Lipopolysaccharide Stimulates p38-dependent Induction of Antiviral Genes in Neutrophils Independently of Paracrine Factors (2003)

Lipopolysaccharide (LPS) induces neutrophils to synthesize and secrete pro-inflammatory cytokines and chemokines, which are regulated at both the transcriptional and translational level. We reported previously that neutrophils stimulated with LPS induce expression of genes typically expressed in response to stimulation with antiviral type I interferons (IFN), such as myxovirus resistance-1 (MX1). However, we present evidence that this response of neutrophils to lipopolysaccharide occurs in the absence of interferon-dependent signaling. Lipopolysaccharide-stimulated neutrophils do not phosphorylate the interferon-associated transcription factors signal transducer and activator of transcription-1 and –3, and medium from lipopolysaccharide-stimulated cells was unable to induceMX1 gene expression, suggesting a soluble factor is not involved. Furthermore, LPS did not alter expression of IFNAand IFNB genes. In contrast to neutrophils, LPS-stimulated human monocyte-derived macrophages induced the expression ofMX1, but IFNB was induced, and medium from LPS-stimulated monocyte-derived macrophages supported MX1induction. An inhibitor of p38 kinase blocked induction ofMX1 by lipopolysaccharide, but not IFNα, in neutrophils, and induction of MX1 was dependent on protein synthesis. LPS, but not IFNα, substantially activated p38. In contrast, the induction of MX1 by LPS in monocyte-derived macrophages was insensitive to p38 inhibition, although p38 is phosphorylated in LPS-stimulated but not IFNα-stimulated monocyte-derived macrophages. The expression of MX1 in neutrophils and monocyte-derived macrophages is mediated by TLR4 but not TLR2. The data presented here indicate that lipopolysaccharide activates novel interferon-independent signaling pathways in neutrophils and that induction of antiviral genes is a consequence of exposure of neutrophils to bacterial products. Lipopolysaccharide (LPS) induces neutrophils to synthesize and secrete pro-inflammatory cytokines and chemokines, which are regulated at both the transcriptional and translational level. We reported previously that neutrophils stimulated with LPS induce expression of genes typically expressed in response to stimulation with antiviral type I interferons (IFN), such as myxovirus resistance-1 (MX1). However, we present evidence that this response of neutrophils to lipopolysaccharide occurs in the absence of interferon-dependent signaling. Lipopolysaccharide-stimulated neutrophils do not phosphorylate the interferon-associated transcription factors signal transducer and activator of transcription-1 and –3, and medium from lipopolysaccharide-stimulated cells was unable to induceMX1 gene expression, suggesting a soluble factor is not involved. Furthermore, LPS did not alter expression of IFNAand IFNB genes. In contrast to neutrophils, LPS-stimulated human monocyte-derived macrophages induced the expression ofMX1, but IFNB was induced, and medium from LPS-stimulated monocyte-derived macrophages supported MX1induction. An inhibitor of p38 kinase blocked induction ofMX1 by lipopolysaccharide, but not IFNα, in neutrophils, and induction of MX1 was dependent on protein synthesis. LPS, but not IFNα, substantially activated p38. In contrast, the induction of MX1 by LPS in monocyte-derived macrophages was insensitive to p38 inhibition, although p38 is phosphorylated in LPS-stimulated but not IFNα-stimulated monocyte-derived macrophages. The expression of MX1 in neutrophils and monocyte-derived macrophages is mediated by TLR4 but not TLR2. The data presented here indicate that lipopolysaccharide activates novel interferon-independent signaling pathways in neutrophils and that induction of antiviral genes is a consequence of exposure of neutrophils to bacterial products. lipopolysaccharide double-stranded RNA interferon IFN-regulatory factor IFN-stimulated gene (S)-5-[2-(1-phenylethylamino)pyrimidin-4-yl]-1-methyl-4-(3-trifluoromethylphenyl)-2-(4-piperidinyl)imidazole myxovirus-resistance gene-1 peptidoglycan dsRNA-dependent protein kinase signal transducer and activator of transcription toll-like receptor glyceraldehyde-3- phosphate dehydrogenase mitogen-activated protein reverse transcription Neutrophils play a vital role in the innate immune response by migrating to sites of infection, ingesting microorganisms, and producing antimicrobial agents. Activation of neutrophils can occur by recognition of structural features of microbes, including the bacterial cell wall component lipopolysaccharide (LPS)1 (1Hoffmann J.A. Kafatos F.C. Janeway C.A. Ezekowitz R.A.B. Science. 1999; 284: 1313-1318Crossref PubMed Scopus (2153) Google Scholar). Exposure of neutrophils and other immune cells to LPS primes the cell to respond to secondary agonists with secretion of antimicrobial peptides and formation of superoxide. In addition, it is now recognized that neutrophils are potent synthetic cells and produce cytokines and chemokines, such as TNFα and IL-1β, which are coded for by theTNFA and IL-1β genes (2Scapini P. Lapinet-Vera J.A. Gasperini S. Calzetti F. Bazzoni F. Cassatella M.A. Immunol. Rev. 2000; 177: 195-203Crossref PubMed Scopus (621) Google Scholar, 3Cassatella M.A. Gasperini S. Russo M.P. Ann. N. Y. Acad. Sci. 1997; 832: 233-242Crossref PubMed Scopus (80) Google Scholar). Production of these immunomodulatory proteins in response to LPS is controlled at both the transcriptional and translational level. The toll-like receptor (TLR) family is implicated in the recognition of bacteria and in the activation of cellular signaling pathways important in host defense (4Muzio M. Polentarutti N. Bosisio D. Prahladen M.K.P. Mantovani A. J. Leukocyte Biol. 2000; 67: 450-456Crossref PubMed Scopus (155) Google Scholar, 5Rock F.L. Hardiman G. Timans J.C. Kastelein R.A. Bazan J.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 588-593Crossref PubMed Scopus (1451) Google Scholar). Genetic and biochemical data suggest that TLR4 is the major LPS receptor (4Muzio M. Polentarutti N. Bosisio D. Prahladen M.K.P. Mantovani A. J. Leukocyte Biol. 2000; 67: 450-456Crossref PubMed Scopus (155) Google Scholar, 6Poltorak A. He X. Smirnova I. Liu M.Y. Huffel C.V. Du X. Birdwell D. Alejos E. Silva M. Galanos C. Freudenberg M. Ricciardi-Castagnoli P. Layton B. Beutler B. Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6450) Google Scholar, 7Takeuchi O. Hoshino K. Kawai T. Sanjo H. Takada H. Ogawa T. Takeda K. Akira S. Immunity. 1999; 11: 443-451Abstract Full Text Full Text PDF PubMed Scopus (2789) Google Scholar, 8Hoshino K. Takeuchi O. Kawai T. Sanjo H. Ogawa T. Takeda Y. Takeda K. Akira S. J. Immunol. 1999; 162: 3749-3752Crossref PubMed Google Scholar, 9Kawasaki K. Akashi S. Shimazu R. Yoshida T. Miyake K. Nishijima M. J. Biol. Chem. 2000; 275: 2251-2254Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar, 10Chow J.C. Young D.W. Golenbock D.T. Christ W.J. Gusovsky F. J. Biol. Chem. 1999; 274: 10689-10692Abstract Full Text Full Text PDF PubMed Scopus (1617) Google Scholar), although TLR2 may also play a role (11Kirschning C.J. Wesche H. Merrill-Ayres T. Rothe M. J. Exp. Med. 1998; 188: 2091-2097Crossref PubMed Scopus (655) Google Scholar, 12Dziarski R. Wang Q. Miyake K. Kirschning C.J. Gupta D. J. Immunol. 2001; 166: 1938-1944Crossref PubMed Scopus (181) Google Scholar). LPS stimulates a pathway leading to the activation of the transcriptional regulator NFκB in many cell types (4Muzio M. Polentarutti N. Bosisio D. Prahladen M.K.P. Mantovani A. J. Leukocyte Biol. 2000; 67: 450-456Crossref PubMed Scopus (155) Google Scholar, 7Takeuchi O. Hoshino K. Kawai T. Sanjo H. Takada H. Ogawa T. Takeda K. Akira S. Immunity. 1999; 11: 443-451Abstract Full Text Full Text PDF PubMed Scopus (2789) Google Scholar, 8Hoshino K. Takeuchi O. Kawai T. Sanjo H. Ogawa T. Takeda Y. Takeda K. Akira S. J. Immunol. 1999; 162: 3749-3752Crossref PubMed Google Scholar, 13Kawai T. Adachi O. Ogawa T. Takeda K. Akira S. Immunity. 1999; 11: 115-122Abstract Full Text Full Text PDF PubMed Scopus (1728) Google Scholar,14Medzhitov R. Preston-Hurlburt P. Kopp E. Stadlen A. Chen C. Ghosh S. Janeway Jr., C.A. Mol. Cell. 1998; 2: 253-258Abstract Full Text Full Text PDF PubMed Scopus (1304) Google Scholar), including neutrophils (15McDonald P.P. Bald A. Cassatella M.A. Blood. 1997; 89: 3421-3422Crossref PubMed Google Scholar). Whereas NFκB plays a pivotal role in regulating the expression of cytokine and chemokine genes, other transcriptional regulators may also be involved in signaling from TLRs. In contrast to many other cell types, transcription factor activation is poorly understood in neutrophils. LPS activates tyrosine and serine/threonine kinases in cells (16Arbibe L. Mira J.P. Teusch N. Kline L. Guha M. Mackman N. Godowski P.J. Ulevitch R.J. Knaus U.G. Nat. Immunol. 2000; 1: 533-540Crossref PubMed Scopus (568) Google Scholar, 17Yang H. Young D.W. Gusovsky F. Chow J.C. J. Biol. Chem. 2000; 275: 20861-20866Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar, 18Weinstein S.L. Sanghera J.S. Lemke K. DeFranco A.L. Pelech S.L. J. Biol. Chem. 1992; 267: 14955-14962Abstract Full Text PDF PubMed Google Scholar, 19Swantek J.L. Cobb M.H. Geppert T.D. Mol. Cell. Biol. 1997; 17: 6274-6282Crossref PubMed Google Scholar, 20Hambleton J. Weinstein S.L. Lem L. DeFranco A.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2774-2778Crossref PubMed Scopus (414) Google Scholar, 21Scherle P.A. Jones E.A. Favata M.F. Daulerio A.J. Covington M.B. Nurnberg S.A. Magolda R.L. Trzaskos J.M. J. Immunol. 1998; 161: 5681-5686PubMed Google Scholar, 22Nahas N. Molski T.F. Fernandez G.A. Sha'afi R.I. Biochem. J. 1996; 318: 247-253Crossref PubMed Scopus (121) Google Scholar, 23Fouda S.I. Molski T.F. Ashour M.S. Sha'afi R.I. Biochem. J. 1995; 308: 815-822Crossref PubMed Scopus (51) Google Scholar). Among these the MAP kinase family of serine/threonine kinases (p42/p44 MAP kinase, c-Jun N-terminal kinase, and p38) plays a major role in cellular activation in a variety of cell types (24Brunet A. Pouyssegur J. Essays Biochem. 1997; 32: 1-16PubMed Google Scholar, 25Minden A. Karin M. Biochim. Biophys. Acta. 1997; 1333: F85-F104PubMed Google Scholar, 26Ono K. Han J. Cell. Signal. 2000; 12: 1-13Crossref PubMed Scopus (1393) Google Scholar). However, in neutrophils the p38 MAP kinase family is predominantly activated in response to LPS and regulates the translation of cytokines, adhesion, and migration (27Nick J.A. Avdi N.J. Gerwins P. Johnson G.L. Worthen G.S. J. Immunol. 1996; 156: 4867-4875PubMed Google Scholar, 28Nick J.A. Avdi N.J. Young S.K. Lehman L.A. McDonald P.P. Frasch S.C. Billstrom M.A. Henson P.M. Johnson G.L. Worthen G.S. J. Clin. Invest. 1999; 103: 851-858Crossref PubMed Scopus (264) Google Scholar, 29Nick J.A. Young S.K. Brown K.K. Avdi N.J. Arndt P.G. Suratt B.T. Janes M.S. Henson P.M. Worthen G.S. J. Immunol. 2000; 164: 2151-2159Crossref PubMed Scopus (229) Google Scholar, 30Zu Y.L. Qi J. Gilchrist A. Fernandez G.A. Vazquez-Abad D. Kreutzer D.L. Huang C.K. Sha'afi R.I. J. Immunol. 1998; 160: 1982-1989PubMed Google Scholar, 31Detmers P.A. Zhou D. Polizzi E. Thieringer R. Hanlon W.A. Vaidya S. Bansal V. J. Immunol. 1998; 161: 1921-1929PubMed Google Scholar). The role of p38 in regulation of gene expression is less well understood, although transcription factors, including activating transcription factor-2, SRF accessory protein-1, and CCAAT-enhancer binding protein-β (26Ono K. Han J. Cell. Signal. 2000; 12: 1-13Crossref PubMed Scopus (1393) Google Scholar), are known substrates of p38. The role of p38 in activating these transcription factors in neutrophils has not been explored. However, inhibitors of p38 have little effect on the expression of cytokine genes in neutrophils (28Nick J.A. Avdi N.J. Young S.K. Lehman L.A. McDonald P.P. Frasch S.C. Billstrom M.A. Henson P.M. Johnson G.L. Worthen G.S. J. Clin. Invest. 1999; 103: 851-858Crossref PubMed Scopus (264) Google Scholar,32Fessler M.B. Malcolm K.C. Duncan M.W. Worthen G.S. J. Biol. Chem. 2002; 277: 31291-31302Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). The cellular response to viral infection includes the induction of genes for the type I interferons, IFNα and IFNβ, which act in an autocrine or paracrine manner to stimulate the induction of interferon-stimulated genes (ISGs) (33Stark G.R. Kerr I.M. Williams B.R.G. Silverman R.H. Schreiber R.D. Annu. Rev. Biochem. 1998; 67: 227-264Crossref PubMed Scopus (3380) Google Scholar). Induction of IFNα/β protein by virus requires the activation of the IRF family of transcriptional regulators by phosphorylation, and subsequent gene expression of IFNA and IFNB (34Mamane Y. Heylbroeck C. Genin P. Algarte M. Servant M.J. LePage C. DeLuca C. Kwon H. Lin R. Hiscott J. Gene (Amst.). 1999; 237: 1-14Crossref PubMed Scopus (464) Google Scholar, 35Hiscott J. Pitha P. Genin P. Nguyen H. Heylbroeck C. Mamane Y. Algarte M. Lin R. J. Interferon Cytokine Res. 1999; 19: 1-13Crossref PubMed Scopus (196) Google Scholar), through poorly understood pathways. IFNα/β binds to the cell surface IFN receptor and activates the Jak1 and Tyk2 tyrosine kinases leading to the phosphorylation of the STAT1 and STAT2 transcription factors and subsequent dimerization (33Stark G.R. Kerr I.M. Williams B.R.G. Silverman R.H. Schreiber R.D. Annu. Rev. Biochem. 1998; 67: 227-264Crossref PubMed Scopus (3380) Google Scholar). The major regulatory factor involved in IFNα/β action, ISGF3, is a complex of STAT1, STAT2, and IRF9-p48-ISGF3γ. ISGF3 binds to a conserved sequence in the 3′ region of IFN-responsive genes known as the interferon-stimulated response element. ISGs, such as MX1, are regulated primarily by transcriptional activation of this element, and the antiviral activity of IFNα/β resides in their ability to induce ISGs. Links between anti-bacterial and antiviral activity have been suggested previously. In particular, the induction of IFNβ by LPS has been well described (36Gessani S. Belardelli F. Pecorelli A. Puddu P. Baglioni C. J. Virol. 1989; 63: 2785-2789Crossref PubMed Google Scholar, 37Maehara N. Ho M. Infect. Immun. 1977; 15: 78-83Crossref PubMed Google Scholar). In this way, antiviral gene expression is accomplished in response to bacterial infection. We recently described (32Fessler M.B. Malcolm K.C. Duncan M.W. Worthen G.S. J. Biol. Chem. 2002; 277: 31291-31302Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 38Malcolm K.C. Arndt P.G. Manos E.J. Jones D.A. Worthen G.S. Am. J. Physiol. 2003; 284: L663-L670PubMed Google Scholar) the regulation of ISGs in LPS-treated neutrophils. Here we investigated the mechanisms by which neutrophils induce ISGs in response to LPS. LPS-stimulated neutrophils do not increaseIFNA or IFNB gene expression, secrete a soluble mediator to induce ISGs, or phosphorylate STAT proteins; in contrast, monocyte-derived macrophages increase IFNB expression, secrete a soluble ISG-inducing factor(s), and phosphorylate STAT proteins. Furthermore, the induction of ISGs by LPS in neutrophils, but not monocyte-derived macrophages, is sensitive to inhibitors of p38, and these effects are mediated by TLR4 but not TLR2. Whereas both IFN-dependent and -independent induction of ISGs by dsRNA and virus has been described (39Hayes M.P. Enterline J.C. Gerrard T.L. Zoon K.C. J. Leukocyte Biol. 1991; 50: 176-181Crossref PubMed Scopus (40) Google Scholar, 40Wathelet M.G. Berr P.M. Huez G.A. Eur. J. Biochem. 1992; 206: 901-910Crossref PubMed Scopus (82) Google Scholar, 41Zhu H. Cong J.P. Shenk T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13985-13990Crossref PubMed Scopus (263) Google Scholar, 42Au W.C. Su Y. Raj B.K. Pitha P.M. J. Biol. Chem. 1993; 268: 24032-24040Abstract Full Text PDF PubMed Google Scholar), to our knowledge this is the first report of IFN-independent ISG induction by LPS. These data suggest a potential new role of neutrophils in innate immunity by inducing an antiviral genotype and suggest that distinct pathways are activated by LPS in neutrophils and monocyte-derived macrophages. LPS from E. coli strain 0111:B4 was purchased from List Biological Laboratories and contained minimal protein contamination as determined by silver staining. Peptidoglycan (Fluka) was prepared by extensive sonication followed by centrifugation. Anti-STAT1 and -STAT3 were obtained from Transduction Laboratories, and anti-phospho-STAT1 (Tyr-701), phospho-STAT3 (Tyr-705), and phospho-p38 were from Cell Signaling. Anti-p38 polyclonal antiserum was produced as described previously (27Nick J.A. Avdi N.J. Gerwins P. Johnson G.L. Worthen G.S. J. Immunol. 1996; 156: 4867-4875PubMed Google Scholar). IFNαA/D was purchased from PBL Biomedical Laboratories. Cycloheximide, lipoteichoic acid (fromStaphylococcus aureus), and polymyxin B were obtained from Sigma. SB203580, SB202474, and PD98059 were from Calbiochem. (S)-5-[2-(1-Phenylethylamino)pyrimidin-4-yl]-1-methyl-4-(3-trifluoromethylphenyl)-2- (4-piperidinyl)imidazole (M39) was from Merck (43Liverton N.J. Butcher J.W. Claiborne C.F. Claremon D.A. Libby B.E. Nguyen K.T. Pitzenberger S.M. Selnick H.G. Smith G.R. Tebben A. Vacca J.P. Varga S.L. Agarwal L. Dancheck K. Forsyth A.J. Fletcher D.S. Frantz B. Hanlon W.A. Harper C.F. Hofsess S.J. Kostura M. Lin J. Luell S. O'Neill E.A. Oreville C.J. Pang M. Parsons J. Rolando A. Sahly Y. Visco D.M. O'Keefe S.J. J. Med. Chem. 1999; 42: 2180-2190Crossref PubMed Scopus (207) Google Scholar). Neutrophils were isolated from citrated blood of healthy donors and purified on a Percoll gradient, as described previously (44Haslett C. Guthrie L.A. Kopaniak M.M. Johnston R.B.J. Henson P.M. Am. J. Pathol. 1985; 119: 101-110PubMed Google Scholar). Contamination with monocytic cells is less than 5%. Cells (20–25 × 106 cells/ml) were resuspended in neutrophil medium (RPMI containing 1% heat-inactivated platelet-poor plasma and 10 mm HEPES, pH 7.6) and divided into 1.5-ml tubes. Heat-inactivated platelet-poor plasma is a necessary source of LPS-binding protein (45Schumann R.R. Leong S.R. Flaggs G.W. Gray P.W. Wright S.D. Mathison J.C. Tobias P.S. Ulevitch R.J. Science. 1990; 249: 1429-1431Crossref PubMed Scopus (1379) Google Scholar). RT-PCR analysis utilized 20 × 106 cells (in 1 ml). Similar RT-PCR results were obtained at lower cell concentrations. Cells were treated with the indicated concentration of LPS, IFNα/D, and PGN and rotated continuously at 37 °C for up to 4 h. In some experiments, cell supernatants were retained for conditioned media experiments or enzyme-linked immunosorbent assay (see below). Human monocyte derived macrophages were prepared by the method of Fadoket al. (46Fadok V.A. Bratton D.L. Konowal A. Freed P.W. Westcott J.Y. Henson P.M. J. Clin. Invest. 1998; 101: 890-898Crossref PubMed Scopus (2562) Google Scholar). Briefly, peripheral monocytic cells from healthy human volunteers were washed in Hanks' balanced salt solution with calcium and magnesium, and 12 × 106 cells in X-vivo medium (BioWhittaker) per well were seeded in 6-well plates. One hour after plating, non-adherent cells were removed by washing, and adherent cells were grown in X-vivo, 10% heat-inactivated human serum. The medium was changed every 3 days thereafter, and experiments were performed 7 or 8 days after plating. RAW264.7 cells, obtained from the American Type Culture Collection, were cultured as described (47Xiao Y.Q. Malcolm K. Worthen G.S. Gardai S. Schiemann W.P. Fadok V.A. Bratton D.L. Henson P.M. J. Biol. Chem. 2002; 277: 14884-14893Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). Preparation of total RNA (5 μg) for analysis by oligonucleotide array (Affymetrix) was performed as described (32Fessler M.B. Malcolm K.C. Duncan M.W. Worthen G.S. J. Biol. Chem. 2002; 277: 31291-31302Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Data were analyzed using Affymetrix GeneChip software. Time courses from three donors were analyzed. cDNA was prepared by reverse transcription using 2 μg of total RNA, derived from 20 × 106 neutrophils, or 0.5–2 μg of RNA from monocyte-derived macrophages and treated as indicated. PCRs were performed using specific primers for MX1, PKR,TNFA, IFNA (48Marie I. Durbin J.E. Levy D.E. EMBO J. 1998; 17: 6660-6669Crossref PubMed Google Scholar), IFNB, andGAPDH. Murine-specific primers were used in experiments with RAW264.7 cells. Secretion of IFNα and IFNβ was quantified by enzyme-linked immunosorbent assay kits as directed by the manufacturer (R & D Systems). Supernatants were isolated from neutrophils or monocyte-derived macrophages stimulated for 4 with LPS or cells neutrophils × were in of neutrophil medium and of or with 10 polymyxin B. macrophages were stimulated for 4 with or with 10 polymyxin and was per well of monocyte-derived macrophages cultured in a 6-well Cells were for 4 at 37 neutrophils were rotated RT-PCR was performed as described Neutrophils × were stimulated for the indicated washed in pH and resuspended in of 20 mm pH mm mm mm 10 10 1 1 mm 2 mm 10 mm and on for cell were using a for 10 with mm for 20 at 4 and by at × for 20 at 4 macrophages were treated as washed in and in were by to and with the indicated cell from stimulated cells or μg from 20 × 106 were with of 1% in mm pH on for 10 and for an 10 The supernatants from this were with for 1 and protein was for an The were and kinase activity was determined at °C in 20 mm pH 10 mm 2 μg of 1 mm and 10 mm were after 20 by of of was after on a and was performed using We recently in gene expression in human neutrophils treated with LPS for 4 using oligonucleotide (32Fessler M.B. Malcolm K.C. Duncan M.W. Worthen G.S. J. Biol. Chem. 2002; 277: 31291-31302Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar) and cDNA K.C. Arndt P.G. Manos E.J. Jones D.A. Worthen G.S. Am. J. Physiol. 2003; 284: L663-L670PubMed Google Scholar) of genes were that in other are with activation by the type I interferons, IFNα or IFNβ S.D. Zhou A. Williams B.R.G. Silverman R.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar). analysis of the of expression of these ISGs by oligonucleotide array indicated that ISG induction after a ISG induction was 4 of LPS of the expression of three such genes are in 1 A. In contrast, expression of and IL-1β, genes for cytokines known to be expressed in LPS-stimulated neutrophils, and was for 4 1 of ISGs, MX1 and and of and were by RT-PCR 1 Induction of MX1 and the of expression determined by oligonucleotide with little expression after 2 but induced 4 after LPS. In contrast, expression was as as after LPS exposure and was at an the 4 of LPS stimulation 1 The expression of MX1 and was and at LPS as as 1 1 of the ISG MX1 induced by LPS were to induced by IFNα 1 a known of MX1, IFNα little of 1 Furthermore, the induction of MX1 by LPS was blocked by with polymyxin an inhibitor of LPS signaling not the of type I interferons by LPS-stimulated cells (36Gessani S. Belardelli F. Pecorelli A. Puddu P. Baglioni C. J. Virol. 1989; 63: 2785-2789Crossref PubMed Google Scholar, 37Maehara N. Ho M. Infect. Immun. 1977; 15: 78-83Crossref PubMed Google Scholar), a response that may on TLR4 V. Jones K. M.J. S. Williams J. M.J. Nat. Immunol. 2002; PubMed Scopus Google Scholar). our knowledge LPS is known to stimulate ISGs through the induction of type I we the that LPS induced IFNA or IFNB in neutrophils. In neutrophils, RT-PCR analysis indicated a and of IFNB a exposure to LPS, neutrophils to the of cytokine 2 Furthermore, IFNB and were from neutrophils and by LPS by gene expression analysis not Cells with the induction of the expression of IFNα/β protein is regulated by IFN gene expression, the that neutrophils IFN protein stimulation by LPS. However, IFNα and IFNβ were not in the supernatants of and LPS-stimulated neutrophils not these data indicate that induction of ISGs in LPS-stimulated neutrophils is of the transcription and of type I the data indicate that type I interferons are not produced in LPS-treated neutrophils, the that LPS stimulation of neutrophils induces genes suggested that IFN-stimulated signaling pathways are The interferon-independent induction of by dsRNA is regulated in by S.K. Jr., T. G.R. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). IFNα/β the STAT family of transcriptional regulators by phosphorylation of on tyrosine (36Gessani S. Belardelli F. Pecorelli A. Puddu P. Baglioni C. J. Virol. 1989; 63: 2785-2789Crossref PubMed Google Scholar, 37Maehara N. Ho M. Infect. Immun. 1977; 15: 78-83Crossref PubMed Google Scholar). cell were for STAT1, and STAT1 and and were the in neutrophils. Exposure of neutrophils to LPS for and 4 did not increase the of or phospho-STAT3 Exposure of neutrophils to LPS for was to the phosphorylation of both STAT proteins. In contrast, IFNα stimulation of cells to a phosphorylation of both STAT proteins 2 of neutrophils with IFNα, but not LPS, for 4 in the of STAT1 but not can STAT proteins M. A.J. C. J.M. Cell. Mol. Biol. 2001; Scholar), we investigated LPS stimulated the of a factor in neutrophils that ISG expression data and the data of A. M. A. M. G. M.A. S. Blood. 1999; 93: PubMed Google Scholar) not indicated that LPS regulates expression of for M. with activation of is also from A. M. A. M. G. M.A. S. Blood. 1999; 93: PubMed Google Scholar). However, human to induce MX1 not the that factors were for supernatants from and LPS-stimulated neutrophils were to neutrophils for 4 and expression of MX1, and was by medium from LPS-stimulated neutrophils induced but not that of MX1 We have not factor from LPS-stimulated neutrophils. although conditioned medium from LPS-stimulated neutrophils is it not an ISG-inducing These data indicate that LPS is to induceMX1 but in contrast to other cells types paracrine factors are not for induction of In macrophages, IFNβ by LPS ISG induction (36Gessani S. Belardelli F. Pecorelli A. Puddu P. Baglioni C. J. Virol. 1989; 63: 2785-2789Crossref PubMed