Potential conflict of interest: Nothing to report.
Although the role of CD4 T cells in tissue inflammation and organ injury resulting from ischemia and reperfusion injury (IRI) has been well documented, it remains unclear how CD4 T cells are activated and function in the absence of a specific antigen (Ag). We used a murine liver warm IRI model to determine first whether de novo Ag-specific CD4 T cell activation was required and then what its functional mechanism was. The critical role of CD4 T cells in liver immune activation against ischemia and reperfusion (IR) was confirmed in CD4 knockout mice and CD4 depleted wild-type mice. Interestingly, the inhibition of CD4 T cell activation without target cell depletion failed to protect livers against IRI, and this suggested that T cells function in liver IRI without Ag-specific de novo activation. To dissect the T cell functional mechanism, we found that CD154 blockade, but not interferon γ (IFN-γ) neutralization, inhibited local immune activation and protected livers from IRI. Furthermore, agonist anti-CD40 antibodies restored liver IRI in otherwise protected CD4-deficient hosts. Finally, fluorescence-activated cell sorting analysis of liver CD4 T cells revealed the selective infiltration of effector cells, which constitutively expressed a higher level of CD154 in comparison with their peripheral counterparts. IR triggered a significant liver increase in CD40 expression but not CD154 expression, and macrophages responded to toll-like receptor 4 and type I IFN stimulation to up-regulate CD40 expression. Conclusion: These novel findings provide evidence that CD4 T cells function in liver IRI via CD154 without de novo Ag-specific activation, and innate immunity–induced CD40 up-regulation may trigger the engagement of CD154-CD40 to facilitate tissue inflammation and injury. (HEPATOLOGY 2009.)
Although tissue damage resulting from ischemia and reperfusion injury (IRI) can develop in the absence of an exogenous antigen (Ag), studies in liver and kidney murine models have documented the key role of T cells in the activation of local immune responses.1, 2 The observation that systemic immunosuppression (cyclosporine A and tacrolimus) attenuates hepatocellular damage provided initial indirect evidence for T cell involvement in the mechanism of IRI.3 In addition, the inhibition of lymphocyte adherence to endothelium, previously thought to affect primarily neutrophils, was shown to target T cells. Finally, results from T cell–deficient and CD4-deficient mice have provided direct proof that the T cell, particularly the CD4 phenotype, is the key player in the early IRI phase.4–7 Indeed, adoptive transfer of T cells or the CD4+ T subset readily restored IRI in T cell–deficient mice.4, 6 Thus, the question arises of how T cells function in this innate immunity-dominated response and in the absence of exogenous Ag stimulation?
T cells may function in an Ag-independent manner by secreting cytokines and up-regulating costimulatory molecules. The role of T cell–derived CD28, CD154, and interferon γ (IFN-γ) in IRI has been demonstrated in both mouse and rat models.8–10 We have shown the importance of CD28 and CD154 expression for the activation of the liver proinflammatory response leading to ischemia and reperfusion (IR)–triggered hepatocellular damage. Indeed, livers in CD154 knockout (KO) mice, CD28 KO mice, and wild-type (WT) mice treated with anti-CD154 antibody (Ab) or cytotoxic t-lymphocyte antigen 4 immunoglobulin were protected from IRI.6 Our recent study on type I IFN receptors versus type II IFN receptors indicated that IFN-γ might be dispensable in liver IRI.11 Although the role of CD154 in IRI has been attributed to its T cell activation, the nature of IR-triggered T cell activation remains elusive. Because no specific Ags are required, we hypothesize that T cells become activated during IR by an Ag-nonspecific proinflammatory milieu independently of Ag-specific first signaling and that CD154 triggers CD40 to facilitate innate immune activation. In this study, we first confirmed the role of CD4 T cells in liver immune activation and then determined the nature of T cell activation in liver IRI. The CD4 blocking Ab YTS177.9,12, 13 which inhibits Ag-specific CD4 T cell activation without target cell depletion,14–18 was used and contrasted with the CD4 depleting Ab GK1.5. To determine the functional mechanisms of CD4 T cells, we first analyzed the effects of CD154 blockade and IFN-γ neutralization in liver IRI followed by CD40 triggering in CD4 KO mice. Our novel findings indicate that CD4 T cells function in liver IRI without the requirement of de novo Ag-specific activation and are dependent on CD154-CD40 signaling but not IFN-γ signaling.
Male WT, nude (B6.Cg-Foxn1nu/J), CD4-deficient (B6.129S2-Cd4tm1Mak/J), and CD8-deficient (B6.129S2-Cd8atm1Mak/J) mice (C57BL/6 strain, 8–12 weeks old) were used (Jackson Laboratory, Bar Harbor, ME). Animals were housed in the University of California–Los Angeles animal facility under specific pathogen-free conditions and received humane care according to the criteria outlined in Guide for the Care and Use of Laboratory Animals (prepared by the National Academy of Sciences; National Institutes of Health publication 86–23, revised 1985).
Mouse Warm Hepatic IRI Model.
We used a warm partial hepatic IRI model in mice.6, 19, 20 After 90 minutes of local ischemia, animals were sacrificed serially at reperfusion. Serum alanine aminotransferase (sALT) levels, an indicator of hepatocellular injury, were measured with an autoanalyzer (ANTECH Diagnostics, Los Angeles, CA). Liver, spleen, and peripheral blood samples were collected. Liver specimens, fixed in 10% buffered formalin and embedded in paraffin, were stained with hematoxylin and eosin and then analyzed blindly. For molecular biology, liver specimens were rinsed in phosphate-buffered saline (PBS) prior to freezing in liquid nitrogen. Sham WT controls underwent the same procedure but without vascular occlusion.
CD4 blocking (YTS177) or depleting (GK1.5) Abs were administered (1 mg/mouse intravenously) 24 hours prior to the experiment. Anti-CD154 and anti–IFN-γ Abs were given (500 μg/mouse intravenously) prior to the onset of liver ischemia. Anti-CD40 Ab was infused (250 μg/mouse) at the onset of reperfusion via the portal vein.
CD4 T Cell Activation In Vitro.
Splenocytes from naïve B6 mice cells were labeled with 4 mM carboxy-fluorescein diacetate succinimidyl ester (CFSE; Molecular Probe, Eugene, OR)21 and incubated with irradiated B6 (syngeneic) or B/c (allogeneic) stimulator cells (2 × 106/mL) or concanavalin A (ConA; 2 U/mL; Sigma) in the absence or presence of anti-CD4 Abs, GK1.5 or YTS177 (10 μg/mL). On day 4, cells were harvested and stained with anti-mouse CD4-phycoerythrin (PE; eBiosciences, San Diego, CA). Topro 3 (1 nM) was added as a viable dye. Flow cytometry was performed on a FACSCalibur dual-laser cytometer (Becton Dickinson). Cells in the lymphocyte gate, Topro 3–negative (viable cells) and CD4-positive, were analyzed for CFSE intensities.
Macrophage Activation In Vitro and CD40 Staining.
Murine bone marrow macrophages were differentiated from the marrow of 6- to 10-week-old C57B/6 mice by culturing in 1× Dulbecco's modified Eagle's medium, 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 30% L929 conditioned medium for 6 days.22 The cell purity was assayed to be 94% to 99% CD11b+. In addition, we used mouse leukemic macrophage line RAW264.7 (TIB-71; American Type Culture Collection, Manassas, VA), which was maintained in Dulbecco's modified Eagle's medium supplemented with 10% FBS. Cells were treated with lipopolysaccharide (1 ng to 10 μg/mL; Sigma, St. Louis, MO) or IFN-β (10–1000 U/mL; R&D Systems, Minneapolis, MN) for 1 to 24 hours. Treatment did not affect macrophage viability (>95%). Cells were collected and stained with FITC–anti-CD11b and PE–anti-CD40 (eBiosciences).
Liver Lymphocyte Isolation and FACS Analysis.
We used a mechanical method to separate intrahepatic lymphocytes from liver parenchymal cells. Livers, perfused in situ with 10 mL of cold PBS to remove circulating peripheral blood lymphocytes, were pressed through a sterile stainless steel screen in 30 mL of Roswell Park Memorial Institute medium with 5% FBS. The hepatocytes were removed by low-speed centrifugation. The supernatant was collected and centrifuged, and the pellet was resuspended. The cell suspension was then layered on top of a density cushion of 25%/50% discontinuous Percoll (Pharmacia) and centrifuged to obtain the lymphocyte fraction at the interface. Lymphocytes were collected, washed, and subjected to FACS analysis. Approximately 500,000 to 1,000,000 liver resident lymphocytes were obtained from one mouse liver by this method. Spleen and liver lymphocytes were first stained with CD4-FITC, CD8-allophycocyanin, and CD62L–cyanine 5. After being washed, cells were fixed with 1% paraformaldehyde-PBS, and this was followed by permeabilization with 0.5% saponin. CD154 (both cell surface and intracellular) was detected by anti-CD154–PE.
RNA (2.5 μg) was reverse-transcribed into complementary DNA with the SuperScript III first-strand synthesis system (Invitrogen, Carlsbad, CA). Quantitative polymerase chain reaction was performed with the DNA engine with the Chromo 4 detector (MJ Research, Waltham, MA). To a final reaction volume of 25 μL, the following were added: 1× SuperMix (Platinum SYBR Green quantitative polymerase chain reaction kit, Invitrogen), complementary DNA, and 0.5 mM of each primer. The amplification conditions were 50°C for 2 minutes, 95°C for 5 minutes, and 50 cycles of 95°C for 15 seconds and 60°C for 30 seconds. Primers to amplify specific gene fragments, tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and chemokine (C-X-C motif) ligand 10 (CXCL10) were described.20 Target gene expressions were calculated by their ratios to the housekeeping gene hypoxanthine-guanine phosphoribosyl transferase.
All values are expressed as the mean ± standard deviation. Data were analyzed with an unpaired, two-tailed Student t test. P < 0.05 was considered to be statistically significant.
CD4 T Cells Are Critical for the Proinflammatory Immune Response in Liver IRI.
To document the role of CD4 T cells in the liver proinflammatory immune response against IR, CD4 KO and WT mice pretreated with CD4 depleting Ab were subjected to 90 minutes of warm ischemia; the hepatocellular damage and gene induction were measured at 6 hours of reperfusion. Indeed, both groups of mice were protected from IRI as their sALT levels were lower (Fig. 1A; 3775 ± 621 for WT, n = 10, versus 445.8 ± 111.9 for CD4 KO, n = 6, and 161.8 ± 27 for anti-CD4, n = 6, P < 0.04), and the liver architecture was better preserved (Fig. 1B) in comparison with WT controls and was similar to that in nude mice. Intrahepatic proinflammatory gene induction by IR was suppressed in the absence of CD4 T cells, as shown by TNF-α and CXCL10 transcript levels (Fig. 1C). Unlike CD4 T cells, CD8 T cells or natural killer (NK) cells were not essential, as their deficiency in respective KO mice or WT mice pretreated with depleting Abs failed to protect livers from IRI. Both sALT levels (Fig. 1A; 1960 ± 976 for CD8 KO, 2784 ± 1355 for anti-CD8 Ab, and 2634 ± 978 for anti-NK Ab, P > 0.1 versus WT, n = 4–8/group) and histology (hematoxylin and eosin staining) revealed comparable tissue damage in CD8 KO depleted, NK depleted, and WT mice (Fig. 1B). In parallel, intrahepatic levels of TNF-α and CXCL10 were up-regulated in CD8 or NK depleted mice, and they were comparable with those of WT mice (Fig. 1C). Thus, CD4 T cells are critical for the IR-triggered proinflammatory response leading to the hepatocellular damage.
De Novo Ag-Specific CD4 Activation Is Not Required for Liver IRI.
To determine the nature of CD4 T cell activation by IR, we used a blocking CD4 Ab capable of inhibiting Ag-specific CD4 activation without target cell depletion. We have shown that treatment of cardiac allograft recipients with YTS177 Ab inhibits CD4-dependent alloreactive CD8 activation.23 To directly demonstrate that this blocking Ab did inhibit Ag-specific CD4 T cell activation, mixed lymphocyte responses (MLRs) were set up in the absence or presence of the CD4 Ab. As shown in Fig. 2A, the addition of YTS177 inhibited CD4 T cell proliferation against allo-Ag stimulation in the culture. The CD4 depleting Ab GK1.5 showed similar capability for suppressing T cell proliferation in MLRs. Interestingly, neither of the CD4 Abs inhibited ConA-stimulated CD4 proliferation (Fig. 2A). In vivo, the administration of GK1.5 depleted CD4 T cells, whereas YTS177 preserved CD4 T cells in both the spleen and liver (Fig. 2B).
Having confirmed its effects on CD4 T cells in vitro, we then analyzed the impact of nondeletional CD4 inhibition in vivo. In contrast to CD4 depletion, CD4 blockade failed to prevent IRI. Indeed, sALT levels (Fig. 3A) and liver pathology (Fig. 3B) showed similar degrees of tissue damage in YTS177-treated mice and controls (10,150 ± 2193 for controls versus 6989 ± 2168 for YTS177 mice, n = 4–6, P > 0.3), and this was in sharp contrast to the effect of CD4 depletion (445.8 ± 111.9 for the GK1.5 group, n = 6, P < 0.01 versus the YTS177 group). Correlated with hepatocellular injury, local TNF-α/CXCL10 levels remained up-regulated following CD4 blockade and were comparable to those in controls yet were higher than those in CD4-depleted mice (Fig. 3C). Thus, inhibition of de novo Ag-specific CD4 T cell activation did not interfere with their function in liver IRI.
CD154, but Not IFN-γ, Is Critical for Liver IRI.
Although both IFN-γ and costimulatory CD154 have been implicated in the mechanism by which CD4 T cells promote IRI, controversial data exist,10, 11 and the function of the two molecules has never been tested in parallel in the same model system. Both CD4 and CD8 T cells can produce IFN-γ upon stimulation, but only CD4 T cells up-regulate CD154 expression (data not shown). This suggests that CD154, but not IFN-γ, may represent the key mediator in the disease process, with CD8 T cells dispensable in liver IRI. To directly compare the functions of the two molecules, groups of WT mice were infused with IFN-γ neutralizing Ab or CD154 blocking Ab. Figure 4 shows that untreated mice and those treated with anti–IFN-γ Ab suffered similar degrees of liver injury, as evidenced by sALT levels (5993 ± 943.2 and 6972 ± 1549, respectively, n = 6-8/group, P = not significant) and histology. Moreover, intrahepatic TNF-α and IL-1β transcript levels remained up-regulated in both recipient groups. In contrast, mice treated with anti-CD154 Ab were protected from liver IRI, as evidenced by lower sALT levels (1407 ± 336.2, n = 4, P < 0.02 versus the anti-IFN group), preservation of liver architecture, and reduced local proinflammatory gene levels (TNF-α, IL-1β, and CXCL10). These results are supportive of our hypothesis that CD154, but not IFN-γ, mediates CD4 T cell function in liver IRI.
Ab-Mediated CD40 Ligation Restores Liver IRI in CD4 KO Mice.
As de novo Ag-specific activation is not required for CD4 T cell function in liver IRI, we then reassessed the role of CD154. We tested the hypothesis that, instead of activating CD4 T cells, CD154 may trigger CD40 on liver innate immune cells to facilitate the proinflammatory immune response against IR. An agonist anti-CD40 Ab was infused into CD4 KO mice at the onset of liver reperfusion. This Ab by itself did not trigger a liver proinflammatory response or hepatocellular injury if infused into naïve animals (data not shown). However, treatment with anti-CD40 Ab readily re-created the hepatocellular damage following IR in CD4 KO mice, as evidenced by increased sALT levels (Fig. 5A; 4050 ± 1189 for controls, n = 9, versus 10,350 ± 1035 for anti-CD40, n = 10, P < 0.001) and liver histology (Fig. 5B), in comparison with untreated CD4 KOs. Moreover, liver immune activation by IR was also restored, as both TNF-α and CXCL10 gene levels markedly increased after CD40 Ab infusion in comparison with controls (Fig. 5B; P < 0.05). Thus, crosslinking of CD40 re-created liver inflammation/damage in otherwise protected CD4 KO mice, and this implies that CD4 T cells may function by activating CD40 in liver IRI.
Liver CD4 T Cells Are Enriched with Effector Cells and Constitutively Express CD154.
Having confirmed the critical roles of CD4 T cells and CD154-CD40 signaling in immune activation against IR, we next addressed the question of how liver CD4 T cells engage the CD154-CD40 pathway without de novo activation. Liver lymphocytes were isolated and subjected to multiparameter FACS analysis of their functional status in parallel with splenocytes of the same animal. As shown in Fig. 6A, liver T cells, both CD4 and CD8 subsets, were highly enriched with proinflammatory effector type cells (CXCR3+CD62Llow): the percentages of the CD4+CXCR3+CD62Llow subset in total CD4 and CD8 were 44% and 51% versus 16.9% and 17.1% in spleens. Interestingly, liver CD4 memory populations, represented by CD4+CD44high, and regulatory populations, represented by CD4+CD25+, were not different from spleen CD4, and this indicates that effector T cells, rather than memory/regulatory T cells, are selectively sequestered in the liver. To show that liver CD4 T cells are capable of engaging CD154-CD40, we directly measured CD154 expression in these T cells without further in vitro stimulation. As cell surface CD154 has a very fast turnover rate, with its majority stored intracellularly,24–26 we performed intracellular staining of CD154 in combination with cell surface CD4/CD62L staining. In spleens, CD4 T cells, but not CD8 T cells, and the CD62Llow effector subset of CD4, but not the CD62Lhigh subset, expressed CD154 (Fig. 6B). As the liver CD4 T cells were enriched with the effector type, they expressed significantly higher levels of CD154 than their spleen counterparts. In fact, their CD154 levels were comparable to those of the spleen CD4+CD62Llow subset. These results indicate that liver CD4 T cells are highly enriched with effector type cells that constitutively express CD154.
Liver IR Triggers CD40 Up-Regulation.
The question arises of the mechanism that triggers CD154-CD40 activation in liver IRI. First, we determined gene expression profiles of CD40 and CD154 in livers during IR byquantitative reverse-transcription polymerase chain reaction. Indeed, CD154 levels were low, with no significant changes observed throughout the 6-hour reperfusion period. In contrast, CD40 levels increased in livers undergoing IR and peaked at 2 to 4 hours post-reperfusion (Fig. 7A). As macrophage CD40 represents the major receptor of CD154 on CD4 T cells for immune activation, we next determined whether CD40 macrophage expression was responsive to toll-like receptor 4 (TLR4) stimulation. In addition, as type I IFNs represent the major functional pathway downstream of TLR4 activation in liver IRI,11 we also attempted to determine the effect of IFN-β on macrophage CD40 expression. As shown in Fig. 7B, both lipopolysaccharide and IFN-β up-regulated CD40 expression in macrophages. Thus, CD40 up-regulation in response to TLR4-type I IFN activation may represent the triggering event in the activation of the CD154-CD40 pathway during liver IR.
Although the role of CD4 T cells in the pathogenesis of organ IRI has been well documented,4, 7 the actual mechanisms remain elusive. The proinflammatory role of T cells in IRI has been recently questioned by the observation that CD4-deficient mice suffered more severe hepatic IRI in comparison with WT controls, despite decreased local neutrophil accumulation.27 Furthermore, recombination activation gene 1 KO mice, lacking both T and B cells, were found to be susceptible to renal IRI, whereas T cell reconstitution in these KO mice exerted cytoprotection.28 In this study, we first confirmed the pathogenic role of CD4 T cells in the murine warm hepatic IRI model. Our results document the selective impact of CD4 cells, but not CD8 or NK1.1 cells, in promoting tissue proinflammatory immune response and injury. As no specific Ags are associated with IRI, we then tested whether de novo Ag-specific CD4 T cell activation is required for their function. The CD4 blockage interrupts CD4 T cell signaling, and this effectively inhibits CD4 T cell activation in transplant recipients23 and CD4 T cell proliferation in MLRs. However, in contrast to the effects of CD4 depletion, CD4 blockade failed to suppress liver proinflammatory response against IR in our study. These data provide direct evidence that CD4 T cells function in liver IRI without the requirement of de novo Ag-specific activation. This conclusion is supported by a recent study in which anti–major histocompatibility complex class II Ab failed to affect liver IRI.29 To dissect the functional mechanism of CD4 T cells in liver IRI, we then differentiated the roles of CD154 and IFN-γ. Consistent with our previous study,11 our present data show that IFN-γ was dispensable for both liver inflammation and injury against IR. To further analyze the role of CD154, we used agonist Ab against CD40 in CD4 KO mice to test the hypothesis that CD4 T cells function by CD154-triggered CD40 signaling in innate immune cells to promote tissue inflammation. Our results confirm that agonist Ab did indeed restore liver proinflammatory response and tissue injury in otherwise protected CD4 KO mice. These data are consistent with our previous findings in CD154 KO mice or after the blockade of CD154 in WT mice.6, 8, 30 However, the mechanism of CD154 in liver IRI suggested by our current data is not the inhibition of T cell activation per se but rather the interruption of T cell help for innate immune activation. Indeed, the ligation of CD40 by soluble or cellular CD154 in macrophages/dendritic cells leads to cell activation with increased TNF-α production in vitro.31–33 Thus, our results indicate that CD4 T cells function in liver IRI via CD154-mediated CD40 signaling without the requirement of de novo Ag-specific activation.
As activated CD4 T cells express CD154,34–36 the absence of de novo CD4 T cell activation during IR suggests that only previously activated CD4 T cells may provide the key help for liver innate immune activation. Indeed, FACS analysis of liver T cells revealed the presence of a higher percentage of a proinflammatory effector (CXCR3+CD62Llow) CD4 subset in liver resident lymphocytes in comparison with splenocytes. Importantly, these effector CD4 T cells in the liver constitutively express higher levels of CD154. Thus, these cells have the capability of triggering CD40 signaling. The question is what triggers the activation of the CD154-CD40 pathway during IR. As liver CD40 expression is up-regulated during IR, we hypothesize that CD40 up-regulation may represent the triggering event of CD154-CD40 activation. Indeed, the agonist anti-CD40 by itself triggered only mild liver injury at later time points, that is, more than 12 hours post-injection,37 and it synergized with IR to activate a liver proinflammatory response within 6 hours in our model. Additionally, we have presented in vitro evidence that both TLR4 ligands and type I IFN can up-regulate CD40 expression in macrophages.
As TLR4 activation (particularly its downstream IFN regulatory factor 3–mediated type I IFN pathway) has been recently shown to represent the major pathway initiating a liver proinflammatory response during IR,20, 38, 39 the obvious question arises of the relationship between CD4 T cell function and TLR4 activation [particularly whether CD154-CD40 signaling may synergize with TLR4 in macrophage (or Kupffer cell) activation]. The demonstration that agonist anti-CD40 Ab up-regulated the TLR4–myeloid differentiation 2 complex in murine dendritic cells without increasing TLR4 transcript levels40 provides a potential synergistic mechanism of CD40 and TLR4 signaling in macrophage activation. This may be particularly relevant to the liver innate immune system, as liver dendritic cells, possibly Kupffer cells, express lower TLR4 levels.41 As TLR4 activation up-regulates macrophage CD40, it may in turn enhance their response to CD4-derived CD154 signaling, which constitutes another putative synergistic mechanism. In vivo, Ag immunization with combined TLR/CD40 stimulation elicited potent cellular immunity exponentially greater than that with any single stimulation.42, 43 In addition, CD40 may synergize with TLR4 in different aspects of liver IRI. As hepatocyte CD40 activation leads to their death, CD40 signaling may also enhance TNF-α or reactive oxygen species–induced liver injury resulting from TLR4 activation.
In summary, this study provides evidence that effector CD4 T cells reside in livers and facilitate liver inflammation/tissue damage during IR by CD154 without de novo Ag-specific activation. These novel findings highlight the unique function of previously activated CD4 T cells in regulating tissue innate immune response.