Regulation of Matrix Metalloproteinases and Their Inhibitor Genes in Lipopolysaccharide-Induced Endotoxemia in Mice
2000; Elsevier BV; Volume: 157; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)64531-2
ISSN1525-2191
AutoresAxel Pagenstecher, Anna K. Stalder, Carrie Kincaid, Benedikt Volk, Iain L. Campbell,
Tópico(s)Blood Coagulation and Thrombosis Mechanisms
ResumoAn imbalance between matrix metalloproteinases (MMPs) and inhibitors of MMPs (TIMPs) may contribute to tissue destruction that is found in various inflammatory disorders. To determine in an in vivo experimental setting whether the inflammatory reaction in the course of lipopolysaccharide (LPS)-induced endotoxemia causes an altered balance in the MMP/TIMP system, we analyzed the expression of a number of MMP and TIMP genes as well as MMP enzymatic activity in the liver, kidney, spleen, and brain at various time points after systemic injection of different doses of LPS in mice. Injection of sublethal doses of LPS led to an organ- and time-specific pattern of up-regulation of several MMP genes and the TIMP-1 gene in the liver, spleen, and kidney, whereas in the brain only TIMP-1 was induced. Injection of a lethal dose of LPS caused similar but more prolonged expression of these MMP genes as well as the induction of additional MMP genes in all organs. In LPS-treated mice in situ hybridization revealed collagenase 3 gene induction in cells resembling macrophages whereas TIMP-1 RNA was detected predominantly in parenchymal cells. Finally, gelatin zymography revealed increased gelatinolytic activity in all organs after LPS treatment. These observations highlight a dramatic shift in favor of increased expression of the MMP genes over the TIMP genes during LPS-induced endotoxemia, and suggest that MMPs may contribute to the development of organ damage in endotoxemia. An imbalance between matrix metalloproteinases (MMPs) and inhibitors of MMPs (TIMPs) may contribute to tissue destruction that is found in various inflammatory disorders. To determine in an in vivo experimental setting whether the inflammatory reaction in the course of lipopolysaccharide (LPS)-induced endotoxemia causes an altered balance in the MMP/TIMP system, we analyzed the expression of a number of MMP and TIMP genes as well as MMP enzymatic activity in the liver, kidney, spleen, and brain at various time points after systemic injection of different doses of LPS in mice. Injection of sublethal doses of LPS led to an organ- and time-specific pattern of up-regulation of several MMP genes and the TIMP-1 gene in the liver, spleen, and kidney, whereas in the brain only TIMP-1 was induced. Injection of a lethal dose of LPS caused similar but more prolonged expression of these MMP genes as well as the induction of additional MMP genes in all organs. In LPS-treated mice in situ hybridization revealed collagenase 3 gene induction in cells resembling macrophages whereas TIMP-1 RNA was detected predominantly in parenchymal cells. Finally, gelatin zymography revealed increased gelatinolytic activity in all organs after LPS treatment. These observations highlight a dramatic shift in favor of increased expression of the MMP genes over the TIMP genes during LPS-induced endotoxemia, and suggest that MMPs may contribute to the development of organ damage in endotoxemia. Sepsis is a disorder with a high lethality even under appropriate treatment with antibiotics and complete eradication of bacteria. This suggests that ongoing responses of the immune system once started may direct the course of the disease. 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The physiological counterregulators of MMPs are the MMP inhibitors. 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However, to date, this issue has rarely been addressed.24Paemen L Jansen PM Proost P Van DJ Opdenakker G Hack E Taylor FB Induction of gelatinase B and MCP-2 in baboons during sublethal and lethal bacteraemia.Cytokine. 1997; 9: 412-415Crossref PubMed Scopus (35) Google Scholar In a recent study, Solorzano and coworkers45Solorzano CC Ksontini R Pruitt JH Auffenberg T Tannahill C Galardy RE Schultz GP MacKay SLD Copeland III, EM Moldawer LL A matrix metalloproteinase inhibitor prevents processing of tumor necrosis factor alpha (TNF alpha) and abrogates endotoxin-induced lethality.Shock. 1997; 7: 427-431Crossref PubMed Scopus (75) Google Scholar demonstrated an ameliorative effect of the synthetic MMP inhibitor GM-6001 in LPS-induced endotoxemia. The MMP inhibitor increased the survival of LPS-treated mice and inhibited plasma levels of TNF-α. However, the effects of the MMP inhibitor on MMP activity were not studied. In the present study, experiments were performed in LPS-responsive and LPS-resistant mice (C3H/HeJ)2Vogel SN Hogan MM Immunophysiology: The Role of Cells and Cytokines in Immunity and Inflammation.in: Oppenheim JJ Shevach EM Oxford University Press, New York1990: 238-258Google Scholar to investigate the expression pattern and the regulation of various MMPs and TIMPs in endotoxemia. To elucidate the role of the complex MMP and TIMP network in this model of inflammation, we used RNase protection assays for the simultaneous and semiquantitative determination of MMP and TIMP gene expression. The cellular localization of RNAs was revealed by in situ hybridization. Furthermore, we analyzed the activity of gelatinases in the organs using gelatin polyacrylamide gel electrophoresis (PAGE) zymography and in situ zymography. C57BL/SJL F1 mice used in this study were maintained under specific pathogen-free conditions in the closed breeding colony of the Scripps Research Institute. Experiments were performed in 8- to 10-week-old mice of both sexes. LPS-resistant C3H/HeJ and LPS-sensitive C3H/FeJ mice as controls were obtained from Jackson Laboratories (Maine, VT) and used at 10 to 14 weeks of age. Endotoxemia was induced by intraperitoneal injection of different doses of LPS (Escherichia coli 026:B6; Sigma, St. Louis, MO) ranging from 20 μg (sublethal dose) to 1 mg (lethal dose). In an initial experiment, two mice at each time point were injected with 20 μg of LPS and were killed at various times ranging from 15 minutes to 24 hours. In another experiment, a lethal dose of 1 mg of LPS was administered and mice were killed 2, 8, or 16 hours later (three mice at each time point). Experiments with LPS-resistant (C3H/HeJ) mice and the respective LPS-sensitive control (C3H/FeJ) mice were performed with a single injection of 20 μg of LPS and the mice were killed 8 hours later. In all experiments, the brain, kidney, spleen, and liver were removed and immediately snap-frozen in liquid nitrogen and stored at −80°C until RNA isolation or protein extraction. For in situ hybridization, mice were injected with 1 mg of LPS and were killed 8 hours later. Organs were removed, fixed in 4% formaldehyde, and embedded in paraffin. For in situ zymography, mice were injected with 100 μg of LPS and were killed 16 hours later, organs were removed and snap-frozen in liquid nitrogen. The production of RPA probe sets for the detection of MMP and TIMP gene expression was described previously.31Pagenstecher A Stalder AK Kincaid CL Shapiro SD Campbell IL Differential expression of matrix metalloproteinase and tissue inhibitor of matrix metalloproteinase genes in the mouse central nervous system in normal and inflammatory states.Am J Pathol. 1998; 152: 729-741PubMed Google Scholar, 46Pagenstecher A Stalder AK Campbell IL RNase protection assays for the simultaneous and semiquantitative analysis of multiple murine matrix metalloproteinase (MMP) and MMP inhibitor mRNAs.J Immunol Methods. 1997; 206: 1-9Crossref PubMed Scopus (36) Google ScholarBriefly, the MMP probe set included probes for stromelysins 1, 2, and 3, matrilysin, metalloelastase, gelatinase A and B, collagenase 3, and membrane type1-MMP (MT1-MMP; MMP probe set). The MMP probe set used for RPAs in the experiment in which 20 μg of LPS was used did not contain the gelatinase A probe. In later experiments, the gelatinase A probe was added to the probe set. These experiments showed that there was no gelatinase A gene expression detectable at the LPS doses tested (20 μg to 1 mg). The probe set for the inhibitors of MMPs (TIMP probe set) included probes for TIMPs 1, 2, and 3, and α2-M. A fragment of the RPL32–4A gene47Dudov KP Perry RP The gene family encoding the mouse ribosomal protein L32 contains a uniquely expressed intron-containing gene and an unmutated processed gene.Cell. 1984; 37: 457-464Abstract Full Text PDF PubMed Scopus (282) Google Scholar served as an internal loading control. Poly(A)+ RNA was prepared as described.48Badley JE Bishop GA St. John T Frelinger JA A simple, rapid method of purification of polyA+ RNA.Biotechniques. 1988; 6: 114-116PubMed Google Scholar RNase protection assays were performed as described previously.49Stalder AK Campbell IL Simultaneous analysis of multiple cytokine receptor mRNAs by RNase protection assay in LPS-induced endotoxemia.Lymphokine Cytokine Res. 1994; 13: 107-112PubMed Google Scholar In situ hybridization was performed as described by Simmons et al50Simmons DM Arriza JL Swanson LW A complete protocol for in situ hybridization of messenger RNAs in brain and other tissues with radiolabeled single-stranded RNA probes.J Histotechnol. 1989; 12: 169-181Crossref Google Scholar with modifications. Briefly, paraffin sections were deparaffinized and rehydrated in graded alcohols. After postfixation in 4% formaldehyde, proteinase K treatment (2.4 mg/100 ml 5× Tris-EDTA buffer at 37°C for 15 minutes), and acetylation in 250 μl acetic anhydride in 100 ml phosphate-buffered saline (PBS) for 10 minutes, the slides were dehydrated in graded alcohol and dried. Templates for the probes for collagenase 3, gelatinase B, stromelysin 1, and TIMP-1 were generated by reverse transcriptase-polymerase chain reaction and cloned into the pGEM-4 plasmid (Promega, Madison, WI). Cloned products were sequenced to confirm their identity with the published sequences. [33P]-labeled sense or antisense probes were hybridized to the tissue overnight at 56 to 59°C. After digestion with RNase A (Promega), slides were washed in decreasing concentrations of standard saline citrate. After dehydration in graded alcohol, slides were air dried and exposed for 5 days to Ultra Vision G film (Sterling, Newark, DE). Then, slides were dipped in Kodak NTB-2 emulsion (Eastman Kodak Co., Rochester, NY), dried, and stored in the dark for 4 weeks. Subsequently, slides were developed, counterstained with Mayer's hematoxylin, and examined by dark- and bright-field microscopy. Protease activity was determined as described previously.31Pagenstecher A Stalder AK Kincaid CL Shapiro SD Campbell IL Differential expression of matrix metalloproteinase and tissue inhibitor of matrix metalloproteinase genes in the mouse central nervous system in normal and inflammatory states.Am J Pathol. 1998; 152: 729-741PubMed Google Scholar Briefly, frozen organs were homogenized (1/10, w/v) on ice in Tris buffer (pH 7.6) containing the serine and cysteine protease inhibitors aprotinin, leupeptin, leustatin, and phenylmethyl sulfonyl fluoride. Lysates were centrifuged and supernatants used for zymography. Samples were electrophoresed on 10% polyacrylamide gels containing 0.1% gelatin (Sigma). After electrophoresis, sodium dodecyl sulfate was removed by soaking the gels for 20 minutes each in buffers containing 50 mmol/L Tris (pH 7.6), 10 mmol/L CaCl, and 2.5% Triton X-100 followed by the same buffer with 1% Triton. Protease activity was restored in the same buffer without Triton. Digestion was performed at 37°C for 36 hours. Gels were stained with Coomassie brilliant blue and destained until clear bands became evident. In situ zymography was performed as described by Oh and co-workers.51Oh LYS Larsen PH Krekoski CA Edwards DR Donovan F Werb Z Yong VW Matrix metalloproteinase-9/gelatinase B is required for process outgrowth by oligodendrocytes.J Neurosci. 1999; 19: 8464-8475Crossref PubMed Google Scholar Briefly, cryostat sections (10 μm) of brain, spleen, liver, and kidney of control mice and of endotoxemic mice were overlaid with assay solution (50 mmol/L Tris-HCl, 5 mmol/L CaCl, 0.2 mmol/L NaN3, pH 7.5) containing 40 μg/ml fluorescein isothiocyanate-labeled DQ gelatin (Molecular Probes, Eugene, OR). Sections were incubated at 37°C for 18 hours and examined by fluorescence microscopy. Gelatinolytic proteases cleave quenched DQ gelatin resulting in fluorescent breakdown products that thereby allow for the localization of net gelatinolytic activity. Addition of 10 mmol/L ethylenediaminetetraacetic acid (EDTA) (a chelating agent that inhibits metal ion dependent proteases such as MMPs) to the assay solution or omission of DQ gelatin served as negative control and abolished the fluorescence. The expression of transcripts for the MMPs: collagenase 3, gelatinase A and B, stromelysins 1, 2, and 3, metalloelastase, matrilysin, and MT1-MMP; and the inhibitors: TIMP-1, 2, and 3 and α2-M was determined after injection of different doses of LPS. After injection of 1 mg of LPS, three of six mice died between 15 and 18 hours. Charts showing the quantitative analysis of MMP and TIMP mRNA expression are given for RNAs that were either induced or were more than twofold up-regulated after challenge with 20 μg or 1 mg of LPS (Figure 1, Figure 2).Figure 2Quantitative analysis of MMP and TIMP gene expression the organs of mice after injection of different doses of LPS (20 μg LPS, fine curve) and after 1 mg of LPS (bold line, three mice were used at each time point, X MMP and TIMP gene expression in three mice that died at 15 to 18 hours after LPS). A: MMP and TIMP gene expression in the spleen after different doses of LPS. B: Quantitative analysis of MMP and TIMP gene expression in the kidney after different doses of LPS. C: Quantitative analysis of MMP and TIMP gene expression in the brain after different doses of LPS. The RPAs and quantitative analysis was performed as described in Materials and Methods and the legend to Figure 1.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In control liver, expression of MT1-MMP and low levels of gelatinase B was observed (Figure 1). After injection of 20 μg of LPS, changes in MMP gene expression first became evident by 1 hour. At 2 hours there was induction of collagenase 3 mRNA that peaked at 8 hours and then declined rapidly during the next 8 hours back to below detectable levels. In contrast, expression of gelatinase B mRNA showed a more delayed response that peaked by 16 hours. MT1-MMP gene expression showed an up-regulation that peaked by 12 hours. A different time course of MMP gene expression was observed after injection of 1 mg of LPS (Figure 1). Whereas the levels of collagenase 3 and gelatinase B gene
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