J Korean Med Sci.  2011 Jul;26(7):919-926. 10.3346/jkms.2011.26.7.919.

CD44 Disruption Attenuates Murine Hepatic Ischemia/Reperfusion Injury

• Kim MS
• Lee KH
• Lee WM
• Jun JH
• Kim DH

• KMID: 1094264
• DOI: http://doi.org/10.3346/jkms.2011.26.7.919

Abstract

Neutrophil adhesion and migration are critical in hepatic ischemia/reperfusion (I/R) injury. Despite very strong preclinical data, recent clinical trials failed to show a protective effect of anti-adhesion therapy in reperfusion injury. Therefore, the aim of this study was to assess the role of CD44 in neutrophil infiltration and liver injury from hepatic I/R. In this study, using a partial hepatic ischemic model in vivo, we determined the potential role of CD44 in neutrophil infiltration and liver injury from I/R. Reperfusion caused significant hepatocellular injury as it was determined by plasma ALT levels and liver histopathology. The injury was associated with a marked neutrophil recruitment and CD44 expression into the ischemic livers. Administration of anti-CD44 antibody to mice reduced the infiltration of neutrophil into the ischemic tissue, associated with liver function preservation. These results support crucial roles of CD44 in neutrophil recruitment and infiltration leading to liver damage in hepatic I/R injury. Moreover, they provide the rationale for targeting to CD44 as a potential therapeutic approach in liver I/R injury.

Keyword

Liver; Ischemia/Reperfusion; CD44; Inflammation; Neutrophils; Recruitment

MeSH Terms

Alanine Transaminase/blood
Animals
Antibodies/immunology/pharmacology
Antigens, CD44/immunology/metabolism/*physiology
Cytokines/metabolism
Disease Models, Animal
Liver/*metabolism/pathology
Male
Mice
Mice, Inbred C57BL
Neutrophils/immunology/physiology
Reperfusion Injury/metabolism/pathology/*prevention & control

Figure

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  Fig. 1 Time-course of plasma ALT levels following hepatic I/R. Mice were subjected to 60 min of ischemia followed by reperfusion with various lengths of time. "Sham" indicates mice that underwent surgical procedure without vascular occlusion followed by reperfusion, while "I/R" denote mice that underwent surgical procedure with vascular occlusion for 60 min followed by reperfusion for various lengths of time. Serum ALT levels were analyzed as a measure of hepatocellular injury. Values are expressed as mean ± SE, n = six mice per each time point/group.

• 

  Fig. 2 Time-course of plasma TNF-α, MCP-1, and IL-6 levels. "Sham" indicates mice that underwent surgical procedure without vascular occlusion followed by reperfusion, while "I/R" indicates mice that underwent surgical procedure with vascular occlusion for 60 min followed by reperfusion at the time point shown. Initial proinflammatory cytokines such as TNF-α and IL-6 levels paralleled ALT plasma levels. Values are expressed as mean ± SE. n = six mice per each time point/group.

• 

  Fig. 3 CD44 expression is up-regulated in the liver after I/R. (A) Western blot analysis for CD44 and beta actin was performed for hepatic protein lysates of the ischemic lobes at the time points shown, with each lane representing a separate animal. Blot shown is representative of three experiments with similar results. (B) The amount of protein was quantified using a densitometer. The values are shown the fold increase relative to the density of the sham animals after normalization to beta actin. Data represents means ± SE, n = 6 mice per group. *P < 0.05 versus mice that were subjected to sham animals and I/R animals.

• 

  Fig. 4 Hepatic histopathology following I/R. Mice were subjected to 60 min of ischemia followed by reperfusion at the time point shown. The ischemic liver sections were prepared and stained with H&E and then immunohistochemical staining of CD44 and neutrophils using specific anti-CD44 monoclonal antibody and anti-neutrophil antibody. (A), (B), and (C) represent the sham mice. There are normal hepatic histology (A). Nornal liver lobule shows mildly positive CD44 immunostaining (B) and negative neutrophil immunostaining (C). (D), (E), and (F) represent the mice subjected to 60 min of ischemia followed by reperfusion for 6 hr. The hepatic lobules show one focus of coagulation necrosis and adjacent ballooning degeneration (D). The liver shows moderately positive CD44 immunostaining (E) and focal a few positive neutrophil cells (F). (G), (H) and (I) represent the mice subjected to 60 min of ischemia followed by reperfusion for 24 hr. There are several large areas of coagulation necrosis and neutrophilic infiltration (G). The liver shows obiously patchy increased CD44 immunostaining (H) and neutrophil influx (I).

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  Fig. 5 Pretreatment of neutralizing antibody to CD44 protects against liver I/R injury. (A) and (B) represent plasma ALT levels from mice that underwent liver I/R injury and mice that were pretreated by neutralizing antibody at the time point shown. Antibody-treated mice received 16 hr before surgery a single intraperitoneal injection of 100 µg of anti-CD44 mAb or control IgG. Serum ALT levels were analyzed as a measure of hepatocellular injury. Values are expressed as mean ± SE, n = six mice per each time point/group. *P < 0.05 versus mice that were subjected to I/R and given anti-CD44 mAb. (C) and (D) represent H&E stained liver sections from mice that were pretreated by control antibody and anti-CD44 mAb before liver I/R injury (original magnification × 400). Images are representative liver sections from six mice per group.

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  Fig. 6 Immunohistochemical detection of CD44 and neutrophil influx in the liver. (A-D) represent immunohistochemical staining of CD44 in the liver sections from control antibody and anti-CD44 mAb-treated mice that were subjected to 60 minutes of ischemia followed by reperfusion at the shown time point. Immunostaining for CD44 reveals decreased CD44 staining in hepatic sinusoidal area of anti-CD44 mAb-treated mice compared with given control antibody after I/R. (E-H) represent immunohistochemical staining of neutrophil in the liver sections adjacent to CD44 immunohistochemical staining. Impaired influx neutrophils into anti-CD44 mAb-treated mice relative to given control antibody was paralleled CD44 immunostianing pattern. Images are representative liver sections from six mice per group.

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