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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 30, 2005 as doi:10.1096/fj.05-3864fje. |
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,1
* Environmental Health Sciences Division, National Institute for Environmental Studies, Tsukuba, Japan;
Inflammation and Immunology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan;
Department of Vaterinary Pathology, Faculty of Agriculture, Tottori University, Tottori, Japan; and
Department of Hygienics, Gifu Pharmaceutical University, Gifu, Japan
1Correspondence: Environmental Health Sciences Division, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, 305-8506, Japan. E-mail: htakano{at}nies.go.jp
SPECIFIC AIM
Metallothionein (MT) is a highly conserved, low molecular weight, cystein-rich protein that plays an important role in cytoprotection. Since proinflammatory cytokines induce MT gene expression in vivo, roles of MT in inflammation have been focused. However, the role of MT in coagulatory and fibrinolytic changes during inflammation has not been elucidated. The aim of the current study was to examine whether MT is protective against coagulatory and fibrinolytic disturbance during severe inflammation induced by lipopolysaccharide (LPS) using MT-deficient (–/–) mice and wild-type (WT) mice.
PRINCIPAL FINDINGS
1. Role of MT in coagulatory and fibrinolytic changes and peripheral blood cell counts after LPS challenge
We first evaluated coagulatory and fibrinolytic parameters 36 h after intraperitoneal challenge with LPS (30mg/kg) or vehicle (Table 1
). LPS challenge caused prolongation of prothrombin time (PT) and activated partial thromboplastin time (APTT) in both genotypes of mice compared with vehicle challenge. In the presence of LPS, PT, and APTT were significantly longer in MT (–/–) mice than in WT mice. Levels of fibrinogen and fibrinogen/fibrin degradation products (FDP) significantly elevated after LPS challenge in both genotypes of mice. After LPS challenge, they were significantly higher in MT (–/–) mice than in WT mice. Compared with vehicle, LPS significantly decreased activated protein C (APC) activity only in MT (–/–) mice. In the presence of LPS, the activity was significantly lower in MT (–/–) mice than in WT mice. Compared with vehicle administration, LPS administration elicited a significant increase in the plasma levels of von Willebrand factor (vWF) only in MT (–/–) mice.
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LPS challenge significantly decreased WBC and platelet counts in both genotypes of mice compared with vehicle challenge. However, there were no significant differences in the counts between both genotypes of mice in the presence or absence of LPS.
2. MT induction in the organs after LPS challenge
We next measured MT protein levels in the lung, kidney, and liver in WT mice. LPS challenge significantly increased MT protein in all the tissues examined compared with vehicle challenge. On the other hand, MT protein was not detected in the tissues of MT (–/–) mice in the presence or absence of LPS.
3. Role of MT in organ damage after LPS challenge
We next evaluated histopathological changes in the lung, kidney, and liver obtained from both genotypes of mice 36 h after LPS challenge. LPS treatment induced more severe inflammatory damage in the lung, kidney, and liver from MT (–/–) mice than in those from WT mice. In addition, thrombus formations and fibrin thrombi were seen only in MT (–/–) mice after LPS challenge. To morphometrically estimate the inflammatory changes, we quantified the number of neutrophils in the lung, kidney, and liver 36 h after LPS challenge. In the presence of LPS, MT (–/–) mice showed significantly increased numbers of neutrophils in the lung, kidney, and liver compared with WT mice (lung: 16.6±2.4/high power field vs. 11.4±1.5; kidney: 3.3±0.5 vs. 1.5±0.2; liver: 33.5±4.4 vs. 22.7±4.7).
4. Role of MT in the levels of proinflammatory proteins related to LPS
To further determine the role of MT in LPS-induced inflammatory responses, protein expressions of proinflammatory cytokines and chemokines in the lung, kidney, and liver 36 h after the LPS administration and serum levels of the proteins 20 h or 52 h after the administration were assessed by enzyme-linked immunosorbent assays. In MT (–/–) mice, LPS caused significant elevations of the lung expression of interleukin (IL)-1ß, IL-6, granulocyte/macrophage-colony-stimulating factor (GM-CSF), macrophage inflammatory protein (MIP)-1
, MIP-2, macrophage chemoattractant protein (MCP)-1, and keratinocyte chemoattractant (KC) compared with vehicle (Table 2
). In WT mice, LPS induced significant enhancement in the expression of IL-1ß, GM-CSF, MIP-1, MCP-1, and KC compared with vehicle. In the presence of LPS, the lung expression of IL-1ß, IL-6, GM-CSF, MIP-1, MIP-2, MCP-1, and KC was significantly greater in MT (–/–) mice than in WT mice. Overall, the protein expressions of theses cytokines and chemokines were greater in MT (–/–) mice than in WT mice after LPS challenge.
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CONCLUSIONS AND SIGNIFICANCE
Fibrinogen is induced by several inflammatory mediators and can result in the activation of coagulation and fibrinolysis. In our study, elevations of fibrinogen and FDP were significantly greater in MT (–/–) mice than in WT mice after LPS challenge. These results suggest that MT can be protective against systemic inflammatory response, and subsequent activation of fibrinolysis and coagulation. APC is an important anticoagulatory factor. In our study, APC was less in MT (–/–) mice than in WT mice after LPS challenge, but levels of vWF are considered to be a marker of endothelial damage. In the present study, vWF was higher in MT (–/–) mice than in WT mice after LPS challenge. Our recent work also demonstrated that MT is protective against endothelial integrity in acute lung vascular injury induced by LPS. Taken together, MT can be protective against endothelial damage during severe systemic inflammation. Endothelial cells can reportedly modulate platelet function, coagulation, and fibrinolysis, resulting in resistance to intravascular coagulation and subsequent organ damages. In fact, clotting times such as PT and APTT were significantly longer in MT (–/–) mice than in WT mice after LPS challenge in the present study. It is well recognized that global clotting times prolong as consequences of the consumption and depletion of various coagulation factors after LPS challenge. On the basis of the present results and previous reports, MT should play protective roles in the coagulatory disturbance related to LPS, possibly maintaining endothelial cell integrity and by inhibition of the activated coagulation and fibrinolysis.
The present study also demonstrated the protective effects of MT on organ damages elicited by LPS in vivo. LPS challenge induced inflammatory organ damages in the liver, lung, and kidney in both genotypes of mice; however, damages including neutrophil infiltration were more prominent in MT (–/–) mice than in WT mice in the presence of LPS. LPS injection induced MT protein in the lung, kidney, and liver from WT mice. Our results suggest that MT is protective against organ damages related to LPS. Thrombus formations could be seen only in the liver from MT (–/–) mice after LPS challenge. This finding also supports the protective role of MT against activated coagulation during systemic inflammation.
Proinflammatory cytokines and chemokines cause the inflammatory tissue injury. Few in vivo studies have indicated the suppressive role of MT in cytokine releases during inflammation. In our study, the protein levels of IL-1ß, GM-CSF, MIP-1, MIP-2, MCP-1, and KC in the lungs, kidneys, and livers were greater in MT (–/–) mice than in WT mice after LPS challenge. We also confirmed that kinetic assay for circulatory levels of the cytokines and chemokines was parallel to the levels of these proteins in the organs in overall trend. Our experiments should be the first in vivo demonstration that the antiinflammatory effects of MT on the LPS-related organ damages should be mediated at least in part via inhibition of the protein expression of these proinflammatory cytokines and chemokines in the multiple organs as well as in the circulation.
In conclusion, the present study has shown that MT (–/–) mice reveal significant prolongation of PT and APTT, a significant increase in fibrinogen and FDP levels, and a significant decrease in APC after LPS treatment compared with WT mice. LPS induces inflammatory organ damages in the lung, kidney, and liver in both genotypes of mice. The damages including neutrophil infiltration were more prominent in MT (–/–) mice than in WT mice after LPS treatment. LPS enhances the protein expression of IL-1ß, GM-CSF, MIP-1, MIP-2, MCP -1, and KC in the lung, kidney, and liver and circulatory levels of IL-1ß, MIP-2, and KC in both genotypes of mice. In overall trends, the levels of these proinflammatory proteins were greater in MT (–/–) mice than in WT mice after LPS challenge. These results suggest that MT protects against systemic inflammation with endothelial damage and coagulo-fibrinolytic disturbance followed by organ damages related to bacterial endotoxin, at least partly, via inhibition of the enhanced expression of proinflammatory cytokines and chemokines (Fig. 1
). MT can be a novel therapeutic molecule for systemic inflammatory response syndrome including disseminated intravascular coagulation and multiple organ dysfunction syndrome.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3864fje;
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