- Email: [email protected]
Effects of secretory leukocyte protease inhibitor on the tumor necrosis factor-alpha production and NF-κB activation of lipopolysaccharide-stimulated macrophages
www.elsevier.com/locate/jn/abr/ycyto Cytokine 21 (2003) 38–42
Short communication
Effects of secretory leukocyte protease inhibitor on the tumor necrosis factor-alpha production and NF-jB activation of lipopolysaccharide-stimulated macrophages Chiaki Sano, Toshiaki Shimizu, Haruaki Tomioka* Department of Microbiology and Immunology, Shimane Medical, University, Izumo, Shimane 693-8501, Japan Received 18 September 2002; accepted 12 November 2002
Abstract It has been reported that lipopolysaccharide (LPS)-hyporesponsiveness of macrophages (M/s) of C3H/HeJ mice with a mutated Lps gene (Lpsd) is related to high-level expression of secretory leukocyte protease inhibitor (SLPI) in response to LPS, causing suppression of NF-jB activation and tumor necrosis factor-a (TNF-a) production. We thus examined the effects of SLPI on the TNF-a production by LPS-stimulated M/s. Neither intact SLPI nor half-sized SLPI (1/2 SLPI) down-regulated M/ TNF-a production. 1/2 SLPI weakly increased M/ TNF-a production in response to LPS signaling and potentiated the LPS-induced activation of NF-jB, especially the binding of p65–p50 heterodimers to the DNA jB sites, suggesting that LPS-hyporesponsiveness of Lpsd M/s is not due to the overexpression of SLPI. Ó 2003 Elsevier Science Ltd. All rights reserved. Keywords: Lipopolysaccharide; Macrophages; Nuclear factor-jB; Secretory leukocyte protease inhibitor; Tumor necrosis factor-a
1. Introduction Secretory leukocyte protease inhibitor (SLPI), a potent serine protease inhibitor in lungs, which is secreted by bronchial and alveolar epithelial cells [1,2] and which inhibits a wide range of proteases, plays important roles in protection of the airway epithelial surface in the lungs from attack by neutrophil-derived proteases generated at the sites of inflammatory reactions [1,2]. Jin et al. [3,4] reported that M/s from LPS-non-responder C3H/HeJ mice carrying a mutated Lps gene (Lpsd) [5] expressed high levels of SLPI responding to LPS signals and that this caused a lack in tumor necrosis factor-a (TNF-a) production by Lpsd M/s in response to LPS stimulation. Moreover, transfection of M/s with SLPI gene suppressed LPS-induced activation of NF-jB, causing the reduction in the production of TNF-a and the expression of inducible nitric oxide synthase (iNOS),
thereby NO production. On the basis of these findings, they proposed that LPS-hyporesponsiveness of Lpsd M/s is principally due to the overexpression of SLPI. On the other hand, Zhang et al. [6] reported a contradictory finding that SLPI failed to modulate TNF-a production by human monocytes although it suppressed the production of prostaglandin H synthase-2, prostaglandin E2, and matrix metalloproteinases. Therefore, the effects of SLPI on M/ production of TNF-a is controversial. In the present study, we studied profiles of TNF-a production and NF-jB activation in M/s from mice with normal Lps gene (Lpsn) in response to LPS stimulation, with special reference to the effects of SLPI on these M/ functions.
2. Results 2.1. Effects of SLPI on TNF-a production by LPS-stimulated M/s
* Corresponding author. Tel.: +81-853-20-2146; fax: +81-853-202145. E-mail address: [email protected] (H. Tomioka).
First, we examined the effects of SLPI on TNF-a production by LPS-stimulated Lpsn M/s. Fig. 1 shows
1043-4666/03/$ - see front matter Ó 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1043-4666(02)00485-4
C. Sano et al. / Cytokine 21 (2003) 38–42
the effects of intact SLPI and half-sized human SLPI (1/2 SLPI), which contains the C-terminal domain (Arg58–Ala107) of intact SLPI [7], on the TNF-a production by M/s during the first 24-h cultivation in response to LPS. The protease activity of intact SLPI is completely retained in the 1/2 SLPI except that the latter is more specific for elastase than for trypsin [7,8]. In addition, human and mouse SLPIs share high amino acid homology with each other [3], and human SLPI is highly active against serine proteases of animals other than humans [8] and also efficacious in ameliorating chemically induced pulmonary fibrosis in hamsters [9]. Thus, human SLPI and 1/2 SLPI exerted its action not only in humans but also in mice even in the cross-species testing. Intact SLPI even at 1 lg/ml did not affect M/ production of TNF-a in response to LPS stimulation (Fig. 1A). On the other hand, 1/2 SLPI at 1 lg/ml weakly but significantly up-regulated the TNF-a production by LPS-stimulated M/s (Fig. 1B). These findings indicate that both the half domain (Ser1–Thr57) of SLPI deleted in 1/2 SLPI and the half domain (Arg58– Ala107) retained by 1/2 SLPI do not inhibit LPSmediated up-regulation of M/ TNF-a production, and the latter domain alone is somewhat stimulatory of M/ TNF-a production. In separate experiments using enzyme-linked immunosorbent assay (ELISA), the
Fig. 1. Effects of SLPI on the production of TNF-a by LPS-stimulated M/s. Murine peritoneal M/s were cultured in the medium with or without the addition of LPS (10 lg/ml) in the presence or absence of indicated concentrations of intact SLPI (A) or 1/2 SLPI (B) for 24 h. Each bar indicates the mean standard errors of the mean (n ¼ 4). *: Significantly larger than the values of M/s which were not given SLPI treatment (P < 0:05).
39
degradation of TNF-a due to M/-derived protease was insignificant when exogenously added TNF-a was incubated in the 12-h culture fluid of LPS-stimulated M/s for 24 h. That is, less than 10% of initially added TNF-a was degraded. This indicates that the 1/2 SLPI-mediated increase in M/ TNF-a production was not due to the anti-protease activity of 1/2 SLPI but mainly due to its up-regulatory effect on the de novo synthesis of TNF-a by M/s. Indeed, as shown in Fig. 2, the ELISPOT assay indicated that 1/2 SLPI slightly increased the number of TNF-a-producing M/ populations in LPS-stimulated M/s. 2.2. Effect of 1/2 SLPI on the NF-jB activation in LPS-stimulated M/s Next, we examined the effect of 1/2 SLPI on the profile of NF-jB activation in LPS-stimulated M/s by an electrophoretic mobility shift assay (EMSA). As shown in Fig. 3, LPS stimulation caused an increase in the binding ability of p65–p50 heterodimers to the DNA jB sequences. Notably, 1/2 SLPI potentiated the LPSinduced increase in the binding activity of the p65–p50 complex to the jB sequences. As indicated in the table inserted in Fig. 3, the extent of LPS-induced increase in the p65–p50 binding to the jB sites in the case of SLPItreated M/s (6.5-fold induction) was about twice as much greater than that in the case of M/s given no SLPI treatment (3.1-fold induction). On the other hand, the M/s exhibited a significant level of the DNA
Fig. 2. Effects of SLPI on the induction of TNF-a producing cell populations in LPS-stimulated M/s. M/s were cultured in the medium with or without LPS (10 lg/ml) in the presence or absence of 1/2 SLPI (0.1 lg/ml). After 24-h cultivation, the number of TNF-a producing cells on the culture wells was determined by the ELISPOT method. Each bar indicates the mean standard errors of the mean (n ¼ 3).
40
C. Sano et al. / Cytokine 21 (2003) 38–42
Fig. 3. Effects of SLPI on profiles of LPS-induced NF-jB activation in M/s. M/s were incubated in the medium with or without the addition of LPS (10 lg/ml) in the presence or absence of 1/2 SLPI (1 lg/ml) for 2 h. The resultant M/s were harvested and subjected to EMSA. DNA binding reaction of extracted nuclear protein fractions from test M/s was performed in the reaction mixture with excess amounts of cold specific competitor oligonucleotide (lanes 4–6) or cold non-specific competitor oligonucleotide (lanes 7–9), or without competitor oligonucleotide (lane 1 to 3). Shown is a representative of two independent experiments. The inserted table shows the relative intensities of p65–p50 and p50–p50 binding to the DNA jB sequences when the value of p65–p50 from the control M/s (without LPS stimulation) was fixed to 1.0.
binding activity of p50–p50 homodimers even before LPS stimulation. Notably, LPS stimulation caused a marked decrease in the binding ability of the p50– p50 complex to the jB sequences. 1/2 SLPI did not significantly affect the p50–p50 binding to the jB sites in LPS-stimulated M/s. Thus, it appears that SLPI may potentiate the activity of p65–p50 complex as a transcriptional factor [10,11] but not the activity of p50–p50 complex as a repressor of the iNOS promoter and enhancer [12].
3. Discussion Previously, Jin et al. [3] reported that M/s from LPS-non-responder C3H/HeJ strain mice carrying a mutated Lps gene (Lpsd) produced high levels of SLPI responding to LPS stimulation, causing the lack in their TNF-a producing ability in response to LPS. They also reported that transfection of the SLPI gene in murine M/s reduced their TNF-a production in response to LPS stimulation, presumably by decreasing NF-jB
C. Sano et al. / Cytokine 21 (2003) 38–42
expression induced by LPS signaling [3]. In the present study, we have found that neither intact SLPI nor 1/2 SLPI down-regulated the TNF-a production by LPSstimulated M/s, when extracellularly acted on M/s. In addition, 1/2 SLPI caused small but significant increase in the TNF-a production by LPS-stimulated M/s and augmented the binding ability of p65–p50 heterodimers of NF-jB to DNA jB sites. Since p65 but not p50 contains transcription activation domain [10,11], this result suggests that SLPI may up-regulate the LPSinduced activation of M/ NF-jB in terms of its transcriptional function. In this context, previous findings by Ding et al. [13] should be noted. They reported that LPS-induced NF-jB activity could be detected in Lpsd M/s, although LPS concentrations required to induce a significant level of NF-jB activation in Lpsd M/s were about 100 times higher than those of M/s with normal Lps gene (Lpsn). These findings, including ours, indicate that LPS-hyporesponsiveness in Lpsd M/s is not due to a lack of NF-jB activating capacity. Since TNF-a and iNOS activities were detected only in Lpsn M/s [12], NF-jB activity may be necessary but not sufficient for induction of these genes by LPS. In addition, our findings support the concept that overexpression of SLPI is not the primary cause of the LPS-hyporesponsiveness of Lpsd M/s. Indeed, recent studies have demonstrated that such LPS-hyporesponsiveness is substantially due to the lack of functional TLR4 due to missense mutation in Lps gene [14,15]. Recently, Zhu et al. [16] have reported that nonsecretory form of SLPI suppressed M/ responses to LPS stimulation, presumably by inhibiting the ubiquitin–proteosome pathway which is needed for the processing of p105 to p50 and the degradation of the I-jB, resulting in the activation of NF-jB. However, this hypothesis cannot explain the reasons why Lpsd M/s respond to cell wall preparations from group B streptococci with TNF-a production and NF-jB activation [17] and why Treponema maltophilum-mediated activation of Lpsd M/s via TLR2 causes the increase in the iNOS activity, which is regulated by NF-jB-dependent signal pathways [18]. Therefore, it appears that the hypothesis of Zhu et al. [16] would refer to only the minor mechanism for the LPS-hyporesponsiveness in Lpsd M/s, even if it were the case.
4. Materials and methods 4.1. Special agents Intact human SLPI (R & D Systems Inc., Minneapolis, MN) and recombinant 1/2 SLPI containing the Cterminal domain (Arg58–Ala107) of human SLPI [7] (Teijin Limited, Tokyo) were used.
41
4.2. Measurement of M/ TNF-a production M/ monolayer culture on a 14-mm plastic culture sheet prepared by seeding 2107 of Zymosan A-induced peritoneal exudate cells from BALB/c mice was cultured in 1.0 ml of RPMI 1640 medium containing 10% heat inactivated fetal bovine serum (FBS) with or without the addition of LPS (Escherichia coli O111: B4) in the presence or absence of SLPI or 1/2 SLPI at 37 C in a CO2 incubator. After 24-h cultivation, M/ culture fluids were removed and measured for TNF-a concentration by ELISA as previously described [19], by using rat anti-mouse TNF-a monoclonal antibody (mAb) (Pharmingen Co., San Diego, CA) and biotinylated rat anti-mouse TNF-a mAb (Pharmingen) as capture and detecting antibodies (Abs), respectively. After staining with alkaline phosphatase (ALP)-conjugated streptavidin (ALP-SA) (Life Technologies Co., Gaitherburg, MD), color development was performed using p-nitrophenyl phosphate as the substrate. 4.3. ELISPOT assay for TNF-a-producing M/s ELISPOT assay for TNF-a-producing M/s was performed by the method of Vesteegen et al. [20] with slight modifications. Briefly, M/ monolayer culture on a plastic culture well (Immulon 4 plate; Dynatech Laboratories, Chantilly, VA), which had been coated with rat anti-mouse TNF-a mAb, was cultivated in 10% FBS-RPMI medium with or without either LPS or 1/2 SLPI alone or both at 37 C for 24 h. The cultured wells were washed with phosphate-buffered saline (PBS), incubated with 0.1 ml of biotinylated rat anti-mouse TNF-a mAb for 2 h, and then stained with ALP-SA for 2 h. The TNF-a secreted from attached M/s was visualized by color development by ALP using 5-bromo4-chloro-3-indolyl phosphate/nitro blue tetrazolium as the substrate. The number of blue spots developed on the M/ wells were counted by microscopy. 4.4. Assay for NF-jB activation LPS-induced NF-jB activation was measured according to the method of Vincenti et al. [21]. Briefly, RAW264.7 M/s grown in 10% FBS-RPMI medium were treated or not treated with 10 lg/ml of LPS at 37 C for 2 h, rinsed with PBS, scraped off with a rubber policeman, and collected by centrifugation (10,000 g, 5 min). After lysis of the obtained cells with 0.6% Nonidet P-40, the released nuclei were collected by centrifugation (13,000 g, 30 s) and then lysed with 0.4 M NaCl–20 mM HEPES buffer (pH 7.9) containing 1 mM each of ethylenediaminetetraacetic acid (EDTA), EGTA, DTT, and PMSF. The resultant nuclear protein fraction was subjected to EMSA [22] using Gel Shift
42
C. Sano et al. / Cytokine 21 (2003) 38–42
Assay Systems (Promega Corporation, Madison, WI) according to the instruction manual of the manufacturer. In this study, an oligonucleotide with a nucleotide sequence, 59-agt tga ggg gac ttt ccc agg c-39/39-tca act ccc ctg aaa ggg tcc g-59, was used as NF-jB consensus oligonucleotide. This oligonucleotide was used as a NFjB binding probe after labeling with 32P using T4 polynucleotide kinase, and moreover used as a coldspecific competitor in a control reaction for the binding specificity of NF-jB to its consensus oligonucleotide. AP2 consensus oligonucleotide, 59-gat cga act gac cgc ccg cgg ccc gt-39/39-cta gct tga ctg gcg ggc gcc ggg ca-59, was used as a cold non-specific competitor in another control reaction for the binding specificity. Acknowledgements We thank Teijin Limited for providing 1/2 SLPI. References [1] Smith CF, Johnson A. Human bronchial leukocyte protease inhibitor. Biochem J 1985;225:463–72. [2] Thompson RC, Ohlsson K. Isolation, properties, and complete amino acid sequence of human leukocyte protease inhibitor, a potent inhibitor of leukocyte elastase. Proc Natl Acad Sci USA 1986;83:6692–6. [3] Jin F, Nathan C, Radzioch D, Ding A. Secretory leukocyte protease inhibitor: a macrophage product induced by and antagonistic to bacterial lipopolysaccharide. Cell 1997;88:417–26. [4] Jin F, Nathan CF, Radzioch D, Ding A. Lipopolysacchariderelated stimuli induce expression of the secretory leukocyte protease inhibitor, a macrophage-derived lipopolysaccharide inhibitor. Infect Immun 1998;66:2447–52. [5] Watson J, Kelly K, Largen M, Taylor BA. The genetic mapping of a defective LPS response gene in C3H/HeJ mice. J Immunol 1978;120:422–4. [6] Zhang Y, DeWitt DL, McNeely TB, Wahl SM, Wahl LM. Secretory leukocyte protease inhibitor suppresses the production of monocyte prostaglandin H synthase-2, prostaglandin E2, and matrix metalloproteinases. J Clin Invest 1997;99:894–900. [7] Renesto P, Balloy V, Kamimura T, Masuda K, Imaizumi A, Chignard M. Inhibition by recombinant SLPI and half-SLPI (Asn55–Ala107) of elastase and cathepsin G activities: consequence for neutrophil–platelet cooperation. Br J Pharmacol 1993;108: 1100–6. [8] Masuda K, Kamimura T, Watanabe K, Suga T, Kanesaki M, Tekeuchi A, et al. Pharmacological activity of the C-terminal and N-terminal domains of secretory leukoprotease inhibitor in vitro. Br J Pharmacol 1995;115:883–8.
[9] Mitsuhashi H, Asano S, Nonaka T, Hamamura I, Masuda K, Kiyoki M. Administration of truncated secretory leukoprotease inhibitor ameliorates bleomycin-induced pulmonary fibrosis in hamsters. Am J Respir Crit Care Med 1997;153:369–74. [10] Schmitz ML, dos Santos Silva MA, Baeuerle PA. Transactivation domain 2 (TA2) of p65 NF-jB. Similarity to TA1 and phorbol ester-stimulated activity and phosphorylation in intact cells. J Biol Chem 1995;270:15576–84. [11] Wang D, Baldwin SA. Activation of nuclear factor-jB-dependent transcription by tumor necrosis factor-a is mediated through phosphorylation of RelA/p65 on serine 529. J Biol Chem 1998; 273:29411–6. [12] Goldring CE, Reveneau S, Pinard D, Jeannin J-F. Hyporesponsiveness to lipopolysaccharide alters the composition of NFjB binding to the regulatory regions of inducible nitric oxide synthase gene. Eur J Immunol 1998;28:2960–70. [13] Ding A, Hwang S, Lander HM, Xie Q. Macrophages derived from C3H/HeJ (Lpsd) mice respond bacterial lipopolysaccharide by activating NF-jB. J Leukoc Biol 1995;57:174–9. [14] Poltorak A, He X, Smirnova I, Liu M-Y, Van Huffel C, Du X, et al. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in the Tlr gene. Science 1998;282:2085–8. [15] Hoshino K, Takeuchi O, Kawai T, Sanjo H, Ogawa T, et al. Tolllike receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J Immunol 1999;162:3749–52. [16] Zhu J, Nathan C, Ding A. Suppression of macrophage responses to bacterial lipopolysaccharide by a non-secretory form of secretory leukocyte protease inhibitor. Biochim Biophys Acta 1999;1451:219–23. [17] Medvedev AE, Flo T, Ingalls RR, Golenbock DT, Teti G, Vogel SN, et al. Involvement of CD14 and complement receptors CR3 and CR4 in nuclear factor-jB activation and TNF production induced by lipopolysaccharide and group B streptococcal cell walls. J Immunol 1998;160:4535–42. [18] Schroder NWJ, Opits B, Lamping N, Michelsen KS, Zahringer U, Gobel UB, et al. Involvement of lipopolysaccharide binding protein, CD14, and Toll-like receptors in the initiation of innate immune responses by Treponema glycolipids. J Immunol 2000; 165:2683–93. [19] Sano C, Shimizu T, Sato K, Kawauchi H, Tomioka H. Effects of secretory leucocyte protease inhibitor on the production of the anti-inflammatory cytokines, IL-10 and transforming growth factor-beta (TGF-b), by lipopolysaccharide-stimulated macrophages. Clin Exp Immunol 2000;121:77–85. [20] Vesteegen JMT, Logtenberg T, Ballieux RE. Enumeration of IFN-c producing human lymphocytes by spot-ELISA. J Immunol Methods 1988;111:25–9. [21] Vincenti MP, Burrell TA, Taffet SM. Regulation of NF-kappaB activity in murine macrophages: effect of bacterial lipopolysaccharide and phorbol ester. J Cell Physiol 1992;150:204–13. [22] Buratoeski S, Chodosh LA. Mobility shift DNA-binding assay using gel electrophoresis. In: Ausubel FM, Brent RE, Kingston RE, Moore D, Seidman JG, Smith JA, Struhl K, editors. Current protocols in molecular biology, vol. 2. New York: Wiley, 1989. 12.2.1–12.2.11.
Short communication
Effects of secretory leukocyte protease inhibitor on the tumor necrosis factor-alpha production and NF-jB activation of lipopolysaccharide-stimulated macrophages Chiaki Sano, Toshiaki Shimizu, Haruaki Tomioka* Department of Microbiology and Immunology, Shimane Medical, University, Izumo, Shimane 693-8501, Japan Received 18 September 2002; accepted 12 November 2002
Abstract It has been reported that lipopolysaccharide (LPS)-hyporesponsiveness of macrophages (M/s) of C3H/HeJ mice with a mutated Lps gene (Lpsd) is related to high-level expression of secretory leukocyte protease inhibitor (SLPI) in response to LPS, causing suppression of NF-jB activation and tumor necrosis factor-a (TNF-a) production. We thus examined the effects of SLPI on the TNF-a production by LPS-stimulated M/s. Neither intact SLPI nor half-sized SLPI (1/2 SLPI) down-regulated M/ TNF-a production. 1/2 SLPI weakly increased M/ TNF-a production in response to LPS signaling and potentiated the LPS-induced activation of NF-jB, especially the binding of p65–p50 heterodimers to the DNA jB sites, suggesting that LPS-hyporesponsiveness of Lpsd M/s is not due to the overexpression of SLPI. Ó 2003 Elsevier Science Ltd. All rights reserved. Keywords: Lipopolysaccharide; Macrophages; Nuclear factor-jB; Secretory leukocyte protease inhibitor; Tumor necrosis factor-a
1. Introduction Secretory leukocyte protease inhibitor (SLPI), a potent serine protease inhibitor in lungs, which is secreted by bronchial and alveolar epithelial cells [1,2] and which inhibits a wide range of proteases, plays important roles in protection of the airway epithelial surface in the lungs from attack by neutrophil-derived proteases generated at the sites of inflammatory reactions [1,2]. Jin et al. [3,4] reported that M/s from LPS-non-responder C3H/HeJ mice carrying a mutated Lps gene (Lpsd) [5] expressed high levels of SLPI responding to LPS signals and that this caused a lack in tumor necrosis factor-a (TNF-a) production by Lpsd M/s in response to LPS stimulation. Moreover, transfection of M/s with SLPI gene suppressed LPS-induced activation of NF-jB, causing the reduction in the production of TNF-a and the expression of inducible nitric oxide synthase (iNOS),
thereby NO production. On the basis of these findings, they proposed that LPS-hyporesponsiveness of Lpsd M/s is principally due to the overexpression of SLPI. On the other hand, Zhang et al. [6] reported a contradictory finding that SLPI failed to modulate TNF-a production by human monocytes although it suppressed the production of prostaglandin H synthase-2, prostaglandin E2, and matrix metalloproteinases. Therefore, the effects of SLPI on M/ production of TNF-a is controversial. In the present study, we studied profiles of TNF-a production and NF-jB activation in M/s from mice with normal Lps gene (Lpsn) in response to LPS stimulation, with special reference to the effects of SLPI on these M/ functions.
2. Results 2.1. Effects of SLPI on TNF-a production by LPS-stimulated M/s
* Corresponding author. Tel.: +81-853-20-2146; fax: +81-853-202145. E-mail address: [email protected] (H. Tomioka).
First, we examined the effects of SLPI on TNF-a production by LPS-stimulated Lpsn M/s. Fig. 1 shows
1043-4666/03/$ - see front matter Ó 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1043-4666(02)00485-4
C. Sano et al. / Cytokine 21 (2003) 38–42
the effects of intact SLPI and half-sized human SLPI (1/2 SLPI), which contains the C-terminal domain (Arg58–Ala107) of intact SLPI [7], on the TNF-a production by M/s during the first 24-h cultivation in response to LPS. The protease activity of intact SLPI is completely retained in the 1/2 SLPI except that the latter is more specific for elastase than for trypsin [7,8]. In addition, human and mouse SLPIs share high amino acid homology with each other [3], and human SLPI is highly active against serine proteases of animals other than humans [8] and also efficacious in ameliorating chemically induced pulmonary fibrosis in hamsters [9]. Thus, human SLPI and 1/2 SLPI exerted its action not only in humans but also in mice even in the cross-species testing. Intact SLPI even at 1 lg/ml did not affect M/ production of TNF-a in response to LPS stimulation (Fig. 1A). On the other hand, 1/2 SLPI at 1 lg/ml weakly but significantly up-regulated the TNF-a production by LPS-stimulated M/s (Fig. 1B). These findings indicate that both the half domain (Ser1–Thr57) of SLPI deleted in 1/2 SLPI and the half domain (Arg58– Ala107) retained by 1/2 SLPI do not inhibit LPSmediated up-regulation of M/ TNF-a production, and the latter domain alone is somewhat stimulatory of M/ TNF-a production. In separate experiments using enzyme-linked immunosorbent assay (ELISA), the
Fig. 1. Effects of SLPI on the production of TNF-a by LPS-stimulated M/s. Murine peritoneal M/s were cultured in the medium with or without the addition of LPS (10 lg/ml) in the presence or absence of indicated concentrations of intact SLPI (A) or 1/2 SLPI (B) for 24 h. Each bar indicates the mean standard errors of the mean (n ¼ 4). *: Significantly larger than the values of M/s which were not given SLPI treatment (P < 0:05).
39
degradation of TNF-a due to M/-derived protease was insignificant when exogenously added TNF-a was incubated in the 12-h culture fluid of LPS-stimulated M/s for 24 h. That is, less than 10% of initially added TNF-a was degraded. This indicates that the 1/2 SLPI-mediated increase in M/ TNF-a production was not due to the anti-protease activity of 1/2 SLPI but mainly due to its up-regulatory effect on the de novo synthesis of TNF-a by M/s. Indeed, as shown in Fig. 2, the ELISPOT assay indicated that 1/2 SLPI slightly increased the number of TNF-a-producing M/ populations in LPS-stimulated M/s. 2.2. Effect of 1/2 SLPI on the NF-jB activation in LPS-stimulated M/s Next, we examined the effect of 1/2 SLPI on the profile of NF-jB activation in LPS-stimulated M/s by an electrophoretic mobility shift assay (EMSA). As shown in Fig. 3, LPS stimulation caused an increase in the binding ability of p65–p50 heterodimers to the DNA jB sequences. Notably, 1/2 SLPI potentiated the LPSinduced increase in the binding activity of the p65–p50 complex to the jB sequences. As indicated in the table inserted in Fig. 3, the extent of LPS-induced increase in the p65–p50 binding to the jB sites in the case of SLPItreated M/s (6.5-fold induction) was about twice as much greater than that in the case of M/s given no SLPI treatment (3.1-fold induction). On the other hand, the M/s exhibited a significant level of the DNA
Fig. 2. Effects of SLPI on the induction of TNF-a producing cell populations in LPS-stimulated M/s. M/s were cultured in the medium with or without LPS (10 lg/ml) in the presence or absence of 1/2 SLPI (0.1 lg/ml). After 24-h cultivation, the number of TNF-a producing cells on the culture wells was determined by the ELISPOT method. Each bar indicates the mean standard errors of the mean (n ¼ 3).
40
C. Sano et al. / Cytokine 21 (2003) 38–42
Fig. 3. Effects of SLPI on profiles of LPS-induced NF-jB activation in M/s. M/s were incubated in the medium with or without the addition of LPS (10 lg/ml) in the presence or absence of 1/2 SLPI (1 lg/ml) for 2 h. The resultant M/s were harvested and subjected to EMSA. DNA binding reaction of extracted nuclear protein fractions from test M/s was performed in the reaction mixture with excess amounts of cold specific competitor oligonucleotide (lanes 4–6) or cold non-specific competitor oligonucleotide (lanes 7–9), or without competitor oligonucleotide (lane 1 to 3). Shown is a representative of two independent experiments. The inserted table shows the relative intensities of p65–p50 and p50–p50 binding to the DNA jB sequences when the value of p65–p50 from the control M/s (without LPS stimulation) was fixed to 1.0.
binding activity of p50–p50 homodimers even before LPS stimulation. Notably, LPS stimulation caused a marked decrease in the binding ability of the p50– p50 complex to the jB sequences. 1/2 SLPI did not significantly affect the p50–p50 binding to the jB sites in LPS-stimulated M/s. Thus, it appears that SLPI may potentiate the activity of p65–p50 complex as a transcriptional factor [10,11] but not the activity of p50–p50 complex as a repressor of the iNOS promoter and enhancer [12].
3. Discussion Previously, Jin et al. [3] reported that M/s from LPS-non-responder C3H/HeJ strain mice carrying a mutated Lps gene (Lpsd) produced high levels of SLPI responding to LPS stimulation, causing the lack in their TNF-a producing ability in response to LPS. They also reported that transfection of the SLPI gene in murine M/s reduced their TNF-a production in response to LPS stimulation, presumably by decreasing NF-jB
C. Sano et al. / Cytokine 21 (2003) 38–42
expression induced by LPS signaling [3]. In the present study, we have found that neither intact SLPI nor 1/2 SLPI down-regulated the TNF-a production by LPSstimulated M/s, when extracellularly acted on M/s. In addition, 1/2 SLPI caused small but significant increase in the TNF-a production by LPS-stimulated M/s and augmented the binding ability of p65–p50 heterodimers of NF-jB to DNA jB sites. Since p65 but not p50 contains transcription activation domain [10,11], this result suggests that SLPI may up-regulate the LPSinduced activation of M/ NF-jB in terms of its transcriptional function. In this context, previous findings by Ding et al. [13] should be noted. They reported that LPS-induced NF-jB activity could be detected in Lpsd M/s, although LPS concentrations required to induce a significant level of NF-jB activation in Lpsd M/s were about 100 times higher than those of M/s with normal Lps gene (Lpsn). These findings, including ours, indicate that LPS-hyporesponsiveness in Lpsd M/s is not due to a lack of NF-jB activating capacity. Since TNF-a and iNOS activities were detected only in Lpsn M/s [12], NF-jB activity may be necessary but not sufficient for induction of these genes by LPS. In addition, our findings support the concept that overexpression of SLPI is not the primary cause of the LPS-hyporesponsiveness of Lpsd M/s. Indeed, recent studies have demonstrated that such LPS-hyporesponsiveness is substantially due to the lack of functional TLR4 due to missense mutation in Lps gene [14,15]. Recently, Zhu et al. [16] have reported that nonsecretory form of SLPI suppressed M/ responses to LPS stimulation, presumably by inhibiting the ubiquitin–proteosome pathway which is needed for the processing of p105 to p50 and the degradation of the I-jB, resulting in the activation of NF-jB. However, this hypothesis cannot explain the reasons why Lpsd M/s respond to cell wall preparations from group B streptococci with TNF-a production and NF-jB activation [17] and why Treponema maltophilum-mediated activation of Lpsd M/s via TLR2 causes the increase in the iNOS activity, which is regulated by NF-jB-dependent signal pathways [18]. Therefore, it appears that the hypothesis of Zhu et al. [16] would refer to only the minor mechanism for the LPS-hyporesponsiveness in Lpsd M/s, even if it were the case.
4. Materials and methods 4.1. Special agents Intact human SLPI (R & D Systems Inc., Minneapolis, MN) and recombinant 1/2 SLPI containing the Cterminal domain (Arg58–Ala107) of human SLPI [7] (Teijin Limited, Tokyo) were used.
41
4.2. Measurement of M/ TNF-a production M/ monolayer culture on a 14-mm plastic culture sheet prepared by seeding 2107 of Zymosan A-induced peritoneal exudate cells from BALB/c mice was cultured in 1.0 ml of RPMI 1640 medium containing 10% heat inactivated fetal bovine serum (FBS) with or without the addition of LPS (Escherichia coli O111: B4) in the presence or absence of SLPI or 1/2 SLPI at 37 C in a CO2 incubator. After 24-h cultivation, M/ culture fluids were removed and measured for TNF-a concentration by ELISA as previously described [19], by using rat anti-mouse TNF-a monoclonal antibody (mAb) (Pharmingen Co., San Diego, CA) and biotinylated rat anti-mouse TNF-a mAb (Pharmingen) as capture and detecting antibodies (Abs), respectively. After staining with alkaline phosphatase (ALP)-conjugated streptavidin (ALP-SA) (Life Technologies Co., Gaitherburg, MD), color development was performed using p-nitrophenyl phosphate as the substrate. 4.3. ELISPOT assay for TNF-a-producing M/s ELISPOT assay for TNF-a-producing M/s was performed by the method of Vesteegen et al. [20] with slight modifications. Briefly, M/ monolayer culture on a plastic culture well (Immulon 4 plate; Dynatech Laboratories, Chantilly, VA), which had been coated with rat anti-mouse TNF-a mAb, was cultivated in 10% FBS-RPMI medium with or without either LPS or 1/2 SLPI alone or both at 37 C for 24 h. The cultured wells were washed with phosphate-buffered saline (PBS), incubated with 0.1 ml of biotinylated rat anti-mouse TNF-a mAb for 2 h, and then stained with ALP-SA for 2 h. The TNF-a secreted from attached M/s was visualized by color development by ALP using 5-bromo4-chloro-3-indolyl phosphate/nitro blue tetrazolium as the substrate. The number of blue spots developed on the M/ wells were counted by microscopy. 4.4. Assay for NF-jB activation LPS-induced NF-jB activation was measured according to the method of Vincenti et al. [21]. Briefly, RAW264.7 M/s grown in 10% FBS-RPMI medium were treated or not treated with 10 lg/ml of LPS at 37 C for 2 h, rinsed with PBS, scraped off with a rubber policeman, and collected by centrifugation (10,000 g, 5 min). After lysis of the obtained cells with 0.6% Nonidet P-40, the released nuclei were collected by centrifugation (13,000 g, 30 s) and then lysed with 0.4 M NaCl–20 mM HEPES buffer (pH 7.9) containing 1 mM each of ethylenediaminetetraacetic acid (EDTA), EGTA, DTT, and PMSF. The resultant nuclear protein fraction was subjected to EMSA [22] using Gel Shift
42
C. Sano et al. / Cytokine 21 (2003) 38–42
Assay Systems (Promega Corporation, Madison, WI) according to the instruction manual of the manufacturer. In this study, an oligonucleotide with a nucleotide sequence, 59-agt tga ggg gac ttt ccc agg c-39/39-tca act ccc ctg aaa ggg tcc g-59, was used as NF-jB consensus oligonucleotide. This oligonucleotide was used as a NFjB binding probe after labeling with 32P using T4 polynucleotide kinase, and moreover used as a coldspecific competitor in a control reaction for the binding specificity of NF-jB to its consensus oligonucleotide. AP2 consensus oligonucleotide, 59-gat cga act gac cgc ccg cgg ccc gt-39/39-cta gct tga ctg gcg ggc gcc ggg ca-59, was used as a cold non-specific competitor in another control reaction for the binding specificity. Acknowledgements We thank Teijin Limited for providing 1/2 SLPI. References [1] Smith CF, Johnson A. Human bronchial leukocyte protease inhibitor. Biochem J 1985;225:463–72. [2] Thompson RC, Ohlsson K. Isolation, properties, and complete amino acid sequence of human leukocyte protease inhibitor, a potent inhibitor of leukocyte elastase. Proc Natl Acad Sci USA 1986;83:6692–6. [3] Jin F, Nathan C, Radzioch D, Ding A. Secretory leukocyte protease inhibitor: a macrophage product induced by and antagonistic to bacterial lipopolysaccharide. Cell 1997;88:417–26. [4] Jin F, Nathan CF, Radzioch D, Ding A. Lipopolysacchariderelated stimuli induce expression of the secretory leukocyte protease inhibitor, a macrophage-derived lipopolysaccharide inhibitor. Infect Immun 1998;66:2447–52. [5] Watson J, Kelly K, Largen M, Taylor BA. The genetic mapping of a defective LPS response gene in C3H/HeJ mice. J Immunol 1978;120:422–4. [6] Zhang Y, DeWitt DL, McNeely TB, Wahl SM, Wahl LM. Secretory leukocyte protease inhibitor suppresses the production of monocyte prostaglandin H synthase-2, prostaglandin E2, and matrix metalloproteinases. J Clin Invest 1997;99:894–900. [7] Renesto P, Balloy V, Kamimura T, Masuda K, Imaizumi A, Chignard M. Inhibition by recombinant SLPI and half-SLPI (Asn55–Ala107) of elastase and cathepsin G activities: consequence for neutrophil–platelet cooperation. Br J Pharmacol 1993;108: 1100–6. [8] Masuda K, Kamimura T, Watanabe K, Suga T, Kanesaki M, Tekeuchi A, et al. Pharmacological activity of the C-terminal and N-terminal domains of secretory leukoprotease inhibitor in vitro. Br J Pharmacol 1995;115:883–8.
[9] Mitsuhashi H, Asano S, Nonaka T, Hamamura I, Masuda K, Kiyoki M. Administration of truncated secretory leukoprotease inhibitor ameliorates bleomycin-induced pulmonary fibrosis in hamsters. Am J Respir Crit Care Med 1997;153:369–74. [10] Schmitz ML, dos Santos Silva MA, Baeuerle PA. Transactivation domain 2 (TA2) of p65 NF-jB. Similarity to TA1 and phorbol ester-stimulated activity and phosphorylation in intact cells. J Biol Chem 1995;270:15576–84. [11] Wang D, Baldwin SA. Activation of nuclear factor-jB-dependent transcription by tumor necrosis factor-a is mediated through phosphorylation of RelA/p65 on serine 529. J Biol Chem 1998; 273:29411–6. [12] Goldring CE, Reveneau S, Pinard D, Jeannin J-F. Hyporesponsiveness to lipopolysaccharide alters the composition of NFjB binding to the regulatory regions of inducible nitric oxide synthase gene. Eur J Immunol 1998;28:2960–70. [13] Ding A, Hwang S, Lander HM, Xie Q. Macrophages derived from C3H/HeJ (Lpsd) mice respond bacterial lipopolysaccharide by activating NF-jB. J Leukoc Biol 1995;57:174–9. [14] Poltorak A, He X, Smirnova I, Liu M-Y, Van Huffel C, Du X, et al. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in the Tlr gene. Science 1998;282:2085–8. [15] Hoshino K, Takeuchi O, Kawai T, Sanjo H, Ogawa T, et al. Tolllike receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J Immunol 1999;162:3749–52. [16] Zhu J, Nathan C, Ding A. Suppression of macrophage responses to bacterial lipopolysaccharide by a non-secretory form of secretory leukocyte protease inhibitor. Biochim Biophys Acta 1999;1451:219–23. [17] Medvedev AE, Flo T, Ingalls RR, Golenbock DT, Teti G, Vogel SN, et al. Involvement of CD14 and complement receptors CR3 and CR4 in nuclear factor-jB activation and TNF production induced by lipopolysaccharide and group B streptococcal cell walls. J Immunol 1998;160:4535–42. [18] Schroder NWJ, Opits B, Lamping N, Michelsen KS, Zahringer U, Gobel UB, et al. Involvement of lipopolysaccharide binding protein, CD14, and Toll-like receptors in the initiation of innate immune responses by Treponema glycolipids. J Immunol 2000; 165:2683–93. [19] Sano C, Shimizu T, Sato K, Kawauchi H, Tomioka H. Effects of secretory leucocyte protease inhibitor on the production of the anti-inflammatory cytokines, IL-10 and transforming growth factor-beta (TGF-b), by lipopolysaccharide-stimulated macrophages. Clin Exp Immunol 2000;121:77–85. [20] Vesteegen JMT, Logtenberg T, Ballieux RE. Enumeration of IFN-c producing human lymphocytes by spot-ELISA. J Immunol Methods 1988;111:25–9. [21] Vincenti MP, Burrell TA, Taffet SM. Regulation of NF-kappaB activity in murine macrophages: effect of bacterial lipopolysaccharide and phorbol ester. J Cell Physiol 1992;150:204–13. [22] Buratoeski S, Chodosh LA. Mobility shift DNA-binding assay using gel electrophoresis. In: Ausubel FM, Brent RE, Kingston RE, Moore D, Seidman JG, Smith JA, Struhl K, editors. Current protocols in molecular biology, vol. 2. New York: Wiley, 1989. 12.2.1–12.2.11.