![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA
The Department of Immunology, The Scripps Research Institute, La Jolla, California
Received March 7, 2008; accepted April 28, 2008.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
, monocyte chemotactic protein 1 (MCP1)/JE, and keratinocyte chemoattractant, and decreased mortality. The PI3K inhibitor wortmannin abolished the insulin-mediated activation of Akt and the reduction of chemokine and interleukin-6 levels. We conclude that insulin reduces LPS-induced inflammation in mice in a PI3K/Akt-dependent manner without affecting blood glucose levels.
(TNF-) and interleukin 6 (IL-6), and chemokines, such as monocyte chemotactic protein 1 (MCP1)/JE and keratinocyte chemoattractant (KC) (Casey et al., 1993
). However, over-reaction of the pro-inflammatory response to infection and injury can contribute to the development of sepsis, septic shock, multiple organ failure, and death. Hyperglycemia and insulin resistance followed by hypoglycemia and hypoinsulinemia can also occur during sepsis as a consequence of the metabolic effects of stress hormone and cytokine production (Maitra et al., 2000; Marik and Raghavan, 2004; Van den Berghe, 2004; van Waardenburg et al., 2006). Despite considerable progress in our understanding of the pathologic pathways that contribute to sepsis and septic shock, pharmacologic interventions are currently limited to insulin and activated protein C (Shapiro et al., 2006). Insulin is administered to septic patients with hyperglycemia to normalize glucose levels (Russell, 2006; Shapiro et al., 2006). Reducing glucose levels with insulin therapy is associated with decreased inflammation and endothelial cell damage (van den Berghe et al., 2001, 2006; Van den Berghe, 2004; Marik and Raghavan, 2004; Langouche et al., 2005). Recently, however, it has been shown that insulin-induced hypoglycemia may counteract the beneficial effects of aggressive insulin therapy in patients with severe sepsis (Brunkhorst et al., 2008).
Currently, insulin is not initiated in normoglycemic septic patients or to correct hypoinsulinemia during sepsis (Mitchell et al., 2006). However, results from animal studies indicate that insulin may have direct anti-inflammatory effects. In nonhyperglycemic animal models of endotoxemia, continuous infusion of insulin while maintaining blood glucose in the normal range with dextrose or administering a bolus injection of insulin results in decreased inflammation and morbidity (Brix-Christensen et al., 2004; Jeschke et al., 2004, 2005). However, the mechanism by which insulin reduces inflammation in the absence of hyperglycemia is unknown. One possibility is that insulin reduces inflammation by activating anti-inflammatory signaling pathways, such as the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway. It is well established that insulin activates PI3K/Akt signaling in many cell types (Taniguchi et al., 2006). It is noteworthy that we and others (Guha and Mackman, 2002; Schabbauer et al., 2004; Liew et al., 2005; Martin et al., 2005) have shown that this pathway negatively regulates LPS-induced signaling and pro-inflammatory cytokine production in monocytic cells. Furthermore, activation of PI3K enhanced survival, whereas inhibition of PI3K reduced survival of endotoxemic mice (Schabbauer et al., 2004; Williams et al., 2004). Taken together, these studies suggest that the protective effects of insulin in endotoxemia models may be mediated by activation of the PI3K/Akt pathway.
In this study, we used a pharmacological inhibitor of PI3K to test the hypothesis that exogenous insulin decreases inflammation in endotoxemic mice by activating the PI3K/Akt pathway and that this anti-inflammatory effect is independent of its affect on blood glucose levels.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
). C57BL/6J male mice of 6 to 16 weeks of age were used for all experiments.
Mouse Endotoxemia Model and Insulin Administration. We used a mouse model of endotoxemia consisting of an intraperitoneal injection of LPS (Escherichia coli serotype O111:B4; Sigma-Aldrich, St Louis, MO). Doses of 5 or 10 mg/kg were used in experiments. These doses corresponded to the LD50 for two different lots of LPS. Accordingly, LPS produced similar levels of cytokine expression and lethality in each experiment. Human insulin (Humulin 70/30; Eli Lilly, Indianapolis, IN) was administered to mice either as a bolus intraperitoneal injection or via Alzet micro-osmotic pumps (model 1003D; Durect Corp, Cupertino, CA) that were implanted subcutaneously 16 h before administration of LPS. These pumps deliver 1 µl/h for 72 h. Human insulin was used to allow us to measure low levels of exogenous insulin and to distinguish it from endogenous mouse insulin in the endotoxemic mice. Furthermore, human insulin has been used previously in studies examining the effect of acute insulin treatment in endotoxemic rodents (Jeschke et al., 2004). To inhibit PI3K activity, mice were given wortmannin (Sigma-Aldrich) at a dose of 0.06 mg/kg or its vehicle [Ringer's solution containing 10% (v/v) dimethyl sulfoxide] three times by retro-orbital injection at –90, +90, and + 360 min relative to LPS administration. Blood samples were collected from the retro-orbital sinus (final concentration 0.32%) at various times after LPS administration (0–24 h). Plasma was collected and stored at –80°C until analysis.
Measurement of Glucose, Insulin, and Cytokine Levels. Glucose levels in the plasma were determined using a Glucometer Elite XL (Bayer Corporation, Elkhart, IN). Human insulin levels in the plasma were determined using a commercial ELISA that detects only human insulin (10-1132-01; Mercodia, Uppsala, Sweden). Total insulin (mouse + human) levels in the plasma were determined using a different commercial ELISA kit (10–1150–01; Mercodia). The levels of TNF-, IL-6, JE, and KC in plasma were determined using commercial ELISA kits (R&D Systems, Minneapolis, MN).
Western Blotting. Muscle and epididymal adipose tissue were collected and quick-frozen in liquid nitrogen and stored at –80°C until processed. Tissues were homogenized in 1.5 ml of a buffer [10 mM HEPES, 10 mM KCl, 300 mM sucrose, 1.5 mM MgCl2, 0.5 mM DTT, 0.5 mM PMSF, half of a tablet, and complete EDTA-free protease inhibitor cocktail (Roche, Indianapolis, IN)], spun at 550g for 2 min at 4°C, and supernatants were frozen. Protein concentration was measured using a colorimetric assay according to manufacturer's instructions (Bio-Rad Laboratories, Hercules, CA). Proteins were separated by SDS-polyacrylamide gel electrophoresis and then transferred to Millipore polyvinylidene fluoride membranes (Millipore, Bedford, MA). Levels of phosphorylated (Ser473) and nonphosphorylated Akt were detected using primary rabbit antibodies (Cell Signaling, Beverly, MA) and a secondary antibody conjugated to horseradish peroxidase. Antibodies were developed using a chemiluminescence reagent assay (Super Signal West Femto; Pierce, Rockford, IL), according to manufacturer's instructions, and exposed to radiographic film (Kodak, Rochester, NY).
|
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
|
|
, IL-6, JE, and KC and improved the survival of endotoxemic mice compared with mice treated with saline (Fig. 2, A–F). It is interesting that we observed a negative correlation between plasma levels of human insulin and IL-6 (data not shown).
Exogenous Insulin Activates Akt in Insulin-Sensitive Tissues in Endotoxemic Mice. We hypothesized that the protective effects of insulin during endotoxemia are mediated by activation of PI3K/Akt. We chose to analyze the activation of Akt in muscle and adipose tissue because glucose transport in theses tissues is regulated by insulin, and they express inflammatory mediators during endotoxemia (Chang et al., 2004; Brix-Christensen et al., 2005; Bultinck et al., 2006). It is noteworthy that Akt phosphorylation was increased in the muscle and maintained in adipose tissue of insulin-treated endotoxemic mice (Fig. 3, A and B).
|
levels at 1.5 h (Fig. 4D).
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
, IL-6, JE, and KC and decreased mortality in endotoxemic mice without exacerbating hypoglycemia. We then used a pharmacologic approach to analyze the role of PI3K/Akt signaling in the anti-inflammatory effects of insulin. The low dose of human insulin used in this study enhanced phosphorylation of Akt in muscle and adipose tissue during the hypoglycemic, hypoinsulinemic phase of endotoxemia. Wortmannin abolished the insulin-induced phosphorylation of Akt in these mice indicating inhibition of PI3K. It is noteworthy that inhibition of insulin-mediated activation of PI3K/Akt with wortmannin reversed the insulin-dependent reduction in LPS-induced IL-6, JE, and KC expression. These data indicate that insulin activation of the PI3K/Akt pathway inhibits the production of these cytokines in endotoxemic mice. However, because wortmannin inhibits other cell signaling pathways, we cannot exclude the possibility that additional mechanisms also contribute to the anti-inflammatory effects of insulin in these mice (Ding et al., 1995).
It is interesting to note that insulin-dependent reduction in TNF- production was not reversed by wortmannin. This observation may, in part, relate to the complexity of TNF- release in endotoxemia. For example, previous studies have shown that the cleavage of the pro-form of TNF- from the membrane by TNF--converting enzyme contributes significantly to plasma TNF- levels in endotoxemic mice (Zhang et al., 2004). Further studies are required to determine whether insulin-mediated activation of PI3K plays a role in reducing TNF- levels in endotoxemic mice.
Other studies indirectly support our observation that activation of PI3K/Akt signaling mediates many of the anti-inflammatory effects of insulin during endotoxemia. For instance, insulin has been shown to increase the expression of mitogen-activated protein kinase phosphatase (MKP-1) in a PI3K-dependent manner (Desbois-Mouthon et al., 2000; Takehara et al., 2000), and this phosphatase negatively regulates IL-6 and TNF- production in endotoxemic mice (Chi et al., 2006). Furthermore, a recent study showed that insulin and a glycogen synthase kinase 3β inhibitor had similar anti-inflammatory effects in a rat model of sepsis (Dugo et al., 2006). It is noteworthy that Akt-dependent phosphorylation decreases the activity of glycogen synthase kinase 3β (Guha and Mackman, 2002; Martin et al., 2005). These studies, together with the findings reported here, support the notion that activation of the PI3K/Akt pathway mediates the protective effects of insulin during endotoxemia.
Both increased levels of IL-6 and hypoinsulinemia have been associated with increased morbidity and decreased survival during endotoxemia and sepsis (Ciancio et al., 1991; Casey et al., 1993; van Waardenburg et al., 2006). In this study, endogenous insulin levels decreased dramatically at a time when IL-6 levels were maximal. In insulin-treated mice, the maximal amount of human insulin in plasma 8 h after LPS was 8.9 mU/liter. However, as a consequence of LPS-induced hypoinsulinemia, this small amount of insulin constituted a large portion of total insulin levels 8 h after the administration of LPS. It is noteworthy that we observed a strong negative correlation between human insulin levels and plasma IL-6 levels in insulin-treated mice. These data suggest that small amounts of insulin can significantly affect the inflammatory response without affecting plasma glucose levels.
Glucose transport into muscle and adipose is partially dependent on insulin-mediated activation of the PI3K/Akt pathway. Insulin-mediated effects on glucose metabolism in the liver are also dependent on activation of this pathway. The dose of insulin used in this study increased levels of phosphorylated Akt in muscle and adipose tissue during endotoxemia. It is interesting that we did not observe increased levels of phosphorylated Akt in the liver (data not shown). Therefore, tissue-specific differences in the activation of the PI3K/Akt pathway may explain how a low dose of insulin can affect the inflammatory response without affecting glucose metabolism.
The cellular target(s) mediating the protective effects of insulin in this model remain to be determined. Insulin-dependent tissues, such as skeletal muscle and adipose tissue, contribute to IL-6 expression during endotoxemia (Brix-Christensen et al., 2005; Bultinck et al., 2006), suggesting that the anti-inflammatory effects of insulin on IL-6 production during endotoxemia may be the result of direct activation of PI3K in these tissues. Hematopoietic cells, such as monocytes and macrophages, have been shown to be the major source of cytokine production during endotoxemia (Michalek et al., 1980). Insulin may directly activate PI3K in monocytes and thereby reduce inflammatory cytokine production in response to LPS in vivo. Insulin signaling has not been studied extensively in macrophages and mononuclear cells. Although they do not require insulin for glucose transport, these cells express insulin receptors and insulin increases glucose transport (Daneman et al., 1992; Estrada et al., 1994; Welham et al., 1997). A recent study showed that high concentrations of insulin (104 µM) decreased TNF- and IL-1β production and inhibited apoptosis in LPS-treated THP-1 cells in vitro (Leffler et al., 2007). The anti-apoptotic effects of insulin were mediated by activation of PI3K. However, this study did not determine whether the anti-inflammatory effects of insulin were PI3K-dependent.
Apoptosis of cells, particularly endothelial cells, may contribute to inflammation and organ damage during endotoxemia (Power et al., 2002; Bannerman and Goldblum, 2003). It is interesting that insulin inhibits apoptosis in a variety of cell types in a PI3K/Akt-dependent manner and may reduce endothelial apoptosis in endotoxemic mice (Hermann et al., 2000; Conejo and Lorenzo, 2001; Leffler et al., 2007). Further studies are required to determine the cellular targets of insulin during endotoxemia.
In summary, we demonstrated that continuous administration of small amounts of insulin decreased inflammation in endotoxemic mice in a PI3K-dependent manner without affecting blood glucose levels. These findings may ultimately have important implications regarding the use of insulin for treating normoglycemic, hypoinsulinemic patients with sepsis.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: LPS, lipopolysaccharide; PI3K, phosphatidylinositol 3-kinase; ELISA, enzyme-linked immunosorbent assay; Akt, protein kinase B; KC, keratinocyte chemoattractant; TNF-, tumor necrosis factor-; IL-6, interleukin-6.
Address correspondence to: Dr. Nigel Mackman, Division of Hematology/Oncology, University of North Carolina, Chapel Hill, NC 27599. E-mail: nmackman{at}med.unc.edu
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Bannerman DD and Goldblum SE (2003) Mechanisms of bacterial lipopolysaccharide-induced endothelial apoptosis. Am J Physiol Lung Cell Mol Physiol 284: L899–L914.
Brix-Christensen V, Andersen SK, Andersen R, Mengel A, Dyhr T, Andersen NT, Larsson A, Schmitz O, Orskov H, and Tonnesen E (2004) Acute hyperinsulinemia restrains endotoxin-induced systemic inflammatory response: an experimental study in a porcine model. Anesthesiology 100: 861–870.[CrossRef][Medline]
Brix-Christensen V, Gjedsted J, Andersen SK, Vestergaard C, Nielsen J, Rix T, Nyboe R, Andersen NT, Larsson A, Schmitz O, et al. (2005) Inflammatory response during hyperglycemia and hyperinsulinemia in a porcine endotoxemic model: the contribution of essential organs. Acta Anaesthesiol Scand 49: 991–998.[CrossRef][Medline]
Brunkhorst F, Engel C, Bloos F, Meier-Hellmann A, Ragaller M, Weiler N, Moerer O, Gruendling M, Oppert M, Grond S, et al. (2008) Intensive insulin therapy and pentastarch resuscitation in severe sepsis. N Engl J Med 358: 125–139.
Bultinck J, Brouckaert P, and Cauwels A (2006) The in vivo contribution of hematopoietic cells to systemic TNF and IL-6 production during endotoxemia. Cytokine 36: 160–166.[CrossRef][Medline]
Casey LC, Balk RA, and Bone RC (1993) Plasma cytokine and endotoxin levels correlate with survival in patients with the sepsis syndrome. Ann Intern Med 119: 771–778.
Chang L, Chiang SH, and Saltiel AR (2004) Insulin signaling and the regulation of glucose transport. Mol Med 10: 65–71.[Medline]
Chi H, Barry SP, Roth RJ, Wu JJ, Jones EA, Bennett AM, and Flavell RA (2006) Dynamic regulation of pro- and anti-inflammatory cytokines by MAPK phosphatase 1 (MKP-1) in innate immune responses. Proc Natl Acad Sci U S A 103: 2274–2279.
Ciancio MJ, Hunt J, Jones SB, and Filkins JP (1991) Comparative and interactive in vivo effects of tumor necrosis factor alpha and endotoxin. Circ Shock 33: 108–120.[Medline]
Conejo R and Lorenzo M (2001) Insulin signaling leading to proliferation, survival, and membrane ruffling in C2C12 myoblasts. J Cell Physiol 187: 96–108.[CrossRef][Medline]
Daneman D, Zinman B, Elliott ME, Bilan PJ, and Klip A (1992) Insulin-stimulated glucose transport in circulating mononuclear cells from nondiabetic and IDDM subjects. Diabetes 41: 227–234.[Abstract]
Desbois-Mouthon C, Cadoret A, Blivet-Van Eggelpoel MJ, Bertrand F, Caron M, Atfi A, Cherqui G, and Capeau J (2000) Insulin-mediated cell proliferation and survival involve inhibition of c-Jun N-terminal kinases through a phosphatidylinositol 3-kinase- and mitogen-activated protein kinase phosphatase-1-dependent pathway. Endocrinology 141: 922–931.
Ding J, Vlahos CJ, Liu R, Brown RF, and Badwey JA (1995) Antagonists of phosphatidylinositol 3-kinase block activation of several novel protein kinases in neutrophils. J Biol Chem 270: 11684–11691.
Dugo L, Collin M, Allen DA, Murch O, Foster SJ, Yaqoob MM, and Thiemermann C (2006) Insulin reduces the multiple organ injury and dysfunction caused by coadministration of lipopolysaccharide and peptidoglycan independently of blood glucose: role of glycogen synthase kinase-3beta inhibition. Crit Care Med 34: 1489–1496.[CrossRef][Medline]
Estrada DE, Elliott E, Zinman B, Poon I, Liu Z, Klip A, and Daneman D (1994) Regulation of glucose transport and expression of GLUT3 transporters in human circulating mononuclear cells: studies in cells from insulin-dependent diabetic and nondiabetic individuals. Metabolism 43: 591–598.[CrossRef][Medline]
Guha M and Mackman N (2002) The phosphatidylinositol 3-kinase-akt pathway limits lipopolysaccharide activation of signaling pathways and expression of inflammatory mediators in human monocytic cells. J Biol Chem 277: 32124–32132.
Hermann C, Assmus B, Urbich C, Zeiher AM, and Dimmeler S (2000) Insulin-mediated stimulation of protein kinase Akt: A potent survival signaling cascade for endothelial cells. Arterioscler Thromb Vasc Biol 20: 402–409.
Institute of Laboratory Animal Resources (1996) Guide for the Care and Use of Laboratory Animals 7th ed. Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council, Washington DC.
Jeschke MG, Klein D, Bolder U, and Einspanier R (2004) Insulin attenuates the systemic inflammatory response in endotoxemic rats. Endocrinology 145: 4084–4093.
Jeschke MG, Rensing H, Klein D, Schubert T, Mautes AE, Bolder U, and Croner RS (2005) Insulin prevents liver damage and preserves liver function in lipopolysaccharide-induced endotoxemic rats. J Hepatol 42: 870–879.[CrossRef][Medline]
Langouche L, Vanhorebeek I, Vlasselaers D, Vander PS, Wouters PJ, Skogstrand K, Hansen TK, and Van den BG (2005) Intensive insulin therapy protects the endothelium of critically ill patients. J Clin Invest 115: 2277–2286.[CrossRef][Medline]
Leffler M, Hrach T, Stuerzl M, Horch RE, Herndon DN, and Jeschke MG (2007) Insulin attenuates apoptosis and exerts anti-inflammatory effects in endotoxemic human macrophages. J Surg Res 143: 398–406.[CrossRef][Medline]
Liew FY, Xu D, Brint EK, and O'Neill LA (2005) Negative regulation of toll-like receptor-mediated immune responses. Nat Rev Immunol 5: 446–458.[CrossRef][Medline]
Maitra SR, Wojnar MM, and Lang CH (2000) Alterations in tissue glucose uptake during the hyperglycemic and hypoglycemic phases of sepsis. Shock 13: 379–385.[Medline]
Marik PE and Raghavan M (2004) Stress-hyperglycemia, insulin and immunomodulation in sepsis. Intensive Care Med 30: 748–756.[CrossRef][Medline]
Martin M, Rehani K, Jope RS, and Michalek SM (2005) Toll-like receptor-mediated cytokine production is differentially regulated by glycogen synthase kinase 3. Nat Immunol 6: 777–784.[CrossRef][Medline]
Michalek SM, Moore RN, McGhee JR, Rosenstreich DL, and Mergenhagen SE (1980) The primary role of lymphoreticular cells in the mediation of host responses to bacterial endotoxim. J Infect Dis 141: 55–63.[Medline]
Mitchell I, Knight E, Gissane J, Tamhane R, Kolli R, Leditschke IA, Bellomo R, and Finfer S (2006) A phase II randomised controlled trial of intensive insulin therapy in general intensive care patients. Crit Care Resusc 8: 289–293.[Medline]
Power C, Fanning N, and Redmond HP (2002) Cellular apoptosis and organ injury in sepsis: a review. Shock 18: 197–211.[CrossRef][Medline]
Russell JA (2006) Management of sepsis. N Engl J Med 19;355: 1699–1713.
Schabbauer G, Tencati M, Pedersen B, Pawlinski R, and Mackman N (2004) PI3K-Akt pathway suppresses coagulation and inflammation in endotoxemic mice. Arterioscler Thromb Vasc Biol 24: 1963–1969.
Shapiro NI, Howell MD, Talmor D, Lahey D, Ngo L, Buras J, Wolfe RE, Weiss JW, and Lisbon A (2006) Implementation and outcomes of the Multiple Urgent Sepsis Therapies (MUST) protocol. Crit Care Med 34: 1025–1032.[CrossRef][Medline]
Takehara N, Kawabe J, Aizawa Y, Hasebe N, and Kikuchi K (2000) High glucose attenuates insulin-induced mitogen-activated protein kinase phosphatase-1 (MKP-1) expression in vascular smooth muscle cells. Biochim Biophys Acta 1497: 244–252.[Medline]
Taniguchi CM, Emanuelli B, and Kahn CR (2006) Critical nodes in signalling pathways: insights into insulin action. Nat Rev Mol Cell Biol 7: 85–96.[CrossRef][Medline]
Van den Berghe G (2004) How does blood glucose control with insulin save lives in intensive care? J Clin Invest 114: 1187–1195.[CrossRef][Medline]
Van den Berghe G, Wilmer A, Hermans G, Meersseman W, Wouters PJ, Milants I, Van Wijngaerden E, Bobbaers H, and Bouillon R (2006) Intensive insulin therapy in the medical ICU. N Engl J Med 354: 449–461.
van den Berghe G, Wouters P, Weekers F, Verwaest C, Bruyninckx F, Schetz M, Vlasselaers D, Ferdinande P, Lauwers P, and Bouillon R (2001) Intensive insulin therapy in the critically ill patients. N Engl J Med 345: 1359–1367.
van Waardenburg DA, Jansen TC, Vos GD, and Buurman WA (2006) Hyperglycemia in children with meningococcal sepsis and septic shock: the relation between plasma levels of insulin and inflammatory mediators. J Clin Endocrinol Metab 91: 3916–3921.
Welham MJ, Bone H, Levings M, Learmonth L, Wang LM, Leslie KB, Pierce JH, and Schrader JW (1997) Insulin receptor substrate-2 is the major 170-kDa protein phosphorylated on tyrosine in response to cytokines in murine lymphohematopoietic cells. J Biol Chem 272: 1377–1381.
Williams DL, Li C, Ha T, Ozment-Skelton T, Kalbfleisch JH, Preiszner J, Brooks L, Breuel K, and Schweitzer JB (2004) Modulation of the phosphoinositide 3-kinase pathway alters innate resistance to polymicrobial sepsis. J Immunol 172: 449–456.
Zhang Y, Xu J, Levin J, Hegen M, Li G, Robertshaw H, Brennan F, Cummons T, Clarke D, Vansell N, et al. (2004) Identification and characterization of 4-[[4[(2-butynyloxy)phenyl]sulfonyl]-N-hydroxy-2,2-dimethyl-(3S) thiomorpholinecarboxamide] (TMI-1), a novel dual tumor necrosis factor-alpha-converting enzyme/matrix metalloprotease inhibitor for the treatment of rheumatoid arthritis. J Pharmacol Exp Ther 309: 348–355.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
