Ischemia-reperfusion (I/R) injury remains the main reason for hepatic dysfunction after liver transplantation and major surgery. Although liver I/R injury occurs in the absence of exogenous T cell antigens in syngeneic recipients, and may proceed in the sterile environment, CD4+ T cells are instrumental in promoting proinflammatory liver immune response.1 Recent studies provide evidence that T cells play a critical role in the development of hepatic I/R injury.2–7 After their diapedesis, T cells migrate through the hepatic parenchyma to the afflicted sites during the postischemic inflammatory reaction. The “fate” as well as the pathophysiologic role, of emigrated CD4+ T cells during alloantigen-independent hepatic inflammation in the postischemic liver parenchyma remains not fully understood. Recent studies of viral hepatitis described interaction between T cells (CD4+, NK cells, regulatory T cells) and hepatic stellate cells (HSCs), which seems to be critical for inflammatory response, regeneration, and fibrosis formation,8-10 reviewed in.11 The HSCs are pericytes found in the space of Disse. They are the major cell type involved in liver fibrosis in response to liver damage. The HSCs have immune regulatory functions and are thought to exert an immunomodulatory effect on lymphocyte reactions in vitro and in vivo.12,13 In fibrotic murine livers, lymphocytes were seen in proximity to HSCs, mainly in the periportal area and along fibrotic septa, which suggests a direct interaction.10 In another study by these authors, the CD4-to-CD8 ratio and NK cells were significantly decreased because of the ingestion of lymphocytes by HSCs in human livers with advanced hepatitis C virus/hepatitis B virus–induced fibrosis.9 Interestingly, hypoxia alters the sensitivity of HSCs to certain activators and chemotaxins through activation of HIF-1α and also regulates the expression of genes that are important for angiogenesis and collagen synthesis.14 Whether CD4+ T cells interact with HSCs during I/R-induced inflammation remains unclear.
According to a recent data in literature, HSC-T cell interactions have varying immunomodulatory effects which strongly depend on the character of the inflammatory reaction. Indeed, HSCs are potent antigen presenting cells (APCs) and can activate natural killer T cells and conventional T lymphocytes.15 Such activation would enhance the immune response after liver transplantation, accelerate the T cell– induced I/R injury, and even increase the graft rejection rate. On the other hand, HSCs were also reported to prevent activation of naive T cells by dendritic cells or artificial APCs in a cell contact-dependent mechanism.16 The pathophysiologic relevance of HSC-CD4+ T cell interaction during hepatic I/R has not been investigated so far.
In the present study, we answered the questions of (i) whether HSCs interact with CD4+ T cells during I/R of the liver and (ii) whether modulation of HSC activity influences CD4+ T cell migration and hepatic I/R injury in vivo. The activity of HSCs was modulated by specific agonists of endocannabinoid receptors, CB-1 and CB-2.17-21 Recent studies show that during inflammation or fibrosis, HSCs are controlled by CB-1 and CB-2. Both receptors are expressed on HSCs and have opposite functions. CB-1 stimulation leads to HSC activation and enhances fibrosis formation, whereas CB-2 activation causes HSC apoptosis.22 The current literature discusses CB-1 and CB-2 as attractive and promising targets for clinical application.22-25
Colocalization Between HSC and CD4+ T Cells
In our first experiments, we tackled the question of whether CD4+ T cells are colocalized with HSC in the postischemic liver. Freshly isolated and fluorescence-labeled CD4+ T cells were infused into heterozygote Cx3CR1(gfp/gfp) mice (mice exhibiting green fluorescent protein-labeled HSCs), and the interaction between CD4+ T cells and HSCs was visualized in the hepatic microcirculation using intravital microscopy. In sham-operated controls, no CD4+ T-cell-HSC colocalizations were observed. After I/R, 26% ± 3% of all accumulated CD4+ T cells were colocalized with HSCs in sinusoids (Figure 1A). This suggests a direct interaction between both cell types. There were more colocalizations (31%±5%) after a prolonged reperfusion time (140 min vs. 120 min, Fig. 1B). The CD4+ T cell-HSC colocalizations were also shown using the intravital two-photon microscopy. This technique is a superior alternative to intravital fluorescence microscopy and confocal microscopy because of its deeper tissue penetration, efficient light detection, and reduced phototoxicity. Figure 1(C) to (E) demonstrates proximity and attachment of both cell types in the liver microcirculation.
Immunostaining for α-Smooth Muscle Actin as a Marker of HSC Activation
In the next set of experiments, we modulated the HSC activity by stimulating CB-1 and CB-2 receptors with specific antagonists. As mentioned above, a stimulation of CB-2 leads to HSC apoptosis, whereas CB-1 agonists activate HSCs. Expression of α-smooth muscle actin (SMA) is a recognized marker of HSC activation in the liver tissue. Using immunostaining, we have shown that α-SMA expression was enhanced in the untreated group after I/R as compared to the sham-operated controls (Figure 2). In contrast, α-SMA expression was almost absent in the I/R group pretreated with CB-2 agonist JWH-133. In the postischemic group, pretreated with the CB-1 agonist arachidonylcyclopropylamide (ACPA), the α-SMA expression was strongly enhanced. Taken together, the HSC activity was negatively influenced by the CB-2 agonist and stimulated by the CB-1 agonist in our model of hepatic I/R.
CD4+ T Cell Recruitment
In an attempt to answer the question of whether the targeting of HSCs through the CB receptors can affect T-cell migration, the recruitment of CD4+ T cells in the hepatic microvasculature was analyzed using intravital microscopy. As shown in Figure 3, only few CD4+ T cells were found accumulated in sinusoids of sham-operated mice (2.8 ± 0.2/acinus). In contrast, CD4+ T cell recruitment was significantly enhanced in the vehicle-treated group after I/R (8.4 ± 0.4/acinus). In mice undergoing HSC depletion with JWH-133, the postischemic CD4+ T-cell accumulation was reduced by about 60% (P < 0.05). The activation of HSCs using ACPA did not significantly influence the postischemic T cell recruitment as compared with the I/R group treated with the vehicle solution. As observed in an additional set of experiments using Cx3CR1(gfp/gfp) mice, the percentage of CD4+ T cell colocalized with HSCs remained almost unchanged on pretreatment with ACPA (28% ± 5% and 30% ± 3% after 120 min and 140 min of reperfusion, respectively) as compared to the vehicle-treated I/R group. Thus, HCS depletion attenuates the I/R-induced CD4+ T cell migration, whereas HSC activation does not affect it.
Microvascular and Hepatocellular I/R Injury
Sinusoidal perfusion failure was quantified using intravital microscopy after plasma labeling with FITC-dextran. The data are presented as a percentage of nonperfused sinusoids to all sinusoids visible per acinus. In the sham-operated group, only 7% ± 1% of all sinusoids were not perfused. In contrast, sinusoidal perfusion failure was 27% ± 3% in the vehicle-treated I/R group. The pretreatment with JWH-133 significantly improved the postischemic perfusion, whereas the perfusion failure was even higher (53% ± 2%) in the ACPA pretreated group as compared to the vehicle-treated I/R group (Figure 4).
The liver enzyme activities were determined in serum as markers of hepatocellular necrotic injury. In line with the data on sinusoidal perfusion, we observed a dramatic increase of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities in the vehicle-treated I/R group. The HSC depletion with JWH-133 significantly reduced the liver enzyme activity (AST by ∼2.5-fold, ALT by ∼3-fold). In the I/R group, which underwent HSC stimulation with ACPA, the AST-ALT activities were comparable to those of the I/R vehicle group (Figure 5).
There is growing evidence that CD4+ T cells are critically involved in the induction of I/R injury of the liver. The exact mechanisms that control CD4+ T cell migration during alloantigen-independent postischemic inflammation remain unclear. We tested the hypothesis that CD4+ T cells interact with HSCs during I/R.
One of the key findings of our study is a colocalization between adherent CD4+ T cells and HSC observed during hepatic I/R in vivo. Such proximity suggests a direct interaction between these cell types. A direct interaction between T cells and HSCs requires a binding between an adhesion receptor and a corresponding counter-receptor or a ligand on the cell surface. It is known that on antigen stimulation, the binding of the antigen-major histocompatibility complex to the T cell receptor complex and CD4 helps the APC and the CD4+ T cell adhere during T cell activation but the integrin protein LFA-1 on the T cell and Intercellular Adhesion Molecule-1 (ICAM-1) on the APC are the primary molecules of adhesion in this cell interaction 26. Indeed, ICAM-1, a member of the immunoglobulin superfamily, is expressed and upregulated on activated HSCs and therefore may be responsible for HSC-CD4+ T cell interactions. Notably, the HSC-T cell proximity was more frequently observed after prolongation of reperfusion time. This is not surprising because we also see more adherent CD4+ T cells in the hepatic microvasculature after longer reperfusion times. Moreover, HSCs are able to move toward certain stimuli. Such chemotaxis of HSCs might also increase the frequency of T cell-HSC interactions.
As shown by the immunostaining, hepatic I/R leads to HSC activation during early reperfusion. We used α-SMA expression in the liver tissue as a parameter of HSC activation. Induction of α-SMA is the single most reliable marker of stellate cell activation because it is absent from other resident liver cells in normal or injured liver except for the smooth muscle cells surrounding large vessels.12 Our results of α-SMA expression were also supported by immunostaining for reelin (data not shown), an additional marker of HSC activation.27 Still the modulation of HSC activity has been a difficult task. In particular, HSCs activated in culture do not fully reproduce the changes in gene expression observed in vivo, making it difficult to correlate in vitro results with HSC behaviors in vivo.28 Several models have been established to deplete HSCs in vivo thus far, such as the use of gliotoxin.29 or gliotoxin-coupled antibodies.30 However, gliotoxin also has broad actions in vivo and in culture, targeting not only HSCs but also immune and endothelial cells and hepatocytes.31 Recently, Puche et al.32 described a new model for depleting mouse HSCs. Transgenic mice expressing the herpes simplex virus thymidine kinase gene driven by the mouse GFAP promoter were used to render proliferating HSCs susceptible to killing in response to ganciclovir. In our study, we modulated HSC activity by using CB-1 and CB-2 agonists. Both cannabioid receptors are highly expressed on HSCs, and their activation leads to opposite effects on HSC activity.22 Indeed, we observed in our study that CB-2 stimulation resulted in HSC depletion in vivo because only a low expression of α-SMA was detectable in the postischemic liver tissue. In contrast, pretreatment with a CB-1 agonist induced a (hyper-) activation of HSC as compared to the sham-operated animals without ischemia and even to the vehicle-treated mice undergoing I/R.
The main finding of our study is that depleting HSCs attenuates the I/R-induced CD4+ T cell recruitment and reduces tissue injury. In contrast, hyperactivation of HSCs does not affect CD4+ T cell migration and even enhances I/R injury, in particular microvascular perfusion failure. There is significant evidence that HSCs can modulate the hepatic immune response. They are known to have various immune functions, which range from immunogenic antigen presentation to the induction of T cell apoptosis. Indeed, activated HSCs can stimulate T lymphocytes and cause lymphocyte proliferation due to their function as professional APC.15,33,34 Furthermore, activated HSCs produce chemokines, such as monocyte chemotactic peptide, CCL21, RANTES, and CCR5,12 which could play a role for T-cell activation during inflammation or I/R. In contrast, activated HSCs also have a mechanism for inhibiting T-cell–mediated cytotoxicity and conversely can induce T-cell apoptosis as shown in models of liver cancer or transplantation.35,36 Activated, but not resting, HSCs express the costimulatory molecule B7-H1, which can bind to counter-receptor programmed deathligand-1 (PD1) on T cells. The PD1 is expressed on a range of immune cells including CD4+ T cells and, at low levels, PD1 activation is sufficient to inhibit the earliest stages of T-cell activation.12,37 An immunotolerizing role is also suggested by experimental models in mice in which transplanted stellate cells protect islet allografts from rejection.38 and enhance engraftment of transplanted hepatocytes.39
Depletion of HSCs with a CB-2 agonist not only attenuated CD4+ T cell migration but also reduced the microvascular and hepatocellular injuries as shown in our study by measurement of the liver enzyme activity and by analysis of the sinusoidal perfusion in vivo. Indeed, the significant role of CD4+ T cells during alloantigen-independent postischemic hepatic inflammation has been demonstrated previously, and interventions targeted at CD4+ T cell activation or migration have had a clear therapeutic impact.2,4,6 However, CB-2 agonists can also protect the liver through other pathways. Activation of CB-2 receptors by specific agonists, such as JWH133 and HU-308, protected against I/R damage by decreasing neutrophil infiltration, tissue and serum tumor necrosis factor-α, chemokines macrophage-inflammatory protein-1α and macrophage- inflammatory protein-2 levels, caspase-3 activity, tissue lipid peroxidation, and expression of adhesion molecule ICAM-1.40,41 These agonists also decreased the tumor necrosis factor-α–induced expressions of endothelial adhesion molecules ICAM-1 and vascular cell adhesion molecule-1 in human liver sinusoidal endothelial cells in vitro.
Hyperactivating HSCs with the CB-1 agonist ACPA does not additionally enhance hepatocellular injury (determined by the liver enzyme activity) compared with the I/R-induced activation. It did however markedly increase sinusoidal perfusion failure. The postischemic shutdown of the hepatic microcirculation is triggered by sinusoidal narrowing caused by endothelial cell edema,42 by activated Kupffer cells, and by HSC-mediated vasoconstriction.43,44 Therefore, it seems likely that HSC hyperactivation through CB-1 receptors increases sinusoidal resistance which further deteriorates postischemic tissue perfusion.
In summary, our in vivo data suggest that (i) CD4+ T cells colocalize and interact with HSCs on their migration into the hepatic parenchyma; (ii) a selective depletion or deactivation of HSCs through CB-2 activation reduces CD4+ T cell–dependent I/R injury, (iii) whereas a HSC hyperactivation through CB-1 receptors is not protective and even enhances perfusion failure. Thus, HSCs might represent a potential target for future therapeutic strategies against T cell–mediated I/R injury during liver transplantation.
MATERIAL AND METHODS
For experiments, 5-week-old to 7-week-old female C57BL/6 wild-type mice (Charles River, Sulzfeld, Germany) were used. Cx3CR1(gfp/gfp) mice were obtained from the European Mouse Mutant Archive (Monterotondo, Italy). All experiments were carried out according to the German legislation on animal protection.
Under inhalation anesthesia with isoflurane-N2O, a catheter was inserted into the left carotid artery for measurement of mean arterial pressure and application of fluorescence dyes as described previously.45 In C57BL/6 mice, a warm (37°C) reversible ischemia of the left liver lobe was induced for 90 min by clamping the supplying nerve vessel bundle using a microclip. The total reperfusion time was 120 min. Sham-operated animals underwent a short (3 sec) clamping of the left liver lobe and afterward were monitored under anesthesia for a total time of 210 min (90 min + 120 min). Tissue and plasma samples were collected at the end of the experiment. All experiments were carried out according to the German legislation on animal protection.
Isolation and Labeling of CD4+ T Cells
For the intravital microscopic studies, CD4+ T cells were isolated from spleens of syngeneic mice using a magnetic cell sorting system with anti-mouse CD4 antibody according to the manufacturer’s instructions (miniMACS; Miltenyi Biotec, Bergisch-Gladbach, Germany). Isolated CD4+ T cells were labeled with the fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE, 5 μM; Molecular Probes, Eugene, OR). A total of 1 × 107 CFSE-labeled CD4+ T cells was infused intraarterially after 120 min of reperfusion and then visualized using intravital microscopy as described previously.4,7 For two-photon microscopy, isolated CD4+ T cells were labeled with eFluor660 (eBioscience, San Diego, CA). The isolation procedure did not lead to T cell activation, and the purity of the CD4+ T cells was routinely greater than 95% as determined by fluorescence assisted cell sorting analysis. The viability of CD4+ cells after the isolation procedure was approximately 94%.
Intravital fluorescence microscopy was performed using a modified Leitz-Orthoplan microscope as described previously.4 CD4+ T cells were isolated from spleens of syngeneic mice by magnetic cell sorting, labeled with the fluorescent dye CFSE (carboxyfluorescein diacetate succinimidyl ester, 5 μM, Molecular Probes, Eugene, OR). Fluorescence-labeled CD4+ T cells (1 × 107) were slowly infused in a total volume of 200 μL PBS via the carotid catheter into the aortic arch and visualized in 7–10 randomly chosen acini. Thereafter, FITC-labeled dextran (MW 150000; 0.1 mL, 5%, Sigma-Aldrich, Taufkirchen, Germany) was infused, and sinusoidal perfusion was visualized in sinusoids within seven to 10 acini. Intravital microscopy was started immediately during the T-cell infusion after 120 min of reperfusion and lasted approximately 20 min. All videotaped images were quantitatively analyzed offline in blinded fashion using CAPIMAGE software (Zeintl, Heidelberg, Germany).
Intravital Two-Photon Microscopy
For in vivo two-photon microscopy, we used an upright microscope with a water immersion objective (20×, 0.95 NA; Olympus, Tokyo, Japan) which was connected with a TriM Scope II two-photon microscope (LaVision Biotech, Gottingen, Germany). The two-photon microscope unit that was equipped with a Mai Tai laser tuned at 870nm. The fluorescence signal emitted from the tissue (filters LP495, BP525/50, LP560, LP665) was detected by four photomultiplier tubes. The software system ImSpector (LaVision Biotech) was used for image acquisition and processing. The three-dimensional scans per time point were flattened and visualized in two dimensions as projections onto the X-Y axes over time. In this experimental set, isolated CD4+ T cells were labeled with eFluor 660 (eBioscience).
Blood samples were taken from the carotid artery at the end of the experiment (90 min of reperfusion), immediately centrifuged at 2,000×g for 10 min, and stored at −80°C. Serum AST and ALT activities were determined at 37°C with an automated analyzer (Hitachi 917; Roche-Boehringer, Mannheim, Germany) using standardized test systems (HiCo GOT and HiCo GPT; Roche-Boehringer).
Staining for α-Smooth Muscle Actin
Paraffin-fixed liver sections were incubated with Animal Research Kit Peroxydase (Dako, Carpinteria, CA). As the primary antibody, we used anti–α-SMA monoclonal antibody (dilution 1:200; Abcam, Cambridge, UK). Next, the slices were incubated with horseradish peroxidase-conjugated streptavidin and diaminobenzidine, and then counterstained with hemalaun. As negative controls, primary antibody was replaced with nonimmune immunoglobulin at the same concentration. No staining was observed in the negative controls.
(I) Colocalization Between HSC and CD4+ T Cells in the Postischemic Liver
In this set of experiments, we analyzed whether CD4+ T cells are colocalized with HSC in the hepatic microcirculation after I/R (I: 90 min, R: 120 min). To visualize HSC in vivo, we used heterozygote Cx3CR1(gfp/gfp) mice exhibiting green fluorescent protein in HSC.46 CFDA-SE–labeled CD4+ T cells were injected into Cx3CR1 mice after I/R and interactions between both cell types were analyzed using in vivo microscopy. Sham-operated animals served as controls (n = 3 each group). The percentage of colocalized T cells was calculated in 7 to 10 acini per experiment as follows: adherent CD4+ T cells colocalized with HSC/adherent CD4+ T cells per acinus × 100%.
In a separate set of experiments, we visualized T cell-HSC interactions with two-photon microscopy in a sham-operated mice as well as in mice after I/R (90/120 min, n = 3 each group).
(II) Effect of HSC Depletion or Activation on CD4+ T-Cell Recruitment and I/R Injury
In an attempt to analyze the effect of HSC targeting (depletion vs. activation), a sham-operated group and three I/R groups (I: 90 min, R: 120 min) were analyzed (n = 7 each): an I/R group pretreated with Tocrisolve 100 solution (200 μL, intraperitoneal [IP], 24 hr before the experiment; Tocris Bioscience, Bristol, UK) as vehicle, an I/R group pretreated with CB-2 agonist JWH-133.41,47 (IP, 24 hr before the experiment, 0.2 mg/kg body weight in 200 μL Tocrisolve; Tocris Bioscience) to reach HSC depletion, and an I/R group pretreated with CB-1 agonist ACPA.48 (IP, 24 hr before the experiment, 1 mg/kg body weight in 200 μL Toscisolve; Tocris Bioscience) for HSC activation. In an additional I/R group (n = 3), the impact of HSC activation with APCA on HSC-CD4+ T cell colocalizations was analyzed in Cx3CR1(gfp/gfp) mice as described above in (I) Colocalization Between HSC and CD4+ T Cells in the Postischemic Liver.
Tocrisolve is a water-soluble emulsion composed of a 1:4 ratio of soya oil and water that is emulsified with the block copolymer Pluronic F68. JWH-133 (6aR,10aR)-3-(1,1-Dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo [b,d]pyran; MW 312, Cat. No 1343) is a potent CB-2 selective agonist (Ki = 3.4 nM). approximately 200-fold selective over CB-1 receptors. Arachidonylcyclopropylamide (MW 344, Cat No.1781) is a potent and selective CB1 agonist (Ki = 2.2 nM) that displays 325-fold selectivity over CB-2 receptors. The applied concentrations of JWH133 and ACPA were established in separate dose-finding experiments and are in line with published studies in mice.
Analysis of variance on ranks followed by Student-Newman-Keuls method was used for the estimation of stochastic probability in intergroup comparison (SigmaPlot 12; Jandel Scientific, Erkrath, Germany). Mean values±SEM are given. P values less than 0.05 were considered significant.
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