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IL-1 Induced Chemokine Production Through the Association of Syk with TNF Receptor-Associated Factor-6 in Nasal Fibroblast Lines¹

Takechiyo Yamada^*, Shigeharu Fujieda^2,*, Shigeru Yanagi, Hirohei Yamamura, Ryoko Inatome, Hideyuki Yamamoto^*, Hideki Igawa^* and Hitoshi Saito^*

^* Department of Otorhinolaryngology, Fukui Medical University, Fukui, Japan; and Department of Biochemistry, Kobe University School of Medicine, Kobe, Japan.

	Abstract

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The fibroblasts stimulated by cytokines released the chemokine and recruited the infiltrating cells, including eosinophils, that play a key role in the pathogenesis of airway disease. We established the human fibroblast lines showing high Syk expression and the lines showing low Syk expression from pieces of nasal polyp. IL-1 induces the interaction of TNFR-associated factor (TRAF) 6 with IL-1R-associated kinase, which is rapidly recruited to the IL-1R after IL-1 induction, whereas TRAF2 participates in TNF--signaling. In the present study, we found that Syk played a different role in IL-1- and TNF--induced chemokine production through a signaling complex involving Syk and TRAF6. Overexpression of wild-type Syk by gene transfer enhanced RANTES production from nasal fibroblasts stimulated with IL-1. The decrease of Syk expression by the administration of Syk antisense inhibited RANTES production in response to IL-1. However, the change of Syk expression did not affect RANTES production by TNF- stimulation. We concluded that Syk is required for the IL-1-induced chemokine production through the association with TRAF-6 in fibroblasts of nasal polyps.

	Introduction

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Fibroblasts, a rich source of chemokines, interact with eosinophils and play a key role in the pathogenesis of airway disease. The fibroblasts released the chemokine RANTES or GM-CSF and recruited the infiltrating cells, including eosinophils (1). High amounts of RANTES were found in the nasal polyp specimens (2). Cultured nasal fibroblasts expressed RANTES mRNA and secreted RANTES protein (3). We established the human fibroblast lines showing high Syk expression and the lines showing low Syk expression from pieces of nasal polyp (4), although Syk is widely expressed and plays an important role in intracellular signal transduction in hemopoietic cells, B cells (5), mast cells (6), platelets (7), eosinophils (8), and so on.

RANTES production requires the transcription factor NF-B and the activation of mitogen-activated protein kinases (MAPKs;³ Refs. 9, 10, 11, 12, 13). IL-1 induces the interaction of TNFR- associated factor 6 (TRAF6) with IL-1R-associated kinase (IRAK), which is recruited rapidly to the IL-1R after IL-1 induction, whereas TRAF2 participates in TNF- signaling (14, 15). Recently, Wong et al. (16) demonstrated a signaling complex involving tyrosine kinase c-Src and TRAF6. Additionally, Li et al. (17) revealed that tyrosine kinase inhibitor substantially alleviated the cytokine gene expression and decreased the level and activity of IRAK.

In the present study, we examined whether Syk is involved in the RANTES production by IL-1 and TNF- via TRAFs. We also looked at the phosphorylation state of c-Jun N-terminal kinase (JNK), extracellular signal-related kinase (ERK), and p38 MAPK in response to IL-1 when Syk expression was artificially changed by oligonucleotides and vectors. Further, IL-1-induced IB degradation was investigated as a reporter of NF-B-signaling, a transcription factor essential for RANTES expression (18, 19).

	Materials and Methods

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Reagents

Anti-Syk Ab, anti-c-Src Ab, anti-TRAF6 Ab, anti-TRAF 2 Ab, anti-JNK Ab, anti-phosphorylated JNK Ab, anti-p38 Ab, anti-phosphorylated p38 Ab, anti-ERK Ab, and anti-phosphorylated ERK Ab were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Recombinant human IL-1 was obtained from Cistron Biotechnology (Pine Brook, NJ). Recombinant human TNF- and the ELISA kits for RANTES were purchased from Biosource (Camarillo, CA). The adenovirus expression vector kit was purchased from Takara Biomedicals (Tokyo, Japan).

Cell preparation

Nasal polyps were obtained during surgery from patients with chronic paranasal rhinosinusitis. Chronic rhinosinusitis was strictly diagnosed via endoscopic findings, paranasal sinus x-ray tomography, clinical history, and symptoms by a specialist of otorhinolaryngology. Nasal polyps were fluid-filled sacks formed from the upper part of the nasal cavity.

Human nasal fibroblast lines were grown from small pieces of nasal polyp and regular passages (20). Fibroblasts were used at passage numbers 4–10. No contamination of epithelial cells was confirmed by immunohistochemical examination with cytokeratin marker. There was wide variation in the expression of Syk by nasal fibroblast lines as described previously (4). Before starting the experiments, Western blot analysis was performed to be determined Syk expression in fibroblast cell line. In this study, we used the human fibroblast line with the highest Syk expression and the line with the lowest Syk expression in our library. We could not find any difference in growth characteristics or morphology between fibroblast line with the highest and that with the lowest Syk expression.

Cell stimulation and chemokine assay

The fibroblasts were stimulated by IL-1 and TNF- in RPMI 1640 medium supplemented with 10% FCS and in humidified atmosphere of 10% CO₂ in air at 37°C for 24 h. The supernatants were harvested and stored at -80°C. Amounts of RANTES in the cell culture supernatant were measured with an ELISA kit. Measurements were performed according to the manufacturers’ protocol.

Coimmunoprecipitation

Cells were collected by centrifugation and lysed in 1 ml of immunoprecipitation buffer (1% digitonin, 0.15 M NaCl, 5 mM , 100 mM Na₃VO₄, 10 mg/ml leupeptin, 1 mM PMSF, and 50 mM Tris-HCl, pH 7.5). The lysate was clarified and incubated with excess protein A-Sepharose 4B (50% slurry). The cleared sample was immunoprecipitated with Abs and protein A-Sepharose 4B at 4°C. The immune complexes were processed for immunoblot as described above.

Gel electrophoresis and Western blots

The fibroblasts or immunoprecipitants were boiled with electrophoresis sample buffer for 3 min and separated by SDS-PAGE. The separated proteins were transferred electrophoretically to polyvinylidene difluoride membranes at the same protein concentration per lane. The membranes were blocked at room temperature for 1 h in pH 7.4 PBS with 10% BSA and were probed with Abs as described previously (4).

Preparation of recombinant adenoviruses

An adenovirus vector encoding Syk was constructed with the use of an adenovirus expression kit (Takara). A 2.7-kb cDNA fragment containing the entire coding sequence of Syk was ligated into the pAxCAwt cosmid vector, which contains the modified chicken -actin promoter with cytomegalovirus-IE enhancer (CAG promoter). Then, the recombinant adenovirus pAxCAwt-Syk was prepared by homologous recombination of the expression cosmid cassette and parental viral genome and amplified to achieve a stock with a titer of 10⁹ PFU/ml. A control recombinant adenovirus pAxCAwt-LacZ was used. Fibroblasts were equally infected with 10 multiplicity of infection of pAxCAwt-Syk or pAxCAwt-LacZ, respectively. Syk expression were detected from day 2 to day 8 after the infection.

Antisense oligodeoxynucleotides

Human nasal fibroblasts were cultured in RPMI 1640 in the presence of phosphorothioate oligodeoxynucleotides (Nisshinbo Industries, Tokyo, Japan) at 10 µM. The sequences used were as follows: antisense Syk, CATGCTTCAGGGGCCGG; sense Syk, CCGGCCCTGAAGCATG (4, 7).

Statistical analyses

Statistical analysis was performed using the Mann-Whitney U test and Wilcoxon signed-ranks test to assess the differences in RANTES production levels. Macintosh computers (Apple Computer, Cupertino, CA) with Statview software (Abacus Concepts, Berkeley, CA) were used for all statistical analyses.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Syk antisense oligodeoxynucleotides attenuated IL-1-induced RANTES production from human nasal fibroblasts

We obtained the human fibroblast line showing high Syk expression. Production of RANTES by the fibroblasts was measured by ELISA. As shown in Fig. 1A, the levels of RANTES production by the fibroblasts in the presence of IL-1 (1 ng/ml) or TNF- (1 ng/ml) was significantly elevated compared with those in the absence of IL-1 or TNF- (p < 0.01).

View larger version (16K): [in this window] [in a new window]   FIGURE 1. Syk antisense oligodeoxynucleotides attenuated IL-1-induced RANTES production from human nasal fibroblasts. A, Nasal fibroblasts with high Syk expression (1 x 106/ml) were cultured in the presence of 10 µM Syk antisense, 10 µM Syk sense, or in the absence of oligodeoxynucleotide for 6 h and then stimulated with IL-1 (1 ng/ml) or TNF- (1 ng/ml). Supernatants were harvested for analysis of RANTES secretion by ELISA 24 h after the stimulation. The pretreatment of Syk antisense-inhibited RANTES production by fibroblasts stimulated with IL-1 (*, p < 0.01), but did not inhibit those stimulated with TNF-. B, Nasal fibroblasts were transfected with Syk antisense, Syk sense, or no oligodeoxynucleotide. Harvested proteins (30 µg) from cultured cells were reacted with anti-Syk Ab and anti-c-Src Ab and then processed for immunoblotting. The arrows indicate the positions of Syk and c-Src.

IL-1 failed to induce RANTES production from fibroblasts that did not exhibit Syk protein (Fig. 1A). Sense oligodeoxynucleotides did not alter IL-1-induced RANTES production. There was a significant difference in IL-1-induced RANTES production between fibroblasts treated with Syk antisense oligodeoxynucleotides and those with sense oligodeoxynucleotides (p < 0.01).

In contrast, there was no difference in TNF--induced RANTES production between fibroblasts treated with Syk antisense oligodeoxynucleotides and those with sense oligodeoxynucleotides (Fig. 1A). Interestingly, the role of Syk for IL-1 signaling might be different from that of TNF- signaling in RANTES production.

IL-1-dependent association of Syk with TRAF6

To investigate IL-1 or TNF--induced signaling, we examined the relationship between Syk and the TRAF family with digitonin as a detergent in lysis buffer, which has been reported to maintain the associations after cell lysis (21). It has been determined that endogenous Syk, c-Src, and TRAF6 form a signaling complex in nasal fibroblasts after simulation of IL-1.

As shown in Fig. 2A, immunoblot analysis revealed the existence of Syk in the immune complex precipitated by anti-TRAF6 Ab. IL-1 stimulation substantially increased the association of TRAF6 with Syk in nasal fibroblasts. Also, we found the existence of c-Src in the immune complex precipitated by anti-TRAF6 Ab after the stimulation of IL-1. In contrast, TNF- stimulation caused no association of TRAF2 with Syk or with c-Src (Fig. 2A). When samples were immunoprecipitated with TRAF6 or TRAF2 and blotted with corresponding TRAF6 or TRAF2, respectively, there were no differences in the levels of TRAF6 and TRAF2 immunoprecipitates before and after stimulation. Also, we found no association of TRAF6 with Syk in the fibroblasts after TNF--stimulation (data not shown).

View larger version (20K): [in this window] [in a new window]   FIGURE 2. IL-1-dependent association of Syk with TRAF6. A, Fibroblasts with high Syk expression were cultured without (lanes 1 and 3), or with 1 ng/ml IL-1 (lane 2) and TNF- (lane 4) for 10 min. The cells were collected and lysed in 1 ml of immunoprecipitation buffer. The sample was immunoprecipitated with anti-TRAF6 Ab (lanes 1 and 2) and anti-TRAF2 Ab (lanes 3 and 4). The sample then was blotted with anti-Syk and anti-Src Ab. The same samples were blotted with anti-TRAF6 Ab (lanes 1 and 2) and anti-TRAF2 Ab (lanes 3 and 4). B, After the treatment as shown in A, the same amount of lysate (30 µg) before immunoprecipitation was applied to each lane and blotted with anti-Syk Ab, anti-c-Src Ab, anti-TRAF6 Ab, and anti-TRAF2 Ab.

The effect of reduced Syk expression on the phosphorylation of JNK, p38, and ERK

Because IL-1 was reported to cause the activation of JNK (14, 15), we investigated IL-1-induced phosphorylation of JNK, ERK, and p38 MAPK. To analyze whether Syk plays some roles in regulating the phosphorylation of these molecules in response to IL-1, the fibroblasts were exposed to Syk sense or Syk antisense oligodeoxynucleotides, and then treated with IL-1 for 10 min. As shown in Fig. 3, cell lysates were subjected to immunoblotting with anti-phosphorylated JNK Ab, anti-phosphorylated p38 Ab, and anti-phosphorylated ERK Ab.

View larger version (24K): [in this window] [in a new window]   FIGURE 3. The effect of reduced Syk expression on the phosphorylation of JNK, p38, and ERK. Nasal fibroblasts with high Syk expression were transfected with 10 µM Syk sense (lanes 1 and 2) or antisense (lanes 3 and 4) for 6 h, and then stimulated without IL-1 (lanes 1 and 3) or with 1 ng/ml IL-1 (lanes 2 and 4) for 10 min. The same amount of lysate (30 µg) was applied to each lane and blotted with anti-phosphorylated JNK Ab (top) and anti-JNK1 Ab (bottom) as shown in Fig. 3A, with anti-phosphorylated p38 Ab (top) and with anti-p38 Ab (bottom) in Fig. 3B, with anti-phosphorylated ERK Ab (top) and with anti-ERK Ab (bottom) in Fig. 3C, and with anti-Syk Ab in Fig. 3D. The antisense treatment decreased IL-1-induced JNK and p38 phosphorylation in nasal fibroblasts.

The treatment with Syk sense oligodeoxynucleotides had no effect on the JNK and p38 phosphorylation induced by IL-1. However, IL-1 failed to induce JNK phosphorylation of the fibroblasts treated with Syk antisense oligodeoxynucleotides (Fig. 3A). Syk antisense oligodeoxynucleotides attenuated p38 phosphorylation induced by IL-1 (Fig. 3B) The semiquantitative densitometric analysis in p38 phosphorylation showed that the value in the Syk antisense-treated fibroblast was half of that in the Syk sense-treated fibroblast (0.38 vs 0.77).

In contrast, ERK was constantly phosphorylated on tyrosine 204 before the stimulation in the fibroblast lines and was not changed by stimulation of IL-1 (Fig. 3C). Syk antisense oligodeoxynucleotides did not affect the phosphorylation of ERK in the fibroblasts.

Wild-type Syk vector transfection magnified IL-1-induced RANTES production by nasal fibroblasts

We also established the human fibroblast line showing low Syk expression and transfected expression vector from wild-type Syk into the fibroblasts. As shown in Fig. 4A, there was no difference in the spontaneous RANTES production in the absence of IL-1 or TNF- among the fibroblasts transfected with the different vectors. Although TNF- caused the high levels of RANTES production from the fibroblasts showing low Syk expression, IL-1 induced small amounts of RANTES production from the same fibroblasts. However, the fibroblasts transfected with the wild-type Syk vector produced significantly higher levels of RANTES after stimulation with IL-1 than those transfected with the control vector did (p < 0.01). Wild-type Syk vector transfection did not affect the TNF--induced RANTES production by nasal fibroblasts. No difference was found in the morphology and cell growth among four fibroblasts even after the transfection of virus vector (data not shown).

View larger version (16K): [in this window] [in a new window]   FIGURE 4. Wild-type Syk vector transfection magnified IL-1-induced RANTES production by nasal fibroblasts. A, After the transfection with wild-type Syk vector or control vector or no vector into the fibroblasts with low Syk expression for 48 h, the cells were incubated in the presence or absence of IL-1 (1 ng/ml) or TNF- (1 ng/ml), and supernatants were harvested for analysis of RANTES secretion by ELISA. IL-1-induced RANTES levels from fibroblasts transfected with wild-type Syk vector were significantly higher than those from fibroblasts transfected with control vector (*, p < 0.01), whereas there was no difference between TNF--induced RANTES levels from fibroblasts transfected with wild-type Syk vector and those from fibroblasts transfected with control vector. B, Nasal fibroblasts with low Syk expression are used in this experiment. The prepared proteins (30 µg) were processed and reacted with anti-Syk Ab or anti-c-Src Ab. Anti-Syk immunoblotting showed the increased Syk expression in the fibroblasts transfected with wild-type Syk vector but did not in those transfected with control vector.

The effect of increased Syk expression on the phosphorylation of JNK, p38, and ERK

After the fibroblasts showing low Syk expression were transfected with wild-type Syk vector or control vector, we investigated IL-1-induced phosphorylation of JNK, p38 MAPK, and ERK. The fibroblasts were transfected with wild-type Syk vector or control vector and then treated with IL-1 for 10 min. The cell lysates were subjected to immunoblotting with anti-phosphorylated JNK Ab, anti-phosphorylated p38 Ab, and anti-phosphorylated ERK Ab (Fig. 5).

View larger version (18K): [in this window] [in a new window]   FIGURE 5. The effect of increased Syk expression on the phosphorylation of JNK, p38, and ERK. Nasal fibroblasts with low Syk expression were transfected with control vector (lanes 1 and 2) or wild-type Syk vector (lanes 3 and 4) for 48 h and then stimulated without IL-1 (lanes 1 and 3) or with 1 ng/ml IL-1 (lanes 2 and 4) for 10 min. The same amount of lysate (30 µg) was applied to each lane and blotted with anti-phosphorylated JNK Ab (top) and with anti-JNK1 Ab (bottom) as shown in A, with anti-phosphorylated p38 Ab (top) and with anti-p38 Ab (bottom) in B, with anti-phosphorylated ERK Ab (top) and with anti-ERK Ab (bottom) in C, and with anti-Syk Ab in D. Stimulation of IL-1 induced JNK phosphorylation and enhanced p38 phosphorylation in fibroblast transfected with wild-type Syk vector.

The effect of increased Syk expression on the degradation of IB

Because IL-1 was reported to cause the activation of NF-B (17), we investigated the effects of Syk expression on the degradation of IB-, which regulates NF-B (Fig. 6). The fibroblasts showing low Syk expression were transfected with wild-type Syk vector or control vector and then stimulated with IL-1. IL-1 induced the degradation of IB- in the fibroblasts transfected with wild-type Syk vector, although it did not induce any degradation IB- in those transfected with control vector, suggesting that IL-1 induced NF-B activation in the fibroblast showing high Syk expression.

View larger version (12K): [in this window] [in a new window]   FIGURE 6. The effect of increased Syk expression on the degradation of IB. The fibroblasts with low Syk expression were transfected with control vector (lanes 1 and 2), or wild-type Syk vector (lanes 3 and 4) for 48 h, and then stimulated without IL-1 (lanes 1 and 3), or with 1 ng/ml IL-1 (lanes 2 and 4) for 20 min. The same amount of lysate (30 µg) was applied to each lane and blotted with IB as shown in A, and with anti-Syk Ab in B. The degradation of IB after IL-1 stimulation was shown in the fibroblast transfected with wild-type Syk vector.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The IL-1 and TNF- signals are transduced through TRAF6 and TRAF2, respectively (15). IL-1 induced the interaction of TRAF6 with IRAK, although IRAK did not associate with TRAF2 after the stimulation of IL-1 (14). The engagement of cytokine receptors with TRAF6 activates the antiapoptotic serine/threonine kinase (Akt/protein kinase B) through a signaling complex involving Src family kinases and TRAF6 (16). Tyrosine kinases could regulate the cytokine gene expression and IRAK activity (17). We revealed that IL-1 induced chemokine production from the nasal fibroblasts through a signaling complex involving Syk and TRAF6. IL-1 also induced the interaction of c-Src with TRAF6. Src homology (SH) 3 domains contain proline-rich motifs containing PXXP consensus sequence (24), and TRAF6 contains a SH3-interacting sequence (16). Therefore, Src family kinases have SH3 domains with the ability to interact with TRAF6. In human platelets, Syk associated transiently with c-Src, and Src family kinases participated in the signaling (25). In our system, Syk could take an important role with Src family kinases and TRAF6 in IL-1 signaling. However, we cannot exclude the possible interaction with unknown adaptor proteins to mediate this signaling.

IL-1 stimulated transcription factors activating protein-1 and NF-B through activation of JNK and the IB kinase (15). In response to IL-1, MAPK was activated in a concentration-dependent manner, and its activation was attenuated by tyrosine kinase inhibitor (13). In this report, we demonstrated that IL-1 induced the phosphorylation of JNK mainly and p38 partially in nasal fibroblast lines. RANTES mRNA expressions were induced through the activation of MAPKs (11, 12, 13) or through JNK and NF-B kinase cascades in macrophages (26). The Syk-generated signal cooperates to enhance Rac-induced JNK activation in T lymphocytes (27). MAPKs activation was compromised in the macrophages of Syk^-/- mice after Fc- receptor stimulation (28). The cytokine production was produced by an early tyrosine phosphorylation of Syk in murine resident peritoneal macrophages (29). Here, we found that Syk expression mainly affected JNK activation in response to IL-1 in nasal fibroblast lines. Transcriptional activation of the human RANTES promoter was dependent on specific activating protein-1, which was regulated by JNK (26). Syk has two SH2, instead of SH3, and the C-terminal SH2 domain of Syk was required for induction of JNK-activation (30).

Regulation of RANTES chemokine gene expression requires NF-B through IB (9). Recently, Das (31) reported that JNK mediated NF-B activation after degradation of IB. We demonstrated that Syk was related to JNK phosphorylation and degradation of IB. Phosphoinositide 3-kinase and Akt were involved in NF-B signaling (32). TRAF6 induced Akt activation through Src family kinases (16). Syk also is required for the activation of the phosphoinositide 3-kinase and Akt in B cells (33).

Toll-like receptor has been proven to mediate LPS-induced cellular signaling (34). TRAF6 mediated both IL-1- and LPS-induced signaling. However, TRAF2 did not mediate in the IL-1- and LPS-signaling while it was involved in TNF-signaling (35). We also found that pretreatment of antisense oligodeoxynucleotides to Syk inhibited RANTES production by fibroblasts stimulated with LPS (4).

The pathogenetic findings of nasal polyps shows an increase in infiltrating cells including eosinophils (36). Eosinophils and fibroblasts strongly interacted with each other in the pathogenesis of airway diseases, because fibroblasts are a rich source of chemokines, cytokines, and inflammatory mediators. Recently, Stenton et al. (37) reported that Syk antisense oligodeoxynucleotides delivered by aerosol to lungs in vivo depressed Syk expression and pulmonary inflammation. In conclusion, Syk proteins are endogenously expressed in human nasal fibroblasts. Syk antisense oligodeoxynucleotides depressed Syk expression and chemokine production in nasal fibroblasts that originated from nasal polyps. The regulation of Syk expression may prove useful as one of the strategies for the treatment of airway diseases such as asthma or nasal polyps.

	Acknowledgments

 
We thank I. Funatsu for excellent technical assistance.

	Footnotes

 
¹ This study was supported by grants-in-aid for Scientific Research from Ministry of Education, Culture, Sports, Science and Technology, Japan, and by the Uehara Foundation.

² Address correspondence and reprint requests to Dr. Shigeharu Fujieda, Shimoaizuki, Matsuoka, Fukui 910-1193, Japan. E-mail address: sfujieda{at}fmsrsa.fukui-med.ac.jp

³ Abbreviations: MAPK, mitogen-activated protein kinase; TRAF, TNFR-associated factor; IRAK, IL-1R-associated kinase; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-related kinase; Akt, antiapoptotic serine/threonine kinase; SH, Src homology.

Received for publication November 20, 2000. Accepted for publication April 23, 2001.

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