Demetris, AJ

Murase, N

Rao, AS

Starzl, TE

Thomson, AW

Tsamandas, A

Ye, Q

English

Name not available

Multilineage hematopoietic reconstitution of supralethally irradiated rats by syngeneic whole organ transplantation: with particular reference to the liver

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0041-1337/96/6101-1$03.0010 TRA:'-iSPL·\''iTATION l'op,'r!i!ht:D 1996 by Williams & Wilkins Vol. 61. 1-4. No. 1. January 15. 199fi Prtnted In u.s.A. Transplantation® RAPID COMMUNICATIONS MULTILINEAGE HEMATOPOIETIC RECONSTITUTION OF SUPRALETHALLY IRRADIATED RATS BY SYNGENEIC WHOLE ORGAN TRANSPLANTATION WITH PARTICULAR REFERENCE TO THE LIVERl NORIKO MURASE, THOMAS E. STARZL,2 QING YE, ATHANASSIOS TSAMANDAS, ANGUS W. THOMSON, ABDUL S. RAo, AND ANTHONY J. DEMETRIS Pittsburgh Transplantation Institute, Uniuersity of Pittsburgh Medical Center, Pittsburgh, Pennsyluania 15213 The migration of multilineage "passenger leukocytes" from transplanted organs and their ubiquitous survival in success-fully treated human recipients for as long as 30 years (mi-crochimerism) has been postulated to be an essential condi-tion for organ allograft acceptance and the explanation for a number of previously enigmatic posttransplant events (1, 2). Following organ revascularization in either humans or ro-dents, donor leukocytes appear in large numbers in the re-cipient blood but usually diminish over a few days to a level undetectable with flow cytometry (2-5). We have construed the persistence of donor cells thereafter as prima facie evi-dence of the presence of hematopoietic stem and precursor cells (1,2, 6). When the blood compartment is serially sampled in long-surviving human organ recipients, the levels of donor cells fluctuate (7), presumably reflecting cyclic activity of these stem cells. Taniguchi et al. (8) showed that primitive CD45 + cells purified from adult mouse livers unfailingly reconsti-tuted all hematolymphopoietic lineages in supralethally ir-radiated adult mouse recipients. We present here direct ev-idence that supralethally irradiated adult rats can be consistently rescued by syngeneic liver transplantation, and that heart transplantation has a less dramatic but signifi-cant therapeutic effect. Inbred Lewis (RT1!, LEW) rats weighing 200-250 g (8-10 weeks old) were purchased from Harlan Sprague Dawley (Indianapolis, IN) and kept in a laminar-flow specific-patho-gen-free environment. Orthotopic liver and heterotopic (ab-dominal) heart transplantation were performed as described previously (5). After death of the donor by exsanguination, the grafts were flushed intravascularly with chilled lactated Ringer's solution until the venous effiuent was clear. Bone marrow cells were harvested from the tibias and femurs, and processed in RPM I 1640 supplemented with 25 mM Hepes buffer, 2 mM L-glutamine, 50 D/ml penicillin, and 50 J..lg/ml streptomycin (all from Gibco, Grand Island, NY) (5). I This study was supported by project grant DK 29961 from the National Institutes of Health, Bethesda. MD. 2 Address correspondence to: Thomas E. Starzl. MD. PhD, 3601 Fifth Avenue. 4C Falk Clinic, Pittsburgh, PA 15213. Trypan blue exclusion testing uniformly showed >95% cell viability_ The cell suspensions were administered into the recipient penile vein. A reagent panel containing monoclonal antibodies against all of the principal leukocyte subsets was used to determine lineages of the control and reconstituted animals (panel available on request), Most experiments were performed with male donors and recipients. However, in 2 liver recipi-ents, male ~ female transplantation was performed and the tissues and organs of the hematopoietically reconstituted recipient were studied with polymerase chain reaction and Southern hybridization, usiiig rat V-chromosome (sex deter-mining region Y [SRY]) specific primers (9). This provided proof of hematopoietic sex change in the reconstituted ani-mals as opposed to recovery of the cytoablated recipient stem cells. Recipients were irradiated with 9.5 Gy, delivered from a cesium source. All cell or organ transplantations were done 4-8 hr after completion of irradiation. Survival was deter-mined of animals receiving 4 different doses of syngeneic bone marrow, and of those given liver and heart grafts (Table 1). In addition, 6 irradiated animals of similar size were given 3 ml of fresh whole blood, which was an estimated 251Jt of their total blood volume. This amount of transfusion ex-ceeded by an estimated thousand-fold or more any residual blood in the thoroughly flushed organs used for transplanta-tion. All untreated animals died within 12 days. Heart trans-plantation significantly prolonged survival (P<0.05). One of 6 cardiac recipients was permanently rescued and 4 of the 5 others lived 2-6 days beyond the longest surviving non-treated control. These results were at least as good as those obtained with a suboptimal dose of 0.5x 106 bone marrow cells (P=0.109 vs. control) and with the infusion of 3 ml of whole blood (P=0.007 vs. controll. Liver transplantation re-sulted in permanent survival of 5 of 6 irradiated recipients with full multilineage reconstitution. a rescue effect that was not significantly different from that following infusion of 1,5, or lOX 106 bone marrow cells (Table 1). Because frequent blood sampling and other manipulations I , ~I ,I , :1 :1 J ,I' II: 2 TRANSPLAc"<'TATION Vol. 61. No.1 TABLE 1. Survival of lethally irradiated (9,5 Gyl LEW rats after different types of syngeneic organ and cell transplantation Group Orgall/cells n Survival (daysl Survival rate Median (daysJ po 1 2 3 4 5 6 7 8 None 6 10. 10. 10. 11, 12. 12 0/6 10.5 Heart 6 11, 14. 14, 16. 18. > 100 116 15,0 <0.05 Liver 6 16. >100X5 5/6 >100 <0,005 Bone marrow 0,5 x 106 6 10. 11. 12. 15, 16. 17 0/6 13,5 NS Bone marrow 1 x 106 6 >100x6 6/6 >100 <0.005 Bone marrow 5X106 6 >100X6 6/6 >100 <0.005 Bone marrow 10 x 106 6 >100x6 6/6 >100 <0.005 Whole blood 3 ml 6 12. 13. 13. 13, 13. 13 0/6 13.0 <0,01 a Versus group 1 (Mann-Whitney U test). Group 2 vs, groups 4 and 8: NS; Group 3 vs, groups 5. 6, and 7: NS, jeopardized survival, hematologic studies were done in a separate cohort of irradiated animals, with subgroups that were untreated, infused with 1 X 106 or 1 X 107 unmodified bone marrow cells, or submitted to liver transplantation. The results, including weight gain, were compared with those in naive unaltered animals in the same environment (Figs, 1-3). The rate and the extent of circulating leukocyte (Fig. 1) and hematocrit restoration (Fig. 2) were similar after liver trans-plantation and infusions of 1 X 106 bone marrow cells, Rats given a lO-fold higher dose of bone marrow cells (lX107 ) recovered more quickly (Figs. 1 and 2), The sequence of lineage recovery was similar in the liver and bone marrow recipients, and appeared to be dependent on the number of progenitor cells, The FACS profile of animals after various kinds of hematopoietic reconstitution is shown in Figure 4. The reconstituted cells in cross-sex liver transplantations (male ---+ female) were predominantly donor (Fig. 5) and were, of course, least densely represented in the heart, The initial delay in postoperative weight gain of liver com-pared with bone marrow recipients (Fig, 3) was explained by the greater stress and metabolic depletion associated with the hepatic replacement compared with the less traumatic penile vein infusion of bone marrow cells. After 3 weeks, the liver recipients had catch-up weight gain, and they achieved parity with all other reconstituted groups by day 50 (Fig. 3). These experiments suggest that the difference between the chimerism (and tolerance) following classical bone marrow transplantation and that produced by the donor ("passen-ger") leukocytes of whole organs is purely semantic. While 18000 1t1OOO ~ E 14OCJO E VI 12000 e :s 1000II 0 " 'i 8000 " OJ 6000 ~ 4OCJO 2ODO 10 20 30 40 50 60 70 (days) FIGURE 1. Total white cell counts (mm3 J in normal and irradiated LEW rats (9.5 GyJ and rats given syngeneic transplants, after treat-ment with red blood cell lysing buffer, Cells were counted in a hemacytometer .•. Normal Lewis (n=3); 0, radiation alone (n=3); ., 1X106 bone marrow (n=6); D. lOx 106 bone marrow (n=4); &, OLTX (n=3). ooK-------------~----------------------~ 70 60 50 30 20 10 10 20 30 40 Days No"""1 L""II ( .. 3) RoGIOtion olano (no3) 1.10' Bono m ...... (_I 1 OX1 0 • eone IMrrow (n.4) OLTX("31 50 60 70 FIGURE 2. Hematocrit (tail vein blood. microcentrifuge method) in same samples as Figure L 60 50 c 40 iii CI 30 :c Q 20 .. iI: 10 >-'C 0 0 III ~ . -10 ·20 10 20 30 40 50 Days FIGURE 3. Body weight changes in animals of Figures 1 and 2. ., Normal LEW (n-==3); 0, radiation alone (n=3); ., 1X106 bone marrow (n=6); D, 10X106 bone marrow (n=4); &. OLTx (n=3). describing the multilineage character of the microchimerism in human and animal organ recipients (1, 2, 6, 10. 11), we have emphasized the invariable histopathologic prominence of dendritic cells because their presence was so much in conflict with the literature preceding 1992. The previous literature had associated these cells almost exclusively with rejection rather than graft acceptance. We have recently summarized elsewhere the impressive evidence acquired since then of a key tolerogenic role played by the dendritic cells, particularly when iIi their precursor stages (J2). How-ever, preoccupation with a single leukocyte lineage could obscure an understanding of the complex cell interactions that we are seeking to understand and manipulate. We be-b January 15. 1996 MURASE ET AL. 3 25-30 days • NOfrrwIL .... o 10.,0680ne".rrow • ,.,06 Bonem.row CLiver 49-56 days 100 100 ~----------------I FIGURE 4. Lineage ofperipheralleuko-cytes shown in Figure l. Granulocytes were determined on blood smear fol-lowing a-naphthyl acetate esterase and hematoxylin staining. Other FACS analysis of percentage of subpopula-tions (mean::': SD) was by direct immu-nofluorescence with antibodies to sur-face markers, including OX1 (CD45). R7.3 (uj3 TCRl, W3/25 (CD4). OXS (CDS), OX33 (B-cells), and NK3.2.3 (NK cells). 80 80 60 60 if. 40 40 20 20 0 Gr.nulo· o Gr.Mulo. COOS T ... " co. COB NK eell (3.2.3) eyt •• cyI" (OX') (A7.3) (W3125) (OXl) Heart PBL ~ Liver Ty SP BM CLN FIGURE 5. Polymerase chain reaction and Southern hybridization of organs and tissues 50 days after lethal irradiation and syngeneic liver transplantation. PBL, peripheral blood lymphocytes; Ty. thy-mus; SP, spleen; BM, bone marrow; CLN, cervical lymph nodes. Male and female (negative) controls are included. lieve it unlikely that transplantation tolerance can be fully explained, reliably induced, or efficiently sustained by any single lineage (5, 6, 13). In the experiments reported herein, heart transplantation also had a significant effect on postirradiation survival, less dramatic than the liver but similar to that of a suboptimal dose of donor bone marrow or a large blood transfusion. The pennanent hematopoietic reconstitution of one of the heart recipients and prolongation of survival of 4 of 5 others strengthen our earlier contention that tolerogenicity is an inherent capability common to all organized tissues and or-gans, with variations in outcome dictated by the quantity and lineage profile of bone marrow-derived leukocytes contained in the graft (), 2, 5, 6, 13). No record could be found in the literature suggesting that these simple and direct reconstitution experiments had been performed previously, presumably because of the assumption that organ transplantation involves a unidirectional host-versus-graft immune reaction (the one-way paradigm). Leszczynski et a1. (}4) showed that lXl06 recipient inflam-matory cells extracted from rejecting rat kidney allografts contained enough host stem cells to fully reconstitute su-pralethally irradiated animals of the recipient strain. The experiments reported herein showed that the transplanted organ (especially the liver) brings enough donor stem cells to provide the basis of the sustained bidirectional immune re-action that we have postulated to be the basis of allograft acceptance and the usual form of transplantation tolerance (the two-way paradigm [5, 15]). Acknowledgments. We thank Stanley H. Chia and Christopher S. Doughton for providing excellent technical assistance, and Dr. Lianfu Wang for help in conducting molecular assay. ADDENDUM Two historical contributions in addition to Reference 8 were brought to our attention after submission of this article, adding evidence that stem cells are present in the adult mouse liver. The first, by Hays, Hays, and Golde (1978), described multiline age reconstitution following supralethal irradiation with cultured syngeneic hepatic NPCs obtained from adult mouse livers, especially if the liver was regener-ating (following partial hepatectomy). The second publica-tion, by Decker, Lohmann-Matthes, and Baccarrine (1988), also emphasized that cultured syngeneic liver NPCs could substitute for bone marrow cells for rescue after irradiation. Hays EF, Hays DM, Golde DW. Hemopoietic stern cells in mouse liver. Exp Hematol 1975; 6: IS. Decker T, Lohmann-Matthes ML. Baccarrini M. Liver-associated macrophage precursor cells proliferate under impairment of regu-lar hemopoiesis. Eur J Immunol 19S5; IS: 697. REFERENCES 1. Starzl TE. Demetris AJ. Murase N. I1dstad S, Ricordi C, Trucco M. Cell migration, chimerism. and graft acceptance. Lancet 1992; 339: 1579. 2. Starzl TE. Demetris AJ, Trucco M, et a1. Cell migration and chimerism after whole-organ transplantation: the basis of graft acceptance. Hepatology 1993; 17: 1127. 3. Iwaki y, Starzl TE. Yagihashi A, et a1. Replacement of donor ! i · " 4 qoAkpmiAkqAqfl~ Fol. 61, No.1 lymphoid tissue in human small bowel transplants under FK 506 immunosuppression. Lancet 1991; 337: 818. 4. Murase N, Demetris AJ. Woo J, et al. Graft versus host disease (GVHD) after BN to LEW compared to LEW to BN rat intes-tinal transplantation under FK 506. Transplantation 1993; 55: l. 5. Murase N, Starzl TE, Tanabe M, et a!. Variable chimerism, graft versus host disease. and tolerance after different kinds of cell and solid organ transplantation from Lewis to Brown-Norway rats. Transplantation 1995; 60: 158. 6. Starzl TE. Demetris AJ, Murase N, Thomson AW. Trucco M. Ricordi C. Donor cell chimerism permitted by immunosuppres-sive drugs: a new view of organ transplantation. Immunol Today 1993; 14: 326. 7. Schlitt HJ. Hundrieser J. Hisanaga M, et al. Patterns of donor-type microchimerism after heart transplantation. Lancet 1994; 343: 1469. 8. Taniguchi H, Toyoshima T, Fukao K, Nakauchi H. Evidence for the presence of hematopoietic stem cells in the adult liver. Transplant Proc 1995; 27: 196. 9. Tashiro H, Fukuda Y, Kimura A, et al. Monitoring for rejection and engraftment following rat orthotopic liver transplantation by polymerase chain reaction. Transplantation 1994; 58: 745. 10. Demetris AJ, Murase N, Fujisaki S, Fung JJ, Rao AS. Starzl TE. 0041-1337/96/6101·4$03.00/0 TRANSPLANTATION Copyright © 1996 by Williams & Wilkins Hematolymphoid cell trafficking. microchimensm. and GVHD reactions after liver. bone marrow. and heart transplantation. Transplant Proc 1993; 25: 3337. 11. Qian S. Demetris AJ. Murase N. Rao AS. Fung JJ. Starzl TE. Murine liver allograft transplantation: tolerance and donor cell chimerism. Hepatology 1994: 19: 916. 12. Thomson AW, Lu L. Murase N. Demetris AJ. Rao AS. Starzl TE. Microchimerism. dendritic cell progenitors and transplanta-tion tolerance. Stem Cells 1995; 13: 622. 13. Demetris AJ. Murase N, Rao AS. Starzl TE. The role of pass en-ger leukocytes in rejection and "tolerance" after solid organ transplantation: a potential explanation of a paradox. In: Touraine JL. Ed. Rejection and tolerance. Vol 25. The Nether-lands: Kluwer Academic Publisher, 1994: 325. 14. Leszczynski D, Halttunen J, Tiisala S, Ustinov J, Renkonen R, Hayry P. Properties of B cells and thy-I-antigen expressing cells infiltrating rat renal allografts. Hum Immunol 1990; 29: 103. 15. Starzl TE, Demetris AJ. Transplantation milestones: viewed with one- and two-way paradigms of tolerance. JAMA 1995; 273: 876. Received 11 October 1995. Accepted 17 October 1995. Vol. 61, 4-9, No.1, January 15, 1996 Printed in U.S.A. CD40-gp39 INTERACTIONS PLAY A CRITICAL ROLE DURING ALLOGRAFT REJECTION SUPPRESSION OF ALLOGRAFI' REJECTION BY BLOCKADE OF THE CD40-GP39 PATHWAy l CHRISTIAN P. LARSEN,2,3 DIANE Z. ALEXANDER,2 DIANE HOLLENBAUGH,4 ERIC T. ELWOOD,2 SHANNON C. RITCHIE,2 ALEJANDRO ARUFFO,4 ROSE HENDRIX,2 AND THOMAS C. PEARSON2.3 Department of Surgery, Emory University School of Medicine, Atlanta. Georgia 30322, and Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121 Studies in vivo have documented the importance of CD40-gp39 interactions in the development of T-de-pendent antibody responses to foreign and auto-anti-gens. In this report, we demonstrate that allograft re-jection is also associated with strong induction of CD40 and gp39 transcripts. When treatment was initi-ated at the time of transplant, MRl, a mAb specific for gp39, induced markedly prolonged survival of fully disparate murine cardiac allografts in both naive and sensitized hosts. However, when therapy was delayed until postoperative day 5, anti-gp39 failed to prolong 1 This study was supported by NIH grants 1R29 Al33588-01Al, AR4268 7 , and 1F32 HL09226 - 0 1A1, an American Heart Association grant-in-aid, and an Emory University research committee grant. 2 Department of Surgery, Emory University School of Medicine. 3 Address correspondence to: Dr. Christian P. Larsen or Dr. Tho-mas C. Pearson, Box M-ll Surgery Research, Emory University Hospital. 1364 Clifton Road, N.E., Atlanta. GA 30322 4 Bristol-Myers Squibb Pharmaceutical Research Institute. graft survival. Allografts from recipients treated with MRl from the time of transplantation showed de-creased expression of transcripts for the macrophage effector molecule, inducible nitric oxide synthase, but essentially unaltered expression of B7 molecules and T cell cytokine transcripts (interleukin [ILJ-2, interfer-on-y, IL-IO, and IL-4) relative to control allografts. In addition, alloantibody responses in the MRl-treated mice were profoundly inhibited. However, our studies using B cell-deficient mice indicated that the ability of MRl to prolong allograft survival was not dependent on B cells. These data suggest that blockade of CD40-gp39 interactions may inhibit allograft rejection primarily by interfering with T cell help for effector functions, rather than by interference with T cell activation. Activated T cells play a pivotal role in the rejection of allografts (1,2), Activation ofT cells to proliferate and secrete cytokines requires both recognition of major histocompatibil-

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Multilineage hematopoietic reconstitution of supralethally irradiated rats by syngeneic whole organ transplantation: with particular reference to the liver

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