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The Journal of Immunology, 2001, 166: 5619-5628.
Copyright © 2001 by The American Association of Immunologists

IL-12 Antagonism Enhances Apoptotic Death of T Cells Within Hepatic Allografts from Flt3 Ligand-Treated Donors and Promotes Graft Acceptance1

Wei Li, Lina Lu, Zhiliang Wang, Lianfu Wang, John J. Fung, Angus W. Thomson and Shiguang Qian2

Thomas E. Starzl Transplantation Institute and Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA 15213


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mouse livers are accepted across MHC barriers and induce donor-specific tolerance without immunosuppressive therapy. By contrast, livers from donors treated with Flt3 ligand, which dramatically increases hepatic interstitial dendritic cells, are rejected acutely (median survival time 5 days). This switch from tolerance to rejection is associated with a marked reduction in apoptotic activity of graft-infiltrating cells. We hypothesized that IL-12 production by enhanced numbers of donor APC might inhibit apoptosis, promote expansion of Th1 cells, and play a key role in liver rejection. Therefore, C3H (H2k) recipients of liver grafts from Flt3 ligand-treated B10 donors were given neutralizing anti-IL-12 mAb (200 or 500 µg) on days 0 and 2 after transplant. Graft survival was markedly prolonged at the higher mAb dose, with 50% of grafts surviving >100 days. This effect was associated with reductions in IFN-{gamma} gene transcripts within the graft-infiltrating cell population and with reductions in circulating IFN-{gamma} and IL-10 levels, donor-specific CTL and NK cell activities, and circulating alloantibody levels. At the same time, there were marked increases in apoptotic (TUNEL+) CD4+ and especially CD8+ cells, both within the grafts and in spleens of anti-IL-12 mAb-treated mice. In vitro, exogenous IL-12 inhibited apoptotic death induced in naive allogeneic T cells by liver nonparenchymal cells. These findings suggest that suppression of rejection by IL-12 antagonism, linked to restoration of apoptotic activity within the peripheral alloreactive T cell population, is important for liver allograft survival and tolerance induction.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mouse liver allografts, unlike those of heart or skin, are accepted across MHC barriers and induce donor-specific tolerance without anti-rejection therapy (1). The mechanism(s) underlying liver transplant tolerance remains unclear. It has been argued that immunomodulatory effects of soluble donor MHC class I Ag, immunosuppressive hepatocyte products, and the regenerative capacity of the liver may be contributory factors (2, 3). Recently, attention has focussed on the possible role of donor hematopoietic cells, in particular dendritic cells (DC),3 in the modulation of host responses to hepatic allografts (1, 4, 5, 6). Thus, donor liver-derived DC progenitors, deficient in surface costimulatory molecules (e.g., CD40, CD80, and CD86), which are poor allostimulators (7, 8, 9), can prolong allograft survival (10). This regulatory effect may be linked to the capacity of DC lacking adequate costimulatory activity to enhance the apoptotic death of alloreactive T cells (11) or to promote immune deviation (12). In contrast, fms-like tyrosine kinase 3 ligand (FL; Ref. 13)-induced increases in donor interstitial DC that mature ex vivo into potent APC (14) precipitate acute liver graft rejection (15). These findings point to a pivotal role for donor DC in determining the outcome of liver transplantation. In both mice and rats, there is evidence that "spontaneous" liver tolerance is associated with the apoptotic death of graft-infiltrating cells (GIC) and of alloreactive T cells within secondary lymphoid tissue (16, 17). Although a role for donor-derived DC and their immunomodulatory products has been implicated in these events, their function has not been elucidated.

IL-12 is a heterodimeric proinflammatory cytokine produced primarily by DC and macrophages that drives the preferential induction of Th1 immune responses (18). It binds to a unique, high-affinity receptor on activated Th cells and NK cells, enhances the expression of antiapoptotic factors (bcl2 and bclxl), and facilitates activated T cell and NK-lymphokine activated killer cell expansion (19). IL-12 indirectly promotes Th1 and inhibits Th2 development by inducing the secretion of IFN-{gamma} by Th1 and NK cells (20, 21, 22, 23). In addition, IL-12 exerts direct stimulatory and inhibitory effects on Th1 and Th2 cells, respectively (24). In animal models, IL-12 promotes profound anticancer effects. Thus, its in vivo administration prevents the development of transplanted tumors, inhibits growth of established primary or metastatic tumors, and induces anti-tumor immunity (25, 26, 27). IL-12 also promotes cellular immunity against alloantigens. In mice, it appears to be a central mediator of acute graft-vs-host disease (GVHD; Refs. 28, 29), whereas neutralization of bioactive IL-12 enhances allogeneic myoblast survival (30). In contrast, IL-12 antagonism has been reported to exacerbate murine cardiac allograft rejection (31). These and other findings point to a key role for IL-12 in the regulation of alloimmune responses.

It has been reported that immature, costimulatory molecule-deficient DC, which resemble normal interstitial liver DC and fail to secrete IL-12, can inhibit heart allograft rejection (32). We hypothesized that the acute rejection of liver allografts from FL-treated donors (15) might be mediated by IL-12 produced by enhanced numbers of donor-derived DC and other APC. Liver DC mature rapidly in vitro into potent inducers of Th1 responses (14). A comparison of livers from FL-treated and control mice confirmed that FL markedly up-regulated expression of IL-12 in situ, in association with large numbers of interstitial DC. Systemic administration of neutralizing anti-IL-12 mAb inhibited the rejection of liver allografts from FL-treated donors. This was associated with restoration of comparatively high levels of apoptotic activity, both in the liver T cell infiltrate and the spleen, and with suppression of anti-donor CTL and NK cell activities. Thus, in this model, donor APC-derived IL-12 appears to play a key role in inhibiting the apoptotic death of donor-reactive T cells, and consequently, in determining the balance between liver transplant tolerance and rejection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Male C57BL/10 (B10; H2b), C3H (H2k), and BALB/c (H2d) mice 8–12 wk of age were purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained in a specific pathogen-free facility of the University of Pittsburgh Medical Center (Pittsburgh, PA). The mice were provided with Purina rodent chow and tap water ad libitum. Animal care was in compliance with the "Principles of Laboratory Animal Care" and the "Guide for the Care and the Use of Laboratory Animals" published by the National Institutes of Health (Bethesda, MD).

Liver transplantation

Orthotopic liver transplantation, in which revascularization was with a combination of suture and cuff technique, was performed from B10 donors to C3H recipients under inhalation anesthesia with methoxyflurane (Medical Developments, Melbourne, Australia) as described previously (33). The hepatic artery was not reconstructed. Allograft survival was determined by recipient survival and rejection was confirmed histologically.

FL and anti-IL-12 mAb administration

Recombinant human FL derived from Chinese hamster ovary cells (10 µg in 200 µl of sterile HBSS; Immunex Research and Development, Seattle, WA; Ref. 34) was administered by daily i.p. injection from days -10 to -1 before liver transplantation on day 0. Control animals received PBS (200 µl) by an identical regimen. Neutralizing hamster anti-mouse IL-12 mAb generated from the hybridoma "Red-T" (a gift from Dr. E. R. Unanue, Washington University, School of Medicine, St. Louis, MO; Ref. 35) was injected (200 or 500 µg/day) on day 0 and on day 2 after transplantation via the lateral tail vein. Control animals received equivalent amounts of hamster IgG (BD PharMingen, San Diego, CA).

Liver nonparenchymal cell (NPC) isolation

NPC were isolated from liver tissue by collagenase digestion followed by Percoll centrifugation as described previously (8). The NPC were comprised predominantly (at least 95%) of CD45+ leukocytes in both control and FL-treated animals.

Immunohistochemistry

IL-12 expression in tissues was determined by an avidin-biotin-alkaline phosphatase complex (ABC) staining procedure. Cryostat sections were fixed in acetone at -20°C for 10 min followed by protein blocking buffer (Shandon, Pittsburgh, PA). Endogenous peroxidase activity was quenched in 2% H2O2 before addition of goat anti-mouse IL-12 (p40) Ab (1:200; 1 h at room temperature (RT); Santa Cruz Biotechnology, Santa Cruz, CA) followed by donkey anti-goat IgG (1:400; 30 min, RT). Goat polyclonal IgG was used as isotype control. ABC (Vector Laboratories, Burlingame, CA) then was added. Aminoethylcarbazole AEC (ScyTek, Logan, UT) was used as the substrate, and sections were counterstained with hematoxylin. Two-color staining of CD11c+ cells and IL-12 p40 in liver sections was performed by sequential avidin-biotin peroxidase and avidin-biotin alkaline phosphatase labeling, respectively, as described (36).

Determination of CTL and NK cell activities

Freshly isolated liver NPC or spleen cells from either allograft recipients or control C3H mice were used as effectors at various E:T cell ratios in 4-h 51Cr release assays, as described (16). The EL4 (H2b) lymphoma cell line (TIB39) and the P815 (H2d) cell line (TIB64; both obtained from American Type Culture Collection, Manassas, VA) were used as sources of donor-specific and third-party targets; YAC-1 tumor cells were used as NK cell targets. The percentage of specific cytotoxicity was calculated with the formula: % cytotoxicity = 100 x [experimental (cpm) - spontaneous (cpm)]/[maximum (cpm) - spontaneous (cpm)]. Results are expressed as means ± SD of % specific 51Cr release in triplicate cultures.

Apoptosis

Apoptotic cells in tissue sections were detected with the In Situ Apoptosis Detection kit (Intergen, Purchase, NY) as described previously (37) with some modifications. Cryosections (4 µm) were mounted on precleaned slides, air-dried overnight at RT, and then fixed for 10 min at RT in 10% neutral-buffered formaldehyde (pH 7.4) followed by two washes (5 min each) in PBS. Endogenous peroxidase activity was quenched in 2% H2O2 before exposure to TdT at 37°C for 60 min. After washing in stop wash buffer (37°C, 60 min), anti-digoxigenin-peroxidase was added (RT; 30 min). AEC was used for color development, and the sections were counterstained in Harris’ hematoxylin (Biomeda, Foster City, CA). DNA strand breaks in fixed cytocentrifuge preparations were identified by TUNEL as described previously (11). For analysis of apoptosis in cell suspensions, DNA strand breaks were identified by TUNEL. After immunofluorescence staining for specific surface markers (5), the cells were fixed in 4% paraformaldehyde and then permeabilized with 0.1% Triton X-100 and 0.1% sodium citrate. Cell death detection kit TUNEL reaction mixture (Roche Diagnostics, Indianapolis, IN) then was added according to the manufacturer’s instructions. Cells incubated with label solution in the absence of terminal transferase were used as negative controls. Quantitative analysis was performed with an EPICS ELITE flow cytometer (Coulter, Hialeah, FL) with 10,000 events acquired from each sample.

Cytokine RNase protection assay

Total RNA was extracted by the guanidinium isothiocyanate-phenol-chloroform method with TRI reagent (Sigma, St. Louis, MO) as described previously (38). Cytokine mRNA expression was determined with the RiboQuant multiprobe RNase protection assay system (BD PharMingen) following the manufacturer’s instructions. Briefly, 5 µg of total RNA were hybridized to 32P-labeled RNA probes overnight at 56°C, followed by treatment with RNase for 45 min at 30°C. The murine L32 and GADPH riboprobes were used as controls. Protected fragments were submitted to electrophoresis through a 7.0 M urea/5% polyacrylamide gel, and then exposed to Kodak X-omat film for 72 h.

Cytokine quantitation by ELISA

Cytokine concentrations in serum samples were determined by ELISA, using Quantikine M kits (R&D Systems, Minneapolis, MN) following the manufacturer’s instructions and using a microplate reader (450 nm).

Complement-dependent cytolytic (CDC) Ab activity

Samples of complement-inactivated sera (1 µl) were diluted serially and placed in V-bottom microtiter plates (Nagle Nunc International, Naperville, IL) in HBSS containing 0.1% w/v BSA (Sigma). Target cells (5 x 103 in 1 µl) obtained from the spleens of B10 (donor; H2b), BALB/c (third-party; H2d), or C3H (syngeneic; H2k) mice were added to each well, and incubated for 30 min at RT. The cells then were washed (twice) in RPMI 1640 medium, and 2 µl of baby rabbit complement (Cedarlane, Hornby, Ontario) was added to each well before incubation for 30 min at 37°C in 5% CO2 in air. The cells then were washed (twice) with RPMI 1640 and one drop of trypan blue added. After 6 min at RT, cytotoxicity was determined as the percentage of dead cells.

Statistical analyses

Graft survival times between groups of transplanted animals were compared by the nonparametric Wilcoxon rank sum test. Other comparisons were made with the Student’s t test. A p value < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Systemic administration of FL markedly up-regulates IL-12 expression in the liver

Liver allografts from FL-treated B10 donors are rejected acutely in C3H recipients (median survival time 5 days), whereas normal B10 livers survive indefinitely (median survival time >100 days; Refs. 15 and 39). To determine the role of IL-12 in the rejection of B10 hepatic allografts from FL-treated donors, we first examined the expression of the cytokine in livers of B10 mice treated with FL by the standard 10-day protocol. Specific anti-mouse-IL-12 mAb was used for immunohistochemical staining. IL-12 expression was strikingly up-regulated in livers from FL-treated mice, compared with those from control animals (Fig. 1GoA). The augmented IL-12 production was associated with pronounced increases in interstitial mononuclear cells, in particular CD11c+ DC, both in portal areas and throughout the liver parenchyma. Two-color immunohistochemical labeling of IL-12 p40 and CD11c in liver sections from FL-treated donors confirmed that numerous CD11c+ cells (presumptive DC) coexpressed IL-12 (Fig. 1GoB). Other interstitial leukocytes (CD11c-) also expressed IL-12. We have reported elsewhere that DC freshly isolated from livers of FL-treated mice are capable of rapid maturation ex vivo, that they traffic to host secondary lymphoid tissue, and that they promote predominantly Th1 responses in vitro and in vivo (14, 15).



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  		FIGURE 1. A, Enhanced expression of immunoreactive IL-12 (red) in both the graft and the spleen of recipients of murine liver allografts from FL-treated donors was completely reversed by host treatment with anti-IL-12 mAb. Tissues were removed from C3H mice 4 days after transplantation of B10 livers from normal control, or FL-treated donors, with or without anti-IL-12 treatment (500 µg i.v., day 0 and day 2). Controls received hamster IgG instead of anti-IL-12 mAb. Cryostat sections were stained with specific goat anti-mouse IL-12 p40 Ab, using the ABC method. Goat IgG was used as isotype control. Magnification, x200. B, Two-color immunohistochemical staining for CD11c (DC; brown) and IL-12 p40 (blue) in liver from an FL-treated donor was performed as described in Materials and Methods. Left, numerous CD11c+ cells in portal areas (right; detail) also stain for IL-12 (and consequently appear purple); other interstitial leukocytes (CD11c-) also appear IL-12+. Left, magnification, x200; right, (detail) magnification, x1000. The results are representative of three experiments, with three animals in each experimental group.

 
Administration of neutralizing anti-IL-12 mAb reverses the rejection of liver allografts from FL-treated donors

To further ascertain the role of IL-12 in this model of liver allograft rejection in which expression of IL-12 is significantly up-regulated within greatly increased numbers of graft interstitial leukocytes, neutralizing anti-IL-12 mAb was injected systemically (i.v.) at 200 or 500 µg/day on day 0 and day 2 after transplantation. Control FL-treated mice received equivalent amounts of hamster IgG. The effectiveness of neutralization of IL-12 in the liver grafts was determined by immunohistochemical staining of tissue sections with specific anti-IL-12 p40 mAb. Fig. 1GoA shows clearly that immunostaining for IL-12 was blocked effectively by the 200-µg mAb regimen. In addition, anti-IL-12 mAb significantly prolonged the survival of FL-treated liver allografts in a dose-related manner. Compared with hamster IgG-treated control mice that rejected grafts from FL-treated donors within 5 days, 50% of mice treated with 500 µg anti-IL-12/day survived "indefinitely" (>100 days; Fig. 2Go). These data indicated that marked increases in IL-12 production by APC within the FL liver allografts played an important role in the switch from tolerance to acute graft rejection.



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  		FIGURE 2. IL-12 neutralization significantly reverses the rejection of liver allografts from FL-treated donors in a dose-related manner. C3H mice received B10 liver grafts from normal control or FL-treated donors with or without anti-IL-12 treatment (200 or 500 µg i.v., day 0 and day 2). Controls received hamster IgG instead of anti-IL-12 mAb. n = 6 mice in each group.

 
IL-12 neutralization inhibits both donor-specific cytotoxic and NK cell activities

To elucidate the mechanisms by which rejection of FL-liver allografts was reversed by in vivo administration of anti-IL-12 mAb, the influence of IL-12 neutralization on the cytotoxic activity of GIC, as well as spleen cells from C3H recipients of hepatic allografts from FL-treated B10 donors were examined. Both donor-specific CTL and NK cell activities of freshly isolated GIC and spleen cells were augmented significantly (p < 0.01) in the FL treatment group (Fig. 3Go). Anti-IL-12 mAb administration markedly reduced (p < 0.01) the donor-specific CTL activity of GIC, and to a lesser but significant extent (p < 0.05), the low level exhibited by host spleen cells. Neutralization of IL-12 also suppressed the low level of splenic NK cell activity, but not that exhibited by GIC (Fig. 3Go). These data demonstrate that rejection of FL-treated liver allografts, associated with enhanced IL-12 production, is linked to potentiated specific and, to a lesser degree, nonspecific cell-mediated cytotoxic responses.



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  		FIGURE 3. IL-12 neutralization inhibits donor-specific CTL and NK cell activities in both (A) GIC and (B) spleen cells from recipients of liver allografts from FL-treated donors. Cytotoxic activity of freshly isolated lymphocytes isolated from grafts or spleens of C3H (H2k) recipients 4 days after transplantation of B10 (H2b) liver allografts from normal control or FL-treated donors with or without anti-IL-12 (or control hamster IgG) treatment (500 µg i.v., day 0 and day 2) was determined in a 4-h 51Cr release assay with EL-4 (H2b) or YAC-1 cells as targets. The data are expressed as means ± SD. There was <15% cytotoxic activity against third-party (p815; H2d) targets at E:T ratios up to 100:1 in all experimental groups (data not shown). The results are representative of three separate experiments, with three mice in each experimental group.

 
IL-12 neutralization augments lymphocyte apoptosis in both liver allografts and recipient lymphoid tissue

Although the precise mechanism(s) remains to be resolved, spontaneous acceptance of liver allografts in mice has been associated with high levels of apoptosis in the GIC population (16). In contrast, FL liver allografts that are rejected acutely show reduced apoptotic activity in GIC within portal areas but enhanced apoptotic death of hepatocytes (39). These data suggest a critical immunoregulatory role for apoptosis in determining the outcome of hepatic allografts. To determine whether the prolonged survival of liver allografts from FL-treated donors achieved by anti-IL-12 mAb administration was associated with enhanced apoptotic death of alloreactive lymphocytes, apoptotic activity in both the graft and spleen was examined by TUNEL staining with immunohistochemistry or two-color flow cytometric analysis, respectively. In situ TUNEL staining of cryostat sections revealed that compared with controls, anti-IL-12 mAb administration augmented apoptosis of GIC within portal areas of FL liver grafts, whereas apoptosis of parenchymal cells was reduced (Fig. 4Go). Apoptosis also was enhanced in T cell areas of the spleen after IL-12 neutralization (Fig. 4Go). Two-color immunostaining of GIC by TUNEL and T cell subset-directed mAb demonstrated that apoptosis of both CD4+ and CD8+ cells was inhibited in FL liver grafts. However, anti-IL-12 administration significantly reversed this effect, and indeed enhanced apoptotic death, particularly in CD8+ cells (Fig. 5Go). Taken together, these data strongly suggest that regulation of apoptotic death of activated alloreactive T cells may be critical in prolonging the survival of murine hepatic allografts.



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  		FIGURE 4. Systemic administration of anti-IL-12 mAb enhances apoptosis of NPC in portal areas of liver grafts, and in periarterial lymphatic sheaths of spleens of recipients of liver allografts from FL-treated donors. A, Cryostat sections obtained 4 days after transplantation of B10 liver allografts from normal control or FL-treated donors with or without anti-IL-12 (or control hamster IgG) treatment (500 µg i.v., day 0 and day 2) were stained by TUNEL with antidigoxigenin peroxidase. AEC was used for color development and counterstaining. The results are representative of three separate experiments with three mice in each group. Magnification, x200. B, TUNEL+ mononuclear cells in portal areas (liver graft) and periarterial lymphatic sheaths (spleen) were enumerated in 20 random high power fields (hpf). Results are means ± SD obtained from three mice in each group.

 


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  		FIGURE 5. Increased incidence of TUNEL+ (apoptotic) CD4+ and CD8+ GIC from recipients of livers from FL-treated donors. GIC were isolated from C3H recipients 4 days after transplantation of B10 liver allografts from normal control or FL-treated donors with or without anti-IL-12 treatment (500 µg i.v., day 0 and day 2). Cells were double-stained with PE-conjugated anti-CD4 or -CD8 mAb and FITC-conjugated TUNEL and then analyzed by flow cytometry. The results are representative of three separate experiments with three mice per group.

 
IL-12 inhibits the apoptotic death of alloactivated T cells induced by liver NPC in vitro

IL-12 has been shown to inhibit activation-induced and Fas-mediated apoptosis of T cells (40, 41, 42), whereas anti-IL-12 enhances these events in vitro and in vivo (43, 44, 45). To address more directly the role of IL-12 in the regulation of apoptotic death of T cells activated by allogeneic liver-derived APC, nylon wool-purified naive C3H splenic T cells were cocultured for 72 h (1:1) with {gamma}-irradiated normal B10 hepatic NPC in the presence or absence of exogenous mouse r IL-12 p70 (10 ng/ml; a gift from Dr. M.T. Lotze, Smith-Kline Beecham, King of Prussia, PA). By 72 h, >90% of the cultured cells expressed responder phenotype (H2Kk+) as determined by flow cytometric analysis. As shown in Fig. 6Go, the addition of IL-12 from the start of the cultures significantly reduced the incidence of TUNEL+ cells (p < 0.01), suggesting that IL-12 may be an important regulator of host T cell apoptosis after stimulation by allogeneic hepatic APC in vivo.



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  		FIGURE 6. Exogenous IL-12 inhibits activation-induced death of alloantigen-stimulated naive T cells. C3H (H2k) splenic T cells were cocultured for 72 h with {gamma}-irradiated B10 (H2b) normal liver NPC in the presence or absence of mouse r IL-12 p 70 (10 ng/ml) added at the start of cultures. Cytocentrifuge preparations were TUNEL-stained as described in Materials and Methods, and the incidence of apoptotic cells determined. Data are expressed as means ± SD and are representative of three separate experiments.

 
IL-12 neutralization inhibits IFN-{gamma} gene expression and cytokine production

To determine the relation of specific cytokine production to the outcome of liver transplantation, we next examined cytokine mRNA expression in GIC and spleen lymphocytes 7 days after liver transplantation by using the RNase protection assay. Expression of gene transcripts for both IL-10 and IFN-{gamma} was markedly up-regulated, whereas message for IL-2 and IL-15 was enhanced only modestly in GIC from FL livers compared with those from control grafts (Fig. 7Go). By contrast, IL-6 gene expression was reduced. Administration of anti-IL-12 mAb inhibited expression of these cytokines, although not to the levels observed in control liver grafts (Fig. 7Go). Thus, the expression of specific cytokines (IFN-{gamma} and IL-10) in GIC appeared to correlate with transplant outcome. Collectively, these data suggest that IL-12 produced mainly by interstitial liver APC generated in response to FL administration is an important factor in inducing allograft rejection in this model. Although IFN-{gamma}, IL-6, and IL-10 gene transcripts also were detected in spleen cells of normal liver graft recipients, changes in cytokine gene expression in the experimental treatment groups were not as marked as those in the GIC population (data not shown). Compared with circulating cytokine levels in recipients of normal livers, those in mice given liver grafts from FL-treated donors showed significant elevations in both IFN-{gamma} and IL-10. These increases were at least partially reversed by IL-12 neutralization (Fig. 8Go). Taken together, these results demonstrate that rejection of liver allografts from FL-treated donors is associated with up-regulation of various cytokines, in particular IFN-{gamma} and IL-10, and that neutralization of IL-12, associated with reductions in IFN-{gamma} and IL-10 production, is associated with prolongation of survival and, in 50% of mice, restoration of >100 day graft survival (Fig. 2Go).



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  		FIGURE 7. IL-12 neutralization inhibits the expression of cytokine mRNA in GIC. GIC were isolated from C3H recipients 4 days after transplantation of B10 livers from normal or FL-treated donors with or without anti-IL-12 treatment (500 µg i.v., day 0 and day 2). Cytokine mRNA expression was determined by RNase protection assay. GAPDH was used as a housekeeping gene. The results are representative of three separate experiments.

 


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  		FIGURE 8. IL-12 neutralization results in inhibition of increased serum IFN-{gamma} and IL-10 levels in recipients of liver allografts from FL-treated donors. Serum was obtained from C3H recipients 4 days after transplantation of B10 liver allografts from normal control or FL-treated donors with or without anti-IL-12 treatment (500 µg i.v., day 0 and day 2). Cytokine levels were determined by ELISA. The data are expressed as means ± SD. The results are representative of three separate experiments with three mice per group.

 
IL-12 neutralization inhibits CDC Ab activity

Levels of CDC Ab activity were determined in the sera of liver graft recipients 4 days after transplantation. Compared with the control liver transplant group, CDC activity against (donor) B10 spleen cells was substantially higher in recipients of FL liver grafts. Anti-IL-12 completely reversed this effect and restored CDC Ab levels to those observed in the control group (Fig. 9Go). These results indicate that IL-12 is a critical cytokine for the generation of cytotoxic alloantibody in this acute liver rejection model.



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  		FIGURE 9. CDC Ab titers against B10 (H2b) (donor) and BALB/c (H2d) (third-party) targets in sera of normal C3H (H2k) mice, and in C3H recipients of normal control B10 livers with or without anti-IL-12 treatment (500 µg i.v., day 0 and day 2). The results are representative of three separate experiments with three animals per group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It has been shown previously that tolerance to mouse liver allografts that is achieved without immunosuppressive therapy (1) can be broken by systemic treatment of donors with the hematopoietic growth factor FL (15). FL affects the growth of primitive multipotent progenitor cells, as well as myeloid- and lymphoid-committed progenitors (13). It dramatically augments the number of interstitial myeloid and lymphoid DC when administered to experimental animals (34). After isolation from the liver, these DC mature during overnight culture into potent allostimulatory cells (14). The migration of large numbers of potential allostimulatory DC into secondary lymphoid tissue of liver graft recipients has been implicated as a crucial factor in the instigation of rejection (15, 39). The findings of the present study suggest that IL-12, which is produced by DC and macrophages in response to various stimuli (18, 19) and shown herein to be produced by abundant DC and other leukocytes within FL liver grafts, is a critical cytokine in mediating the rejection of liver transplants from FL-treated donors. Thus, IL-12 production was markedly up-regulated in FL liver grafts in association with pronounced increases in DC, both in portal triads and throughout the parenchyma. This contrasted to normal livers, which exhibited very few IL-12-positive cells, in keeping with the presence only of rare immature DC restricted to portal areas (7, 14). Direct evidence for a crucial role of IL-12 in inducing the rejection of FL liver allografts was provided by in vivo neutralization of IL-12 activity. This effectively reversed acute liver graft rejection (i.e., survival of animals for only 5 days) in a dose-related manner (Fig. 2Go). Thus, 20 and 50% of recipients of FL liver grafts treated with 200 and 500 µg FL, respectively, survived >100 days. Prolonged graft survival was associated with markedly diminished anti-donor immune effector mechanisms. In addition to these observations, the present study provides further, novel insight into the role of programmed cell death of both host T cells within the graft and in secondary lymphoid tissue in determining liver transplant survival. Thus, IL-12 antagonism restored the incidence of apoptotic T cells within FL liver grafts to the comparatively high levels observed in normal liver transplants. In these latter grafts, the apoptotic activity is associated with progressive diminution of anti-donor CTL responses and the establishment of a tolerant state (16). In the present study, we also show that addition of IL-12 to cocultures of irradiated hepatic NPC and naive allogeneic T cells reduces the incidence of apoptotic cell death. This provides direct evidence, consistent with previous findings (40, 41, 42, 43, 44, 45), for a role of IL-12 in regulation of activation-induced T cell death.

IL-12 is critically involved in the regulation of cell-mediated immune responses by promoting Th1 lymphocyte development and by inhibiting that of Th2 cells (24, 46, 47). It promotes Th1 development by inducing IFN-{gamma} production by activated T cells and NK cells (48, 49, 50, 51, 52, 53). This has been confirmed in IL-12 knockout mice, that are deficient in generating a normal Th1 response and in producing IFN-{gamma} (54). Th1 cells have been shown to be associated with allograft rejection, as they promote both delayed-type hypersensitivity and CTL responses, thought to be the principal effectors of acute transplant rejection (55, 56).

Prolongation of liver allografts from FL-treated donors (in some instances indefinitely) by administration of anti-IL-12 was associated in this study with suppressed anti-donor CTL and NK cell activities (Fig. 3Go, A and B). The data clearly demonstrate that in vivo IL-12 antagonism significantly inhibits production of IFN-{gamma} (Figs. 6Go and 7Go). Notably, the IL-12-Th1-rejection theory has been challenged by conflicting results obtained in animal models. Thus, IL-12 is not mandatory for acute rejection of murine cardiac allografts (31, 57). In contrast, IL-12 appears to have a central role in the progression of acute GVHD in mice (28, 29). In these latter studies, neutralization of IL-12 with a polyclonal anti-IL-12 Ab resulted in the amelioration of acute GVHD (29). By contrast, treatment with exogenous IL-12 converted chronic GVHD into exacerbated acute GVHD (28, 29). Although IL-12 antagonism in mouse recipients of vascularized cardiac allografts promoted intragraft Th2 cytokine (IL-4 and IL-10) gene expression, these grafts were rejected in an accelerated fashion compared with untreated recipients (31). Such findings clearly suggest that complex mechanisms may underlie the role of IL-12 in alloimmune responses in different experimental models. It also is possible that Th1 development can occur independent of IL-12 (31). Other levels of regulation and control of liver graft rejection at the level of cytokine and costimulatory molecule expression are likely to exist. Thus, our related studies have shown that blockade of the B7-CD28 costimulatory molecule pathway with the chimeric fusion protein CTLA4Ig significantly prolongs the survival of livers from FL-donors (W.L. and S.Q., unpublished observations).

We attempted to elucidate the mechanism(s) whereby IL-12 antagonism might regulate alloreactive T cell responses and restore the tolerance to the liver. The data show that reduction in cytokine production in the anti-IL-12-treated mice was not restricted to IFN-{gamma}. Thus, production of IL-10, secreted by Th2 cells and other cells, was also suppressed, suggesting that inhibition of Th1 differentiation might not be the only mechanism leading to subversion of anti-donor immune reactivity. Indeed, there is evidence that IL-10 can exacerbate organ allograft rejection and that its neutralization can modestly prolong transplant survival (58, 59, 60).

Given that acute rejection of FL livers is associated with diminution in apoptotic activity within the GIC and that extinction of IL-12 signaling promotes T cell apoptosis, we speculated that enhanced deletion of donor-reactive T cells might contribute to the efficacy of IL-12 antagonism. In keeping with this hypothesis, immunohistochemical TUNEL staining of grafts and spleens from anti-IL-12-treated FL liver recipients showed strikingly increased numbers of apoptotic cells in the GIC population and T cell areas, respectively. This finding was supported by the augmented incidence of TUNEL-positive CD4+ and more especially, CD8+ GIC in anti-IL-12-treated FL liver recipients (Fig. 5Go). Thus, it appears that peripheral alloreactive T cell deletion in recipients of FL liver grafts is enhanced by systemic IL-12 neutralization and that this mechanism underlies reversion toward to the tolerant state.

Because IL-12 is an activator of T cell and NK cells, we investigated its role in enhancement of donor-specific cytotoxic alloantibody responses. IL-12 has been shown to enhance Ag-specific Ab production, an effect mediated by IFN-{gamma} (61, 62, 63), as well as by IFN-{gamma}-independent mechanisms mediated by other intermediary cytokines. IL-12 also may stimulate B cells directly (64). There also is evidence that NK cells play a major role in stimulation of T-independent IgG responses through the release of IFN-{gamma}, because IgG secretion induced in vivo or in vitro can be increased by NK cell activation and inhibited by NK cell depletion (65, 66). Neutralization of IFN-{gamma} reverses the effect of NK cells. Recently, a role for endogenous IL-12 in T-independent responses was proposed (65), based on the finding that Ab responses were inhibited by IL-12 neutralization. In the present study, the observed inhibition of donor-specific CDC Ab levels that resulted from neutralization of IL-12 in recipients of FL livers was consistent with a role of IL-12 in the promotion of CTL and NK cell activities leading to enhanced alloantibody production.

These observations support our view (16) that programmed cell death of immunoreactive T cells within the graft and host secondary lymphoid tissue plays a pivotal role in determining the balance between liver transplant tolerance and rejection. They are also consistent with recent findings that prevention of apoptosis of alloreactive T cells blocks the induction of peripheral transplant tolerance (67). The precise molecular mechanisms underlying regulation of alloreactive T cell death remain uncertain. In preliminary studies, we have observed that 75% of liver grafts from gld (FasL-deficient) mice survive indefinitely (W.L. and S.Q., unpublished observations), indicating that the Fas pathway does not play a critical role in regulation of liver allograft survival and tolerance induction in nonimmunosuppressed recipients. In summary, our data suggest that inhibition of apoptosis of alloreactive T cells by IL-12 derived from enhanced numbers of stimulatory donor APC helps promote amplification of immune effector rather than protective mechanisms that lead to graft destruction.


   Acknowledgments
 
We thank Dr. Emil R. Unanue for providing the anti-IL-12-producing hybridoma, Immunex for FL, Alison Logar for flow cytometric analyses, Zeeshan Babar for color print preparation, and Shelly L. Conklin forsecretarial support.


   Footnotes
 
1 This study was supported by National Institutes of Health Grants DK 29961 and DK 49745. Back

2 Address correspondence and reprint requests to Dr. Shiguang Qian, Thomas E. Starzl Transplantation Institute and Department of Surgery, University of Pittsburgh Medical Center, E1540 Biomedical Science Tower, 200 Lothrop Street, Pittsburgh, PA, 15213. Back

3 Abbreviations used in this paper: DC, dendritic cells; FL, Flt3 Ligand; B10, C57BL/10; NPC, nonparenchymal cells; RT, room temperature; CDC, complement-dependent cytolytic; GIC, graft-infiltrating cells; GVHD, graft-vs-host disease; AEC, aminoethylcarbazole. Back

Received for publication September 14, 2000. Accepted for publication February 23, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Marked Prolongation of Cardiac Allograft Survival by Dendritic Cells Genetically Engineered with NF-{kappa}B Oligodeoxyribonucleotide Decoys and Adenoviral Vectors Encoding CTLA4-Ig
J. Immunol., September 15, 2002; 169(6): 3382 - 3391.
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T. Teshima, P. Reddy, K. P. Lowler, M. A. KuKuruga, C. Liu, K. R. Cooke, and J. L. M. Ferrara
Flt3 ligand therapy for recipients of allogeneic bone marrow transplants expands host CD8alpha + dendritic cells and reduces experimental acute graft-versus-host disease
Blood, March 1, 2002; 99(5): 1825 - 1832.
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