Volume 10 Issue 6 - September 11, 2009 PDF
Lectin-like domain of thrombomodulin binds to its specific ligand Lewis Y antigen and neutralizes lipopolysaccharide-induced inflammatory response
Chung-Sheng Shi1,2, Guey-Yueh Shi1,2,*, Shi-Ming Hsiao1,2, Yuan-Chung Kao1,2, Kuan-Lin Kuo1,2, Chih-Yuan Ma1,2, Cheng-Hsiang Kuo1,2, Bi-Ing Chang1,2, Chuan-Fa Chang3,4, Chun-Hung Lin3,4, Chi-Huey Wong3,4, and Hua-Lin Wu1,2,*

1Department of Biochemistry and Molecular Biology, College of Medicine, and 2Cardiovascular Research Center, National Cheng Kung University, Taiwan
3Genomics Research Center and 4Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, Republic of China

Blood. 2008 November 1; 112(9): 3661–3670.

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Thrombomodulin (TM), a widely expressing glycoprotein originally identified in vascular endothelium, is an important cofactor in anticoagulant system. We presently have demonstrated that recombinant N-terminal lectinlike domain of TM (rTMD1) functions as a protective agent against sepsis, a leading cause of death in hospitalized patients, caused by Gram-negative bacterial infections. rTMD1 inhibited lipopolysaccharide (LPS), a component of Gram-negative bacterial outer membrane, -induced inflammatory mediator production via interference with CD14 and LPS binding. rTMD1 bound to the Klebsiella pneumoniae (K. pneumoniae) and LPS by specifically interacting with Lewis Y antigen. Moreover, administration of rTMD1 protected the host by suppressing inflammatory responses induced by LPS and Gram-negative bacteria, and enhanced LPS and bacterial clearance in sepsis. Thus, rTMD1 can be used to defend against bacterial infection and inhibit LPS-induced inflammatory responses, suggesting that rTMD1 may be valuable in the treatment of severe inflammation in sepsis, especially in Gram-negative bacterial infections.

Fig. 1. Effects of rTMD proteins on LPS-induced inflammatory mediator productions via interference with CD14 and LPS binding. (A, B) rTMD1 protein was pre-incubated with LPS before adding to RAW 264.7 cells. After 6 hours incubation, culture media were collected for the measurement of (A) TNF-αand (B) NO. (C) rTMD1 blockage of the binding of CD14 to LPS. LPS was coated onto wells and incubated with indicated concentrations of rTMD1 and CD14. The binding of CD14 to LPS was detected using CD14 antibody.
To test whether TMD1 has anti-inflammatory property, we prepared rTMD proteins using both Pichia pastoris and mammalian protein expression systems. We first tested the anti-inflammatory effect of rTMD proteins in murine macrophages RAW 264.7 cells stimulated with LPS. rTMD1 dose-dependently inhibited tumor necrosis factor-alpha (TNF-α) production and nitric oxide (NO) secretion in RAW 264.7 cells stimulated with LPS (Fig. 1, A and B). It was conceivable that the binding of rTMD1 to LPS might block the interaction of LPS with LPS-binding molecule such as CD14. As shown in Fig. 1C, the binding of CD14 to LPS was inhibited by rTMD1 in a dose-dependent manner

To test whether TMD1 is capable of specific binding to Gram-negative bacteria and LPS, rTMD1 and recombinant TM domains 2 and 3 (rTMD23) were used for binding with K. pneumoniae, LPS, or BSA. rTMD1 but not rTMD23 could specifically bind to K. pneumoniae (Fig. 2A). Because rTMD1 could bind to Gram-negative bacteria, LPS of Gram-negative bacteria was assumed a potential candidate ligand of rTMD1. Appropriately, binding of rTMD1 and rTMD23 to LPS or BSA was measured. rTMD1 but not rTMD23 specifically bound to LPS (Fig. 2B). To identify the ligand specificity of TMD1, a panel of carbohydrate ligands (Fig. 2C) was tested for rTMD1 affinity using the AlphaScreen method. The result showed that Lewis Y (Ley) antigen was a specific ligand for rTMD1 (Fig. 2C).
Fig. 2. rTMD1 bound to the K. pneumoniae and LPS by specifically interacting with Lewis Y antigen. (A) K. pneumoniae or BSA. (B) E. coli O111:B4 LPS or BSA. (A and B) K. pneumoniae, LPS, or BSA was coated onto wells. Equimolar amounts of rTMD proteins were added to each well. The binding of rTMD proteins was detected. (C) AlphaScreen assay results. Values are the mean ±SD (n=4). Sugar binding specificity of rTMD1 is indicated by relative intensities (with reference to the highest absorbance unit). The sugar identities are designated by numbers as listed.

Fig. 3. rTMD1 reduces LPS- and K. pneumoniae -induced inflammatory response and lethality. (A and B) rTMD1 was i.v.-administered before i.p. injection of LPS (20 mg/kg). Sera were collected 6 hours after administration of LPS for assay of (A) TNF-α and (B) NO production. (C) Mice received LPS (40 mg/kg) and rTMD1 (four i.v. doses of 2 mg/kg at 0, 6, 12, and 24 hours after LPS injection). Survival was determined. For each experimental group, n=20. (D and E) rTMD1 was administered before injection of K. pneumoniae (5×102 CFU/mouse) to mice. Sera were collected 12 hours after administration of K. pneumoniae for assay of (D) TNF-α and (E) NO production. Values are the mean ± SD (n=10). (F) rTMD1 (10 mg/kg) was administered before injection of K. pneumoniae (5×103 CFU/mouse) to mice. Survival was determined. For each experimental group, n=20.
Because rTMD1 could inhibit LPS-induced inflammatory mediator productions, we proposed that rTMD1 might function as a therapeutic agent to reduce the inflammatory response and lethality induced by LPS in vivo. TNF-α and NO levels were increased in mice 6 hours after i.p. administration of 20 mg/kg LPS, relative to control mice. Mice receiving an i.v. injection of rTMD1 (1–5 mg/kg, [43.66– 218.3 nmole/kg] had significantly decreasing levels of TNF-α and NO (Fig. 3, A and B). To study whether this effect protects against lethality, we treated mice with four i.v.-administered doses of rTMD1 (2 mg/kg) or PBS and observed the mice until all mice in either experimental group died. rTMD1 treatment possessed significant protection against lethality and improved survival during endotoxemia (rTMD1-treated group survival, 100%; PBS-treated group survival, 10%, 1 day after LPS challenge; and rTMD1-treated group survival, 60%; PBS-treated group survival, 0%, 4 days after LPS challenge) (Fig. 3C), suggesting that rTMD1 may have therapeutic potential. To test whether rTMD1 protects against lethality induced by Gram-negative bacteria, we induced systemic sepsis in mice by injecting them i.p. with K. pneumoniae and treating with rTMD1 (Fig. 3 D-F). TNF-α and NO levels were increased within 12 hours in mice receiving K. pneumoniae (5×102 CFU). rTMD1 treatment (525 mg/kg; i.v.) effectively reduced the TNF-α and NO production (Fig. 3, D and E). For the survival experiment, all mice received K. pneumoniae (5×103 CFU) died within 18 hours, whereas 50% of the mice received a single i.v. dose of rTMD1 (10 mg/kg) survived more than 24 hours (Fig. 3F).

The injected LPS (20 mg/kg) reached a maximum level 2 hours after i.p.-administration and was cleared from the circulation with an approximate 6- to 8-hour half-life (Fig. 4A). rTMD1 administration significantly enhanced LPS clearance (Fig. 4A). To explore whether the effect of K. pneumoniae-induced mortality results from rTMD1 promoted bacterial clearance, the amount of viable bacteria in the blood was determined. FVB mice were each i.p. injected with K. pneumoniae (5×102 CFU) without or with rTMD1 treatment (10 mg/kg; i.v.). rTMD1 significantly enhanced K. pneumoniae clearance in circulation at 12, 24, and 36 hours after injection (Fig. 4B).
Fig. 4. rTMD1 enhances LPS and bacterial clearance in vivo. (A) Clearance of LPS in circulation without or with rTMD1 treatment. LPS (20 mg/kg) was i.p.-administrated to male FVB mice without or with rTMD1 (10 mg/kg; i.v.), and serum samples were collected at various time intervals and the amount of LPS was determined by the Limulus amebocyte lysate test. Values are the mean ±SD. For each time interval group, n=5. (B) rTMD1 enhanced K. pneumoniae clearance in circulation. FVB mice were i.p.-injected with K. pneumoniae (5×102 CFU/mouse) without or with rTMD1 (10 mg/kg; i.v.). The blood samples from each group were collected at various time intervals and assayed for viable bacterial CFU counts. Values are the mean ±SD. For each time interval group, n=5

In conclusion, the specific interaction of rTMD1 with Ley highlights a novel mechanism in modulating LPS-mediated inflammatory responses. The therapeutic potential of rTMD1 in treating acute Gram-negative septicemia should be noticed. The identification of Ley as the specific ligand of rTMD1 also paves the way for investigating the roles of TMD1- Ley couple in cell-cell interaction and their biological functions in vivo.
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