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(Journal of Leukocyte Biology. 2002;72:800-809.)
© 2002 by Society for Leukocyte Biology

IL-10 expression profiling in human monocytes

Lynn Williams*, Gabor Jarai{dagger}, Alexandra Smith{dagger} and Peter Finan{dagger}

* Kennedy Institute of Rheumatology, London, United Kingdom; and
{dagger} Novartis Horsham Research Centre, West Sussex, United Kingdom

Correspondence: Dr. Lynn Williams, Kennedy Institute of Rheumatology, Imperial College of Science, Technology and Medicine, Charing Cross Campus, ARC Building, 1 Aspenlea Road, Hammersmith, London, W6 8LH, UK. E-mail: lynn.williams{at}ic.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine with numerous immunomodulatory effects, including the inhibition of proinflammatory cytokine production. The mechanisms by which IL-10 exerts these effects still remain largely unknown. As there is evidence that suggests IL-10-mediated cytokine suppression requires the induction of an intermediate gene, we have used gene-chip technology to identify IL-10-inducible genes in human monocytes. We have been able to identify a total of 19 genes that are up-regulated in response to IL-10. Three of these genes had been identified previously: IL-1ra, suppressors of cytokine signaling-3, and CD163; however, the other 16 represent newly identified IL-10-responsive genes. Further analysis of the regulation of eight of these genes showed a remarkable specificity to regulation by lipopolysaccharides (LPS) and IL-10, but not by other anti-inflammatory mediators such as IL-4 and transforming growth factor-ß, suggesting that two diverse stimuli such as IL-10 and LPS may engage common signaling mechanisms.

Key Words: LPS • IFN • lipopolysaccharide • transmembrane • TGF-ß


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interleukin-10 (IL-10) is a pleiotrophic cytokine that has an important role in regulating the immune response [1 ]. This cytokine potently inactivates macrophages, inhibiting the expression of proinflammatory cytokines [e.g., tumor necrosis factor {alpha} (TNF-{alpha}) and IL-6] and disabling antigen presentation/T cell activation by inhibiting expression of major histocompatibility complex class II, B7-1, and B7-2 [2 3 4 ]. The anti-inflammatory activity of IL-10 is augmented by enhancing the release of soluble(s) TNF receptors (R) and IL-1R antagonist [5 ]. In contrast to its activities on macrophages, IL-10 induces the proliferation of mast cells, B and T cells, and enhances T cell responses to IL-2 [6 7 8 ].

Numerous studies have shown that IL-10 treatment can decrease the severity of inflammatory processes in vivo. Specifically, IL-10 has been shown to reduce disease activity in numerous animal models of inflammation such as sepsis [9 ], collagen-induced arthritis [10 ], and insulitis [11 ] and in some models of experimental autoimmune encephalomyelitis [12 ]. Given its efficacy in animal models, recombinant IL-10 treatment has been developed as a candidate therapy for several immune diseases. However, phase II trial data from Crohn’s disease and rheumatoid arthritis suggested only a mild amelioration of disease activity [13 , 14 ]. More encouraging data are emerging from phase II trials of systemic administration of IL-10 in the treatment of psoratic skin lesions [15 ].

The intracellular mechanism by which IL-10 mediates its anti-inflammatory and other effects remains largely unknown. This subject, however, is of more than academic interest, given the potential of IL-10 as a therapeutic agent. IL-10 mediates these diverse activities via a high affinity cell surface receptor [16 17 18 ] composed of two chains, IL-10R1 and the recently identified CRF4 (IL-10R2 or IL-10Rß) [19 , 20 ]. Both chains of the receptor have been classified as members of the class II subgroup of cytokine receptors, called the interferon receptor (IFNR) family. Like other members of this family, IL-10 activates the JAK kinases, Jak-1 and Tyk-2 [21 ]. In addition, IL-10 has been reported to activate signal transducer and activator of transcription (STAT)-1, STAT-3, STAT-5 [22 23 24 25 ], phosphatidylinositol-3 kinase, and p70 S6 kinase [26 ].

A major focus of IL-10 research has been to identify the mechanism by which IL-10 mediates suppression of cytokine synthesis. This remains a controversial field; specifically, the ability of IL-10 to inhibit lipopolysaccharide (LPS)-induced gene expression has been shown to be transcriptionally mediated via the inhibition of the nuclear factor-{kappa}B pathway [27 , 28 ]. However, further evidence also suggests that IL-10 can act through a post-transciptional mechanism via destabilizing mRNA. In the case of TNF-{alpha} and the chemokine KC, this effect requires the AU-rich elements in the 3' untranslated region [29 , 30 ]. Furthermore, these reports suggest that the effects of IL-10 are indirect and that IL-10 is inducing a gene whose product is responsible for mediating the destabilization of mRNA [30 31 32 ]. The generation of macrophage-specific STAT-3 knockout mice has provided further evidence supporting a role for IL-10-induced de novo protein synthesis in the anti-inflammatory response of IL-10. Macrophages isolated from these mice are no longer sensitive to IL-10-mediated suppression of TNF release [33 ]. Only a limited number of genes have been shown to be up-regulated by IL-10; these include CD16, CD64, TIMP-1, monocyte chemoattractant protein-1 (MCP-1), CCR5, IL-1ra, TNF-R2, suppressors of cytokine signaling-3 (SOCS3), and CD163, as reviewed by Donnelly et al. [34 ]. Although some of these proteins have anti-inflammatory properties, it is not thought that they could be responsible for the suppression of cytokine synthesis.

We have used microarray analysis to identify IL-10-inducible genes in the presence and absence of the powerful pro-inflammatory stimulus LPS. These studies have identified 19 IL-10-inducible genes. Three of these genes, IL-1ra, SOCS3, and CD163, have previously been shown as being regulated by IL-10; however, the other 16 represent novel IL-10-inducible genes first identified in this study.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells
Single donor platelephoresis residues were purchased from the South London Blood Transfusion Service (Tooting, UK). Mononuclear cells were isolated by Ficoll-Hypaque centrifugation (specific density, 1.077 g/ml) preceding T cell/monocyte separation in a Beckman JE6 elutriator. T cell purity was assessed by flow cytometry using directly conjugated anti-CD3 (Becton Dickinson, Oxford, UK), and monocyte purity was assessed using anti-CD45 and anti-CD14 antibodies (LeucoGATE, Becton Dickinson) and was routinely greater than 90%. All media and sera are routinely tested for endotoxin using the limulus amoebocyte lysate test (BioWhittaker Inc., Walkersville, MD) and are rejected if the endotoxin concentration exceeded 0.1 U/ml. Monocytes isolated by elutriation were cultured in six-well plates in RPMI supplemented with 10% human AB serum (Sigma Chemical Co., Dorset, UK) for 7 days. Cells were stimulated with IL-10, transforming growth factor-ß (TGF-ß), IL-4 (R&D Systems, Oxon, UK), TGF-ß, dexamethasone (Dex; Sigma Chemical Co.), or Salmonella typhimurium LPS (Sigma Chemical Co.). Anti-IL-10 from R&D Systems isotype control was provided from D. Mason (Oxford, UK).

RNA isolation
Following stimulation, cells were pelleted (14,000 rpm/30 s), supernatants were discarded, and 1 ml TRIZOL (Gibco, Scotland) was used to extract RNA from the cell pellets as per the manufacturer’s protocol. In total, RNA isolated from five donors was pooled and normalized between donors prior to mRNA purification using Oligotex-dt (Qiagen, Mississauga, Ontario, Canada) as described [35 ].

Microarray analysis
To generate fluorescently labeled probes, 200 ng mRNA was used to make first-strand cDNA in the presence of fluorescent nucleotide analogues [35 ]. Labeled probes were hybridized to the Synteni UniGemV2.12 human microarray containing 10,000 genes and expressed sequence tags (ESTs) and represent most well-characterized genes. This chip was chosen out of a set of six chips encompassing 60,000 ESTs as it allows the collection of the most informative subset of the expression data. However, the analysis of our samples on the complete chip set would identify additional, regulated genes and ESTs. Images were analyzed as described [35 ]. Hybridizations were performed in duplicate for control purposes, and each probe was labeled with two different dyes. Data were analyzed using the software GemTools, using a modified algorithm for normalization. For the computation of the normalized differential expression values, a background subtraction was performed. Essentially, the background intensity values were subtracted for each probe for each data point, and the values obtained were used for the generation of differential expression values. Only genes with statistically significant differential expression values (significance P<0.05) were considered for further analyses.

cDNA synthesis
Synthesis of cDNA from total RNA samples was performed using the Multiscribe reverse transcriptase (RT) and random hexamers according to the manufacturer’s protocol (PE Applied Biosystems, Foster City, CA). Briefly, 40 ng total RNA was used to prepare a master solution containing 5.5 mM MgCl2, 500 µM (each) dNTP, 2.5 µM random hexamers, 0.4 U/µl RNase inhibitor, 1.5 U/µl Multiscribe RT, and 1x TaqMan RT buffer. The mixture was incubated at 25°C for 10 min and 48°C for 30 min, and the reaction was terminated by 95°C for 5 min. After synthesis, cDNA was stored at 4°C.

Quantitation of gene expression by real-time RT-polymerase chain reaction (PCR; TaqMan)
TaqMan probes and primers for quantitative detection of each gene transcript were designed using Primer Express computer software (PE Applied Biosystems). A TaqMan PCR core reagent kit was used to prepare a master mix for each experiment. Primers, forward (F) and reverse (R), and probes (P), were purchased from (PE Biosystems, Warrington, Cheshire); concentrations were optimized on an individual basis and are shown in Table 1 . An ABI PRISM 7700 detector sequence was programmed for the initial step of 2 min at 50°C and 10 min at 95°C, followed by 45 cycles of 15 s at 95°C and 1 min at 58°C. Each measurement was set up in triplicate, and three independent experiments were performed.


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  		Table 1. TaqMan Primers and Probes Used in This Study

 
Calculations
The CT value is defined as the cycle number in which the detected fluorescence exceeds the threshold value. In all experiments, the threshold value is kept constant. The "fold" difference between unstimulated and stimulated was calculated, where CT is normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Relative quantitation of gene expression was determined using comparative CT-multiplex PCR in the same tube after validating that the efficiencies of target gene and GAPDH PCR were approximately equal. All calculations followed procedures outlined in ABI PRISM 7700 sequence detective system bulletin #2.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
IL-10 induces a small subset of genes in human monocytes
We were interested in identifying genes that were up-regulated in response to IL-10, or alternatively, genes that were sensitive to IL-10-mediated suppression under proinflammatory conditions. Human monocytes were left unstimulated or stimulated with LPS alone, IL-10 alone, or LPS and IL-10 together for 4 h. After RNA isolation and cDNA synthesis, fluorescently labeled, first-strand cDNA probes were hybridized to cDNA microarrays, which contained sequences corresponding to a total of 10,000 human genes and EST clusters. The mean "fold" induction of repression of each sample was expressed as a ratio of intensities between stimulated over unstimulated in two parallel experiments performed. In total, the expression of 70 known genes and two EST sequences was found to be significantly (P<0.05) increased in response to LPS stimulation (at least a threefold increase in expression compared with unstimulated) as shown in Table 2 .


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  		Table 2. LPS Induced RNA Expression Profile

 
In contrast, only 19 known genes were shown to be significantly up-regulated in response to IL-10 (Table 3 ). Surprisingly, eight genes were common to both stimuli: IL-1R antagonist, IL-7R, IFN-induced transmembrane (TM) protein-3, glycerol kinase, pre-B cell colony-enhancing factor, SOCS3, acid spingomyelinase-like phosphodiesterase, and immunoglobulin (Ig)-like receptor subfamily A (Ig-like 4; with TM domain) and Ig-like 6 (without TM domain). Three of the IL-10-specific genes (IL-1ra, SOCS3, and CD163) had previously been shown to be up-regulated in response to IL-10, thus validating the experimental approach.


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  		Table 3. IL-10-Induced RNA Expression Profile

 
IL-10 down-regulates a large proportion of LPS-stimulated genes
Comparison of the LPS-induced gene profile to the LPS/IL-10 profile (Table 4 ) revealed the extent of IL-10-mediated suppression of cytokine synthesis. Many well-characterized, IL-10-sensitive, proinflammatory genes such as MCP-2, IL-1, IL-8, and prostagladin-endoperoxidase synthase 2 (COX2) were all down-regulated in response to IL-10 (i.e., they were no longer present in the IL-10/LPS profile), again validating experimental conditions. Perhaps most interesting was the ability of IL-10 to suppress the expression of such a diverse range of LPS-inducible genes. Only 12 LPS-specific genes (i.e., genes not induced in response to IL-10) were still significantly expressed in the presence of IL-10: aquaporin 9, trytophanyl-tRNA synthetase, carboxypeptidase, B-factor (properdin), BCL-related protein A1, syndecan 2, TNF-induced protein 3, EH domain containing 1, egf-like molecule containing mucin-like hormone receptor sequence, indoeamine-pyrrole2, 3 dioxygenase, Vav1, and superoxide dismutase. Further analysis comparing the 5' promoter regions and the 3' untranslated regions of these IL-10-resistant genes to the IL-10-sensitive genes may reveal which sequences specifically predispose these mRNAs to regulation by IL-10.


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  		Table 4. IL-10 and LPS-Induced RNA Expression Profile

 
Stimulation with IL-10 alone and LPS alone resulted in the suppression of a limited number of genes and ESTs. With the exception of the gene encoding ecotopic viral integration site 2A, the presence of LPS alone (Table 5 ) or IL-10 alone (Table 6 ) seems to prevent the down-regulation of the same set of genes. It is interesting that a distinct set of genes was down-regulated by the combination of the two stimuli as opposed to stimulating individually with IL-10 or LPS (Table 7 ).


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  		Table 5. Genes that Are Down-regulated in Response to LPS

 

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  		Table 6. Genes that Are Down-regulated in Response to IL-10

 

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  		Table 7. Genes that are Down-regulated in Response to IL-10 and LPS

 
Kinetics of IL-10 gene induction
As the gene-chip experiment was performed at one time point, it was important to further investigate the kinetics of gene induction. Of the 19 genes that were shown to up-regulated in response to IL-10 stimulation, we chose to look at eight in more detail using real-time PCR. These genes i) had not previously been shown to regulate these genes, or ii) their function was unknown or could potentially be involved in an IL-10 anti-inflammatory response. The experiments were performed on two different donors who gave similar results. Of the eight genes that had previously been shown to be up-regulated by IL-10 in the microarray experiments, we were only able to confirm such regulation of seven genes by real-time PCR. This may be a consequence of using RNA from pooled (n=5) as opposed to single donors. However, we tested three further donors and found that IL-10 was able to induce KIA0390 mRNA accumulation in one donor (data not shown). As shown in Figure 1 , IL-10 induces significant accumulation of mRNA of the remaining seven genes: S100A9, T cell protein tyrosine phosphatase (TCPTP), Ig-like 4 and Ig-like 6, 15+ NAD dehydroprostagladin dehyrogenase (PGDH), TLRI and signaling lymphocyte activation molecule (SLAM). With the exception of Ig-like 6, mRNA accumulation is detected within 60 min of IL-10 stimulation, suggesting that this is a direct effect of IL-10. Interestingly, with the exception of S100A9, IL-10-induced mRNA accumulation remained sustained for up to 24 h.



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  		Figure 1. Kinetics of IL-10 gene induction. Monocytes were stimulated 0–24 h with IL-10 (25 ng/ml). After isolation of RNA, the relative expression of each gene’s mRNA was determined using TaqMan real-time PCR. The housekeeping gene GAPDH was used for normalization. Data are presented as mean ± SD of triplicate reactions. The figure is representative of three independent experiments (using three separate donors). Calculations were performed as described in Materials and Methods. The statistical significance of the "fold" induction compared with unstimulated is indicated as follows: *, significant (0.01<P<0.05), or **, highly significant (0.01<P<0.001), as determined by Student’s t-test.

 
Comparison of expression of IL-10-inducible genes in T cells, monocytes, and macrophages
Given that TCPTP and SLAM have previously been shown to be expressed in T cells [36 , 37 ], it was a concern that these events were not occurring in monocytes but were a result of IL-10 gene induction in a population of contaminating T cells. To clarify this issue, T cells and monocytes were isolated from the same donor and stimulated with IL-10 for 0–24 h. In each case, no greater then a 2% contaminating population was present. Furthermore, monocytes were driven into a macrophage-like population by incubating monocytes with human AB+ serum for 7 days. At the end of this period, CD3+ T cells were no longer detectable (by CD3 staining). Cells were stimulated for 0, 1, 4, or 24 h with IL-10. The data generated after 4 h of IL-10 stimulation are shown in Table 8 . IL-10 induced a small but significant increase in SLAM (1.94±0.38) in T cells. However, in monocytes, the "fold" increase was 30 ± 10.73, and in macrophages, the "fold" increase was 10.22 ± 0.11. These data would suggest that this induction could be assigned to the monocyte rather than the T cell population.


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  		Table 8. IL-10 Induction of Gene Expression in T Cells, Monocytes, and Macrophages of Expression

 
Surprisingly, no increase in TCPTP mRNA was observed in T cells stimulated with IL-10 at any of the time points studied. In contrast, IL-10 induced similar levels of TCPTP mRNA in macrophages. Conversely, IL-10 induced a low level 15+ PGDH mRNA in T cells but not in macrophages. This discrepancy may be because steady-state 15+ PGDH mRNA levels were much higher in macrophages than in T cell or monocytes (data not shown). S100A9 was not regulated in T cells in response to IL-10, but macrophages showed very different expression patterns to those observed in monocytes (Table 8 ): at early time points, 1–4 h, IL-10 high levels of S100A mRNA (14.53±4.5). However, after 24 h, the level of expression increased to 58-fold ± 10 (data not shown), which may suggest that IL-10 induces another protein that acts alone as a potent inducer of S100A9 mRNA expression or strongly synergizes with IL-10 to enhance expression to such a high level. Ig-like 4 or Ig-like 6 mRNA expression did not appear to be regulated by IL-10 in T cells or macrophages. Like 15+ PGDH, much higher steady-state levels of mRNA were observed in macrophages compared with monocytes. Again IL-10 could not induce KIA0390 expression in T cells or macrophages.

Comparison of IL-10 gene induction to other anti-inflammatory mediators
We were interested to know if any of the genes we had shown as being regulated by IL-10 were also regulated by other anti-inflammatory stimuli. Surprisingly, with the exception of IL-1ra [38 , 39 ], most of the previously known genes induced by IL-10 are not induced by other anti-inflammatory stimuli. We chose to look at three other anti-inflammatory stimuli, which all have the ability to inhibit LPS-induced TNF-{alpha} production, namely IL-4, TGF-ß, and dexamethasone. As shown in Figure 2 , four out of the six genes (S100A9, TCPTP, Ig-like 4, and 15+ PGDH) were not regulated by other anti-inflammatory stimuli. Dexamethasone, and to a lesser degree IL-4, also induced Ig-like 6 and SLAM expression. It is most interesting that dexamethasone was able to significantly enhance KIA0390 mRNA expression. At this moment, it is unclear why dexamethasone was able to induce expression in the same donor in which IL-10 had no effect.



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  		Figure 2. Comparison of IL-10 gene induction to other anti-inflammatory mediators. Monocytes were left unstimulated or stimulated for 4 h with IL-10 (25 ng/ml), IL-4 (25 ng/ml), TGF-ß (25 ng/ml), or Dex (100 nM). After isolation of RNA, the relative expression of each gene’s mRNA was determined using TaqMan real-time PCR. The housekeeping gene GAPDH was used for normalization. Data are presented as mean ± SD of triplicate reactions. Calculations were performed as described in Materials and Methods. The statistical significance of the "fold" induction compared with unstimulated is indicated as follows: *, significant (0.01<P<0.05), or **, highly significant (0.01<P<0.001), as determined by Student’s t-test.

 
Effects of the presence of LPS on IL-10-induced gene induction
We were also interested in looking at the regulation of these eight genes in the presence of a strong inflammatory stimulus such as LPS. It has previously been observed that LPS can synergize with IL-10 to further enhance expression of TNF-R2 [40 ] and IL-1ra [41 ]. However, we did not observe any synergy between IL-10 and LPS. In five of the eight genes studied (Fig. 3a 3b 3c 3d and 3h ), LPS and IL-10 induced similar levels of induction. In all these genes, inclusion of anti-IL-10 antibody did not inhibit LPS-induced gene induction. This would suggest that this effect is specific to LPS rather than by any IL-10 produced by the monocytes in response to LPS. Only Toll-like receptor 1 (TLR1) was uniquely induced by IL-10 (Fig. 3g) . In this particular donor, IL-10 induced a small but significant level of expression of KIA0390 (1.5±0.2) as did LPS (3.1±0.4).



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  		Figure 3. Effects of the presence of LPS on IL-10-induced gene induction. Monocytes were left unstimulated or stimulated for 4 h with IL-10 (25 ng/ml) or LPS (10 ng/ml) in the absence or presence of anti-IL-10 (10 µg/ml) or an isotype (Ig) control (10 µg/ml) or a combination of LPS and IL-10. After isolation of RNA, the relative expression of each gene’s mRNA was determined using TaqMan real-time PCR. The housekeeping gene GAPDH was used for normalization. Data are presented as mean ± SD of triplicate reactions. The figure is representative of three independent experiments (using three separate donors). Calculations were performed as described in Materials and Methods. The statistical significance of the "fold" induction compared with unstimulated is indicated as follows: *, significant (0.01+P<0.05), or **, highly significant (0.01<P<0.001), as determined by Student’s t-test.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, our aim was to identify novel genes regulated by IL-10. Using microarrays representing 10,000 human genes and ESTs, we were able to identify 19 genes whose expression increased in the presence of IL-10. Only three of these genes have previously been shown to be regulated by IL-10, leaving 16 newly identified IL-10-regulated genes. Furthermore, we have identified many new genes whose expression is down-regulated in the unique environment of combined LPS and IL-10 stimulation.

We chose to study further a limited number of genes. One of the genes up-regulated in response to IL-10 in monocytes and macrophages was TCPTP. This is an attractive candidate anti-inflammatory mediator, as the signaling pathways activated by LPS are well-characterized, and stimulation of monocytes by LPS results in activation of numerous tyrosine kinases (as reviewed in ref [42 ]). However, LPS also caused a mild elevation of TCPTP mRNA levels. TCPTP is an intracellular, non-TM tyrosine phosphatase initially isolated from a T cell cDNA library [36 ] but has subsequently been shown to be expressed in many tissues. Two splice variants are expressed: one, a 48-KD protein, is localized within the endoplasmic reticulum, whereas a 45-kDa (TC45) form, if found, is in basal conditions in the nucleus. At present, we are unsure as to which splice variant IL-10 induces. Recently, TCPTP has been shown to act as a negative regulator of Jak1 and Jak3 [43 ], so analogous to SOCS3, IL-10 may induce TCPTP to dampen down IL-10-induced JAK/STAT signaling. We have constructed adenoviral vectors containing a dominant negative form of TC45 and are studying its role in macrophage biology.

Another protein, whose expression had previously been linked to T cells, is SLAM. A member of the CD2 subfamily of Ig superfamily, SLAM is rapidly induced upon T cell activation, and engagement of this receptor leads to IFN-{gamma} production [37 ]. It has subsequently been shown to be expressed on B cells, natural killer cells, and CD86+ dendritic cells, and the receptor is believed to mediate entry of the measles virus into immune cells [44 ]. SLAM exists in multiple isoforms—namely, cytoplasmic, soluble, membrane, and variant membrane [45 ]. The TaqMan PCR primers, which were used in this study, were designed to recognize all isoforms, and it is intriguing to speculate which, if any, are regulated at the level of protein expression. To date, IL-10 has been shown to down-regulate the expression of the membrane isoform of SLAM in T cells [46 ], which is in keeping with its immunosuppressive properties.

Like TCPTP and SLAM, S100A9 mRNA was also induced in macrophages and monocytes, an important characteristic for any candidate gene if it is to mediate IL-10-induced suppression of cytokine synthesis. This protein was initially identified as a calcium-binding protein isolated from infiltrate macrophages of rheumatoid arthritis [47 ]. Along with a closely related protein S100A8, these proteins were found to be elevated in plasma of patients with chronic inflammatory diseases. Several S100 proteins are known to be chemotactic, but a conclusive role for S100A9 has yet to be defined. Recently, IL-10 has been shown to synergize with LPS to induce S100A8 expression in the murine macrophages cell line RAW 264 [48 ]. In macrophages, we observed a substantial increase in S100A9 expression after 24 h of IL-10 stimulation, suggesting that this may be a secondary event meditated by induction of another gene product, which we may not have identified in our initial profiling study in monocytes.

Both IL-10 and LPS induced the Ig-like genes 4 and 6, but it is interesting that no synergy between the two stimuli was observed. The emerging family of Ig-like receptors can bind to human leukocyte antigen class I molecules and is believed to modulate function in a positive and negative manner [49 ]. Ig-like 4 is believed to act as an inhibitory receptor, and Ig-like 6 is stimulatory. However, the gene product we observed to be regulated by IL-10 lacks a TM domain and exists as a soluble receptor. Unlike any other anti-inflammatory stimuli tested, IL-10 induced TLR1 mRNA accumulation in monocytes. Upon maturation in macrophages, IL-10 was no longer capable of up-regulating Ig-like receptor 4 or 6 or the TLR1, probably because the maturation process seems to up-regulate steady-state levels of these three receptors. Given that the Toll family of receptors serves to recognize a diverse array of microbial products that mediate innate immune defenses [50 ], it is somewhat surprising that this particular TLR appears to be up-regulated by IL-10 and down-regulated by LPS. However, it is now becoming clear that TLRs can form heterodimers, and TLR1, when found in a heterodimer with TLR2, can act as an inhibitory receptor preventing modulin, a soluble factor released by Staphylococcus epidermidis-induced responses in macrophages [51 ]. Any elevation of TLR1 expression at the protein level may serve to enhance the levels of inhibitory TLRs on monocytes.

The expression of 15+ PGDH followed a unique pattern. It was expressed in T cells and monocytes but not macrophages, and, like many of the other genes, it was also induced by LPS. dexamethasone also induced significant expression, which is at odds with previous data that had shown that dexamethasone actually inhibited phorbol 12-myristate 13-acetate-induced PGDH expression in U937 cells [52 ]. However, there is a precedent for a role of IL-10 in the regulation of PGDH. Pomini et al. [53 ] described that IL-10 alone was unable to induce PGDH mRNA expression, but it was able to reverse TNF- and IL-1ß-mediated suppression. PGDH expression is tightly regulated, and its expression has only been detected in a limited number of tissues. It is a key catabolic enzyme involved in the inactivation of prostagladins. The ability of IL-10 to inhibit prostaglandin E2 production is believed to be mediated by the inhibition of COX1 and COX2 gene expression [54 ]. However, induction of PGDH by IL-10 may represent an additional mechanism by which this potent, anti-inflammatory cytokine exerts its effects.

In summary, two main conclusions may be drawn from this study. First, IL-10 appears to induce only a limited number of genes in human monocytes. Surprisingly, LPS also induced seven out of the eight genes identified within this study. It would therefore seem unlikely that these particular genes are responsible for mediating the anti-inflammatory response of IL-10. Second, and of equal interest, is the apparent ability of IL-10 to inhibit the expression of such a large array of LPS-inducible genes. We find this intriguing, as this suppression seems not to be limited to the expression of well-characterized genes. In addition to suppressing proinflammatory mediators, this group of genes down-regulated by IL-10 also included numerous genes whose function is not believed to be involved in the inflammatory process, further emphasizing the wide array of IL-10 functions in addition to the conventional anti-inflammatory role ascribed to this intriguing cytokine.


   ACKNOWLEDGEMENTS
 
We thank Dr. Smith and Prof. B. Foxwell for their helpful reading of this manuscript.

Received November 20, 2001; revised April 17, 2002; accepted April 18, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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