Oxidative Stress Reprograms Lipopolysaccharide Signaling via Src Kinase-dependent Pathway in RAW 264.7 Macrophage Cell Line

Oxidative stress generated during ischemia/reperfusion injury has been shown to augment cellular responsiveness. Whereas oxidants are themselves known to induce several intracellular signaling cascades, their effect on signaling pathways initiated by other inflammatory stimuli remains poorly elucidated. Previous work has suggested that oxidants are able to prime alveolar macrophages for increased NF-κB translocation in response to treatment with lipopolysaccharide (LPS). Because oxidants are known to stimulate the Src family of tyrosine kinases, we hypothesized that the oxidants might contribute to augmented NF-κB translocation by LPS via the involvement of Src family kinases. To model macrophage priming in vitro, the murine macrophage cell line, RAW 264.7, was first incubated with various oxidants and then exposed to low dose LPS. These studies show that oxidant stress is able to augment macrophage responsiveness to LPS as evidenced by earlier and increased NF-κB translocation. Inhibition of the Src family kinases by either pharmacological inhibition using PP2 or through a molecular approach by cell transfection with Csk was found to prevent the augmented LPS-induced NF-κB translocation caused by oxidants. Interestingly, while Src kinase inhibition was able to prevent the LPS-induced NF-κB translocation in oxidant-treated macrophages, this strategy had no effect on NF-κB translocation caused by LPS in the absence of oxidants. These findings suggested that oxidative stress might divert LPS signaling along an alternative signaling pathway. Further studies demonstrated that the Src-dependent pathway induced by oxidant pretreatment involved the activation of phosphatidylinositol 3-kinase. Involvement of this pathway appeared to be independent of traditional LPS signaling. Together, these studies provide a novel potential mechanism whereby oxidants might prime alveolar macrophages for altered responsiveness to subsequent inflammatory stimuli and suggest different cellular targets for immunomodulation following ischemia/reperfusion. Oxidative stress generated during ischemia/reperfusion injury has been shown to augment cellular responsiveness. Whereas oxidants are themselves known to induce several intracellular signaling cascades, their effect on signaling pathways initiated by other inflammatory stimuli remains poorly elucidated. Previous work has suggested that oxidants are able to prime alveolar macrophages for increased NF-κB translocation in response to treatment with lipopolysaccharide (LPS). Because oxidants are known to stimulate the Src family of tyrosine kinases, we hypothesized that the oxidants might contribute to augmented NF-κB translocation by LPS via the involvement of Src family kinases. To model macrophage priming in vitro, the murine macrophage cell line, RAW 264.7, was first incubated with various oxidants and then exposed to low dose LPS. These studies show that oxidant stress is able to augment macrophage responsiveness to LPS as evidenced by earlier and increased NF-κB translocation. Inhibition of the Src family kinases by either pharmacological inhibition using PP2 or through a molecular approach by cell transfection with Csk was found to prevent the augmented LPS-induced NF-κB translocation caused by oxidants. Interestingly, while Src kinase inhibition was able to prevent the LPS-induced NF-κB translocation in oxidant-treated macrophages, this strategy had no effect on NF-κB translocation caused by LPS in the absence of oxidants. These findings suggested that oxidative stress might divert LPS signaling along an alternative signaling pathway. Further studies demonstrated that the Src-dependent pathway induced by oxidant pretreatment involved the activation of phosphatidylinositol 3-kinase. Involvement of this pathway appeared to be independent of traditional LPS signaling. Together, these studies provide a novel potential mechanism whereby oxidants might prime alveolar macrophages for altered responsiveness to subsequent inflammatory stimuli and suggest different cellular targets for immunomodulation following ischemia/reperfusion. Civilian trauma remains a leading cause of disability and mortality in North American society (1MacKenzie E.J. Morris Jr., J.A. Smith G.S. Fahey M. J. Trauma. 1990; 30: 1096-1101Crossref PubMed Scopus (163) Google Scholar). Late deaths are related to the development of multiple organ failure in up to two-thirds of these patients. Many studies have suggested that the global ischemia-reperfusion resulting from resuscitated hemorrhagic shock predisposes to organ injury by priming for an exaggerated immune response to a delayed inflammatory stimulus (2Moore F.A. Moore E.E. Surg. Clin. North Am. 1995; 75: 257-277Abstract Full Text PDF PubMed Scopus (461) Google Scholar). The synergistic effect of sequential stimuli has been coined the “two-hit” hypothesis for the development of organ injury in trauma patients (2Moore F.A. Moore E.E. Surg. Clin. North Am. 1995; 75: 257-277Abstract Full Text PDF PubMed Scopus (461) Google Scholar). We have previously reported an animal model wherein antecedent resuscitated shock was shown to prime for increased LPS 1The abbreviations used are: LPSlipopolysaccharideCINCcytokine-induced neutrophil chemoattractantXOxanthine oxidasePI 3-kinasephosphoinositol 3-kinaseGFPgreen fluorescent proteinEMSAelectrophoretic mobility shift assayPBSphosphate-buffered salineIFimmunofluorescencePP24-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine.-induced lung injury by augmenting alveolar macrophage NF-κB nuclear translocation and gene transcription of the chemokine, cytokine-induced neutrophil chemoattractant (CINC), the rodent orthologue of interleukin-8. The exaggerated generation of CINC by alveolar macrophages in this model was primarily responsible for increased lung neutrophilia and consequent lung injury in animals exposed to the combined stimuli of resuscitated shock followed by LPS (3Fan J. Marshall J.C. Jimenez M. Shek P.N. Zagorski J. Rotstein O.D. J. Immunol. 1998; 161: 440-447PubMed Google Scholar). lipopolysaccharide cytokine-induced neutrophil chemoattractant xanthine oxidase phosphoinositol 3-kinase green fluorescent protein electrophoretic mobility shift assay phosphate-buffered saline immunofluorescence 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine. Ischemia/reperfusion produces oxidative stress through the irreversible conversion of xanthine dehydrogenase to xanthine oxidase (XO) during ischemia with consequent production of oxygen-free radicals and H2O2 during reperfusion (4McCord J.M. N. Engl. J. Med. 1985; 312: 159-163Crossref PubMed Scopus (4999) Google Scholar, 5McCutchan H.J. Schwappach J.R. Enquist E.G. Walden D.L. Terada L.S. Reiss O.K. Leff J.A. Repine J.E. Am. J. Physiol. 1990; 258: H1415-H1419PubMed Google Scholar). Several lines of evidence suggest a role for oxidative stress as contributing to activation of hematopoietic cells. In the in vitro setting, oxidants have been shown to induce activation of multiple signaling pathways in hematopoietic cells. The best studied of these involves signaling cascades culminating in NF-κB translocation and induction of transcription of NF-κB-dependent genes. Early reports documented the ability of oxidants to induce NF-κB translocation and also defined the role of oxidant stress in NF-κB translocation by various inflammatory stimuli including LPS and tumor necrosis factor. In vivo studies have generally supported a role for oxidative stress in the induction of NF-κB translocation following ischemia/reperfusion. Schwartz et al. (6Schwartz M.D. Repine J.E. Abraham E. Am. J. Respir. Cell Mol. Biol. 1995; 12: 434-440Crossref PubMed Scopus (84) Google Scholar) reported that inhibition of xanthine oxidase generation or activity using a tungsten-enriched diet or allopurinol, respectively, prevented induction of NF-κB-dependent gene expression such as tumor necrosis factor in a murine hemorrhagic shock model. The potential priming role of oxidant stress generated by ischemia/reperfusion is well demonstrated in our previous reports. In these studies, neither shock/resuscitation nor low dose LPS was able to induce significant NF-κB translocation in alveolar macrophages, whereas the combined presence of these two stimuli culminated in a robust response, far greater than the additive effect of the separate responses. Importantly, supplementation of resuscitation fluid with the antioxidant N-acetylcysteine prevented the synergistic rise in NF-κB translocation (3Fan J. Marshall J.C. Jimenez M. Shek P.N. Zagorski J. Rotstein O.D. J. Immunol. 1998; 161: 440-447PubMed Google Scholar). These observations are consistent with a role for oxidative stress in the priming of macrophages for increased responsiveness to a delayed second inflammatory stimulus. However, whereas oxidative stress is known to activate a number of signaling pathways in inflammatory cells, the alterations in cell signaling leading to enhanced responsiveness following exposure to oxidants have not been elucidated. The Src family of tyrosine kinases are involved in many signal transduction pathways. The Src tyrosine kinases have been shown to be activated by oxidant stress such as UV irradiation as well as H2O2 (7Aikawa R. Komuro I. Yamazaki T. Zou Y. Kudoh S. Tanaka M. Shiojima I. Hiroi Y. Yazaki Y. J. Clin. Invest. 1997; 100: 1813-1821Crossref PubMed Scopus (630) Google Scholar, 8Devary Y. Gottlieb R.A. Smeal T. Karin M. Cell. 1992; 71: 1081-1091Abstract Full Text PDF PubMed Scopus (796) Google Scholar). Relevant to the activation of NF-κB and induction of NF-κB-dependent proinflammatory genes, Src has also been shown to phosphorylate inhibitory κB (IκB) on tyrosine residues, an event that activates NF-κB without inducing IκB degradation itself (9Imbert V. Farahifar D. Auberger P. Mary D. Rossi B. Peyron J.F. J. Inflamm. 1996; 46: 65-77PubMed Google Scholar). In the present studies, we tested the hypothesis that oxidant stress might contribute to augmented NF-κB translocation in response to LPS through involvement of members of the Src family kinases. To examine this hypothesis, an in vitro culture system was established wherein macrophages were sequentially exposed to an oxidative stress followed by LPS as a means of modeling the in vivo “two-hit hypothesis” of cellular activation and organ injury following shock/resuscitation. The major findings in the present studies are that Src family kinases are not only involved in macrophage priming by oxidants, but also that antecedent oxidant stress reprograms LPS-NF-κB signaling such that it changes from a Src-independent pathway to one that is Src-dependent. Buffers and Reagents—Hydrogen peroxide (30%) (H2O2) was purchased from University of Toronto Medstore (Toronto, ON). Antibodies against p-Lyn, c-Fgr, p-Hck, p-phosphoinositol (PI) 3-kinase, and IκBα were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The polyclonal anti-IκBα, anti-phosphotyrosine-specific monoclonal antibody (G410), PI 3-kinase antibody (clone UB93-3), and an in vitro tyrosine kinase assay kit were purchased from Upstate Biochemical Institute (Lake Placid, NY). An inhibitor of the Src family of proteintyrosine kinases, PP2, was purchased from Calbiochem, Inc. (San Diego, CA). The PI substrate was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). The Protein A-Sepharose beads, horseradish peroxidase-conjugated donkey anti-rabbit IgG secondary antibody, and ECL were purchased from Amersham Biosciences. The 50× protease inhibitor was purchased from (San CA). The and were from and were purchased from was from Cell murine macrophage cell line, RAW was in with and were in a of Cell were in The were exposed to LPS or a of pretreatment with for and then LPS treatment from to the oxidant H2O2 was used of the to LPS were by the on In inhibition studies, RAW were in the presence of PP2 for the on culture were using green fluorescent protein and Csk The Csk was a from I. The of murine Csk was using and transfection with the was using to the were with of well The was in The of to was the the was and with with and of Cell transfection of RAW cell lines Csk were by of T. N. M. T. J. Immunol. PubMed Scopus Google Scholar). The were in with and was from using the P. N. PubMed Scopus Google Scholar). was using a for CINC with the is to of CINC by S. University of J. Immunol. 1996; Google Scholar). were then of and were by in the presence of were and for is a gene to for of was using a and and was to the dehydrogenase were for on with and to protease inhibitor had been Cell were then for and the were then and with protein A-Sepharose of cellular were with of polyclonal anti-IκBα, or antibody for of protein A-Sepharose were to the and incubated for The resulting immune were with the and then in of were and in cell cell were with followed by for were with and were following for and with followed by for from were on and to were then with of anti-phosphotyrosine-specific monoclonal antibody (G410), antibody, or antibody for with the horseradish peroxidase-conjugated donkey IgG secondary antibody for were using an of was as previously The tyrosine kinase assay kit was substrate was incubated with the tyrosine in the presence of PP2 was to the kinase The was then in a with peroxidase-conjugated monoclonal antibody was used to the was used for with a of were used to the of PI were with of and to protease inhibitor had been were on for then cellular was by for The were incubated with of PI 3-kinase antibody and then to of protein A-Sepharose for on a The were with and was then as previously S. 1996; PubMed Scopus Google Scholar). the were in of the to protease inhibitor has been in a the of in of and of and incubated for The was with of and the was with of and on a in phosphatidylinositol were by on Protein protein were from the by the of and Google Scholar). The were in of and The were for and in were incubated on for and for were in of and incubated on for were then by and were with the protein assay used for is a to a to in the the NF-κB J. Immunol. 1996; Google Scholar). was by kinase in the presence of were on a An of of nuclear protein was incubated with the in the presence of of in and for was by of CINC to the nuclear from the with the nuclear The was by on a in were and to were to to in incubated in previously were for in with The were with for with in for and then with in for The were with polyclonal in for with for and incubated with donkey IgG secondary in for were with The were on using from CA). The was using a to a by for cell involved a of to cells, with an of for The of was by the number of to cells. are as the of independent are are of independent the means were using of by of than was of Oxidative on examine the effect of oxidants on LPS H2O2 was for effect on LPS-induced NF-κB translocation. shown in low dose H2O2 itself was able to only a delayed NF-κB enhanced the ability of LPS to induce NF-κB by inducing an earlier response as well as a response during the LPS a dose response of the effect of of H2O2 on LPS-induced NF-κB translocation. of LPS H2O2 from to was able to augment LPS-induced NF-κB translocation. The of H2O2 caused NF-κB translocation in this whereas in The dose of LPS in these studies was on ability to induce and changes in NF-κB as a means of the ability of oxidants to augment this oxidase is a of oxidants following ischemia/reperfusion and was also tested for ability to augment LPS responsiveness. In were exposed to LPS or the of pretreatment with followed by LPS Whereas pretreatment induced NF-κB it augmented the responsiveness to LPS from to were studied and priming the not Because CINC gene expression is on NF-κB translocation and transcription is augmented in vivo by shock LPS (3Fan J. Marshall J.C. Jimenez M. Shek P.N. Zagorski J. Rotstein O.D. J. Immunol. 1998; 161: 440-447PubMed Google we a of the increased NF-κB translocation in this model by following the expression of the of with enhanced of CINC expression following LPS treatment with LPS The of CINC following treatment were increased with Together, these studies established a cellular model of oxidant priming for increased LPS an in vitro of the priming of alveolar macrophages shock/resuscitation in on the studies, we to H2O2 as the priming dose of oxidative stress in subsequent of the Src members of the Src family of kinases might contribute to we tyrosine as a of their The major Src family members in the macrophage are and P. 1996; PubMed Scopus Google Scholar). H2O2 induced in of these kinases following exposure the ability of oxidative stress to cause tyrosine of the Src kinases is consistent with their potential to the activation of signaling M. Biol. Med. 30: PubMed Scopus Google Scholar). To examine the role of Src kinases in the priming of LPS we the PP2, a and pharmacological inhibitor of the Src family of tyrosine kinases E.J. J. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). An in vitro kinase assay was to the ability for PP2 to prevent the activation of Src kinase activity was by the of PP2 pretreatment with PP2 translocation of NF-κB The of PP2, had no inhibitory effect not In pretreatment of macrophages with PP2 to treatment with LPS not LPS-induced NF-κB translocation and was as in These findings in with previous Src kinases are not for NF-κB translocation induced by LPS in the absence of oxidative stress J. Med. 1997; PubMed Scopus Google Scholar). Together, these studies suggest that oxidant pretreatment the LPS signaling pathway such that it involves Src kinases. H2O2 LPS-induced an alternative approach to the effect of oxidant stress on LPS we used immunofluorescence to nuclear translocation of as a for NF-κB translocation. a of the effect of H2O2 on LPS-induced nuclear translocation was used to nuclear of translocation was by in response to LPS H2O2 whereas neither H2O2 nor LPS induced translocation these to translocation was in and cells. These findings for the in that the of induced an earlier translocation of with LPS However, as the also demonstrated an increased of nuclear whereas the immunofluorescence studies is a of the that the of translocation by immunofluorescence is poorly with translocation of in the the major that oxidant exposure for earlier NF-κB translocation is to demonstrated the ability of this model to the findings in the cell several were to the of the Src family kinases in this pathway. were with or without the Src nor with PP2 evidence of of two However, PP2 was able to LPS-induced NF-κB translocation in to H2O2 two this was no translocation in with either H2O2 or LPS and was no effect of PP2 not We also a molecular approach wherein were with the Src kinase an inhibitor of Src family kinases. with was used to cells. in translocation had in with had no effect in and not this Csk transfection prevented nuclear translocation of induced by H2O2 LPS following LPS with or without H2O2 translocation of To the role of Src family kinases on LPS-induced signaling LPS with or without oxidative we the effect of Src inhibition on translocation. In these studies, transfection of RAW with Csk was of LPS translocation had in of with either H2O2 or Csk transfection prevented nuclear translocation in with H2O2 it had no effect on LPS-induced nuclear translocation in the absence of H2O2 Cell these Csk transfection of exposed to H2O2 LPS caused a in the number of with translocation studies with with a studies with in with LPS In these studies suggest that exposure of to oxidant stress an in LPS signaling for such that it on activation of Src family kinases. for PI in a Src-dependent reports have shown that the PI 3-kinase pathway be a of cellular response to oxidative stress Jr., J.F. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). this kinase is on tyrosine residues, and might as a substrate for the Src family kinases Y. M. J. R. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). We hypothesized that LPS signaling following oxidant stress involves the PI 3-kinase pathway and is activated by Src kinases. We first tyrosine of PI 3-kinase following LPS with and without to shown in an increased by with LPS rise was prevented by treatment with the Src tyrosine kinase inhibitor To that this was with increased activation of PI 3-kinase, kinase assay was by the of PI 3-kinase and with phosphatidylinositol and The of H2O2 LPS caused a in PI 3-kinase activity and of LPS with either LPS or H2O2 The Src family kinase PP2, was able to prevent this activation these reported by M. V. M. M. J. Immunol. PubMed Scopus Google Scholar, D. J. Immunol. PubMed Scopus Google LPS was able to cause activation of PI 3-kinase, but this activation was not by PP2 not consistent with the that activation of Src family kinases is of PI 3-kinase following oxidant of by LPS following H2O2 is These studies suggested that the PI 3-kinase pathway is involved in the signaling following Src Because inhibition of the Src family NF-κB we inhibition of the PI 3-kinase pathway might have studies translocation of by for LPS and H2O2 with the involvement of PI 3-kinase in LPS signaling in oxidant-treated cells, had effect on translocation studies with in while a of translocation in studies with in LPS of nuclear translocation is known to be through to demonstrated that oxidant stress related to was able to induce NF-κB activation through a tyrosine of IκBα in D. J.F. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). To examine this pathway might be involved in the Src-dependent LPS signaling in RAW following H2O2 tyrosine of IκBα was shown in whereas LPS and H2O2 LPS induced a of on was no the two consistent with the that NF-κB translocation was augmented through a pathway of degradation was augmented in exposed to oxidant stress LPS with LPS The of oxidative stress to intracellular signaling pathways has a of in the of In to their ability to activate various signaling cascades, oxidants have also been shown to prime for an augmented response to a second inflammatory stimulus. is to the of patients with oxidants generated during ischemia/reperfusion have been shown to the inflammatory response and consequent organ injury to a delayed stimulus (2Moore F.A. Moore E.E. Surg. Clin. North Am. 1995; 75: 257-277Abstract Full Text PDF PubMed Scopus (461) Google Scholar). In the present studies, we have this in vitro to the cellular whereby oxidative stress might this priming The major in this is that antecedent oxidative stress reprograms the LPS signaling pathway leading to NF-κB translocation such that it involves activation of Src family kinase of PI 3-kinase to be a of Src activation and is involved in the signaling leading to NF-κB translocation. with this have shown that members of the Src family induce tyrosine of the leading to an of PI 3-kinase activity Y. M. J. R. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, D. Mol. Cell. Biol. 1997; PubMed Scopus Google Scholar). our studies a novel signaling pathway with priming of macrophages and suggest of alternative cell activation of ischemia/reperfusion. The of LPS signaling the PI 3-kinase pathway to be a of the that oxidant Whereas the mechanism of this effect is one suggested that LPS-induced generation was of PI 3-kinase D. J. Immunol. PubMed Scopus Google Scholar) and be a in the priming is generated from through the of an that of role for in the priming is supported by the observations the of to activation by oxidants. in alveolar cells, activity was shown to be activated by oxidants with other Am. J. Physiol. 1992; Google Scholar). and J. H.J. J. PubMed Scopus Google Scholar) that H2O2 pretreatment of alveolar macrophages was able to prime alveolar macrophages for enhanced activity in response to a subsequent an effect that was prevented by the inhibitor of the Src-dependent activation of PI 3-kinase in the present studies also be consistent with a role for in the priming In cells, PI 3-kinase activation was found to be with tyrosine of and to be by a Src kinase inhibitor P. Am. J. Physiol. Google Scholar). with the these reports suggest a mechanism whereby oxidants might divert LPS signaling such that the induction of in Src activation of PI 3-kinase and NF-κB nuclear translocation. in the present was the that oxidant pretreatment not only LPS signaling to Src family kinases, but to the of the traditional Src-independent LPS signaling of Src inhibition in oxidant-treated caused inhibition of NF-κB with no signaling the Src-independent suggest that oxidant pretreatment might have the to divert the LPS signal from the traditional to NF-κB studies have demonstrated the for of LPS the and to LPS M. J. Cell PubMed Google Scholar). might that oxidative stress such that different are to the LPS signaling and pathways. In this and J. PubMed Scopus Google Scholar) demonstrated that oxidative stress was able to and in cells. Because a role in of such as inhibition of by oxidant stress with LPS signaling via the S. PubMed Scopus Google Scholar). LPS signaling following oxidative stress through the with a Src pathway or through for LPS of IκBα from NF-κB is for the translocation of the the of either on or tyrosine residues, have been shown to to NF-κB translocation. that tyrosine of IκBα was in NF-κB translocation in and that oxidative stress was in this effect D. J.F. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). the present studies suggest that the effect of oxidant treatment followed by LPS culminated in an effect on IκBα that was by LPS and induced of tyrosine on induced degradation of a consistent with induction of of IκBα than with tyrosine the has been reported to cause from but without degradation V. R.A. Farahifar D. Rossi B. Auberger P. Peyron J.F. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). be consistent with the of PI IκB kinase is a well substrate of the one of the potential of PI 3-kinase Jr., J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). The present studies provide a novel mechanism for the priming of oxidative through signaling through a Src pathway activation of PI 3-kinase. The potential of this pathway was suggested in studies using an model of lung injury following PI 3-kinase animals lung neutrophil and NF-κB J. J. R. J.M. T. Abraham E. J. Immunol. PubMed Scopus Google Scholar). Together, these findings suggest that alternative oxidant stress following shock/resuscitation defined signaling pathways might the of trauma for organ

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