Transforming growth factor-b inhibits interleukin-10 synthesis by human monocytic cells.

ARTICLE

INTRODUCTION

In addition to its role in development, cell proliferation, cell differentiation and extracellular matrix production, TGF-beta exerts key anti-inflammatory properties. In vitro studies have demonstrated that TGF-beta1 suppresses the capacity of monocytes/macrophages to release reactive oxygen intermediates [1], reactive nitrogen intermediates [2] and proinflammatory cytokines [3, 4]. In vivo, TGF-beta1-null mutation in mice causes dramatic inflammatory lesions with massive infiltration of lymphocytes and macrophages in tissues such as heart and lungs [5, 6]. Conversely, gene transfer of plasmid DNA encoding TGF-beta1 suppresses inflammatory lesions as seen in an arthritis model [7]. TGF-beta1 appears to share a number of anti-inflammatory properties with IL-10 [8, 9]. The importance of IL-10 in inhibiting inflammation is indicated by the phenotype of IL-10-null mice, which develop inflammatory lesions, mainly in the intestinal tract [10]. In addition, both gene transfer of plasmid DNA encoding IL-10 and administration of recombinant IL-10-blunt inflammatory lesions, for instance in glomerulonephritis models [11].

Recently, TGF-beta1 has been shown to enhance IL-10 synthesis by mouse cells, suggesting an involvement of IL-10 in the anti-inflammatory activities of TGF-beta1. Firstly, studies from our laboratories have demonstrated that TGF-beta1 increases LPS-induced IL-10 generation by mouse mesangial cells [12]. TGF-beta1 has been shown to also up-regulate IL-10 synthesis by mouse macrophages [13, 14] and rat hepatic stellate cells [15]. Whether TGF-beta1 also enhances IL-10 synthesis by human monocytes/macrophages remains to be confirmed.

To this end, we evaluated the influence of TGF-beta1 on IL-10 synthesis by U. 937 cells, a human promonocytic cell line. In addition, we examined the possible mechanisms by which TGF-beta1 may exert its influence. The results provide the first evidence that TGF-beta1, instead of increasing, dramatically decreases IL-10 release from human monocytes/macrophages. This regulation appears to be mediated, at least in part, via inhibition of IL-10 gene transcription.

METHODS

Cells. The promonocytic U. 937 cell line was purchased from the American Type Culture Collection (Rockville, MD, USA) and maintained in RPMI 1640 supplemented with 10% FCS, 10 mM HEPES and antibiotics (culture medium) in a humidified incubator (95% air and 5% CO₂). These cells were differentiated to a mature macrophage-like phenotype, as previously described [16]: 0.5 x 10⁶ cells per ml were pretreated for 24 hours with 1.2% DMSO, washed twice with PBS and resuspended at 10⁶ cells, in 0.5 ml per well of culture medium (24-well plates). They were stimulated by either PMA (50 ng/ml; Sigma Chemical Co., St Louis, Mo, USA), Escherichia coli LPS (serotype 026B6 10 mug/ml; Sigma), TNF-alpha (5 ng/ml; Genzyme, Cambridge, MA, USA) or dibutyryl-cAMP (100 muM; Sigma) with or without ultrapure natural human TGF-beta1 (0-50 ng/ml; Genzyme) and polyclonal neutralizing antibody to TNF-alpha (Genzyme). The effects of agents either on cell growth and cell viability were determined by measuring cell number with a hematocytometer, and cell exclusion of trypan blue dye, respectively. Monocytes were obtained by plastic adherence of peripheral blood mononuclear cells from healthy volunteers, as previously described [17].

ELISA for IL-10. The supernatants were harvested after 24 hours, centrifuged to remove cell debris, and stored at - 70° C until they were analyzed for IL-10. Concentrations of IL-10 were measured by ELISA (PeliKine Compact human IL-10 ELISA kit, CLB, Amsterdam, The Netherlands). The minimum detectable concentration was 3 pg/ml.

Quantitative ELISA-polymerase chain reaction at saturation for IL-10 mRNA. Total cellular RNA was extracted from 0.1 x 10⁶ cells using the RNAble kit (Eurobio, Les Ulis, France) with a co-precipitant (Pellet Paint, Novagen, Madison, WI, USA) according to the protocol of the supplier. RNA was resuspended in 40 mul and reverse transcription carried out on 10 mul of RNA in a volume of 20 mul using random hexamers and oligo-dT and avian myeloblastosis virus transcriptase (Promoga, Madison, WI, USA) as described [18]. Quantitative PCR was carried out using an homologous DNA internal standard (IS) as described in ref 19, with the primers described in ref 18 and 20. Briefly, the IS was spiked in the PCR mix and an external scale, made of serial dilutions of purified cDNA amplicons (GAPDH or IL-10), was amplified in every experiment. After 42 PCR cycles, the amount of cDNA and of IS was quantified by a luminometric ELISA system [18]. The ratios of the cDNA/IS signals of the unknowns were compared to those of the scale by linear regression, allowing absolute values to be found. The ratio of IL-10 to GAPDH were computed for each sample, to normalize for RNA yield and RT efficiency and the duplicate values were averaged.

Cell transfection and reporter gene assay. The effect of TGF-beta1on IL-10 gene transcription was analyzed by transiently transfecting U. 937 cells with an IL-10 promoter-luciferase reporter plasmid (a kind gift of Dr. H.D. Volk, Humboldt University, Berlin, Germany). This plasmid contains a 1,308-bp fragment from the 5' non-coding sequence of the human IL-10 gene that was subcloned into the KpnI and Pst I sites of the luciferase reporter vector pGL2-Basic (Promega) to generate the construct pGL2-IL-10 [21]. After 24 hours of DMSO pretreatment, U. 937 cells in suspension were transfected with 2.5 mug plasmid DNA per 10⁷ cells using the DEAE-dextran procedure, as described elsewhere [22]. At 2 hours after transfection, U. 937 cells were plated onto 6-well plates at a density of 4 x 10⁶ cells/well and stimulated with PMA (50 ng/ml) or dibutyryl cAMP (100 muM) together with or without TGF-beta1 (1 ng/ml) for 24 hours. Luciferase activity was measured as previously described [22].

Statistics. Results are presented as the mean ± SD from n determinations. Differences between groups were assessed by Student's t-test. Statistical significance was set at p < 0.05.

RESULTS

TGF-beta1 inhibits IL-10 production in PMA-differentiated U. 937 cells

To determine if TGF-beta1 affects IL-10 production by PMA-differentiated U. 937 cells, DMSO-treated cells were stimulated with PMA together with or without different concentrations of TGF-beta1 for 24 hours. Supernatants were harvested at this time point and tested for IL-10 using a specific ELISA. The addition of TGF-beta1 dose-dependently decreased the IL-10 concentration (Figure 1). At the highest dose, IL-10 synthesis was suppressed by more than 90%. Under these conditions, cell number and cell viability were not decreased (97.6 ± 1.5% and 95.1 ± 0.8% cell viability after 24 hours exposure to 0 and 50 ng/ml TGF-beta1, respectively). In a further set of experiments, the time dependence of the TGF-beta1 inhibitory effects was examined. TGF-beta1 was effective when added at the time or up to 6 hours after cell stimulation with PMA (Figure 2).

To determine whether different pathways of IL-10 induction were susceptible to inhibition by TGF-beta1, undifferentiated U. 937 cells were stimulated with LPS, TNF-alpha or dibutyryl cAMP (Figure 3). All agents induced IL-10 expression, as shown previously [23]. The addition of TGF-beta1 dose-dependently inhibited this induction. This is consistent with TGF-beta1 having a broad effect on various pathways of IL-10 induction rather than a specific effect on PMA-signaling.

To further investigate the effect of TGF-beta1 on IL-10 production by human monocytes/macrophages, a subset of experiments was repeated using blood monocytes. Exposure of these cells to 10 ng/ml TGF-beta1 for 24 hours caused a 34.1 ± 13.7% decrease in IL-10 production (Table 1).

TGF-beta1 inhibits IL-10 mRNA expression in PMA-differentiated U. 937 cells

To determine whether IL-10 inhibition by TGF-beta1 was regulated at the transcriptional level, total RNA was isolated form U. 937 cells stimulated with PMA together with or without different concentrations of TGF-beta1 for 3 hours, and quantitative kinetic ELISA PCR was performed. The addition of TGF-beta1 dose-dependently decreased IL-10 mRNA expression (Figure 4A). The inhibition reached 92% at 5 ng/ml TGF-beta1 . The mechanisms whereby TGF-beta1 decreased IL-10 mRNA may include a decrease in IL-10 gene transcription and/or a decrease in IL-10 mRNA stability. To address this issue, the half-life of IL-10 mRNA was determined by measuring the IL-10 mRNA level after cell exposure to 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB), a specific RNA polymerase II inhibitor. As shown in Figure 4B, the decay of IL-10 mRNA was fast after DRB addition and obviously not accelerated by TGF-beta1. Thus TGF-beta1 effect on IL-10 mRNA levels may possibly be due to an effect on IL-10 gene transcription.

To further evaluate whether IL-10 mRNA expression was transcriptionally regulated by TGF-beta1, transient transfection assays were performed with a promoter-luciferase gene construct containing a IL-10 promoter fragment (1,308 bp upstream from the start codon). The levels of luciferase activity were determined after stimulation of U. 397 cells with either PMA or dibutyryl cAMP, in the absence or presence of TGF-beta1 (Figure 5). A low level of luciferase activity was detected in unstimulated U. 937 cells. Either PMA or dibutyryl cAMP stimulated the promoter activity of the construct. This response was completely suppressed by the addition of 1 ng/ml TGF-beta1.

TGF-beta1 inhibits IL-10 production in PMA-differentiated U. 937 cells through TNF-alpha-independent mechanisms

TGF-beta1 has previously been demonstrated to blunt the release of TNF-alpha from mononuclear cells [3]. Since TNF-alpha plays a predominant role in human monocyte IL-10 synthesis [24], this suggests that a decrease in TNF-alpha availability might participate in the TGF-beta1-mediated inhibition of IL-10 production. Therefore, in a further set of experiments, this possible action of TNF-alpha was prevented by the addition of neutralizing anti-TNF-alpha antibody (Figure 6). This slightly reduced IL-10 production by PMA-differentiated U. 937 cells but did not affect the response of these cells to TGF-beta1. Thus, TNF-alpha limitation is not involved in IL-10 decrease after cell exposure to TGF-beta1.

DISCUSSION

A number of previous studies have documented the induction of IL-10 in mouse monocytes/macrophages following either in vitro or in vivo exposure to TGF-beta1 [12-14]. Thus, at the start of these studies, we hypothesized that TGF-beta1 would stimulate IL-10 synthesis by human monocytic cells as well. This did not, however, prove to be true. On the contrary, the studies demonstrate that TGF-beta1 rather prevented IL-10 synthesis by U. 937 monocytic cells. Such opposite effects can not be explained by differences in the treatment of cells by TGF-beta1. In the present study as in previous studies [12, 14, 15], effective concentrations of TGF-beta1 ranged between 0.1 and 50 ng/ml. Concentrations of TGF-beta1 ranging between 0.001 and 0.05 ng/ml were without effect (data not shown). In addition, this result does not reflect a cytotoxic effect of TGF-beta1 against U. 937 cells as the viability of these cells was not decreased even at the highest TGF-beta1 concentration tested. This is in agreement with previous studies showing that TGF-beta1 does not affect proliferation and viability of U. 937 cells up to a concentration of 10 ng/ml [25].

The present study also shows that TGF-beta1 suppressed IL-10 mRNA expression in U. 937 cells to the same extent as IL-10 protein release. This is presumably the result of transcriptional inhibition rather than mRNA decay. Indeed, TGF-beta1 which did not modify IL-10 mRNA drops after inhibition of RNA de novo synthesis, decreased IL-10 promoter activity. The molecular mechanisms that account for such a decrease have not been identified. One likely possibility is that TGF-beta1 exerts a direct effect on the regulation of IL-10 gene transcription. Cloning and sequencing of the 5' regulatory region of the human IL-10 gene have revealed numerous sequence elements potentially involved in that regulation [21, 26, 27]. They include TATA box, IFN-enhancer core sequence and responsive elements to glucocorticoid, cAMP, AP-1, AP-2 and Sp1. Since both AP-1 and Sp1 participate in the transcriptional responses to TGF-beta1 [28], the cognate responsive elements could be particularly involved in the suppression of IL-10 gene transcription. Interestingly, the Sp 1 recognition site is located in a promoter region that suppresses IL-10 expression in B cells [26]. Thus, it may be speculated that Sp 1 induction is key event underlying TGF-beta1-suppression of IL-10 gene transcription in U. 937 monocytic cells. Alternatively, TGF-beta1 might interfere with the cAMP signaling pathway that participates in PMA-induced differentiation of U. 937 cells [29]. This mechanism is supported by the results demonstrating that TGF-beta1 also suppressed IL-10 release from U. 937 cells stimulated with dibutyryl cAMP instead of PMA.

A second possibility is that TGF-beta1 suppresses IL-10 mRNA expression indirectly by limiting the availability of other cytokines responsible for IL-10 induction. Prominent in this regard is the importance of TNF-alpha. TNF-alpha has been previously identified as the main inducer of IL-10 in human monocytes [24]. Our studies add to this data by demonstrating that a neutralizing anti-TNF-alpha antibody significantly decreased IL-10 induction in PMA-differentiated U. 937 cells. However, while TGF-beta has been shown to limit TNF-alpha synthesis in human mononuclear cells [3], this inhibition was not mandatory for IL-10 suppression in PMA-differentiated U. 937 cells. Indeed, no change in the response of these cells to TGF-beta1 has been observed upon exposure to neutralizing anti-TNF-alpha antibody. Thus, more experiments are needed to determine if an indirect mechanism is involved in the regulation of IL-10 suppression by TGF-beta1.

The in vitro and in vivo relevance of TGF-beta1-induced IL-10 suppression remains to be established. TGF-beta1 and IL-10 may constitute a cascade of cytokines produced sequentially to limit the development of inflammatory responses. The results of studies analyzing TGF-beta1 and IL-10 expression at different time points of human monocyte differentiation into macrophages have demonstrated a progressive inability of the cells to produce TGF-beta1, in contrast to an intact ability to produce IL-10 [30]. Our study suggests that the lack of TGF-beta1 is potentially critical to that prolonged expression of IL-10.

CONCLUSION

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