RNA loading. (B) IL-13 mRNA decay curves as determined from the densitometric analysis of the above data. (C) TNF-cx mRNA decay curves as determined from the densitometric analysis of the above data. All values were corrected depending on RNA loading. Results are representative of a total of three experiments.
reported that ligation of MHC class II molecules in the human monocytic cell line THP-1 with superantigens or anti-class II antibodies selectively induces IL-13 expression (2). These data clearly support the involvement of superantigens in the severity of the inflammatory reaction and suggest that signaling via MHC class II is distinct from all stimuli known to induce coordinately the expression of IL-1 and IL-lRa. To understand more fully the nature of the superantigeninduced response, we analyzed the effects of DEX and IL-4 on MAM-induced IL-1lB and TNF-o gene expression. Both agents modulated MAM-induced monokine gene expression by common as well as distinct pathways. DEX exerts its inhibitory activity on gene expression via transcriptional and posttranscriptional mechanisms. Our data demonstrated a posttranscriptional effect in the regulation of MAM-induced IL-13 and
TNF-c. The fact that both mRNAs possess A+U-rich sequences (involved in the stability of mRNA [29]) in their 3' untranslated regions (4) and the necessity for ongoing protein synthesis for the action of DEX support the involvement of a DEX-induced A+U-specific mRNA-binding protein or an RNase in its suppressor effect on MAM-induced monokine gene expression. The existence of an AUUUA-specific mRNAbinding protein (19) reinforces this suggestion. On the other hand, our results can neither confirm nor rule out the effect of DEX at the transcriptional level. The complete inhibition of IL-13 and TNF-ac gene expression obtained with i0' M DEX could be the result of the production of regulatory RNases and might reflect a transcriptional repression. Stimulation of the THP-1 cell line with MAM alone or in combination with DEX had no effect on IL-lRa gene expression. These data and those reported by several other investigators clearly indicate that IL-1 and IL-lRa gene expression in human monocytes is differentially regulated. Various studies demonstrated the inhibitory effects of IL-4 on monocyte/macrophage function, including its ability to suppress monokines (16, 23). The MAM-induced IL-113 and TNF-a mRNAs were downregulated and even completely inhibited when THP-1 cells were pretreated with IL-4. At this point, it may be suggested that IL-4 regulates MAM-induced monokine gene expression by activating a preexisting repressor factor or by inducing certain proteins that can affect the IL-113 and TNF-a gene expression. It is noteworthy that this inhibition was not due to a decrease in the level of MHC class II expression, because it is well established that IL-4 augments the expression of these molecules in monocytes (18). This report shows that IL-4 destabilized the MAM-induced TNF-a mRNA and markedly accelerated its turnover and suggests that this action may be via a regulatory protein(s) or RNases, because the inhibitory effect was completely blocked in presence of CHX. It has been reported that the effect of IL-4 on the LPS-induced TNF-a mRNA is mostly at the transcriptional level (9). In contrast, Donnelly et al. (8) suggested that IL-4 suppression of LPSinduced TNF-ao is mediated primarily at the posttranscriptional level. Here its posttranscriptional effect on the TNF-aL mRNA is confirmed, at least for that induced by MAM. Similarly, IL-4 strongly decreased the level of MAM-induced IL-13 gene expression by destabilizing the mRNA and shortening its t112 in a protein-dependent manner, demonstrating the involvement of a regulatory protein in this IL-4-mediated inhibition. Interestingly, the simultaneous incubation of THP-1 cells with MAM and IL-4 repressed IL-13 gene expression and induced IL-lRa mRNA. The detected IL-lRa mRNA is the result of a solo action of IL-4 in THP-1 cells, because MAM does not induce IL-lRa, as we previously demonstrated (2), or act synergistically with IL-4 for its induction. The direct induction of IL-lRa gene expression in purified human monocytes by IL-4 has also been reported by Wong et al. (35). The observation that IL-4 represses the induction of a gene and simultaneously induces the expression of its antagonist poses an important question: is there any molecular relation between these two effects? The results presented do not allow one to draw any conclusion regarding this question. Nevertheless, they do suggest a hypothesis. Similar to glucocorticoid, IL-4 seems to have both negative and positive regulatory effects on gene expression. The manifestation of one or the other can be the function of the presence or absence of "positive or negative elements" in the gene promoter region. Binding of IL-4 to its receptor will initiate a cascade of intracellular events, leading to the induction or activation of certain transcriptional factor(s). If this factor(s) binds a positive element, it will induce
REGULATION OF MAM-INDUCED CYTOKINE GENE EXPRESSION
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the transcription while repressing the expression of a gene possessing a negative element in its regulatory region. Identifying the mechanisms mediating the activation versus inhibition of gene transcription by IL-4 is very important. The system described in this report (THP-1 cells, MAM, and IL-4) presents an interesting model for further investigation of these mechanisms. In conclusion, IL-4 and DXE regulate the proinflammatory cytokine gene expression induced selectively via MHC class II molecules in a manner similar to that observed for LPS- and phorbol 12-myristate 13-acetate-induced IL-lp and TNF-a gene expression.
15. 16.
17. 18.
ACKNOWLEDGMENTS This work was supported in part by grants and fellowships from the MRC, the FRSQ, and the Arthritis Society of Canada to W. Mourad.
19.
REFERENCES
20.
1. Al-Daccak, R, K. Mehindate, J. Hebert, L. Rink, S. Mecheri, and W. Mourad. 1994. Mycoplasma arthritidis-derived superantigen induces proinflammatory monokine gene expression in the THP-1 human monocytic cell line. Infect. Immun. 62:2409-2416. 2. Al-Daccak, R, K. Mehindate, P. E. Poubelle, and W. Mourad. 1994. Signalling via MHC class II molecules induces selectively IL-lp over IL-1 receptor antagonist gene expression. Biochem. Biophys. Res. Commun. 201:855-860. 3. Andersson, J., S. Nagy, L. Bjork, J. Abrams, S. Holm, and U. Andersson. 1992. Bacterial toxin-induced cytokine production studied at the single-cell level. Immunol. Rev. 127:69-96. 4. Caput, D., B. Beutler, K. Hartog, R Thayer, S. Broun-Shimer, and A. Cerami. 1986. Identification of a common nucleotide sequence in the 3'-untranslated region of mRNA molecules specifying inflammatory mediators. Proc. Natl. Acad. Sci. USA 83:16701674. 5. Chatila, T., and R S. Geha. 1993. Signal transduction by microbial superantigens via MHC class II molecules. Immunol. Rev. 131: 43-59. 6. Cole, B. C., J. R Ward, R S. Jones, and J. F. Cahill. 1971. Chronic proliferative arthritis of mice induced by Mycoplasma arthritidis. Infect. Immun. 4:344-355. 7. Crow, M. K., G. Zagon, Z. Chu, B. Ravina, J. R Tumang, B. C. Cole, and S. M. Friedman. 1992. Human B cell differentiation induced by microbial superantigens: unselected peripheral blood lymphocytes secrete polyclonal immunoglobulin in response to Mycoplasma arthritidis mitogen. Autoimmunity 14:23-32. 8. Donnelly, R P., M. J. Fenton, J. D. Kaufman, and T. L Gerrard. 1991. IL-1 expression in human monocytes is transcriptionally and posttranscriptionally regulated by IL-4. J. Immunol. 146:3431-3436. 9. Essner, R K., K. Rhoades, W. H. McBride, D. L Morton, and J. S. Economou. 1989. IL-4 downregulated IL-1 and TNF gene expression in human monocytes. J. Immunol. 142:3857-3861. 10. Friedman, S. M., M. K. Crow, J. R Tumang, Y. Xu, A. S. Hodtsev, B. C. Cole, and D. N. Posnett. 1991. Characterization of human T cells reactive with the mycoplasma arthritidis-derived superantigen (MAM): generation of a monoclonal antibody against VP17 the T cell gene product expressed by a large fraction of MAMreactive human T cells. J. Exp. Med. 174:891-900. 11. Hart, P. H., D. R Burgess, G. F. Vitti, and J. A. Hamilton. 1989. Interleukin-4 stimulates human monocytes to produce tissue-type plasminogen activator. Blood 74:1222-1225. 12. Hart, P. H., G. F. Vitti, D. R Burgess, G. A. Whitty, D. S. Piccoli, and J. A. Hamilton. 1989. Potential anti-inflammatory effects of interleukin 4: suppression of human monocyte tumor necrosis factor a, interleukin 1, and prostaglandin E2. Proc. Natl. Acad. Sci. USA 86:3803-3807. 13. Howard, M., J. Farrar, M. Hilfiker, B. Johnson, K. Takatsu, T. Hamaoka, and W. E. Paul. 1982. Identification of a T cell derived B cell growth factor distinct from interleukin 2. J. Exp. Med. 155:914-923. 14. Howell, M. D., J. P. Diveley, K. A. Lundeen, A. Esty, S. T. Winters, D. J. Carol, and S. W. Brostoff. 1991. Limited T-cell receptor beta chain heterogeneity among interleukin 2 receptor-positive syno-
21. 22.
23.
24.
25. 26. 27.
28.
29. 30.
31. 32.
33. 34. 35.
36.
4721
vial T cells suggests a role for superantigen in rheumatoid arthritis. Proc. Natl. Acad. Sci. USA 88:10921-10925. Kirchner, H., G. Bnehm, W. Nicklas,R Beck, and F. Herbst. 1986. Biochemical characterization of the T-cell mitogen derived from mycoplasma arthritidis. Scand. J. Immunol. 24:245-249. Lew, W., J. J. Oppenheim, and K. Matsushima. 1988. Analysis of the suppression of IL-la and IL-13 production in human peripheral blood mononuclear adherent cells by a glucocorticoid hormone. J. Immunol. 140:1895-1902. Li, Y., G. Zagon, S. M. Friedman, M. K. Crow, and D. N. Posnett. 1993. Oligoclonal V117 T cells in rheumatoid synovial tissue. J. Immunol. 150:25A. Littman, B. H., F. F. Dastuan, P. L. Carlson, and K. M. Sanders. 1989. Regulation of monocyte/macrophage C2 production and HLA-DR expression by IL-4 (BSF-1) and INF--y. J. Immunol. 142:520-525. Malter, J. S. 1989. Identification of an AUUUA-specific messenger RNA binding protein. Science 246:664-666. Marrack, P., and J. Kappler. 1990. The staphylococcal enterotoxins and their relatives. Science 248:705-711. Mourad, W., R Al-Daccak, T. Chatila, and R S. Geha. 1993. Staphylococcal superantigens as inducers of signal transduction in MHC-class II positive cells. Semin. Immunol. 5:47-55. Mourad, W., K. Mehindate, T. J. Scholl, and S. R McColl. 1992. Engagement of major histocompatibility complex class-II molecules by superantigens induces inflammatory cytokine gene expression in human rheumatoid fibroblast-like synoviocytes. J. Exp. Med. 175:613-616. Mukaida, N., C. C. 0. Zachariae, G. L Gusella, and K. Matsushima. 1991. Dexamethasone inhibits the induction of monocyte chemotactic-activating factor production by IL-1 or tumor necrosis factor. J. Immunol. 146:1212-1215. Paliard, X., S. G. West, J. A. Lafferty, J. R Clements, J. W. Kappler, P. Marrack, and B. L Kotzin. 1991. Evidence for the effects of a superantigen in rheumatoid arthritis. Science 253:325329. Posnett, D. N. 1993. Do superantigens play a role in autoimmunity? Semin. Immunol. 5:65-72. Rink, L., and H. Kirchner. 1992. Mycoplasma arthritidis-derived superantigen. Chem. Immunol. 55:137-145. Rink, L., W. Nicklas, L Alvarez-Ossorio, M. Koester, and H. Kirchner. 1994. Differential induction of tumor necrosis factor alpha in murine and human leukocytes by Mycoplasma arthritidisderived superantigen. Infect. Immun. 62:462-467. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Shaw, G., and R Kamen. 1986. A conserved AU-sequence from the 3' untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell 46:659-667. Standiford, T. J., R M. Strieter, S. W. Chensue, J. Westwick, K. Kasahara, and S. L. Kunkel. 1990. IL-4 inhibits the expression of IL-8 from stimulated human monocytes. J. Immunol. 145:1435-1439. Thornhill, M. H., U. Kyan-Aung, and D. 0. Haskard. 1990. IL-4 increases human endothelial cell adhesiveness for T cells but not for neutrophils. J. Immunol. 144:3060-3065. Tobler, A., R Meier, M. Seitz, B. Dewald, M. Baggiolini, and M. F. Fey. 1992. Glucocorticoid downregulates gene expression of GMCSF, Nap-1/IL-8, and IL-6, but not M-CSF in human fibroblasts. Blood 79:45-51. Waage, A., and 0. Bakke. 1988. Glucocorticoids suppress the production of tumor necrosis factor by lipopolysaccharide-stimulated human monocytes. Immunology 63:299-302. Wong, H. L., G. L. Costa, M. T. Lotze, and S. M. Wahl. 1993. Interleukin (IL)4 differentially regulates IL-1 family gene expression and synthesis in vitro and in vivo. J. Exp. Med. 177:775-781. Wong, H. L., M. T. Lotze, L. M. Wahl, and S. M. Wahl. 1992. Administration of recombinant IL-4 to humans regulates gene expression, phenotype and function in circulating monocytes. J. Immunol. 148:2118-2125. Woodland, D. L., and M. A. Blackman. 1993. How do T-cell receptors, MHC molecules and superantigens get together? Immunol. Today 14:208.
