Published online before print July 15, 2003
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Department of Internal Medicine, Division of Digestive and Liver Diseases, University of Texas Southwestern Medical Center at Dallas
1Correspondence: Department of Internal Medicine, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9151. E-mail: dwain.thiele{at}utsouthwestern.edu
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expressing intrahepatic lymphocytes (IHL) were similar in AdCMV-lacZ-infected, TNF-deficient, TNFR1-deficient, TNFR2-deficient, and control mice. However, IHL isolated from AdCMV-lacZ-infected, TNF-deficient or AdCMV-lacZ-infected, TNFR2-deficient mice exhibited decreased levels of FasL expression and adenovirus-specific cytolytic T lymphocyte (CTL) activity. Similar defects in allo-specific killing of Fas-sensitive hepatocyte targets by TNF-deficient or TNFR2-deficient but not TNFR1-deficient CTL were also noted. No defects in generation of allo-specific cytotoxicity directed against perforin-sensitive target cells were noted in TNF-, TNFR1-, or TNFR2-deficient lymphocytes. These findings indicate that TNF/TNFR2 interactions facilitate generation of FasL-dependent CTL effector pathways that play an important role in in vivo antiviral-immune responses in the liver.
Key Words: viral • cytokine receptors • cytokines • cytotoxicity
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In addition to the proinflammatory and direct antiviral effects of TNF, other actions of TNF during adaptive cellular-immune responses have been noted. TNF/TNFR1 and TNF/TNFR2 signaling pathways have been found to potentiate T cell proliferation and T helper 1 (Th1) cytokine responses [12 , 13 ]. As clearance of noncytopathic viruses is largely dependent on CD8+ T cell responses [14 , 15 ], any immunomodulatory effects of TNF on generation of this component of the acquired-immune response are likely to play an important role in the course of antiviral-immune responses that develop during the course of naturally occurring infection by hepatotropic viruses or following liver-directed gene therapies that use recombinant viral vectors.
The present studies were designed to better define the potential role of TNF, TNFR1, and TNFR2 in antiviral-immune responses. As previously reported [6 ], markedly delayed clearance of a ß-galactosidase-encoding recombinant adenovirus (AdCMV-lacZ) was observed in TNF-deficient mice. Less-pronounced delays in AdCMV-lacZ clearance were also observed in TNFR2-deficient but not TNFR1-deficient mice. In contrast to prior inferences derived from histological assessments [6 7 8 ], normal total numbers of liver-infiltrating lymphocytes were isolated from TNF-deficient mice following AdCMV-lacZ infection. However, defects in generation of FasL-dependent cytolytic T lymphocyte (CTL) effector pathways were apparent in intrahepatic CD8+ lymphocytes isolated from AdCMV-lacZ-infected, TNF-deficient, or TNFR2-deficient recipient animals, indicating that TNF/TNFR2 interactions play a previously unrecognized role in the development of other cytopathic immune mechanisms in the liver.
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Adenovirus vectors
The E1-deleted, replication-deficient, ß-galactosidase-encoding recombinant adenovirus AdCMV-lacZ was propagated in 293 cell cultures and purified on a cesium chloride gradient, and titers of infectious virus were determined by plaque assay as previously described [17
]. Mice were injected with 109–1010 plaque-forming units (PFU) AdCMV-lacZ. In each experiment a constant number of PFU/g body weight were administered to each mouse. Target cells for virus-specific cytotoxicity assays were infected with AdCMV-lacZ at a multiplicity of infection of five to 10 overnight. In other experiments, serum was collected from mice infected with a replication-deficient adenoviral vector encoding a chimeric fusion protein consisting of the extracellular domain of the human 55-kD TNFR linked to the hinge and Fc regions of murine immunoglobulin G1 (IgG1; TNFR-Ig; ref. [18
]). TNFR-Ig or control mouse Ig was purified from the sera by adherence to protein G sepharose (Amersham Pharmacia Biotech AB, Uppsala, Sweden) columns.
ß-Galactosidase assay
ß-Galactosidase activity was quantified by measuring the rate of cleavage of 4-methylumbelliferyl-ß-D-galactoside to yield the fluorescent product 4-methylumbelliferone [19
]. Tissue samples were washed in phosphate-buffered saline (PBS). The tissues were then homogenized in a buffer containing 25 mmol/L Tris-HCl at pH 7.5, 125 mmol/L NaCl, and 2 mmol/L MgCl2 and were then centrifuged at 15,000 g. Then, 40 µL supernatant or the supernatant diluted with reaction buffer was incubated at 37ºC for 30 min with 160 µL reaction mixture containing 25 mmol/L Tris-HCl at pH 7.5, 125 mmol/L NaCl, 2 mmol/L MgCl2, 12 mmol/L 2-mercaptoethanol, and 0.3 mmol/L 4-methylumbelliferyl-ß-D-galactoside (Sigma Chemical Co., St. Louis, MO). The reactions were stopped by adding 50 µL 25% trichloroacetic acid. Tubes were cooled on ice for 5–10 min and then centrifuged at high speed for 1–2 min. Thereafter, 100 µL supernatant was added to 1.9 mL glycine-carbonate reagent. Light emission at 460 nm after excitation at 365 nm was compared with emission by standard concentrations of 4-methyl-umbelliferone purchased from Sigma Chemical Co.
Protein assay
Protein concentrations in tissue homogenates were assayed by the bicinchoninic acid method using reagents purchased from Pierce Chemical Co. (Rockford, IL) and using bovine serum albumin as a standard [20
].
Isolation of hepatocytes and [3H] thymidine labeling
Anesthetized mice underwent laparotomy, and a catheter was introduced into the inferior vena cava through the right atrium. The portal vein was then severed, the catheter connected to a peristaltic pump, and the liver perfused at 5 mL/min for 2 min with preperfusion buffer: NaCl (0.14 M), KCl (5.4 mM), Na2HPO4 (0.8 mM), HEPES (25 mM), EGTA (12.5 mM), sodium pyruvate (2.3 mM), L-glutamine (2.3 mM), D-glucose (0.5 mM), pH 7.4. The liver was then perfused for 10 min with the perfusion medium: NaCl (0.14 M), KCl (5.4 mM), Na2HPO4 (0.8 mM), HEPES (25 mM), sodium pyruvate (2.3 mM), L-glutamine (2.3 mM), D-glucose (0.5 mM), CaCl2 (2 mM), MgSO4 (0.8 mM), collagenase A (0.163 U/mL, Boehringer-Mannheim, Mannheim, Germany), and DNAse I (0.004%, Sigma Chemical Co.). The perfused liver was then removed, placed in a sterile dish containing William’s medium (Life Technologies, Inc., Gaithersburg, MD), and then passed through a 100-µ nylon mesh to obtain a single-cell suspension. Hepatocytes were then centrifuged with 50% percoll (Amersham Pharmacia Biotech AB) at 500 rpm for 15 min to separate dead cells. Hepatocytes were cultured in plates coated with 1% collagen type I (Sigma Chemical Co.) in William’s medium supplemented with fetal bovine serum (FBS; 10%), HEPES (25 mM), penicillin (100 U/mL), streptomycin (100 µg/mL), fungisone (5 µg/mL), insulin (10 µg/mL), transferrin (10 µg/mL), selenous acid (10 ng/mL), dexamethasone (1 µM), epidermal growth factor (5 nm/mL), glucagon (0.1 µM), somatotropin (10 µU/mL), and prolactin (20 mU/mL), all purchased from Sigma Chemical Co. and added to the medium immediately before use. For [3H] thymidine labeling, the cells were incubated for 24–48 h in the same medium with 5–10 µCi/ml [3H] thymidine, recombinant human hepatocyte growth factor (15 ng/mL; Calbiochem, La Jolla, CA), and norepinephrine (10-5 M; Sigma Chemical Co.).
Isolation of hepatic lymphocytes
Intrahepatic lymphocytes (IHL) were isolated as previously detailed [9
, 21
]. Briefly, after intraperitoneal injection with 10 U heparin and CO2 narcosis, the abdomen was entered under sterile technique, the portal vein cut, and the abdominal portion of the vena cava perfused with 20 mL Ca2+- and Mg2+-free PBS preheated to 37ºC. The liver was removed and passed through a 40-mesh screen and then a 300-mesh screen (VWR Scientific, West Chester, PA, cat. #EC587-40 and EC589-300, respectively). The cell suspension was centrifuged with 35% percoll at 1500 rpm for 15 min, and the cell pellet was cultured in a 75-cm2 flask in complete medium supplemented with 10% FBS (Life Technologies), 100 U/mL penicillin, 1 µg/mL gentamycin, and 2 mM L-glutamine (Sigma Chemical Co.) for 4 h. Then, the lymphocytes were aspirated and centrifuged again with 35% percoll. Afterwards, cells were used as effectors in cytotoxicity assays or for flow cytometric studies.
Flow cytometric analysis
Spleen cells, IHL, or cultured cell lines were washed and incubated for 30 min at 4ºC with fluorescein isothiocyanate (FITC)-labeled anti-CD4 (L3T4, GK1.5), anti-CD8a (Ly-2, 53-6.7), anti-T cell receptor (TCR)
ß (H57-597), anti-TCR
(GL3), anti-natural killer 1.1 (DX5), anti-H-2Kq (KH114), or the appropriate isotype control. In other experiments, cells were washed and incubated for 30 min at 4ºC with unconjugated anti-H-2Kb (Y-3) or anti-H-2Dq (28-14-8s) or the appropriate isotype control and then washed and incubated for an additional 20 min at 4ºC with FITC-labeled goat anti-mouse IgG. All antibodies used were purchased from BD Pharmingen (San Diego, CA) or were produced as culture supernatant of hybridomas purchased from American Type Culture Collection (ATCC; Manassas, VA). The cells were analyzed by fluorescence-activated flow cytometry on a FACScan. For assessment of intracellular interferon- (IFN-) and FasL expression, cells were incubated in 5% CO2, 37ºC incubator with phorbol 12-myristate 13-acetate (50 ng/ml), A23187 (500 ng/ml), and Brefeldin A (10 µg/ml) for 4 h. Cells were then labeled with the surface-staining antibodies and fixed with 4% formaldehyde at room temperature for 10 min. After fixation, the cells were incubated on ice for 1 h with saponin-containing medium to permeabilize the membranes. The phycoerythrin (PE)-labeled anti-IFN- (XMG1.2) or PE-labeled anti-Fas ligand (FasL, Jo-2) antibodies were added and incubated at 4ºC for 1 h. This was followed by two washes with saponin-containing medium and one final wash with normal staining medium. The cells were analyzed by fluorescence-activated flow cytometry on a FACScan. Representative dot plot results obtained with these flow cytometric techniques have been published previously [9
].
Cell lines
The nontransformed AML-12 hepatocyte cell line (H-2Kb,Dq, [9
]) and the P815 mastocytoma cell line (H-2d) were purchased from ATCC.
Generation of allo-specific CTL
In vitro-activated allo-specific CTL were generated in 5-day mixed lymphocyte cultures (MLC) containing 10–12 million responder spleen cells from B6 mice and an equal number of irradiated FVB or DBA/2J spleen cells to generate anti H-2q- and H-2d-specific CTL, respectively, as previously described [9
, 22
].
Chromium release assay
Targets were labeled with 150 µCi Na2CrO4 for 60–90 min at 37ºC and washed twice before incubation with the different effectors at different effector:target ratios in 200 µL cultures. After 12 or 18 h, 100 µL supernatant was harvested from experimental and control wells and the percentage of specific lysis, as calculated from the formula: % Specific lysis = experimental release (cpm) - spontaneous release (cpm)/maximal release (cpm) - spontaneous release (cpm) x 100.
Virus-specific cytotoxicity was calculated by subtracting the percent-specific lysis of noninfected targets from percent-specific lysis of infected targets [9 , 22 ]. In some assays, 1 µg protein G sepharose-purified TNFR-Ig or control Ig was added to each well of cytotoxicity assays. All the assays were performed in triplicate, and results shown use mean ± SEM.
JAM test for apoptosis (DNA fragmentation)
[3H] Thymidine was added to culture dishes for 12–18 h at a final concentration of 2.5–5 µCi/mL. Targets were harvested and cultured with effector cells as in the chromium release assay. At the end of the assay, cells were aspirated onto fiberglass filters by vacuum suction, DNA dried, and [3H] thymidine-labeled high molecular weight DNA quantitated by scintillation counting. The specific DNA fragmentation was determined using the following formula: % Specific DNA fragmentation = control (cpm) - experimental (cpm)/control (cpm) x 100, and control cpm is the total [3H] thymidine retained in absence of effector cells [23
].
Statistical analysis
Comparison between continuous variables was made by nonparametric ANOVA using Sigma Stat (SPSS Inc., Chicago, IL) software.
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Figure 1. Time course of clearance of AdCMV-lacZ from livers of B6.TNF–/– (TNF-deficient), B6.TNFR1–/– (TNFR1-deficient), B6.TNFR2–/– (TNFR2-deficient), and control B6 mice. Three to four mice per experimental group were infected with AdCMV-lacZ, and ß-galactosidase expression in the liver was assessed at the indicated time points. AFU, Absolute fluorescence units.
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Figure 2. Increase in number of IHL following AdCMV-lacZ infection. Total numbers of IHL isolated from uninfected B6 ( ) and AdCMV-lacZ-infected B6 (•), B6.TNF–/– ( ), B6.TNFR1–/– ( ), and B6.TNFR2–/– ( ) are displayed in the left panel, and the absolute numbers of intrahepatic CD8+ T cells, as determined by flow cytometric analysis, are detailed in the right panel. Results represent the mean ± SEM of three to five mice from each experimental group.
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in intrahepatic CD8+ or CD8- lymphocytes expression, we found that the percentage of CD8+ or CD8- cells expressing IFN- was similar in all strains (left panels), and the total number of IFN--expressing IHL recovered from each strain 7 days after AdCMV-lacZ infection, was not significantly decreased in any of the strains with TNF or TNFR deficiency (data not shown). Of interest, however, because of a lower absolute number of CD8+ IHL in the livers of TNF-deficient mice (Fig. 2)
, there was a modest decrease in number of IFN--expressing CD8+ cells in these mice (Fig. 3
, upper right), but because of a reciprocal increase in the number of CD8 (–) IFN-expressing IHL in these TNF-deficient mice (Fig. 3
, lower right), the total IFN- response was indistinguishable from that of other strains.
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Figure 3. IFN- expression in CD8+ or CD8- IHL following AdCMV-lacZ infection. IHL were isolated from B6 (•), B6.TNF–/– (), B6.TNFR1–/– (), and B6.TNFR2–/– () mice 7 days after infection with AdCMV-lacZ and were double-stained with anti-CD8 and anti-IFN-, as detailed in Materials and Methods. The graphs show the percentages (left panels) and the absolute numbers (right panels) expressed as mean ± SEM of three to four determinations per strain.
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Figure 4. FasL expression in CD8+ IHL following AdCMV-lacZ infection. IHL were isolated from B6 (•), B6.TNF–/– (), B6.TNFR1–/– (), and B6.TNFR2–/– () mice 7 days after infection with AdCMV-lacZ and were double-stained with anti-CD8 and anti-FasL, as detailed in Materials and Methods. The results are seen as mean ± SEM from three staining experiments.
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Figure 5. Virus-specific killing of AdCMV-lacZ-infected B6 hepatocyte targets. IHL isolated from B6.TNF–/– () and B6 (•) livers 7 days after infection with AdCMV-lacZ were assessed for their ability to kill control or AdCMV-lacZ-infected B6 hepatocytes in 18-h assays (Exp #1). Experiment 2 shows the virus-specific killing of B6 hepatocytes by IHL isolated from AdCMV-lacZ-infected B6 (•), B6.TNF–/– (), B6.TNFR1–/– (), and B6.TNFR2–/– () mice. Virus-specific killing was determined by subtracting the background killing of noninfected control targets from that of infected targets. Results detailed in this figure are representative of three separate experiments assessing killing of AdCMV-lacZ-infected hepatocytes by IHL from each mouse strain.
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Figure 6. CTL killing of allo-specific hepatocyte or nonhepatic target cells. B6 (•, ), B6.TNF–/– (), B6.TNFR1–/– (), and B6.TNFR2–/– () effector cells were generated in 5-day MLC with stimulator cells from FVB (H-2q) mice (left panel) or DBA/2J (H-2d) mice (right panel) and were assessed for killing of AML-12 targets in 18-h assays and for killing of P815 targets in 5-h assays. Results are representative of four experiments with similar results.
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The present findings are among only a limited number of observations that provide insight into in vivo functions of TNFR2 [25 ]. Soluble and membrane-bound TNF have similar agonist activity for TNFR1, whereas TNFR2 preferentially responds to membrane-bound TNF. Therefore, it is not surprising that TNF/TNFR1 interactions appear to play a more important role in the proinflammatory and cytotoxic effects of soluble TNF released by macrophages early in the course of innate-immune responses [24 , 25 ]. In contrast, the present findings as well as those from a variety of previous studies support a role for TNF/TNFR2 interactions in adaptive cellular-immune responses. Prior studies have indicated a role for TNF/TNFR2 signaling in thymocyte and T lymphocyte proliferative responses [12 , 13 , 26 ] and in promotion of Th1 cytokine responses during major histocompatibility complex class II disparate graft-versus-host disease (GVHD) [12 ]. Although the present studies are the first to indicate a role for TNF/TNR2 signaling in generation of CD8+ CTL effector function, other investigators have also observed a role for TNF in promotion of allo-specific CTL responses during GVHD [27 ], and other TNF family members, such as CD70 and LIGHT [homologous to lymphotoxins, exhibits inducible expression, competes with herpes simplex virus glycoprotein D for herpes virus entry mediator (HVEM), a receptor expressed by T lymphocytes], promote generation of CD8+ CTL effector function [22 , 28 , 29 ]. Of note, such CTL activation by CD70 and LIGHT is mediated by engagement of TNFR2-like receptors that bind adaptor proteins capable of mediating protean intracellular activation signals. Thus, future studies examining common TNF/TNFR2, CD70/CD27, and LIGHT/HVEM signaling pathways in CD8+ T cells may provide insight into common mechanisms used by TNF/TNFR family members for potentiation of antiviral CTL responses.
Although direct effects of soluble TNF on viral replication and/or virally encoded gene expression have been observed in other viral infection models [4 , 5 ], the kinetics of AdCMV-lacZ transgene expression observed in the present studies in mice with selective deficiencies in TNF, TNFR1, or TNFR2 argues that TNF has no significant direct, noncytopathic effect on virally encoded gene expression in this experimental model. These differences likely relate to differences in adenoviral cytomegalovirus promoter constructs, as direct effects of TNF on adenoviral vector reporter gene expression have been noted to be promoter-dependent [5 ]. Prior studies have observed vigorous TNF responses within 24–48 h of recombinant adenovirus infusion into mice [30 , 31 ]. Yet, 3 days after infection with AdCMV-lacZ, levels of ß-galactosidase expression in TNF-deficient, TNFR1-deficient, and TNFR2-deficient mice were not significantly different than observed in wild-type B6 mice. Only later in the course of AdCMV-lacZ infection, at time points when adaptive CTL responses had been elicited, were statistically significant differences in levels of adenoviral transgene expression observed in mice with deficiencies in TNF and/or TNFR expression.
It has been postulated previously that impaired intrahepatic antiviral-immune responses in TNF-deficient mice may be related to diminished induction of adhesion molecule expression and secondary decreases in leukocyte migration into the liver of infected animals [6 7 8 ]. However, in the present studies, no decrease in total numbers of IHL was detected in B6.TNF–/–, B6.TNFR1–/–, or B6.TNFR2–/– mice 7 days after AdCMV-lacZ infection, a time point previously found to correspond to the peak of intrahepatic T lymphocyte infiltration and expansion in wild-type B6 mice [9 ]. These findings suggest that mechanisms other than defects in lymphocyte migration to the liver likely account for previously noted differences in histologic patterns of inflammation found in the livers of adenovirally infected TNF-deficient mice. Of note, in contrast to the absence of inflammatory responses to apoptotic cell death in other organs, induction of hepatocyte apoptosis, as occurs during CTL killing of virally infected hepatocytes, is associated with an inflammatory response characterized histologically by foci of mixed inflammatory cells that accumulate initially at sites of hepatocyte death [32 , 33 ]. In the present studies, diminished intrahepatic CD8+ T cell FasL expression and decreased IHL cytotoxic effector function were noted in TNF-deficient and TNFR2-deficient mice during adenoviral infection. Thus, defects in CTL killing of adenovirally infected hepatocytes and a secondary diminished inflammatory response afford the best explanation for prior reports of diminished foci of inflammatory cell infiltrates and the present observations indicating normal expansion of intrahepatic T lymphocytes in the livers of adenovirus-infected, TNF-deficient mice.
In the present studies, defects in adenovirus clearance were more pronounced in TNF-deficient mice than in mice with selective deficiency of TNFR1 or TNFR2. One potential explanation for this observation is that abnormalities unique to TNF-deficient mice account for more severe defects in antiviral immunity. TNF-deficient and TNFR1-deficient mice exhibit defects in germinal center formation [16 , 34 ]. However, as TNFR1-deficient mice exhibit no delay in adenoviral clearance (present studies), and inhibition of TNF activity by expression of circulating TNF inhibitor proteins in mature, wild-type mice also leads to prolonged adenoviral gene expression comparable with what is observed in TNF-deficient mice [7 , 8 ], defects in lymphocyte development do not appear to explain impaired antiviral immunity in TNF-deficient animals. An alternative explanation for the present findings is that complementary and overlapping roles of TNF/TNFR1 and TNF/TNFR2 signals in antiviral-immune responses are responsible for the much more severe delay in adenoviral clearance observed in TNF-deficient mice than is observed in animals deficient in only a single TNFR. For instance, in addition to the role for TNF/TNFR2 signaling during T cell activation in generation of FasL-dependent, cell-mediated cytotoxicity, TNF engagement of TNFR1 on hepatocytes represents an independent pathway for cytopathic removal of virally infected hepatocytes [9 , 11 ]. Of note, signaling via TNFR1 and TNFR2 has been reported to be necessary for hepatotoxicity mediated by transmembrane TNF [35 ]. Finally, TNFR2 signaling has been noted to potentiate FasL-mediated cytotoxicity [36 ]. Thus, various TNF/TNFR signaling pathways appear to be important in the generation of activated, cytotoxic effector cells and in the effector phase of antiviral immunity, and the additive and synergistic nature of these immune effector mechanisms likely accounts for the much more dramatic impairment of antiviral immunity in TNF-deficient mice than is noted in mice deficient in only a single TNFR.
Although decreases in T cell-proliferative responses and IFN- expression by TNFR1- and TNFR2-deficient T cells have been observed in other experimental models [12
, 37
, 38
], in the present studies, only modest impairment in expansion of intrahepatic CD8+ T cells was noted in TNF-deficient mice, and no change was in total IHL expansion or IFN- expression. Mice deficient in TNFR1 or TNFR2 exhibited no alterations in intrahepatic T cell expansion or IFN- expression. Yet, in TNF-deficient and TNFR2-deficient mice, impairment of CD8+ T cell FasL expression and FasL-dependent cytotoxicity was apparent. These findings suggest a hierarchy in levels of TNF-mediated, costimulatory signals required for optimal T cell responses in this experimental model, and generation of FasL-dependent CTL effector function is most dependent on TNF costimulation. Such differences in the signaling events needed to generate FasL-dependent CTL effector function versus other T cell effector functions have been reported previously [39
].
In summary, the present studies provide evidence for a previously unrecognized role for TNF/TNFR2 interactions in promotion of FasL-dependent CTL effector functions during in vivo antiviral-immune responses in the liver. Prior studies have found hepatocytes to be resistant to killing via perforin-dependent mechanisms [9 , 11 ] and have identified no significant role for perforin in clearance of viral infections from the liver [9 ]. Thus, it is likely that TNF plays a more prominent role in antiviral-immune responses in the liver, because of the greater dependence on both TNF-mediated and TNF-potentiated, cytopathic mechanisms in clearance of viral infections from this organ.
Received January 22, 2003; revised May 30, 2003; accepted June 11, 2003.
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