©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Interleukin (IL)-10 Inhibits Nuclear Factor B (NFB) Activation in Human Monocytes
IL-10 AND IL-4 SUPPRESS CYTOKINE SYNTHESIS BY DIFFERENT MECHANISMS (*)

Peng Wang , Ping Wu , Marvin I. Siegel , Robert W. Egan , M. Motasim Billah

From the (1) Schering-Plough Research Institute, Kenilworth, New Jersey 07033

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Our previous studies in human monocytes have demonstrated that interleukin (IL)-10 inhibits lipopolysaccharide (LPS)-stimulated production of inflammatory cytokines, IL-1, IL-6, IL-8, and tumor necrosis factor (TNF)- by blocking gene transcription. Using electrophoretic mobility shift assays (EMSA), we now show that, in monocytes stimulated with LPS or TNF, IL-10 inhibits nuclear localization of nuclear factor B (NFB), a transcription factor involved in the expression of inflammatory cytokine genes. Several other transcription factors including NF-IL-6, AP-1, AP-2, GR, CREB, Oct-1, and Sp-1 are not affected by IL-10. This selective inhibition by IL-10 of NFB activation occurs rapidly and in a dose-dependent manner and correlates well with IL-10's cytokine synthesis inhibitory activity in terms of both kinetics and dose responsiveness. Furthermore, compounds such as tosylphenylalanyl chloromethyl ketone and pyrrolidinedithiocarbamate that are known to selectively inhibit NFB activation block cytokine gene transcription in LPS-stimulated monocytes. Taken together, these results suggest that inhibition of NFB activation may be an important mechanism for IL-10 supression of cytokine gene transcription in human monocytes. IL-4, another cytokine that inhibits cytokine mRNA accumulation in monocytes, shows little inhibitory effect on LPS-induced NFB activation. Further examination reveals that, unlike IL-10, IL-4 enhances mRNA degradation and does not suppress cytokine gene transcription. These data indicate that IL-10 and IL-4 inhibit cytokine production by different mechanisms.


INTRODUCTION

Interleukin-10 (IL-10),() originally identified as a cytokine synthesis inhibitory factor, inhibits production of a wide range of cytokines in various cell types (for review, see Ref. 1). For instance, IL-10 suppresses synthesis of IL-1, IL-6, IL-8, and tumor necrosis factor (TNF)- in monocytes/macrophages (2, 3, 4, 5, 6) , of IL-1, IL-8, and TNF in neutrophils (5) , and of IL-2, TNF, interferon-, granulocyte/macrophage colony-stimulating factor, and others in T cells (7) . This inhibition of cytokine production is accompanied by reduced accumulation of mRNAs (2, 3, 4, 5, 6) . Nuclear run-on experiments in human monocytes have demonstrated that IL-10 inhibits lipopolysaccharide (LPS)-induced synthesis of IL-1, IL-6, IL-8, and TNF by acting mainly at the level of gene transcription (5, 6) , raising the possibility of IL-10 affecting the activities of transcription factors involved in cytokine gene expression. Gene promoters for IL-1, IL-6, IL-8, and TNF contain multiple cis-acting motifs including those that bind such transcription factors as nuclear factor B (NFB), NF-IL-6, AP-1, AP-2, CREB, GR, Oct-1, and Sp-1 (8, 9, 10, 11) . Motifs for NFB, NF-IL-6, AP-1, AP-2, and CREB are common to all four cytokine gene promoters, and recent data have clearly indicated that NFB, NF-IL-6, and AP-1 are critical for the induction of these cytokine genes (8, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21) .

NFB was originally identified as a heterodimeric complex consisting of two subunits, p65 and p50 (reviewed in Ref. 22). In most cells including monocytes, NFB is constitutively present in the cytoplasm as an inactive p65-p50-IB ternary complex. Various stimuli including LPS induce rapid dissociation of the inhibitory subunit IB. The resultant heterodimer of p50 and p65 translocates into the nucleus and initiates transcription of appropriate genes in synergy with other transcription factors such as NF-IL-6. Like NFB, NF-IL-6 exists in the cytosol and responds to stimulation by rapidly translocating into the nucleus (23) . By contrast, c-Fos and c-Jun, the two components of AP-1, are synthesized de novo during stimulation (24) . Our present data suggest that IL-10 causes selective inhibition of NFB activation in LPS-stimulated human monocytes, and that this NFB inhibition may be a mechanism by which IL-10 causes the suppression of the cytokine production. Our data further demonstrate that IL-4 inhibits cytokine production by a mechanism that does not involve NFB.


MATERIALS AND METHODS

Cell Preparation and Incubation

Human peripheral blood mononuclear cells (PBMC) were prepared as described (5) . For some experiments, purified monocytes were prepared from PBMC by elutriation (25) . The elutriated monocyte fraction consisted of >95% monocytes as determined by histologic staining and by immunofluorescence assay for CD14 antigen. Cell incubation was performed as described previously (5) .

Assays for Cytokine Proteins

Cytokines released into cell culture media were determined by using commercially available specific enzyme-linked immunosorbent assay kits (IL-6 and TNF kits from Biosource, Camarillo, CA; IL-1 and IL-8 kits from R& Systems, Minneapolis, MN). The assays were performed in duplicate. The data presented are from representative experiments. Data points are the means of two determinations, which were within 10%, mostly 5%, of the mean.

Northern Blot Analysis

Cytokine mRNA levels in cells after various treatments were determined by Northern blotting as described previously (6) . Cytokine cDNA probes were from R& Systems.

Nuclear Extract Preparation and Electrophoretic Mobility Shift Assay (EMSA)

At the end of the specified incubation time, cells (15-20 10per sample) were collected by centrifuging at 350 g for 5 min at 4 °C. After washing once with phosphate-buffered saline, the cell pellet was suspended in 0.5 ml of buffer A (10 m M Hepes-NaOH, pH 7.8, 15 m M KCl, 2 m M MgCl, 0.1 m M EDTA, 1 m M dithiothreitol, and 1 m M phenylmethylsulfonyl fluoride). The suspension was transferred to a microcentrifuge tube then centrifuged at 750 g for 5 min. The supernatant was removed by aspiration, and the pellet was resuspended in 0.2 ml of buffer A. After 10 min on ice, Nonidet P-40 was added to 0.5% and the suspension was centrifuged at 1330 g for 15 min. The resultant nuclear pellet was suspended in 15 µl of buffer B (20 m M Hepes-NaOH, pH 7.9, 1.5 m M MgCl, 0.5 m M dithiothreitol, 0.42 M NaCl, 0.2 m M EDTA, 25% glycerol, and 0.5 m M phenylmethylsulfonyl fluoride). After 15 min on ice with vigorous stirring, the suspension was centrifuged at 16,300 g for 10 min. Fifteen µl of the resultant supernatant was diluted with 75 µl of buffer C (20 m M Hepes-NaOH, pH 7.9, 50 m M KCl, 0.2 m M EDTA, 0.5 m M dithiothreitol, and 0.5 m M phenylmethylsulfonyl fluoride) to obtain the final nuclear extract preparation. EMSAs for NFB and other transcription factors in nuclei were performed by using gel shift assay system (Promega), except that 8% (instead of 4%) gels were used. The reaction mixture contained 2 µl of 5 gel shift binding buffer, 1 µl of P-labeled consensus oligonucleotide (oligo) probe of appropriate specificity, 2 µl of water, and 5 µl of nuclear extract (1 µg of protein).

Nuclear Run-on Assay

Cytokine gene transcription rates were measured by nuclear run-on assays as described previously (6) .

mRNA Stability Analysis

Cytokine mRNA stability was analyzed as described previously (6) .

Other Reagents

Human recombinant IL-10 and IL-4 were from Schering-Plough. Human recombinant TNF was obtained from R& Systems. LPS, TPCK, TLCK, and PDTC were purchased from Sigma. Consensus oligonucleotide probes for various transcription factors, except for NF-IL-6 were from Promega. The probe for NF-IL-6 was synthesized as reported (26) . Antibodies against p65 and p50 subunits of NFB were from Santa Cruz Biotechnology. Phosphorothioate analogs of NFB oligonucleotides, both sense and antisense, were prepared as reported (13) .


RESULTS

IL-10 Selectively Inhibits NFB Activation

In order to determine the effects of IL-10 treatment on transcription factors involved in the synthesis of proinflammatory cytokines, nuclear extracts were prepared from appropriately treated PBMC, and EMSA was performed using specific P-labeled cis-acting DNA elements. Thus, when nuclear extracts from LPS-treated cells were incubated with a P-labeled NFB oligonucleotide probe, a P-labeled protein-oligonucleotide complex was formed (Fig. 1 A). This complex formation was inhibited in the presence of an excess of unlabeled NFB oligonucleotide, but not in the presence of an excess of unlabeled Oct-1 oligonucleotide, showing that the NFB oligonucleotide specifically competes with P-labeled probe. Furthermore, specific antibodies to the NFB components p50 and p65 caused a supershift of the P-labeled complex, confirming the identity of the protein in the complex as NFB (Fig. 1). Hence, the P-labeled oligonucleotide probe used in these experiments complexed with cellular NFB and LPS stimulated NFB translocation into the nucleus. The data that anti-p50 antibody caused the total shift of the P-oligonucleotide-NFB complex while anti-p65 antibody caused only a partial shift show that LPS-stimulated nuclear NFB exists as both p50-p50 homodimer and p50-p65 heterodimer, with the homodimer predominating.


Figure 1: Effect of IL-10 on LPS- and TNF-induced NFB activation in human PBMC. A, the nuclear protein sample from LPS-stimulated cells (see below) was used to examine the specificity of the complex observed by EMSA using a P-labeled NFB oligonucleotide probe as described under ``Materials and Methods.'' Competition experiments were performed using unlabeled NFB and Oct-1 oligonucleotide probes at 100 molar excess. In ``supershift'' experiments, antibodies were used at a concentration of 1 µg per assay. B, cells were stimulated with 80 ng/ml LPS or 20 ng/ml TNF in the absence or presence of 10 ng/ml IL-10 for 1.5 h before the extraction of nuclear protein for EMSA.



Incubation of PBMC with IL-10 alone for up to 1.5 h had no effect on nuclear NFB. However, when IL-10 was added 5 min before LPS, NFB translocation induced by LPS was completely inhibited (Fig. 1 B). Under the same conditions, production of IL-6 and IL-8 was also inhibited (data not shown). Stimulation of PBMC with a different stimulus, TNF, caused nuclear localization of NFB, but to a smaller degree than LPS. TNF-induced NFB translocation (and cytokine synthesis under the same experimental conditions) was inhibited by IL-10 treatment. Thus, IL-10 inhibits NFB activation in PBMC in response to two distinct stimulating agents.

When incubated with P-labeled NF-IL-6 cis-acting element, nuclear extracts from LPS-stimulated PBMC generated a P-labeled protein-oligonucleotide complex, which was competed away with unlabeled NF-IL-6 oligonucleotide but not with unlabeled Oct-1 or NFB oligonucleotide. This complex formation was not affected by IL-10 treatment. LPS-stimulated localization of AP-1 into the nucleus, when assessed under similar conditions, was also not inhibited by IL-10. When incubated with the P-labeled cis-acting elements for AP-2, CREB, GR, Oct-1, and Sp-1, nuclear extracts from resting cells produced the appropriate P-labeled protein-oligonucleotide complexes. None of these complexes was affected by either LPS or IL-10 (data not shown). These results suggest that IL-10 causes selective inhibition of NFB activation in LPS-stimulated PBMC.

Characteristics of IL-10 Inhibition of NFB Activation

The inhibitory effects of IL-10 on NFB activation and cytokine synthesis were dependent on IL-10 concentration (Fig. 2). Nearly complete inhibition of both NFB translocation and cytokine synthesis occurred at 1-10 ng/ml IL-10. However, substantial NFB activity was still detected at 0.1 ng/ml IL-10, a concentration that inhibited TNF and IL-1 synthesis (60-75% inhibition) more effectively than it did IL-6 and IL-8 synthesis (20-35% inhibition) (Fig. 2). This differential inhibition pattern suggests that the threshold levels of NFB necessary for the initiation of cytokine gene expression vary among different cytokines and confirms our previous conclusion that cytokine gene expression is differentially regulated in LPS-stimulated monocytes (6) . This experiment was performed using a PBMC-derived preparation that contained >95% monocytes. The lymphocyte fraction obtained from the same PBMC preparation produced neither cytokines nor NFB translocation. Thus, cytokine production and NFB activation and their inhibition by IL-10 in LPS-stimulated PBMC were due primarily to monocytes and not to lymphocytes, although the lymphocytes accounted for 80% of the PBMC population.


Figure 2: Effects of IL-10 concentrations on LPS-induced NFB activation and cytokine release in human monocytes. A, various concentrations of IL-10 were added 5 min before the addition of 80 ng/ml LPS. After 1.5 h, nuclear protein was extracted and EMSA was performed as described under ``Materials and Methods.'' B, an aliquot of cell suspension from each of the culture samples used for EMSA was further incubated for a total period of 4 h. Cytokines released to the supernatants were measured by enzyme-linked immunosorbent assay. The amounts of IL-1, IL-6, IL-8, and TNF produced by LPS stimulation in the absence of IL-10 were 2.4, 17.0, 120.6, and 3.1 ng/ml, respectively.



In experiments discussed above where a complete inhibition of NFB activation was observed, IL-10 was added 5 min before LPS. The same complete inhibition was noted when IL-10 was added simultaneously with or 2 h before LPS, suggesting that IL-10 acts rapidly to inhibit NFB activity and that the signal(s) generated by IL-10 lasts for at least 2 h (Fig. 3). Similar rapid inhibitions have also been observed with both cytokine protein synthesis and cytokine gene expression (6) , demonstrating a strong correlation between cytokine gene expression and NFB activation. In addition, the nuclear appearance of NFB in response to LPS preceded cytokine mRNA accumulation, and nuclear NFB activity reached a maximum within 30 min of LPS addition and remained virtually unchanged over the next 2.5 h (data not shown). When IL-10 was added 0.5 h or 1 h after LPS during an LPS stimulation period of 1.5 h, substantial inhibition of NFB activity was still observed and was more pronounced at 0.5 h than at 1 h (Fig. 3). This observation supports the view that nuclear NFB undergoes rapid turnover during LPS stimulation (27) .


Figure 3: Effect of IL-10 addition time on inhibition of LPS-induced NFB activation in human PBMC. IL-10 (10 ng/ml) was added 2 h prior to, 0, 0.5, or 1 h after LPS (80 ng/ml). Cells were exposed to LPS for a total period of 1.5 h. For all other details, see ``Materials and Methods.''



Inhibition of NFB May Be Sufficient for Suppressed Synthesis of Inflammatory Cytokines

To determine whether inhibition of NFB activation is sufficient for suppression of cytokine production, two non-peptide inhibitors of NFB activation were investigated for their ability to inhibit cytokine mRNA synthesis. TPCK, a chymotrypsin-like serine protease inhibitor, is a potent inhibitor of NFB activation (28) . In our system, TPCK inhibited LPS-induced NFB activation as well as the accumulation of IL-1, IL-6, IL-8, and TNF mRNAs in a dose-dependent manner, and, at 20 µ M, both responses were completely inhibited (Fig. 4). TLCK, an analog of TPCK with inhibitory activity against trypsin-like serine proteases (28) , inhibited neither NFB activation (data not shown) nor cytokine mRNA accumulation in LPS-stimulated PBMC (Fig. 4). In LPS-stimulated human monocytes, it has been published that the antioxidant, PDTC, inhibits NFB activation (29, 30) and TNF synthesis (30) in a dose-dependent manner. In our experiments, PDTC by itself caused slight accumulation of cytokine mRNAs, but a dose-dependent inhibition of LPS-induced accumulation of various cytokine mRNAs was still observed (Fig. 4). Given the relative specificity with which these two compounds (TPCK in particular) inhibit NFB among various transcription factors tested (28, 29) , our present data are consistent with NFB inhibition being responsible for the inhibition of cytokine mRNA accumulation.


Figure 4: Effects of TPCK, TLCK, and PDTC on LPS-induced NFB activation and cytokine mRNA accumulation in human PBMC. A, cells were treated with TPCK at the specified concentrations for 30 min prior to stimulation with 80 ng/ml LPS for an additional 30 min. Nuclear protein was extracted and EMSA was performed as described under ``Materials and Methods.'' B, various inhibitors as indicated were added to the cell suspension for 30 min before the addition of LPS. Cells were cultured for an additional 2.5 h before extracting total RNA for Northern blot analysis. For details, see ``Materials and Methods.''



An antisense oligonucleotide to the p65 subunit of NFB inhibits IL-8 production in Jurkat T lymphocytes (13) . We have also examined the effect of this antisense oligonucleotide on the production of IL-1, IL-6, and TNF by LPS-stimulated PBMC. Preincubation of cells with the antisense oligonucleotide for 4-8 h resulted in an inhibition of cytokine synthesis by 50% to 70%, probably due to insufficient uptake of the oligonucleotide. Upon longer preincubations, the oligonucleotide itself induced cytokine synthesis. Taken together, these data suggest that inhibition of NFB activation may be sufficient for suppression of the synthesis of various inflammatory cytokines.

IL-4 Does Not Inhibit NFB Activation

The effect of IL-10 on LPS-induced NFB activation was compared with that of IL-4, because IL-4 is a potent inhibitor of cytokine synthesis in human monocytes (5) . Under the present experimental conditions, maximal IL-4 inhibition of LPS-induced synthesis of the cytokines was 80% (IL-1, IL-6, and IL-8) or 70% (TNF). The steady state levels of cytokine mRNAs, as determined by Northern blot analysis, were also similarly inhibited. However, at 10 ng/ml, that was optimal for its inhibitory effects on cytokine synthesis, IL-4 caused no significant suppression of the NFB activation (Fig. 5), suggesting that the inhibition of cytokine synthesis by IL-4 does not involve NFB.


Figure 5: Effect of IL-4 on LPS-induced NFB activation in human PBMC. Cells were stimulated with 80 ng/ml LPS in the presence of 10 ng/ml each of IL-10 or IL-4 for 1.5 h. Nuclear protein was extracted and EMSA was performed as described under ``Materials and Methods.''



Nuclear run-on experiments revealed no inhibition by IL-4 of transcription of cytokine genes in LPS-stimulated monocytes (Fig. 6), while mRNA stability analysis showed that IL-4 treatment did enhance the degradation of various cytokine mRNAs (Fig. 7). These results are consistent with IL-4 not inhibiting NFB activation and indicate that IL-4 and IL-10 inhibit cytokine synthesis in LPS-stimulated human monocytes through distinct mechanisms.


DISCUSSION

We have previously shown in human monocytes that IL-10 inhibits cytokine mRNA accumulation by acting at the level of gene transcription (6) , indicating a possible mechanism of IL-10 action through transcription factors. Several approaches using mutation or deletion of B sequence, overexpression of NFB proteins, antisense oligonucleotides to NFB, and the NFB DNA binding sequence have provided data to strongly implicate NFB in the expression of cytokine genes in monocytes (8, 12, 13, 14, 31, 32) . Additional studies have further suggested that NFB acts synergistically with NF-IL-6 and AP-1 to effect cytokine gene expression (17, 19) . In the present study, we demonstrate that IL-10 inhibits NFB activation in stimulated human monocytes without affecting AP-1, NF-IL-6, or other transcription factors that are believed to be involved in cytokine gene transcription. This selective inhibition of NFB activation occurs rapidly and in a dose-dependent manner and correlates well with the inhibition of cytokine gene transcription in terms of both time course and dose-response, indicating that IL-10 inhibition of cytokine gene expression may be mediated by the blockade of NFB activation.

Although treatment of cells with TPCK and PDTC may elicit broader effects than inhibition of NFB only, it is noteworthy that these compounds do not affect other transcription factors such as AP-1, Oct-1, CREB, and Sp-1 (28, 29) and that a structural analog of TPCK is without effect on NFB activation or on cytokine mRNA accumulation (Fig. 4). This selectivity with regard to NFB inhibition and the inhibition of cytokine mRNA accumulation by TPCK and PDTC in conjunction with the observations that antisense oligonucleotides and the NFB binding sequence inhibit cytokine mRNA synthesis would indicate that the blockade of NFB activation might indeed be sufficient for the inhibition of cytokine gene transcription, thus supporting the view that NFB is a potentially important target for IL-10's inhibitory action in human monocytes.

Our data, however, do not rule out the possibility that IL-10 exerts its inhibitory action on cytokine gene transcription through other transcription factors. While transcription factors such as AP-1, NF-IL-6, Oct-1, Sp-1, and CREB are not affected by IL-10, IL-10 does cause a rapid nuclear localization of the transcription factor, p91, a component of the protein complex which binds to the interferon- response region (33) . However, the importance of this complex in cytokine gene transcription has yet to be determined. Recently, it has been reported that in LPS-stimulated macrophages from mice lacking the p50 subunit of NFB, TNF and IL-1 release are normal (34) , suggesting NFB-independent regulation of the cytokine genes. However, it remains unclear whether the in vivo role of p50 in cytokine gene regulation can be compensated for by other members of the NFB family ( e.g. p65). A definitive assessment of the role of NFB in gene expression of inflammatory cytokines must await evaluation of animals with targeted disruption of both p50 and p65.

The fact that IL-4 inhibits cytokine synthesis and cytokine mRNA accumulation by enhancing cytokine mRNA degradation suggests the existence in human monocytes of a transcription factor-independent mechanism. This mechanism is not stimulated by IL-10 in human monocytes, as suggested by our observation that IL-10 has little or no effect on cytokine mRNA stability. Interestingly, in murine peritoneal (35) and porcine alveolar (36) macrophages, IL-4 inhibits cytokine gene transcription with little or no effect on mRNA stability, whereas, in these same systems, IL-10 appears to act by enhancing mRNA degradation (4) . These observations suggest that IL-4 and IL-10 generate distinct signals affecting distinct steps in cytokine mRNA metabolism and that the mechanisms employed by these two inhibitory cytokines vary depending on the animal species.

IL-10 action on NFB most likely involves the blockade of a reaction required for release of IB from the NFB complex in intact cells. Experiments using cell-free preparations have suggested that protein kinases such as protein kinase C phosphorylate IB causing its release and ensuing activation of NFB (37) . Although phorbol myristate acetate-induced activation of NF-B appears to involve protein kinase C, other NFB activating agents such as TNF and IL-1 appear not to require protein kinase C (38, 39) . A recent study has demonstrated that several NFB activators including LPS, IL-1, TNF and phorbol ester activate a chymotrypsin-like serine protease that rapidly degrades the inhibitory subunit IB (28) . Additionally, the p50 component of the NFB complex is derived from the inactive cytosolic precursor protein p105 through a proteolytic cleavage (27) catalyzed presumably by an ATP-dependent serine protease activity (40) , and this proteolytic cleavage occurs within 15 min after LPS addition (27) . That this latter process might also be a target for IL-10 action is suggested by the fact that the p50 homodimer predominates in the nucleus from LPS-stimulated monocytes and that this translocation is inhibited by IL-10.

The protease activities involved in the processing of p105 and IB are likely regulated by reactive oxygen intermediates such as hydroxyl radical, based on the observation that various NFB inducers produce reactive oxygen intermediates and that antioxidants and radical scavengers inhibit NFB activation (29) . Because IL-10 blocks NFB activation in response to distinct NFB inducers such as LPS and TNF, IL-10-induced signals seem to interfere with the activity of a common messenger produced by different signaling pathways, and this messenger might well be a reactive oxygen intermediate. Therefore, it will be interesting to determine whether IL-10 inhibits metabolism of precursor proteins and formation of reactive oxygen intermediates.

In conclusion, the present study shows that the cytokine synthesis inhibitory activity of IL-10 may be mediated by inhibition of NFB activation in activated human monocytes, and that the major mechanism by which IL-4 inhibits cytokine synthesis in human monocytes is, distinctively, the enhancement of cytokine mRNA degradation.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: IL, interleukin; LPS, lipopolysaccharide; TNF, tumor necrosis factor; PBMC, peripheral blood mononuclear cell; NF, nuclear factor; EMSA, electrophoretic mobility shift assay; TPCK, tosylphenylalanyl chloromethyl ketone; TLCK, N- p-tosyl- L-lysine chloromethyl ketone; PDTC, pyrrolidinedithiocarbamate.


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