critical role of fas/fas ligand interaction in cd28-independent pathway of allogeneic murine...

Critical Role of Fas/Fas Ligand Interaction in CD28-IndependentPathway of Allogeneic Murine Hepatocyte Rejection

TOSHIYASU KAWAHARA,1,2 SHINICHI KASAI,1 HIDEO YAGITA,3 MASAYUKI SAWA,1 KAZUYA KATO,1 MIYUKI AZUMA,3

ATSUO NAKAJIMA,3 KO OKUMURA,3 SHUNJI FUTAGAWA,2 AND MICHIO MITO1

Cytolytic induction of T cells requires both the T-cell recep- munosuppression, allogeneic hepatocytes were rapidly re-jected within a few days after transplantation.3tor (TCR)-mediated antigenic stimulation and the CD28-me-

diated co-stimulatory signal. Blockade of the interactions be- The rejection of allografts is dependent on the activationof alloreactive T cells, which requires T-cell receptor (TCR)tween CD28 and its ligands, CD80 and CD86, prolongs the

survival of allografts in some transplantation models. How- engagement by antigen and co-stimulatory signals deliveredby some T-cell surface molecules.4 Signaling through theever, we found that allogeneic hepatocytes were completely

rejected within 7 days after intrasplenic transplantation, even TCR alone without a co-stimulatory signal induces a state ofT-cell anergy.5,6 Blockade of co-stimulatory signal is usefulwhen treated with monoclonal antibodies (mAbs) against

CD80 and CD86 (anti-CD80/86). Recent studies have shown for transplantation.7-10 Most particularly interesting co-stim-ulatory molecules are CD80 and CD86, which interact withthat there are two main mechanisms of T-cell–mediated cyto-

toxicity, perforin-based and Fas-based ones. It has been shown their receptor (CD28) on T cells to provide a key signal forgeneration of T-cell immunity.11 Activation of the TCR inthat the liver is highly sensitive to induction of apoptosis by

an agonistic anti-Fas mAb. We then investigated the role of the presence of a co-stimulatory signal from CD28 results inT-cell clonal expansion and the induction of effector func-the Fas/Fas ligand (FasL) system in the CD28-independent

allogeneic hepatocyte rejection. With the anti-CD80/86 mAb tions such as the production of lymphokines and the induc-tion of cytotoxic activity.12 Consequently, blockade of thetreatment, hepatocytes from C57BL/6 lpr/lpr (B6 lpr) mice,

which express little Fas antigen, could survive for 7 days after CD28 co-stimulatory pathway by the treatment with anti-CD80/86 monoclonal antibody (mAb) or CTLA4 Immuno-intrasplenic transplantation, and hepatocytes from C57BL/6

(B6) mice could also survive for 7 days in the spleen of C3H/ globulin (CTLA4-Ig), which is a fusion protein comprisingthe extracellular domain of CTLA4 and the Fc domain ofHe gld/gld (C3H gld) mice, which express no functional FasL.

CD28-independent induction of cytotoxicity against alloge- immunoglobulin G1 and binds to CD80/CD86, has beenshown to prolong allograft survival in some transplantationneic hepatocytes was not observed when the effector cells

were derived from C3H gld mice. These results indicated models.9,10

Fas is type I membrane protein in the tumor necrosis factorthat the Fas/FasL system plays a critical role in the CD28-independent pathway of allogeneic hepatocyte rejection. (HEP- receptor superfamily and mediates apoptosis upon ligatation

by an agonistic anti-Fas mAbs or its ligand (FasL) bind-ATOLOGY 1997;26:944-948.)ing.13,14 Molecular cloning of FasL revealed that it is a typeII transmembrane protein and belongs to the tumor necrosis

Hepatocyte transplantation has been expected to be a po- factor family.15 It has been shown that there are two maintential therapeutic modality for congenital hepatic enzyme mechanisms of T-cell–mediated cytotoxicity, perforin-deficiency or acute hepatic failure.1 The advantages of hepa- based16 and Fas-based ones.17-19 The Fas/FasL system istocyte transplantation over liver transplantation are as fol- thought to play an important role not only in T- and B-lows: 1) it requires no vascular anastomosis; 2) long-term lymphocyte homeostasis, but also in T-cell–mediated cyto-preservation of hepatocytes is possible; 3) gene therapy can toxicity.20 Hepatocytes are extremely sensitive to the Fas-be easily applied; and 4) hepatocytes can be transplanted to mediated apoptosis, because intraperitoneal injection of anseveral recipients from a single donor. We previously found agonistic anti-Fas mAb-induced apoptosis of hepatocytesthat syngeneic hepatocytes were preserved up to 18 months leading to fulminant hepatic failure in mice.14

after transplantation into the spleen.2 However, without im- In the present study, we showed the importance of theFasL-mediated cytotoxicity in the CD28-independent path-way of allogeneic hepatocyte rejection.

Abbreviations: TCR, T-cell receptor; mAb, monoclonal antibody; LDH, lactose dehy- MATERIALS AND METHODSdrogenase; MMC, mitomycin C.

From the 1Second Department of Surgery, Asahikawa Medical College, Hokkaido; Animals. C57BL/6 (B6) and C3H/He (C3H) male mice (6 weeks2Second Department of Surgery, Juntendo University School of Medicine; and 3Depart- of age) were purchased from Japan Clea Co. (Tokyo, Japan). C57BL/ment of Immunology, Juntendo University School of Medicine, Tokyo, Japan. 6 lpr/lpr (B6 lpr) male mice (5 weeks of age), which express little

Received March 4, 1997; accepted June 4, 1997.Fas antigen,20 and C3H/He gld/gld (C3H gld) male mice (5 weeks

Address reprint requests to: Toshiyasu Kawahara, M.D., Second Department ofof age), which express no functional FasL,20 were purchased fromSurgery, Juntendo University, School of Medicine, 2-1-1 Hongo, Bunkyo-ku, TokyoJapan SLC (Shizuoka, Japan). All animals received care according113, Japan. Fax: 81-3-3818-7589.to the National Institutes of Health guidelines for care and use ofCopyright q 1997 by the American Association for the Study of Liver Diseases.

0270-9139/97/2604-0022$3.00/0 laboratory animals.

944

AID Hepa 0051 / 5p27$$1001 07-09-98 07:45:50 hepa WBS: Hepatology

HEPATOLOGY Vol. 26, No. 4, 1997 KAWAHARA ET AL. 945

Hepatocyte Isolation and Transplantation. Hepatocytes were isolatedfrom Fas-bearing B6, C3H, or Fas-deficient B6 lpr mice livers by insitu collagenase (Type I, Sigma, St. Louis, MO) perfusion, followedby differential centrifugation.21 The final cell suspension contained85% to 90% viable hepatocytes as determined by Trypan Blue exclu-sion. Each recipient mouse received 2 1 106 hepatocyte suspensioninto the spleen.

mAbs and Administration. The rat mAbs to mouse CD80 (RM80,rat immunoglobulin G2a), CD86 (PO3, rat immunoglobulin G2b)were prepared as described previously.22 Each recipient mouse re-ceived intraperitoneal administration of both anti-CD80 and CD86mAb (100 mg/d) for 5 days, initiated at the time of transplantation.

Histological Examination. The recipient mice were killed at 7 or50 days after transplantation to examine the survival of allogeneichepatocytes in the spleen. The spleens were fixed with 10% neutralformalin, sectioned at 3 mm, stained with periodic acid–Schiff, andexamined under light microscopy.

Cytotoxic Activity of Mouse FasL Transfectant Against Hepatocytes. Asdescribed previously, mouse FasL complementary DNA was trans-fected into a mouse T lymphoma cell line (L5178Y).23 The transfec-tant cells (mFasL/L5178Y) expressed a high level of functionalmouse FasL as estimated by their cytotoxic activity against Fas-bearing targets.23 Various numbers of mFasL/L5178Y cells wereadded to the target hepatocytes (5 1 103 cells/well) from B6 or B6lpr mice in flat-bottomed 96-well plates, and they were incubatedfor 12 hours in Williams’ E medium (Gibco BRL, Gaithersburg MD)containing 10% fetal bovine serum, 20 ng/mL epidermal growthfactor, 1009 mol/L insulin, and antibiotics. Cytotoxic activities weredetermined by lactose dehydrogenase (LDH) release in the superna-tants,24 which was measured by spectrophotometry using LDH-I kit(Serotec, Sapporo, Japan). The percentage of lysis was calculatedby using the following formula:% LDH release Å 100 1 (experimental release

0 spontaneous release)/(maximum release 0 spontaneous release).

Maximum release was obtained by complete solubilization of hepa-tocytes with 1% Triton X-100.

Assessment of Cytotoxic Activity. Nylon-nonadherent splenocytes FIG. 1. Periodic acid-Schiff–stained sections of the recipient spleensafter hepatocyte transplantation. (A) The spleen of anti-CD80/86 mAbs-(splenic T cells) (2 1 106 cells/well) from C3H or C3H gld micetreated C3H mice at 7 days after transplantation of B6 hepatocytes. Therewere cultured for 5 days with mitomycin C (MMC)-treated (finalwere no viable hepatocytes in the spleen, and only macrophages that phago-concentration, 60 mg/mL) B6 splenocytes (2 1 106 cells/well) incytized dead hepatocytes were observed (arrows). (B) The spleen of anti-24-well plates in RPMI 1640 medium (Nissui Pharmaceutical Co.,CD80/86 mAbs-treated C3H mice at 7 days after transplantation of B6 lprTokyo, Japan) supplemented with 10% fetal bovine serum (JR Scien-hepatocytes. The colonies of surviving hepatocytes were detected in the

tific Inc., Woodland, CA), 1 mmol/L L-glutamine, 0.05 mmol/L 2- splenic red pulps and were positive for periodic acid-Schiff staining. (Origi-mercaptoethanol, and antibiotics in the presence or absence of anti- nal magnification [A and B] 1200.)CD80/86 mAbs (10 mg/mL each). After 5 days, stimulated splenicT cells (5 1 105 cells/well) were added to target hepatocytes (5 1103 cells/well) in flat-bottom, 96-well plates, and they were incu-

jected in the spleen of C3H mice within 7 days regardless ofbated for 20 hours in Williams’ E medium at an effector/targetratio of 100. After incubation, the released LDH was measured as the anti-CD80/86 mAb treatment (Fig. 1A). There were nodescribed above. viable hepatocytes in the spleen, and only macrophages that

Proliferation Assay. Purified nylon-nonadherent splenocytes (3 1 phagocytized dead hepatocytes were observed.105 cells/well) from recipient mice at 20 days after transplantation, To investigate the role of the Fas/FasL system in allogeneicin which hepatocytes from B6 lpr mice with or without the anti- hepatocyte rejection, we transplanted hepatocytes from B6CD80/86 mAb treatment, were cultured with MMC-treated (final

lpr mice, which express little Fas antigen, into the spleen ofconcentration, 60 mg/mL) splenocytes (3 1 105 cells/well) from B6allogeneic C3H recipients with or without the anti-CD80/86or C3H mice in 96-well, flat-bottom plates using RPMI 1640 me-mAb treatment. Without the anti-CD80/86 mAb treatment,dium for 5 days. The assay was pulsed with [3H]thymidine (1 mCi/hepatocytes from B6 lpr mice were also rejected within 7 dayswell) (DuPont-NEN, Boston, MA) for 18 hours before harvest using(Table 1). However, with the anti-CD80/86 mAb treatment,a Micro 96 Harvester (Skatron, Lier, Norway). Incorporated radioac-

tivity was measured in a microplate b counter (Micro Beta Plus; hepatocytes from B6 lpr mice could survive for 7 days afterWallac, Turku, Finland). transplantation (Fig. 1B). Viable hepatocytes were still ob-

Statistical Analysis. Statistical significance was evaluated using the served at 50 days after transplantation (data not shown).Mann-Whitney U test. P õ .05 was considered statistically signifi- Furthermore, with the anti-CD80/86 mAb treatment, alloge-cant. All statistical analyses were performed using commercial soft- neic hepatocytes from B6 mice could also survive for 7 daysware (InStat, version 2.00, Berkeley, CA).

in the FasL-deficient C3H gld recipients, while they wereRESULTS rejected without the anti-CD80/86 mAb treatment (Table 1).

Cytotoxic Activity of mFasL/L5178Y Transfectant Against B6Survival of Transplanted Allogeneic Hepatocytes in the Spleen.Allogeneic hepatocytes from B6 mice were completely re- Hepatocytes. To examine the susceptibility of hepatocytes to


946 KAWAHARA ET AL. HEPATOLOGY October 1997

TABLE 1. Hepatocytes in the Recipient Spleenat 7 Days After Transplantation

Graft at 7 Days AfterDonor Recipient Anti-CD80/86 mAbs Transplantation

B6 C3H / RejectedB6 lpr C3H 0 RejectedB6 lpr C3H / AcceptedB6 C3H gld 0 RejectedB6 C3H gld / Accepted

NOTE. Allogeneic B6 hepatocytes were transplanted into the recipientC3H spleen with or without the anti-CD80/86 mAb treatment. With theanti-CD80/86 mAb treatment, Fas-deficient B6 lpr hepatocytes could survivein the spleen of allogeneic recipients, and Fas-bearing B6 hepatocytes couldalso survive in the spleen of FasL-deficient C3H gld mice for 7 days aftertransplantation.

FasL, mFasL/L5178Y cells were added to B6 or B6 lpr hepato-cytes and incubated for 12 hours. As shown in Fig. 2, hepato-

FIG. 3. Cytotoxic activity of splenic T cells from C3H or C3H gld micecytes derived from B6 mice, but not those from B6 lpr mice,against allogeneic hepatocytes. C3H or C3H gld splenic T cells were stimu-were efficiently lysed by mFasL/L5178Y cells in vitro. These lated by MMC-treated B6 splenocytes for 5 days in the presence or absence

results indicated that hepatocytes from B6 mice express func- of anti-CD80/86 mAbs. Cytotoxic activity against B6 or C3H (syngeneic)hepatocytes was tested by 20 hours of LDH release at an effector/target ratiotional Fas that can mediate FasL-cytoxicity.of 100. Data represent means { SD of triplicated samples. In the presenceCD28-Independent Cytolytic Induction of Alloreactive Splenic Tof anti-CD80/86 mAbs, substantial cytotoxicity against B6 hepatocytes wasCells Against Hepatocytes. To clarify whether the Fas/FasL sys-induced when C3H splenic T cells were used as the effector cells, but not

tem is critical for cytotoxic T lymphocyte–mediated cytolysis when FasL-deficient C3H gld was used as the effector cells.against allogeneic hepatocytes and whether FasL is inducedindependently of CD28-mediated co-stimulation, we exam-ined the cytotoxic activity of C3H splenic T cells that had

complete inhibition of cytotoxicity was not achieved (Fig.been stimulated by MMC-treated B6 splenocytes in the pres-3).ence or absence of anti-CD80/86 mAbs. In the presence of

We next investigated the cytotoxic activity of C3H gldanti-CD80/86 mAbs, cytotoxic activity was significantly re-splenic T cells against B6 hepatocytes. In the absence of anti-duced from 95.0% { 5.3% to 34.3% { 8.2% (P õ .001), butCD80/86 mAbs, cytotoxic activity of C3H gld splenic T cellsagainst B6 hepatocytes was lower than that of C3H (46.8%{ 6.2% vs. 95.0% { 5.3%; P õ .001) (Fig. 3). However, inthe presence of anti-CD80/86 mAbs, cytotoxic activity ofC3H gld splenic T cells was greatly reduced to 6.8% { 2.5%,which was almost comparable with the cytotoxicity againstsyngeneic C3H hepatocytes.

Proliferation Responses of Alloreactive T Cells. Splenic T cells,which were purified from the recipient mice at 20 days aftertransplantation of B6 lpr hepatocytes with or without theanti-CD80/86 mAb treatment, were examined in vitro forreactivity to donor-antigen. Without the anti-CD80/86 mAbtreatment, splenic T cells from the recipient mice revealedhigh response to donor-antigen (Fig. 4). However, with theanti-CD80/86 mAb treatment, proliferation responses ofsplenic T cells were significantly reduced from 19,759 {3,389 to 4,880 { 442 cpm (P õ .001), which suggests thatthere was a functional tolerance to donor-antigen.

DISCUSSION

In this study, we showed the importance of the Fas/FasLsystem in allogeneic hepatocyte rejection. Previous studieshave shown that blockade of the interactions between CD28and CD80/CD86 could inhibit the induction of alloreactivecytotoxic T lymphocyte or T-cell proliferation against alloan-tigens, and that it prolonged the survival of allogeneic heart10

FIG. 2. Cytotoxic activity of mFasL/L5178Y transfectant against B6 or or pancreatic islets.9B6 lpr hepatocytes. mFasL/L5178Y cells were co-cultured with (j) B6 or However, we found that blockade of the CD28 co-stimula-(●) B6 lpr hepatocytes for 12 hours at the indicated effector/target ratios.

tory pathway could not prolong the survival of allogeneic B6Fas-bearing B6 hepatocytes, but not Fas-deficient B6 lpr hepatocytes, wereefficiently lysed by mFasL/L5178Y cells in vitro. hepatocytes transplanted into the spleen of C3H mice. This


HEPATOLOGY Vol. 26, No. 4, 1997 KAWAHARA ET AL. 947

effector cells. These results clearly indicate that the CD28-independent cytotoxic induction against allogeneic hepato-cytes is predominantly mediated by the Fas/FasL system andthat FasL derived from the recipient is critical.

It was concluded that both the CD28-dependent cytotoxicmechanism and CD28-independent Fas/FasL system are criti-cal pathways of allogeneic hepatocyte rejection. Therefore,the blockade of both pathways by CTLA4-Ig or anti-CD80/86 mAbs and anti-FasL mAb would be a useful therapeuticstrategy to prolong the survival of transplanted allogeneichepatocytes.

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