Warm Ischemia-induced Alterations in Oxidative and Inflammatory Proteins in Hepatic Kupffer Cells in Rats

Abstract

The aim of the study was to investigate the impact of ischemia/reperfusion injury on the proteome of Kupffer cells. Lean Zucker rats (n = 6 each group) were randomized to 75 min of warm ischemia or sham operation. After reperfusion for 8 h, Kupffer cells were isolated by enzymatic perfusion and density gradient centrifugation. Proteins were tryptically digested into peptides and differentially labeled with iTRAQ (isobaric tags for relative and absolute quantitation) reagent. After fractionation by cation exchange chromatography, peptides were identified by mass spectrometry (ESI-LC-MS/MS). Spectra were interrogated against the Swiss-Prot database and quantified using ProteinProspector®. The results for heat shock protein 70 and myeloperoxidase were validated by ELISA. Quantitative information for more than 1559 proteins was obtained. In the ischemia group proteins involved in inflammation were significantly up-regulated. The ratio for calgranulin B in the ischemia/sham group was 1.81 ± 0.97 (p < 0.0001), for complement C3 the ratio was 1.81 ± 0.49 (p < 0.0001), and for myeloperoxidase the ratio was 1.30 ± 0.32. Myeloperoxidase was only recently documented in Kupffer cells. The antioxidative proteins Cu,Zn-superoxide dismutase (1.34 ± 0.19; p < 0.001) and catalase (1.23 ± 0.43; p < 0.001) were also elevated. In conclusion, ischemia/reperfusion injury induces alterations in the Kupffer cell proteome. Isotope ratio mass spectrometry is a powerful tool to investigate these reactions. The ability to simultaneously monitor several pathways involved in reperfusion stress may result in important mechanistic insight and possibly new treatment options. The aim of the study was to investigate the impact of ischemia/reperfusion injury on the proteome of Kupffer cells. Lean Zucker rats (n = 6 each group) were randomized to 75 min of warm ischemia or sham operation. After reperfusion for 8 h, Kupffer cells were isolated by enzymatic perfusion and density gradient centrifugation. Proteins were tryptically digested into peptides and differentially labeled with iTRAQ (isobaric tags for relative and absolute quantitation) reagent. After fractionation by cation exchange chromatography, peptides were identified by mass spectrometry (ESI-LC-MS/MS). Spectra were interrogated against the Swiss-Prot database and quantified using ProteinProspector®. The results for heat shock protein 70 and myeloperoxidase were validated by ELISA. Quantitative information for more than 1559 proteins was obtained. In the ischemia group proteins involved in inflammation were significantly up-regulated. The ratio for calgranulin B in the ischemia/sham group was 1.81 ± 0.97 (p < 0.0001), for complement C3 the ratio was 1.81 ± 0.49 (p < 0.0001), and for myeloperoxidase the ratio was 1.30 ± 0.32. Myeloperoxidase was only recently documented in Kupffer cells. The antioxidative proteins Cu,Zn-superoxide dismutase (1.34 ± 0.19; p < 0.001) and catalase (1.23 ± 0.43; p < 0.001) were also elevated. In conclusion, ischemia/reperfusion injury induces alterations in the Kupffer cell proteome. Isotope ratio mass spectrometry is a powerful tool to investigate these reactions. The ability to simultaneously monitor several pathways involved in reperfusion stress may result in important mechanistic insight and possibly new treatment options. There is substantial evidence that indicates that Kupffer cells play a central role in the pathogenesis of liver parenchymal cell damage as seen during ischemia/reperfusion injury (1Sakamoto N. Sun Z. Brengman M.L. Maemura K. Ozaki M. Bulkley G.B. Klein A.S. Hepatic reticuloendothelial system dysfunction after ischemia-reperfusion: role of P-selectin-mediated neutrophil accumulation.Liver Transplant. 2003; 9: 940-948Crossref PubMed Scopus (16) Google Scholar). The activation of Kupffer cells that occurs during ischemia results in the production of oxygen radicals and the modulation of proinflammatory and anti-inflammatory cytokines (2Jaeschke H. Molecular mechanisms of hepatic ischemia-reperfusion injury and preconditioning.Am. J. Physiol. 2003; 284: G15-G26Crossref PubMed Scopus (731) Google Scholar). These mediators are implicated in hepatic cell injury and sinusoidal function. It has been demonstrated that modulation of Kupffer cells can protect the liver (3Vajdova K. Heinrich S. Tian Y. Graf R. Clavien P.A. Ischemic preconditioning and intermittent clamping improve murine hepatic microcirculation and Kupffer cell function after ischemic injury.Liver Transplant. 2004; 10: 520-528Crossref PubMed Scopus (53) Google Scholar, 4Zhong Z. Connor H. Mason R.P. Qu W. Stachlewitz R.F. Gao W. Lemasters J.J. Thurman R.G. 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Chem. 1998; 70: 647R-716RCrossref PubMed Scopus (185) Google Scholar). Isotope ratio mass spectrometry with isobaric tags for relative and absolute quantitation (iTRAQ) 1The abbreviations used are: iTRAQ, isobaric tags for relative and absolute quantitation; AIF-1, allograft inflammatory factor-1; C3, complement factor 3; C3a, activated C3; FDR, false discovery rate; IL, interleukin. 1The abbreviations used are: iTRAQ, isobaric tags for relative and absolute quantitation; AIF-1, allograft inflammatory factor-1; C3, complement factor 3; C3a, activated C3; FDR, false discovery rate; IL, interleukin. allows for the quantification and identification of the protein content of up to four samples in one experiment by labeling all peptides in a given sample (14Ross P.L. Huang Y.N. Marchese J.N. Williamson B. Parker K. Hattan S. Khainovski N. Pillai S. Dey S. Daniels S. Purkayastha S. Juhasz P. Martin S. Bartlet-Jones M. He F. Jacobson A. Pappin D.J. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.Mol. Cell. Proteomics. 2004; 3: 1154-1169Abstract Full Text Full Text PDF PubMed Scopus (3680) Google Scholar). Peptides of the samples are modified by incorporating the four different reagents. The derivatized peptides are indistinguishable in MS but exhibit intense low mass MS/MS signature ions that support quantitation. To our knowledge there have been no studies to date that have used a proteomic approach to study the effects of warm ischemia on protein expression in Kupffer cells. We hypothesized that a proteomic approach might identify patterns of proteins that contribute to a more complete understanding of hepatic injury. All animal experiments were carried out at the University of California at San Francisco with approval by the University of California at San Francisco Committee on Animal Research. Animal care was in agreement with the National Institutes of Health guidelines (57National Research CouncilGuide for the Care and Use of Laboratory Animals. 6th Ed. National Academy Press, Washington, D. C.2002Google Scholar). Inbred male lean Zucker rats (Harlan Sprague-Dawley) were used for this Lean Zucker rats (n = 6 each group) were randomized to 75 min of warm ischemia or sham operation. to and were on a After with the liver was exposed a the the rats were with heat and heat to a of than as by min the of and the of ischemia. We a liver ischemia this is a with In to the and were identified and for 75 min using a The and from the To the of the the and were for of ischemia. ischemia the was with and the was with After 75 min of warm ischemia the were to reperfusion for 8 of of the The rats in the sham group the with the that ischemia was to the After of Kupffer cells were isolated to J.J. and Kupffer cells to in the of J. Physiol. Google Scholar). Kupffer cells were isolated by and perfusion and of the liver by density gradient centrifugation. The samples from and from were in iTRAQ and cation exchange the an of protein from isolated Kupffer cells from the was The protein was after with protein and to and for the quantitation of of protein the of PubMed Scopus Google Scholar). at against was used for all protein iTRAQ labeling was using on the by (14Ross P.L. Huang Y.N. Marchese J.N. Williamson B. Parker K. Hattan S. Khainovski N. Pillai S. Dey S. Daniels S. Purkayastha S. Juhasz P. Martin S. Bartlet-Jones M. He F. Jacobson A. Pappin D.J. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.Mol. Cell. Proteomics. 2004; 3: 1154-1169Abstract Full Text Full Text PDF PubMed Scopus (3680) Google Scholar). were by as D. for the of protein in in the of and PubMed Scopus Google Scholar). of and of were to each of the sample of The were were and the were at for The were of was and the were for min at was by the of in The was to at for The were the iTRAQ in 70 of was to the and the samples were for at The was with of was by an of the samples on the mass after with of the samples was in one and mass spectrometry The of the were to for in labeling and protein The differentially labeled and samples were of the sample was into different by cation exchange exchange was using an system with a was was and for with was gradient of min was and were from the the samples were to complete in the and in The were in an using the system with a of the from each of the labeled sample were by The separation of the peptides was by a gradient of in min using as the on a peptides were to analysis on a mass in that was in with the as J. A.L. Mass analysis of protein at low using and Cell. Proteomics. 2003; 2: Full Text Full Text PDF PubMed Scopus Google Scholar). were by using a of the sample was with an from the peptides identified in the with all the of four mass spectrometry were for each To the protein identification and results that were by mass the protein content of myeloperoxidase and heat shock protein 70 were also by ELISA. for these proteins were used myeloperoxidase and 70 have been to proteins J. M. S. J. R. protect against hepatic warm ischemia/reperfusion injury in Surg. 10: PubMed Google and are used in our of each of the samples from rats and rats were used for both were in to the of the The and that were with the were were using the with the iTRAQ for each of the sample a ratio was The for each iTRAQ experiment were and interrogated against the Swiss-Prot protein database using the of California at San Hansen M. A.L. analysis of a mass spectrometry on a mass I. of the is by Cell. Proteomics. 4: Full Text Full Text PDF PubMed Scopus Google Scholar, Huang L. Hansen M. A.L. analysis of a mass spectrometry on a mass in Protein for and analysis of Cell. Proteomics. 4: Full Text Full Text PDF PubMed Scopus Google to of a protein of and of and a of were used for identification Peptides were for to the peptides that were against protein Protein identification and quantification for the proteins was was by the ratio of the the and using the tool from the of of quantitation on the of more than four and in more than sample were each identified protein the and of the from all peptides to a given protein were was using the in for were only the information was from a of peptides and in a of were used to the p for the of the that the ratio of the is no in the the for was to the false discovery by Y. D. N. I. the false discovery in 2001; PubMed Scopus Google using the for made to the at with a of p than the that was using this were the results of this to by using one for the identification the protein with the and only results from at four peptides sample and in at sample were used for quantitation. However, changes in the content of one protein might for proteins that the to results in our were on the protein and proteins were quantified were in both samples and The for this is a to a of identified peptides protein in the ischemia to a of for Quantitative information was for 1559 of these proteins the for quantitation in at sample with quantitation on at four peptides in the quantified proteins were in ± sample and were identified on the of ± from information from more than peptides in sample be for proteins with and proteins in each of the for by analysis in an of After for in or more sample were in Quantitative information for of the was obtained. the of the all of these were in significantly in the Kupffer cells was in a after ischemia (p < and The quantitation of four different an content in and of peptides for and an of and for and 6 that was (p < There was a in the of myeloperoxidase after ischemia of peptides for and different of dismutase were quantified and in Cu,Zn-superoxide dismutase was by (p < The dismutase a with an of (p = 6 6 protein in a new There was an in content of complement C3 in Kupffer cells of after warm ischemia (p < and the content of calgranulin B was after ischemia and was (p = The allograft inflammatory was by (p < and B protein C3 in a new of the proteins with the be quantified and The content of the was by (p < was by (p = There was only a for the (p = of the in a new The myeloperoxidase in a with an of The myeloperoxidase in of sample with an content of ± The ratio of the in was ± mass myeloperoxidase was quantified in sample in an content ratio of 1.30 ± in one sample was myeloperoxidase quantified by both the content ratio for the was with for the iTRAQ quantitation. heat shock protein 70 the in a with an of heat shock protein 70 was quantified in of sample with an content of ± The ratio of the in was ± mass heat shock protein 70 was quantified in the sample in an content ratio of ± The study demonstrated that the proteomic approach is to monitor several different proteins and the protein expression in rats hepatic ischemia and sham were studies changes in the proteomics demonstrated in several inflammatory proteins calgranulin and allograft inflammatory that have been in the of were were the of the iTRAQ as a discovery the changes by iTRAQ a to the by in is a of these of be by of the of protein by and peptides by mass spectrometry that are or to a different protein by the and the approach used for mass spectrometry were to the with both The for by and with a that to and also a the of to into the S.J. and the of PubMed Scopus Google Scholar). 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