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. Author manuscript; available in PMC: 2011 Apr 1.
Published in final edited form as: Hepatology. 2010 Apr;51(4):1363–1372. doi: 10.1002/hep.23442

The Emerging Role of T Cell Ig Mucin-1 (TIM-1) in The Mechanism of Liver Ischemia and Reperfusion Injury*

Yoichiro Uchida 1, Bibo Ke 1, Maria Cecilia S Freitas 1, Haofeng Ji 1, Danyun Zhao 1, Elizabeth R Benjamin 1, Nader Najafian 2, Hideo Yagita 3, Hisaya Akiba 3, Ronald W Busuttil 1, Jerzy W Kupiec-Weglinski 1,#
PMCID: PMC3066468  NIHMSID: NIHMS217846  PMID: 20091883

Abstract

The TIM (T cell Immunoglobulin and Mucin domain-containing molecules) protein family, expressed by T cells, plays a crucial role in regulating host adaptive immunity and tolerance. However, its role in local inflammation, such as innate immunity-dominated organ ischemia and reperfusion injury (IRI) remains unknown. Liver IRI occurs frequently after major hepatic resection or liver transplantation. Using an antagonistic anti-TIM-1 Ab, we studied the role of TIM-1 signaling in the model of partial warm liver ischemia followed by reperfusion. Anti-TIM-1 Ab monotherapy ameliorated the hepatocellular damage and improved liver function due to IR, as compared to controls. Histological examination has revealed that anti-TIM-1 Ab treatment decreased local neutrophil infiltration, inhibited sequestration of T lymphocytes, macrophages, TIM-1 ligand expressing TIM-4+ cells, and reduced liver cell apoptosis. Intrahepatic neutrophil activity and induction of pro-inflammatory cytokines/chemokines were also reduced in the treatment group. In parallel in-vitro studies, anti-TIM-1 Ab suppressed IFN-γ production in Con A-stimulated spleen T cells, and diminished TNF-α/IL-6 expression in macrophage/spleen T cell co-culture system. This is the first study, which provides evidence for the novel role of TIM-1 signaling in the mechanism of liver IRI. TIM-1 regulates not only T cell activation but may also affect macrophage function in the local inflammation response. These results provide compelling data for further investigation of TIM-1 pathway in the mechanism of IRI, to improve liver function, expand the organ donor pool, and improve the overall success of liver transplantation.

Introduction

The T-cell immunoglobulin mucin (TIM) family of cell surface proteins has attracted much attention as potential regulators of the immune system. The TIM family is located on chromosome 11B1.1 in mice and consists of four identified members (TIM-1, -2, -3, and -4) and four punitive members (TIM-5, -6, -7, and -8). In humans, the TIM family is located on chromosome 5q33.2 and consists of three members, TIM-1, TIM-3, and TIM-4. All are predicted to be type I membrane proteins that share a characteristic immunoglobulin V (Ig V), mucin, transmembrane and cytoplasmic domain structure (1).

TIM-1, also known as kidney injury molecule (KIM-1), was first identified as a marker of acute kidney injury. TIM-1 mRNA and protein dramatically increase after ischemic kidney injury while there are low levels in normal kidneys (2, 3). In addition, TIM-1 is expressed on CD4+ T cells after activation and its expression was sustained preferentially in Th2 but not Th1 cells (4). However, TIM-1 is expressed on both Th1 and Th2 cells and TIM-1 blockade prolongs survival of fully MHC-mismatched cardiac transplants (5). TIM-4, expressed by macrophages, is the ligand for TIM-1, and TIM-1 - TIM-4 interactions regulate Th cell responses and modulate Th1/Th2 cytokine balance (6). Moreover, TIM-1 can regulate macrophage activation and alter the co-stimulatory properties of macrophages (7).

Ischemia and reperfusion injury (IRI), an exogenous antigen-independent inflammatory event, remains an important problem in clinical practice, including transplantation. In fact, severe damage related to organ retrieval, preservation, and reperfusion often leads to primary graft nonfunction and may adversely affect the development of acute and chronic rejection (8). Others and we have shown that T lymphocytes, especially CD4+ T cells, are key mediators in IR-triggered liver inflammation (9–11). On the other hand, Kupffer cells, the resident macrophages of the liver, by releasing pro-inflammatory mediators, such as TNF-α and IL-6 are critical in the pathophysiology of IRI (12,13). In addition, interactions between CD4+ lymphocytes and Kupffer cells constitute a key event in the cascade leading to liver IRI and CD4+ T-cells may amplify Kupffer cell activity (14). However, the mechanisms underlying cross talk between T cells and Kupffer cells/macrophages have not been fully elucidated well.

The role of TIM family in local inflammation, such as innate immunity-dominated IRI remains unknown. Here, we provide evidence that TIM-1 signaling is critical for T cell – macrophage cross talk during the course of liver IRI, and its targeting may provide new means to ameliorate tissue innate and adaptive immune responses in the clinics.

Material and Methods

Animals

Male C57BL/6 mice (8–10 weeks old) were used (The Jackson Laboratory, Bar Harbor, ME). Mice were housed in the UCLA animal facility under specific pathogen-free conditions. All animals received human care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of sciences and published by the National Institute of Health (NIH publication 86-23, revised 1985).

Liver IRI model

We used a mouse model of partial warm hepatic IRI (11,15,16). Briefly, mice were anesthetized, injected with heparin (100 U/kg), and an atraumatic clip was used to interrupt the artery/portal venous blood supply to the left/middle liver lobes. After 90 min of ischemia, the clamp was removed, initiating reperfusion. To investigate the role of TIM-1, we used anti-mouse TIM-1 mAb (RMT1-10; rat IgG2a,κ). Based on published data (5), mice were given a single injection of mAb (0.5 mg i.p.) one day prior to the ischemia insult, and then sacrificed at 6 or 24 h after reperfusion. Controls were treated with irrelevant rat IgG Ab or PBS. Sham-operated mice underwent the same procedure, but without vascular occlusion.

Hepatocellular function

Serum alanine aminotransferase (sALT) levels were measured with an autoanalyzer (ANTECH Diagnostics, Los Angeles, CA).

Histology

Liver paraffin sections (5-µm thick) were stained with hematoxylin and eosin. The severity of liver IRI (necrosis, sinusoidal congestion, and centrilobular ballooning) was blindly graded with modified Suzuki’s criteria on a scale from 0–4 (17).

Immunohistochemistry

Primary mAb against mouse Ly-6G (1A8; BD Biosciences, San Jose, CA), CD3 (17A2; BD Biosciences), CD68 (FA-11; AbD Serotec, Raleigh, NC), and human TIM-4 (rabbit polyclonal; LS-B1413; Lifespan Biosciences, Seattle, WA) were used on liver cryostat or paraffin sections. The secondary, biotinylated goat anti-rat IgG (Vector, Burlingame, CA), was incubated with immunoperoxidase (ABC Kit, Vector). Positive cells were counted blindly in 10 HPF/section (×400).

Myeloperoxidase activity assay

The presence of myeloperoxidase (MPO) was used as an index of neutrophil accumulation in the liver (18). The change in absorbance was measured spectrophotometrically at 655 nm (Bio-tek Instruments). One unit of MPO activity was defined as the quantity of enzyme degrading 1 µmol peroxide per minute at 25°C per gram of tissue.

Quantitative RT-PCR

Quantitative PCR was performed using the DNA Engine with Chromo 4Detector (MJ Research, Waltham, MA). In a final reaction volume of 20 µL, the following were added: 1 X SuperMix (Platinum SYBR Green qPCR Kit, Invitrogen, Carlsbad, CA), complementary DNA, and 10 µM of each primer. Amplification conditions were: 50°C (2 min), 95°C (5 min), followed by 45 cycles of 95°C (15 sec), 60°C (30 sec). Primers used to amplify specific gene fragments are listed in Supplementary Table 1. Target gene expressions were calculated by their ratios to the housekeeping gene HPRT.

Western blots

Western blots were performed using liver proteins (30 µg/sample), and polyclonal rabbit anti-mouse cleaved caspase-3 (Cell Signaling Technology, Danvers, MA), Bcl-2, Bcl-xl, NF-κB and β-actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), as described (10, 11). Relative quantities of protein were determined by densitometer and expressed in absorbance units (AU).

Caspase-3 activity

Caspase-3 activity was determined by an assay kit (Calbiochem, Gibbstown, NJ), and measuring the absorbance at a wavelength of 405 nm (Bio-tek Instruments). To determine cellular activity, the inhibitor-treated protein extracts and the purified caspase-3 (as a standard) were used.

Apoptosis assay

Apoptosis in liver sections was detected by the TUNEL method using the In Situ Cell Death Detection Kit (Roche, Indianapolis, IN). Negative control was prepared by omission of terminal transferase. Positive controls were generated by treatment with DNase. TUNEL-positive cells were counted in 10 HPF/section under light microscopy (×400).

Cell isolation/in vitro splenocytes cultures

Single-cell suspensions were treated with Red Cell Lysing Buffer (Sigma-Aldrich, St. Louis, MO) and resuspended in RPMI 1640 medium (Invitrogen, San Diego, CA). Splenocytes, adjusted to 1 × 106 cells/ml, were plated (150 µl) and incubated for 12 h at 37°C. For T cell stimulation, cells were incubated for 48 h with 50 µl of ConA (Sigma-Aldrich, St. Louis, MO; final concentration 5 µg/ml) with or without anti-mouse TIM-1 Ab (final concentration 10 µg/ml) (19).

IFN-γ assay

IFN-γ concentration in cell supernatants was evaluated using IFN-γ ELISA kit (eBioscience, San Diego, CA). Standard curves starting at 1000 pg/ml were performed with serial 2-fold dilutions, and the OD measured with an ELISA reader (BioTek Instruments).

Macrophage/splenocyte co-culture

Mouse macrophages (RAW 264.7; American Type Culture Collection, ATCC, Manassas, VA) were cultured in Dulbucco’s Modified Eagle’s medium (Invitrogen, Carlsbad, CA). Macrophages were co-cultured with C57BL/6 splenocytes at responder : stimulator (1:5) ratios (20). The co-cultured cells were incubated for 48 h with 50 µl of ConA (5 µg/ml) with or without anti-mouse TIM-1 mAb (10 µg/ml). Cell-free supernatants were evaluated for TNF-α and IL-6 by ELISA (eBioscience, San Diego, CA). Standard curves starting at 1000 pg/ml were performed with serial 2-fold dilutions.

Statistical analysis

All data are expressed as means ± SD. Differences between experimental groups were analyzed using one-way analysis of variance or Student’s t test for unpaired data. All differences were considered statistically significant at the P value of <0.05.

Results

TIM-1 signaling is required for IR-triggered hepatocellular damage

We analyzed liver function in the model of partial liver warm ischemia (90 min) followed by reperfusion. Treatment with anti-TIM-1 Ab reduced otherwise fulminant IR-induced hepatocellular damage. The serum ALT levels (IU/L) were significantly suppressed at 6 h and 24 h after reperfusion in the treatment group, as compared with controls (Figure 1a: [6 h] IgG/PBS: 27100 ± 1414 / 26638 ± 1874 vs 10474 ± 3146 and [24 h] IgG/PBS: 6775 ± 742 / 6212 ± 1323 vs 2873 ± 1595; p<0.01).

Figure 1.

Figure 1

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Anti-TIM-1 Ab treatment ameliorates liver IRI. (a) The hepatocellular function, as evidenced by sALT levels after liver warm ischemia (90 min) followed by reperfusion (6 and 24 h), was significantly improved in anti-TIM-1 Ab treatment group, as compared with controls (*p<0.01; n=6-8/group). Means±SD are shown. (b) Representative liver histology (H&E staining; magnification ×100 and ×400 inserts) of ischemic liver lobes after reperfusion (Upper panel: 6 h; Suzuki’s score = 11.5 ± 0.55 vs 8.83 ± 1.47; p<0.01, Lower panel: 24 h; 8.67 ± 0.82 vs 6.67 ± 1.21; p<0.01, n=6-8/group). Arrows depict apoptotic cells.

These data correlated with Suzuki’s histological criteria of the hepatocellular damage. Indeed, control livers showed severe lobular edema, congestion, ballooning and hepatocellular necrosis (Figure 1b: score = [6 h] 11.5 ± 0.55 and [24 h] 8.67 ± 0.82). In contrast, livers in anti-TIM-1 conditioned mice showed largely well-preserved architecture and histological detail after reperfusion (score = [6 h] 8.83 ± 1.47; p<0.01 and [24 h] 6.67 ± 1.21; p<0.01). Fig. 1b inserts depict and identify apoptotic cells in the hepatic necrotic areas.

TIM-1 signaling is needed for IR-mediated neutrophil, T cell and macrophage liver sequestration

We performed MPO assay, an index of liver neutrophil infiltration (Figure 2a). The MPO activity (U/g) was significantly suppressed in the treatment group, as compared with controls ([6 h] 5.27 ± 0.57 vs 1.32 ± 0.24 and [24 h] 4.79 ± 0.42 vs 1.13 ± 0.17; p<0.05). These results were correlated with the number of neutrophils in the livers, and assessed by the immunohistochemistry (Figure 2b). Indeed, neutrophil accumulation in the treated livers was significantly decreased, as compared with controls ([6 h] 13.80 ± 5.30 vs 31.47 ± 6.09 vs; p<0.01 and [24 h] 9.90 ± 3.30 vs 19.33 ± 5.29; p<0.05).

Figure 2.

Figure 2

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Neutrophil accumulation in ischemic lobes harvested at 6 h and 24 h of reperfusion after 90 min of warm ischemia +/− anti-TIM-1 Ab treatment. (a) MPO activity (**p<0.05; n=2-3/group). (b) Left panel: Representative liver sections stained by Ly-6G (×400 magnification). Right panel: Quantification of neutrophil infiltration by immunohistology. Infiltration of polymorphonuclear cells (dark spots) in treated livers was decreased as compared with controls (*p < 0.01, **p < 0.05; n = 2-3/group). Means±SD are shown.

In parallel, we performed immunohistochemical staining for CD3, CD 68 and TIM-4 expression. Although relatively few CD3 positive T cells could be found in control groups, their numbers decreased further after treatment with anti-TIM-1 Ab (Figure 3a: [6 h] 3.6 ± 0.42 vs 1.25 ± 0.21; p<0.05 and [24 h] 1.45 ± 0.35 vs 0.65 ± 0.21; p=n.s.). Moreover, disruption of TIM-1 signaling diminished CD68 macrophage infiltration, as compared with controls (Figure 3b: [6 h] 8.8 ± 1.83 vs 13.7 ± 1.41; p<0.05 and [24 h] 5.5 ± 3.96 vs 9.55 ± 1.34 vs; p=n.s.). Treatment with anti-Tim-1 Ab decreased the number of TIM-4 positive cells (Figure 3c: [6 h] 3.37 ± 0.67 vs 1.23 ± 0.61 p<0.01).

Figure 3.

Figure 3

Figure 3

Figure 3

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Accumulation of: (a) T cells, (b) macrophages, and (c) TIM-4 positive cells in ischemic liver lobes at 6–24 h of reperfusion after 90 min of ischemia. (×400 magnification; (*p < 0.01, **p < 0.05; representative of n=2-3/group).

TIM-1 signaling facilitates IR-mediated cytokine/chemokine programs

We used qRT-PCR to analyze liver expression of cytokines (TNF-α, IL-6, IL-1β and IFN-γ) and chemokines (CXCL-1 [KC: a mouse homolog of human chemokine gro-α] and CXCL-2 [macrophage inflammatory protein-2: MIP-2]) during IRI, and calculated the ratio between post-IR and basal mRNA levels in each animal (Figure 4). Control mice showed significantly increased induction ratios of TNF-α (TNF-α/HPRT) mRNA: [6 h] 2.52±0.49 vs 1.06±0.22, p<0.01; [24 h] 0.34±0.06 vs 0.21±0.03, p<0.05), IL-6 (IL-6/HPRT mRNA: [6 h] 3.19±0.88 vs 0.50±0.30, p<0.05; [24 h] 1.02±0.38 vs 0.10±0.06, p<0.05), IL-1β (IL-β/HPRT mRNA: [6 h] 2.07±0.21 vs 0.60±0.21, p<0.01; [24 h] 0.40±0.19 vs 0.10±0.05) and IFN-γ (IFN-γ/HPRT mRNA: [6 h] 0.23±0.08 vs 0.09±0.03, p<0.05; [24 h] 0.07±0.08 vs 0.01±0.01) as compared with the treated group (Figure 4). CXC chemokines are known to act predominantly on neutrophils (21). In mice, the two CXC chemokines important in the mechanism of liver IRI (22) are CXCL-1 (KC) and CXCL-2 (MIP-2). The expression of CXCL-1 and -2 was increased in unmodified vs anti-TIM-1 mAb treated group (CXCL-1/HPRT mRNA: [6 h] 3.71±0.38 vs 1.23±0.23, p<0.01; [24 h] 1.02±0.34 vs 0.33±0.14, p<0.05, CXCL-2/HPRT mRNA: [6 h] 7.66±1.34 vs 1.13±0.88, p<0.01; [24 h] 1.16±0.46 vs 0.32±0.27, p<0.05)

Figure 4.

Figure 4

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Quantitative RT-PCR-assisted detection of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β, IFN-γ)and chemokines (CXCL-1 and CXCL-2) at 6 h and 24 h of reperfusion after 90 min of warm ischemia with or without anti-TIM-1 Ab treatment. Data were normalized to HPRT gene expression (*p < 0.01, **p < 0.05; n =3-5/group). Means±SD are shown.

TIM-1 signaling promotes liver cell apoptosis during IRI

Western blot analysis (Figure 5a) has revealed that treatment with anti-Tim-1 Ab inhibited the expression of cleaved caspase-3 and NF-kB, as compared with controls (cleaved caspase-3: sham [0.05–0.1 AU], control 6h [1.8–2.0 AU], anti-TIM-1 treated 6h [0.1–0.2 AU], control 24h [2.0–2.2 AU], anti-TIM-1 Ab treated, 24h [0.1–0.2 AU], NF-kB: sham [0.05–0.1 AU], control 6h [2.1–2.2 AU], anti-TIM-1 Ab treated, 6h [0.5–0.7 AU], control 24h [2.0–2.1 AU], anti-TIM-1 Ab treated, 24h [0.5–0.6 AU]). Treatment with anti-TIM-1 Ab simultaneously increased the expression of anti-apoptotic Bcl-2 and Bcl-xl (Bcl-2: sham [0.1–0.2 AU], control 6h [0.7–0.8 AU], anti-Tim-1 6h [1.9–2.1 AU], control 24h [0.6–0.8 AU], anti-TIM-1 24h [1.8–2.0 AU], Bcl-xL: sham [0.05–0.1 AU], control 6h [0.3–0.4 AU], anti-TIM1 6h [1.1–1.2 AU], control 24h [0.4–0.5 AU], anti-TIM-1 Ab 24h [1.6–1.8 AU]). Moreover, the enzymatic activity of caspase-3 was reduced after reperfusion in anti-TIM-1 Ab group, as compared with controls (Figure 5b: [6 h] 3.47±0.19 vs 1.20±0.24, p<0.01; [24 h] 3.03±0.29 vs 0.66±0.17, p<0.05). The disruption of TIM-1 signaling decreased the number of TUNEL positive cells (Figure 5c: [6 h] 25.47 ± 7.03 vs 11.43 ± 7.55; p<0.05 and [24 h] 19.60 ± 6.69 vs 7.30 ± 5.10; p<0.05).

Figure 5.

Figure 5

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Anti-TIM-1 Ab treatment suppresses apoptosis. (a) Western blot analysis of cleaved caspase-3, NF-κB, Bcl-2, Bcl-xl expression at 6 h and 24 h of reperfusion after 90 min of ischemia. β-actin was used as internal control. (b) Caspase-3 activity (*p<0.01; n=3/group). (c) Representative TUNEL-assisted detection of hepatic apoptosis in ischemic liver lobes. Left panel: Stained liver sections (×400 magnification). Right panel: Quantification of hepatic apoptosis. Controls with high frequency of TUNEL positive cells (dark spots), as compared with anti-TIM-1 Ab group. (**p < 0.05; n = 2-3/group). Means±SD are shown.

TIM-1 signaling regulates T cell - macrophage cross talk

We further analyzed the immunomodulatory function of TIM-1 signaling in well-controlled cell culture experiments, designed to mimic in vivo liver IRI model. Addition of anti-TIM-1 Ab significantly suppressed Con A-stimulated IFN-γ production by murine spleen T cells in vitro (Figure 6a: 2774±542.8 vs 401.2±35.8; p<0.0001). Interestingly, in macrophage - T cell co-culture system, anti-TIM-1 Ab supplement decreased Con A-mediated production of both TNF-α and IL-6, the “signature” macrophage-derived mediators of IR-mediated hepatocellular damage (Figure 6b: 729.8±34.12 vs 178.9±41.26; p<0.0001 and 345.5±24.59 vs 68.54±18.92; p<0.0001). In marked contrast, anti-TIM-1 Ab did not affect TNF-α or IL-6 production in pure macrophage cultures devoid of T cells (366.1±9.00 vs 351.8±16.13; and 216.8±16.96 vs 207±15.89). These results document the key regulatory function of T cell-dependent TIM-1 signaling in macrophage activation, which can proceed via indirect or direct pathway, as evidenced by in vitro IFN-γ and TNF-α/IL-6 production profiles, respectively.

Figure 6.

Figure 6

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Cytokine production in murine spleen T cells and RAW 264.7 macrophages. (a) Upregulated IFN-γ production in Con A-stimulated T cells. Marked depression of IFN-γ after treatment with anti-TIM-1 Ab (*p <0.01; n=3/group). (b) Upregulated TNF-α/IL-6 production in Con A-stimulated spleen T cells and RAW 264.7 macrophages co-cultures. Anti-TIM-1 Ab treatment significantly suppressed TNF-α and IL-6 levels (*p < 0.01; n=3/group).

Discussion

This study is the first to document the role of TIM-1 pathway in mouse model of liver warm ischemia followed by reperfusion. Current results provide evidence for a novel mechanism by which disruption of TIM-1 signaling ameliorates the hepatocellular damage in the innate immunity-dominated liver IRI cascade. The beneficial effects were accompanied by reduced neutrophil activity/infiltration; diminished T cell and TIM-4 positive macrophage accumulation; reduced apoptosis via caspase-3 pathway; inhibition of NF-kB activation; and downregulation of pro-inflammatory cytokine/chemokine gene programs. The parallel in vitro co-culture studies support the in vivo data, and suggest that IR-triggered liver damage resulting from activated T cell – macrophage cross talk may proceed via direct and indirect TIM-1 signaling.

The liver IRI consists of a two-phase acute and subacute responses. In the acute phase at 3–6 h after reperfusion, the hepatocellular injury associates with T lymphocyte and Kupffer cell activation and apoptosis (9, 23, 24). In the subacute phase at 18–24 h, the massive neutrophil accumulation takes place (25). The recruited neutrophils lead to cellular responses that culminate in the ultimate tissue damage (12, 26–29). Activated Kupffer cells can increase the oxidative stress by the release of superoxide radicals, TNF-α and IL-1 during the early reperfusion stages (12, 30–33). On the other hand, T-lymphocytes, especially of CD4 phenotype, are the key regulators in initiating IR-induced liver inflammation (9–11). Surprisingly, although T cell - Kupffer cell interactions constitute a key event in the local IR-induced cascade (9, 34–36), no hard data exists to support such a hypothesis.

In this study, we have first documented the therapeutic efficacy of TIM-1 signaling blockade, as evidenced by amelioration of otherwise fulminant IR-triggered hepatocellular damage. This beneficial effect was accompanied by decreased sALT levels, and local cardinal histological features of liver injury, i.e., lobular edema, ballooning, hepatocyte necrosis and sinusoidal congestion. One of the striking effects after TIM-1 Ab treatment was the marked decrease in MPO activity and Ly-6G neutrophil infiltration, in parallel with decreased expression of CXCL-1 (KC) and CXCL-2 (MIP-2), the known neutrophil chemoattractants (22). Further, Th1-derived IFN-γ may also act directly on neutrophils to enhance their sequestration to the damaged liver (9). These data indicates that TIM-1 signaling regulates neutrophil function through cytokines/chemokines produced after liver IRI.

Apoptosis represents a key event after liver reperfusion, the severity of which correlates with the degree of local injury (37). Anti-TIM-1 Ab treated livers showed reduced frequency of TUNEL+ cells, accompanied by increased Bcl-2/Bcl-xl expression. These anti-apoptotic molecules play cytoprotective functions by inhibiting apoptotic cell death, are essential for maintenance of the major organ systems (38), and decreased pro-apoptotic Caspase-3 expression/activity. Activation of NF-κB, an important regulator in the early stages of liver IRI, affects the cell growth and programmed cell death (39). Moreover, the expression of multiple pro-inflammatory cytokines leading to neutrophil-mediated inflammation has been linked to NF-κB activation (40). In this study, and consistent with previous report (41), inhibition of NF-κB after anti-TIM-1 Ab treatment improved the hepatocellular function. Hence, TIM-1 signaling regulates apoptotic pathway, consistent with our present finding of decreased frequency of TIM-4 positive cells in the liver after Ab treatment. Indeed, both TIM-1 and TIM-4 serve as phosphatidylserine (PS) receptors for the engulfment of apoptotic cells (42). We have shown that treatment with Diannexin, which binds to PS and prevents translocation of leukocytes, inhibited attachment of circulating cells and depressed local inflammatory mediator formation, with resultant amelioration of IR-induced liver damage (43). Indeed, it is plausible that by serving as PS receptors, TIM-1 – TIM-4 signaling may exert novel function of modulating leukocyte trafficking patterns.

Anti-TIM-1 Ab treatment significantly decreased pro-inflammatory cytokine (TNF-α, IL-6, IFN-γ and IL-1β) and chemokine (CXCL-1 and CXCL-2) induction ratios in the livers, as compared with controls. These cytokine/chemokine mediators influence T cell/macrophage trafficking patterns. Relatively few liver CD3+ and CD4+ (data not shown) cells were found, consistent with activation/recruitment of CD4+ T cells to the liver within the first hour of reperfusion (9). As disruption of TIM-1 signaling diminished CD68+ macrophage infiltration, IFN-γ might trigger Kupffer cell/macrophage activation in IR inflammatory cascade.

The question arises as to how T cell signaling may regulate Kupffer cell - macrophage cross talk, and how TIM-1 pathway may affect IR-induced liver damage? To mimic in vivo scenario we have employed ConA cell culture system to stimulate lymphocyte TIM-1 expression (6, 44). TIM-1 transcripts are present in high abundance in lymph nodes and kidney, in low levels in the spleen, lung, and thymus, and are largely absent in the heart and liver. These patterns are consistent with TIM-1 RNA expression by spleen CD4+ T cells stimulated by Con A (4). Indeed, TIM-1 expressing Con A-stimulated spleen T cells (predominantly Th1; 45) produced high levels of IFN-γ (Fig. 5A), whereas anti-TIM-1 Ab treatment did suppress that production. This is consistent with our in vivo data where anti-TIM-1 Ab treatment diminished IFN-γ levels at 6 h after reperfusion. We also used RAW 264.7 mouse macrophages and spleen T cells co-culture to investigate direct T cell – macrophage interactions (46). Interestingly, although anti-TIM-1 treatment suppressed TNF-α/IL-6 in the co-culture system, it had little or no effect upon cytokine elaboration profiles when macrophages were cultured alone (Figure 6b). As upon CD4+ T cell polarization, TIM-1 is expressed on Th2 cells at higher levels (4), it is plausible that cross-linking TIM-4 ligand on macrophages results in TNF-α/IL-6 secretion. Our findings, consistent with previous reports (6, 47), suggest not only IFN-γ stimulation, but also TIM-1–TIM-4 interaction on macrophages contributes to IR-induced liver damage.

Figure 7 summarizes putative mechanisms by which TIM-1 expressed by activated T cells mediate local innate immunity-driven inflammation. Liver IR triggers activation of Th1 cells, Th2 cells, and macrophages (9, 48). In the “direct” pathway, TIM-1 on activated Th2 cells cross-links TIM-4 to directly activate macrophages. In the “indirect” pathway, TIM-1 on activated Th1 cells triggers IFN-γ production that results in macrophage activation as well. Regardless of the pathway, activated macrophages elaborate cytokine and chemokine programs that facilitate the ultimate liver damage.

Figure 7.

Figure 7

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Scheme of cross-talk interactions between liver IR and TIM-1 signaling. Both Th1 and Th2 cells express TIM-1, whereas macrophages express TIM-4, the TIM-1 ligand. Liver IR damage leads to the activation of Th1, Th2 and macrophages. TIM-1 on Th2 cells cross-links TIM-4 to directly activate macrophages (“direct pathway”). On the other hand, TIM-1 on activated Th1 cells triggers IFN-γ production that also activates macrophages (“indirect pathway”). As a result, activated macrophages produce cytokine/chemokine programs that ultimately facilitate liver damage.

In summary, liver IRI triggers TIM-1 signaling to activate T cells, which in turn initiates the liver damage. The blockade of TIM-1 pathway ameliorates liver IRI by inhibiting T cell activation, with resultant inhibition of Kupffer cells/macrophage function. This study provides evidence for a novel mechanism by which TIM-1 signaling affects innate immunity-driven pro-inflammatory cascade during the course of liver IRI. Indeed, disruption of TIM-1 – TIM-4 pathway may represent a novel means to improve liver function, expand the organ donor pool, and improve the overall success of liver transplantation.

Supplementary Material

supplementary 1
01

Acknowledgements

The authors thank Dr. Vijai Kuchroo for helpful discussion.

Abbreviations

ALT

alanine aminotransferase

IRI

ischemia/reperfusion injury

MPO

myeloperoxidase

TIM-1

T Cell Ig Mucin-1

TNF

tumor necrosis factor

Footnotes

*

This work was supported by NIH Grants RO1 DK062357, AI23847, AI42223, and The Dumont Research Foundation.

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