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CD14 Contributes to Warm Hepatic Ischemia-Reperfusion Injury in Mice

Cai, Changchun; Shi, Xiaolian; Korff, Sebastian; Zhang, Jinxiang; Loughran, Patricia A.; Ruan, Xiangcai; Zhang, Yong; Liu, Li; Billiar, Timothy R.

doi: 10.1097/SHK.0b013e318299d1a7
Basic Science Aspects
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ABSTRACT: Introduction: Ischemia/reperfusion (I/R) of the liver contributes to the pathobiology of liver injury in transplantation, liver surgery, and hemorrhagic shock. Ischemia/reperfusion induces an inflammatory response that is driven, in part, by Toll-like receptor 4 (TLR) signaling. CD14 is known to participate in the function of TLR4. We hypothesized that CD14 would be involved in the pathobiology of warm hepatic I/R. Methods: Using a 70% liver inflow inclusion model, CD14 knockout and wild-type (WT) mice were subjected to 1-h warm ischemia followed by reperfusion. CD14 mRNA, circulating transaminase, interleukin 6, soluble CD14, and high-mobility group box 1 (HMGB1) levels were measured. CD14 neutralizing antibody or isotype control antibody was given before ischemia or reperfusion for CD14 blockade in WT mice. Recombinant HMGB1 was given before reperfusion in some experiments to test if liver injury worsens. Results: There was an upregulation of CD14 mRNA in reperfused livers together with increased soluble CD14 levels in the circulation. Compared with WT control mice, CD14 knockout mice had much lower alanine aminotransferase and interleukin 6 levels at 6 and 24 h following I/R, and much less liver necrosis by histology. TUNEL (terminal deoxynucleotidyl-transferase dUTP nick end labeling) staining displayed less apoptosis at 24 h in the absence of CD14. CD14 blockage by neutralizing antibody also attenuated liver injury and the inflammatory response in C57BL/6 mice following I/R, but did not provide additional protection to TLR4 mutant C3H/Hej mice. CD14 deficiency did not change circulating HMGB1 levels following I/R (6 h). A dose of recombinant HMGB1, which worsened hepatic injury when given before reperfusion in WT mice, did not increase liver damage in CD14-deficient mice. Conclusions: CD14 is actively involved in hepatic I/R injury. Its deficiency or blockade ischemia attenuates liver injury and inflammatory response. CD14 mediates liver damage and inflammatory responses in the setting of warm hepatic I/R in mice.

Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania

Address reprint requests to Timothy R Billiar, MD, George Vance Foster Professor of Surgery and Chair, Department of Surgery, F1281, Presbyterian University Hospital, PO Box 7533, University of Pittsburgh Medical Center, 200 Lothrop Street, Pittsburgh, PA, 15213. E-mail:

This work was supported by the National Institute of General Medical Sciences (grant P50-GM-53789).

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Hepatic ischemia/reperfusion injury (I/R) plays an important role following liver transplantation, liver surgery, and hemorrhagic shock (1). Evidence has accumulated that Toll-like receptor 4 (TLR4) plays a central role in liver injury and the associated inflammation in warm (2, 3, 4) and cold (5) liver I/R. Endogenous TLR4 activators such as high-mobility group box 1 (HMGB1) account for a significant part of TLR4 signaling in liver I/R (4). During lipopolysaccharide (LPS) signaling, CD14 plays an important role in recognition of LPS by the TLR4/MD2 complex (6). We have recently shown that CD14 is also required for the recognition of HMGB1 by macrophages (7). However, the contribution of CD14 to I/R-induced liver injury or inflammation induced by warm I/R is not known. Here, we show CD14 deficiency or neutralization of CD14 suppressed both injury and inflammation in warm liver I/R. Furthermore, CD14 is required for the recognition of exogenous HMGB1 in the setting of I/R. Thus, CD14 is part of the host response to I/R in the liver.

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Purified NA/LE rat anti–mouse CD14 antibodies were purchased from BD Biosciences (San Jose, Calif), and its isotype control antibody was bought from Biolegend (San Diego, Calif). Mouse CD14 and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) primers were from Qiagen Sciences (Germantown, MD). Mouse interleukin 6 enzyme-linked immune sorbent assay (ELISA) kit was from R&D System (Minneapolis, Minn). Mouse soluble CD14 (sCD14) ELISA kit was purchased from Cell Sciences (Canton, Mass). High-mobility group box 1 ELISA kit was ordered from IBL International (Toronto, Ontario, Canada). Recombinant mouse HMGB1 was kindly provided by Dr. Kevin Tracey. Anti–mouse CD14 and GADPH primary antibodies were purchased from R&D Systems and Abcam (Cambridge, Mass), respectively.

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CD14 knockout (KO) mice were provided by Dr. Mason Freeman and were bred and maintained in the animal facility of the University of Pittsburgh. Wild-type control CD57BL/6, C3H/Hej, and C3H/HeOuj mice were purchased from Jackson Laboratory (Bar Harbor, Me). All mice were male, 8 to 12 weeks old, and weighed 20 to 30 g at the time of the experimental procedures. Animal handling and care complied with the regulations regarding the care and use of experimental animals published by the National Institutes of Health and was approved by the Institutional Animal Use and Care Committee of the University of Pittsburgh. The animals were maintained in the animal facility with a 12-h light-dark cycle and free access to standard laboratory chow and water.

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Warm hepatic I/R injury

Mice 8 to 12 weeks old were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) by intraperitoneal injection. After a midline laparotomy, the hepatic lobes were freed by dividing the surrounding ligaments. The vessels supplying to the left side 70% of the liver were disrupted by a micro–atraumatic vascular clamp, whereas the blood supply to the remaining liver lobes still remained patent to avoid intestinal venous congestion. The incision was then covered with a piece of saline-soaked gauze, together with a piece of plastic film, to reduce dehydration, and mice were placed on a heating pad to maintain a rectal temperature of 37°C. The clamp was subsequently removed after 60 min to initiate hepatic reperfusion, and the abdominal incision was closed with running 4-0 Vicryl-plus (Ethicon, Somerville, NJ) sutures. Sham-treated mice underwent the same manipulation, except that the vascular clamp was not applied.

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Sample collection

At the conclusion of the experiment, animals were anesthetized with isoflurane, and blood was harvested by cardiac puncture. Serum was collected by centrifuging the blood at 5,000 revolutions/min for 10 min at 4°C. Serum was aliquoted and stored at −80°C until further analysis. The same left hepatic lobe was harvested and snap-frozen in liquid nitrogen and then stored at −80°C. The liver was also removed and fixed in 10% neutral formalin for hematoxylin-eosin staining. For immunofluoresence staining, liver was flushed with phosphate-buffered saline (PBS) and then perfused with 2% paraformaldehyde. After fixation for 2 h, the tissues were switched to 30% sucrose for 24 h with sucrose change a total of three times. The samples were cryoperserved in 2-methylbutane and stored at −80°C until ready for sectioning.

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Alanine aminotransferase measurement

Serum alanine aminotransferase (ALT) was measured with HESKA Dri-Chem 4000 (HESKA, Loveland, Colo; slides from Fujifilm, Tokyo, Japan).

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Reverse transcriptase–polymerase chain reaction

The harvested frozen liver lobes were pulverized, and an aliquot of 20 to 30 μg from each mouse was used to extract total RNA with RNeasy kit (Qiagen) following the standard protocol. A total of 1μg RNA was used for cDNA synthesis using iScript Reverse Transcription Supermix (Bio-Rad, Hercules, Calif) according to the manufacturer’s instructions. Predesigned CD14 and GAPDH primers from Qiagen were used. Polymerase chain reaction (PCR) was performed on a conventional thermocycler, and cycle conditions were as follows: 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 35 seconds. The PCR products were run on a 1.2% agarose gel to compare the relative intensity semiquantitatively with Adobe Photoshop CS5 (Adobe Systems, San Jose, Calif).

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Western blot

Western blot analysis was used to assess CD14 protein levels in whole liver. Fifty micrograms of liver homogenate was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane. The membrane was blocked for 1 h in PBS-Tween (0.1%) with 5% milk, followed by immunostaining with optimized dilutions of a polyclonal rabbit anti–mouse CD14 antibody (1:2,000) in 1% milk in PBS-Tween overnight at 4°C. Horseradish peroxidase–conjugated secondary antibodies were given, and membranes were developed with the Super Signal West Pico chemiluminescent kit (Thermo Fisher Scientific, Rockford, Ill) and exposed to film. GADPH was used as the loading controls for each group.

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ELISA for sCD14 and interleukin 6

Soluble CD14 and interleukin 6 (IL-6) levels in serum were detected with ELISA kits according to their standard protocols. To measure IL-6 level in liver tissue, liver homogenates were lysed with cell lysis buffer with protease inhibitors added. After centrifugation at full speed for 15 min at 4°C, supernatants were harvested, and protein levels were measured with BCA kit. Then, the IL-6 level of each ischemic liver was measured by ELISA kit and standardized by its protein concentration.

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Serum HMGB1 level measurement

Serum HMGB1 levels were measured with the HMGB1 ELISA kit developed by Shino-test Corporation (Tokyo, Japan) according to its standard protocol.

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TdT-mediated dUTP digoxigenin nick end labeling assay:

Livers were fixed in 2% paraformaldehyde followed by standardized protocol for cryopreservation. Apoptosis was measured with TUNEL (terminal deoxynucleotidyl-transferase dUTP nick end labeling) assay using a kit purchased from Promega (Madison, Wisc) following manufacturer’s protocol and counterstained with Hoechst nuclear stain. TUNEL-positive cells were quantitated using a Metamorph image acquisition and analysis system (Chester, Pa) incorporating a Nikon microscope (Nikon, Melville, NY). The numbers of positive stained cells per randomized high-power field were calculated and compared.

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Liver samples were fixed in 10% formalin and embedded in paraffin, then cut to 6-μm thick sections. Tissues were stained with hematoxylin-eosin, and slides were assessed for inflammation and tissue damage.

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Statistical analysis

Results are expressed as the mean ± SEM. We used SigmaPlot 11.0 (Systat Software, Inc, Point Richmond, Calif) for the statistical analysis. One-way analysis of variance was used for multiple groups, and comparisons of two groups under the same treatment were performed using the Student t test. Significance was established at the 95% confidence (P < 0.05).

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CD14 expression increases following warm hepatic I/R

We first assessed the level of CD14 expression in the liver and circulation following liver ischemia and reperfusion. Ischemia alone did not change CD14 mRNA expression from baseline (Fig. 1, A and B). However, reperfusion induced a marked upregulation of CD14 mRNA in the liver that was first detected at 1 h of reperfusion. CD14 protein levels in the ischemic lobe of the liver decreased with the start of ischemia but then increased again between 3 and 6 h following reperfusion (Fig. 1C). Control mice showed detectable but low sCD14 levels (Fig. 2). Ischemia alone did not increase serum sCD14 levels. However, serum sCD14 levels were significantly higher than those at baseline at 6 and 24 h following reperfusion (P < 0.01, comparison with baseline levels). These observations show that CD14 gene expression is increased by hepatic I/R and that liver I/R is associated with release of sCD14 into the circulation. It is noteworthy that several cell types in the liver, including hepatocytes, express CD14. Total CD14 levels reflect intracellular and cell surface expression of CD14.

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CD14 contributes to liver injury following I/R

We next carried out experiments to determine if CD14 contributed to I/R-induced liver injury. CD14 KO mice and their wild-type (WT) counterparts were subjected to 1 h of ischemia followed by 6 or 24 h of reperfusion. There was no difference in ALT levels between WT and CD14 KO sham groups. However, circulating ALT levels were significantly higher in WT mice subjected to I/R at both 6 and 24 h than in CD14 KO mice (Fig. 3). Histological analysis confirmed that CD14 KO mice were protected from the extensive central lobular necrosis observed at 6 and 24 h in the WT mice following I/R (Fig. 4). Ischemia/reperfusion was also associated with apoptosis assessed by TUNEL staining at 24 h. Here again, CD14 deficiency was associated with the near absence of apoptosis (Fig. 5, A and B).

To confirm the importance of CD14 to I/R-induced injury, we treated WT mice with a neutralizing anti-CD14 antibody either before the onset of ischemia or just before reperfusion. As shown in Figure 6, pretreatment with anti-CD14 antibody significantly attenuated the injury as measured by circulating ALT levels. Administration of antibody just before reperfusion had minimal protective effects (data not shown). Thus, CD14 participates in the pathobiology of I/R-induced liver injury and most likely is engaged early in the ischemia insult.

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CD14 contributes to the inflammatory response after hepatic I/R

Ischemia/reperfusion–induced liver injury is the result of both ischemic cell death and the inflammatory response that is induced by I/R. To determine if CD14 is involved in I/R-induced inflammation, we measured IL-6 in the liver and circulation as a marker of the inflammatory response. CD14 KO mice showed much lower elevations in IL-6 in the liver (Fig. 7A) and circulation (Fig. 7B) than seen in the WT mice subjected to I/R. Wild-type mice pretreated with anti-CD14 antibody also had lower circulating IL-6 levels following I/R (Fig. 7C). Thus, CD14 is involved in the regulation of I/R-induced inflammation.

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Neutralizing CD14 does not reduce I/R-induced injury in TLR4-deficient mice

We (4) and others (2, 3) have shown that TLR4 is involved in I/R-induced liver damage. Because CD14 is part of the TLR4 receptor complex, we sought evidence that CD14 might participate with TLR4 in liver I/R injury. Anti-CD14 antibody was administered to both WT and TLR4 mutant mice. As expected, TLR4 mutant mice were partially protected from I/R-induced damage (Fig. 8A). No further reduction in liver injury was seen by neutralizing CD14 in these mice (Fig. 8B).

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Recombinant HMGB1 given before reperfusion does not increase liver injury in CD14 KO mice

High-mobility group box 1 is a prototypical danger-associated molecular pattern (DAMP) previously shown to contribute to liver damage in I/R through TLR4. Furthermore, rHMGB1 exacerbates I/R-induced liver damage via a TLR4-dependent mechanism (4). We found no difference in circulating HMGB1 levels between WT and CD14 KO mice at 6 h, suggesting that CD14 is not required for early HMGB1 release (Fig. 8C). We administered 55 μg/mouse rHMGB1 (dose based on a pilot study, not shown), which did not result in any detectable liver injury by itself, to both WT and CD14 KO mice intraperitoneally. This dose of HMGB1 did not induce liver injury in control mice. Whereas rHMGB1 worsened liver damage when administered at reperfusion to WT mice, no increase in damage was seen in CD14 KO mice receiving rHMGB1(Fig. 8D). Taken together, these data suggest that CD14 is involved in sensing extracellular HMGB1 in liver I/R.

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This study was undertaken to determine if CD14 is involved in I/R-induced liver injury. This question is important because CD14 is known to participate in ligand recognition by the TLR4 receptor complex (6), and TLR4 is involved in the injury and inflammation induced by warm I/R in liver (4). Moreover, recently Luan and colleagues (8) reported that I/R could upregulate CD14 expression in Kupffer cells in rats. We show here that CD14 is involved in both the injury and the inflammation induced by liver I/R. Whereas the release of HMGB1 in liver I/R did not require CD14, the recognition of HMGB1 does. These findings suggest that DAMP recognition in liver I/R involves CD14.

CD14 is a 55-kd cell surface glycoprotein that together with TLR4 and MD-2 acts as a receptor for endotoxin (6). CD14 exists in two forms: one is anchored to the cellular membrane by a glycosylphosphatidylinositol tail, i.e., membrane CD14 (mCD14), and the other a soluble form (sCD14). Membrane CD14 is mainly expressed on macrophages, neutrophils, and dendritic cells, but epithelial cells (9), endothelial cells (10), and fibroblasts (11) also express low levels of mCD14. Although mononuclear phagocytes are known to shed their membrane-expressed CD14 (12) and liver can secrete CD14 (13), the origin of sCD14 in plasma is not yet totally clear. Soluble CD14 may confer LPS responsiveness to cells that do not express CD14 (14). Blood and other body fluids contain sCD14, and levels increase during inflammation and infection. Therefore, sCD14 can be regarded as an acute phase protein (15). Here we show that CD14 mRNA levels in the liver increase after the hepatic I/R and that this corresponds to an increase in circulating sCD14 levels. It is unclear if the source of this sCD14 is from the liver. These results do indicate that I/R is a potent stimulus for CD14 upregulation and release.

CD14 KO mice resist 10 times the lethal dose 100% of LPS (16). CD14 neutralizing antibody prevented lethality after in vivo exposure to endotoxin in rabbits and primates (17, 18). Neutralizing antibody to mouse CD14 even protects mice sensitized with galactosamine against LPS challenge (19). Our group found that the essential components for LPS uptake by hepatocytes were CD14, TLR4, MD2, and the β2-integrin CD11b/CD18 of hepatocytes (20). Although LPS is considered as its main ligand, previous studies have shown that CD14 can also recognize other pathogen-associated molecular patterns, including peptidoglycan, lipoteichoic acid, and lipoarabinomannan (21). Recent findings show that CD14 can promote TLR4 endocytosis and interferon expression, and CD14-induced endocytosis occurs independent of TLR4 signaling (22). Moreover, CD14 also enhances TLR2 (23) or TLR3-dependent cellular activation (24). In this study, we have shown that CD14 is actively involved in this sterile model of hepatic I/R injury, and CD14 deficiency or blockade before ischemia attenuates liver injury and inflammatory response in hepatic I/R. Attempts to reduce liver damage through delayed anti-CD14 antibody administration were less successful, suggesting that CD14 is involved in the early events in hepatic I/R.

The proinflammatory properties of HMGB1 were first identified in sepsis models where HMGB1 was shown to be a late mediator of lethality (25). In contrast to sepsis, HMGB1 acts as an early mediator in models of sterile inflammation. For example, we have previously shown that HMGB1 contributes to TLR4-dependent signaling in liver I/R and that rHMGB1 exacerbates I/R-induced injury in the liver in a TLR4-dependent manner (4). Here, we show that CD14 is also required for the exacerbation in I/R injury induced by rHMGB1. We have also reported that CD14 is involved in the injury and inflammation in cold cardiac I/R, a model that would involve no pathogen-associated molecular pattern exposure (26). Therefore, CD14 is likely to be involved in the detection of DAMPs, such as HMGB1 released in I/R injury.

Whether I/R responses depend on sCD14 or mCD14 or both deserves further exploration. Because HMGB1 has been shown to bind to LPS and facilitate transfer of LPS to CD14 and enhance human monocyte activation (27), it is possible that HMGB1 can also bind other DAMPs. One potential candidate is heat shock protein (HSP) 70. Similar to HMGB1, it can be released actively from stressed cells (28) or passively from necrotic cells (29). Asea et al. (30) reported CD14 was a coreceptor for HSP70-mediated signaling in human monocytes. Exogenous HSP70 bound with high affinity to the plasma membrane of monocytes caused monocyte activation in both CD14-dependent and CD14-independent pathways.

Taken together, our findings provide new insights into a novel role for CD14 in I/R. A better understanding of its mechanisms may ultimately lead to the development of new methods to prevent warm hepatic I/R injury.

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The authors thank Qingde Wang, Pei Zhou and Rick Shapiro for technical support in this study.

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I/R: ischemia/reperfusion

HMGB1: high-mobility group box 1

ALT: alanine aminotransferase

IL-6: interleukin 6

TLR: Toll-like receptor

sCD14: soluble CD14

ELISA: enzyme-linked immune sorbent assay

WT: wild type

mCD14: membrane CD14

DAMP: danger-associated molecular pattern

HSP: heat shock protein

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CD14; sCD14; high-mobility group box 1; hepatic ischemia/reperfusion injury; Toll-like receptor 4

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