Graft-versus-host Reaction in Small-bowel Transplantation and Possibilities for Its Circumvention

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1 The Journal of International Medical Research 2001; 29: Graft-versus-host Reaction in Small-bowel Transplantation and Possibilities for Its Circumvention K WATANABE, T YAGI, H IWAGAKI, Y KIMURA, N MITSUOKA, M INAGAKI, S TANAKA AND N TANAKA First Department of Surgery, Okayama University Medical School, Okayama, Japan To study graft-versus-host reaction (GVHR) in small-bowel transplantation and its underlying mechanisms and to find methods for circumventing GVHR, we used an unidirectional GVHR model in which Lewis (LEW) 3 Wistar King A (WKA) hybrid rats received small-bowel transplants from either LEW or WKA parent rats. The survival time of hybrid rats that received full-length small-bowel transplantation from LEW and WKA was 16.3 ± 2.1 days and 18.2 ± 3.4 days, respectively. When one-quarter of LEW small bowel was transplanted to an hybrid recipient, the survival time was significantly longer at 44.0 ± 23.4 days compared with rats that had received fulllength LEW small-bowel transplantation. The survival time of hybrid rats which received an injection of high-dose ( cells) LEW or WKA spleen cells was 11.9 ± 4.0 days and 13.1 ± 3.6 days, respectively. However, when an injection containing a low dose ( cells) of LEW spleen cells was used, survival was > 100 days, showing significance compared with the survival of rats receiving the higher dose LEW spleen-cell injection. Both small-bowel transplantation and spleen-cell injection were compared for the effective period of recipient resistance to donor cell or small-bowel transplantation as second challenge. When the rats given a quarter LEW small-bowel transplant as first challenge were treated with a high-dose of spleen cells 30 days after transplantation, they survived for > 30 days without GVHR. rats that were treated with a low-dose LEW spleen-cell injection, followed 30 days later by full LEW small-bowel transplantation, had a survival time of > 100 days. These results indicate that segmental small-bowel transplantation and spleen-cell injection as first challenge may facilitate the prevention of GVHR, resulting in resistance to subsequent immunological challenge. KEY WORDS: SMALL-BOWEL TRANSPLANTATION; GRAFT-VERSUS-HOST REACTION; IMMUNOLOGICAL CHALLENGE Introduction Graft-versus-host reaction (GVHR) is a frequent and serious complication of bone-marrow transplantation, but is infrequent in solidorgan transplantation. 1 In experimental small-bowel transplantation, however, where the graft includes a large number of 222

2 lymphocytes that are subject to massive exchange migration between the graft and the host, GVHR can be a serious problem. 2 In humans, small-bowel transplantation has recently become feasible through the use of immunosuppressive drugs, such as cyclosporin A and FK506. 3,4 Nevertheless, it is still more difficult to control the immune reaction after small-bowel transplantation than after transplantation of other organs, especially in the late stages of transplantation. 5 We wished to investigate the underlying mechanisms of GVHR in small-bowel transplantation and to identify methods for its circumvention. We utilized an unidirectional GVHR model with parent-to- hybrid transplantation. In this model, cutaneous GVHR, which includes symptoms such as ear and snout redness, is the typical occurrence after transplantation. Systemic GVHR follows, with intestinal damage, splenomegaly and hepatomegaly, leading to the recipient s death. 6 In this study, the immunological state of long-term surviving hosts was evaluated from the viewpoint of resistance to donor immunocompetent cells. In addition, we investigated the specificity of this resistance and compared the resistance effected in the small-boweltransplantation- and spleen-cell-injectioninduced GVHR models. Materials and methods ANIMALS Adult Lewis LEW (LEW) (RT1 l ) and Wistar King A (WKA) (RT1 k ) rats were purchased from Shizuoka-ken Experimental Animal Supply Co. (Shizuoka, Japan) and (LEW WKA) hybrid rats were bred and maintained at Okayama University Medical School Animal Centre (Okayama, Japan). For small-bowel transplantation, male LEW, WKA and hybrids weighing g were used. Male LEW, WKA and rats weighing 250 g were sacrificed for spleen-cell harvesting. STUDY DESIGN The following 12 groups were studied. Groups 1 (n = 22), 2 (n = 20) and 3 (n = 5) were transplantation-induced GVHR model LEW WKA recipients, which received transplants of either LEW full-length smallbowel (LEW full SBT rats), LEW one-quarter small bowel (LEW 1 / 4 SBT rats) or WKA fulllength small bowel (WKA full SBT rats), respectively. Groups 4 (n = 7), 5 (n = 6) and 6 (n = 4) were spleen-cell-induced GVHR model recipients, in which high-dose LEW spleen cells, low-dose LEW spleen cells or high-dose WKA spleen cells, respectively, were intraperitoneally injected. For groups 7 (n = 4) and 8 (n = 4), recipients were challenged for a second time, 30 days following receipt of a quarter LEW small bowel, with high-dose LEW or WKA spleen cells, respectively. Groups 9 (n = 6) and 10 (n = 5) were LEW spleen-cell-induced GVHR models, in which low-dose spleen cells were intraperitoneally injected into the recipients as the first challenge with LEW or WKA full small-bowel transplantation, respectively, administered as second challenge 30 days after spleen-cell injection. Group 11 (n = 6) was the mesenteric lymph-node-cell-induced GVHR model, in which a high dose of LEW lymph-node cells ( ) was intraperitoneally injected into the recipients. In group 12, rats received total LEW small bowel with mesenteric lymphadenectomy from LEW rats. SMALL-BOWEL TRANSPLANTATION Auxiliary heterotopic small-bowel transplantation was performed as previously 223

3 described. Briefly, the entire small bowel, together with an aortic cuff and portal vein, was removed, followed by perfusion with cold heparinized saline. For segmental small-bowel transplantation, the proximal quarter of the small bowel with the mesenteric lymph node was harvested with the preservation of the aortic cuff and portal vein. For revascularization, the graft aortic cuff was anastomosed to the infrarenal abdominal aorta and the graft portal vein was anastomosed to the infrarenal vena cava. The proximal end of the gut lumen was closed and the distal end opened to the abdominal wall using a Thiry Vella loop. Following surgery, the rats received their normal diet and water ad libitum. In total small-bowel transplantation with mesenteric lymphadenectomy, the visible mesenteric lymph node parallel to the superior mesenteric artery was surgically removed and the entire small bowel was transplanted by the same method, as described above. SPLEEN-CELL OR MESENTERIC LYMPH-NODE-CELL INJECTION A single-cell suspension was obtained from the spleen or mesenteric lymph node of LEW or WKA rats by applying gentle pressure. The suspension was then filtered through a 150-µm platinum mesh and treated with Tris NH 4 Cl (ph 7.6) to lyse red blood cells. The remaining cells were washed three times with phosphate-buffered solution (ph 7.4), and were counted and re-suspended in RPMI-1640 culture medium at a concentration of cells/ml. Subsequently, either 2 ml ( cells; highdose spleen cells) or 0.4 ml ( cells; lowdose spleen cells) of the suspension was injected intraperitoneally under general anaesthesia. STATISTICAL ANALYSIS All data were analysed with a one-way ANOVA and either Gehan s generalized Wilcoxon test or Sheffé s test, using STATISTICA for Macintosh (StatSoft, USA). A P-value of < 0.05 was taken to denote statistical significance. Results SURVIVAL AFTER SMALL-BOWEL TRANSPLANTATION OR SPLEEN- CELL INJECTION The mean ± SD survival times of LEW and WKA full SBT rats (groups 1 and 3) were 16.3 ± 2.1 days and 18.2 ± 3.4 days, respectively (Table 1). The mean survival time of LEW 1 / 4 SBT rats (group 2) was 44.0 ± 23.4 days, showing significant prolongation compared with LEW full SBT rats (group 1; P < 0.05). The mean ± SD survival times of high-dose LEW spleen-cell and WKA spleen-cell rats (groups 4 and 6) were 11.9 ± 4.0 days and 13.1 ± 3.6 days, respectively, whereas that of rats treated with low-dose LEW spleen-cell injection (group 5) was > 100 days. This was significantly longer compared with that of rats receiving highdose LEW spleen-cell injection (group 4; P < 0.01). SURVIVAL AFTER SPLEEN-CELL INJECTION OR SMALL-BOWEL TRANSPLANTATION AS SECOND CHALLENGE The survival of LEW 1 / 4 SBT rats followed by high-dose LEW spleen-cell injection (group 7) was > 30 days (Table 1). This shows significant prolongation compared with the rats that received high-dose LEW spleen-cell injection only (group 4; P < 0.01). The survival of LEW 1 / 4 SBT rats that subsequently received high-dose WKA spleen-cell injection (group 8) was also > 30 days, which was significantly longer than that of high-dose WKA spleen-cellinjection recipients (group 6; P < 0.01). The 224

4 TABLE 1: Recipient rat survival following: small-bowel transplantation; spleen-cell injection; spleen-cell injection followed by small-bowel transplantation as second challenge; mesenteric lymph-node-cell injection; or small-bowel transplantation with mesenteric lymphadenectomy Mean survival Group n First challenge Second challenge time ± SD (days) P-value a 1 22 LEW SBT 16.3 ± 2.1 < 0.01 versus (full-length) group LEW 1/ 4 SBT 44.0 ± 23.4 < 0.05 versus (quarter-length) group WKA SBT 18.2 ± 3.4 (full-length) 4 7 LEW Spl 11.9 ± 4.0 < 0.01 versus ( cells) groups 7 and LEW Spl > 100 < 0.01 versus ( cells) group WKA Spl 13.1 ± 3.6 < 0.01 versus ( cells) group LEW 1/ 4 SBT LEW Spl > 30 ( cells) 8 4 LEW 1/ 4 SBT WKA Spl > 30 ( cells) 9 6 LEW Spl LEW SBT > 100 ( cells) 10 5 LEW Spl WKA SBT 18.3 ± 1.0 ( cells) 11 6 LEW MLN > 100 ( cells) LEW full SBT 20.0 ± 14.7 without MLN a P-values were determined by Gehan s generalized Wilcoxon test after a one-way ANOVA. LEW, Lewis; MLN, mesenteric lymph-node cells; SBT, small-bowel transplantation; Spl, spleen cells; WKA, Wistar King A. survival of low-dose LEW spleen-cell-injected rats that subsequently received LEW full small-bowel transplantation (group 9) was > 100 days, which showed significant prolongation compared with LEW full SBT rats (group 1; P < 0.01). The survival of low-dose LEW spleen-cell-injected rats that subsequently received WKA full small-bowel transplantation (group 10) was 18.3 ± 1.0 days, which showed no significant difference compared with the survival of WKA full SBT rats (group 3). 225

5 SURVIVAL AFTER MESENTERIC LYMPH-NODE-CELL INJECTION OR SMALL-BOWEL TRANSPLANTATION WITH LYMPHADENECTOMY The survival of rats that received a high-dose LEW mesenteric lymph-node-cell injection (group 11) was > 100 days (Table 1). This shows significant prolongation compared with high-dose LEW spleen-cell recipients (group 4; P < 0.01). The mean survival of rats who received LEW full small-bowel transplantation with lymphadenectomy (group 12) was 20.0 ± 14.7 days, showing no statistical difference compared with the survival time of LEW full SBT rats (group 1). Discussion This study shows that transplantationinduced GVHR is comparable to GVHR induced by spleen-cell injection in terms of the survival time of the recipient. In addition, in this parent-to- -hybrid transplantation model, segmental transplantation and low-dose spleen-cell injection did not induce fatal GVHR. This represented a significant prolongation in survival compared with recipients of full small-bowel transplantation and high-dose spleen-cell injection, respectively. When high-dose LEW spleen cells were injected into the surviving rats after segmental LEW transplantation, survival was prolonged significantly compared with rats that received high-dose LEW spleen-cell injection only. Likewise, when high-dose WKA spleen cells were injected into rats after segmental LEW small-bowel transplantation, the recipients showed a significant increase in survival time compared with the group that received highdose WKA spleen cells only. These results suggest that pre-sensitization with segmental small-bowel transplantation can induce resistance to high-dose spleen-cell injection as second challenge. This resistance was considered non-specific. The survival of LEW full SBT rats after low-dose LEW spleen-cell injection was significantly prolonged compared with the survival of the group that received LEW full small-bowel transplantation only. In contrast, when WKA full small-bowel transplantation was performed after lowdose LEW spleen-cell injection, its survival was not significantly different from that of the group that received WKA full smallbowel transplantation only. This indicates that pre-sensitization with low-dose splenocytes induces donor-specific immunehyporesponsiveness to secondary smallbowel transplantation. This difference in resistance observed between the small-bowel-transplantationand spleen-cell-injection-induced GVHR models may be due to quantitative factors such as inoculated volume of lymphocytes, or to qualitative factors such as spleen cell versus mesenteric lymph-node cell or the existence of small bowel in the recipient. This present research suggests that the origin and dose of cells responsible for GVHR or resistance differed according to the nature of the donor cells. The small bowel contains immunocompetent cells in Peyer s patches, gut wall and mesenteric lymph nodes. The origin of immunocompetent cells and the structure in which they exist warrants investigation. A previous study reported that small-bowel-transplantation-induced GVHR was mediated by mesenteric lymph nodes and did not require the bowel per se in the parent-to- hybrid model. 7 Another report demonstrated that removal of the mesenteric lymph nodes uniformly prevented small-bowel-transplantation-induced GVHR, whereas intraperitoneal mesenteric lymphnode grafting induced GVHR, suggesting that the mesenteric lymph node is the major 226

6 lymphoid tissue responsible for small-boweltransplantation-induced GVHR. 8 Our results, however, showed that highdose LEW mesenteric lymph-node-cell injection could not induce GVHR and that the survival of rats receiving LEW full small-bowel transplantation without mesenteric lymph-node-cell injection was not significantly different from that of LEW full SBT rats. Therefore, the mesenteric lymph-node cells are not the major lymphnode cells responsible for GVHR in our model. This result is consistent with a previous report that graft mesenteric lymphadenectomy prevented GVHR only in certain strain combinations. 9 The present results suggest that the intestinal mucosal layer has an important immunological role in small-bowel transplantation-induced GVHR. Indeed, it has already been reported that small-bowel-transplantation-induced GVHR depends on the length and site of origin. 10 In the current study, we demonstrated that rats receiving segmental small-bowel transplantation avoided GVHR in 18 of 20 cases and pathological examination revealed that normal structures were completely preserved (data not shown). In contrast, rats receiving LEW full small-bowel transplantation, either with or without mesenteric lymph-node cells, did not avoid GVHR. This suggests that the gut itself, rather than the mesenteric lymph nodes, plays an important role in the induction of GVHR. In conclusion, we found that segmental small-bowel transplantation from parent to rats does not induce lethal GVHR and establishes non-specific resistance to lethal GVHR by secondary challenge with parental strain immunocompetent cells. Provided the appropriate site and segment of small bowel is used for small-bowel transplantation, the recipient would avoid GVHR and maintain a normal physiological status. In addition, rats receiving low-dose Spl injection from parent can avoid small-bowel transplantation-induced GVHR, and this resistance is donor-specific. This suggests that presensitization with low-dose donor antigens can induce tolerance to secondary organ transplantation. Further research is warranted to elucidate the underlying mechanism by which this tolerance is acquired. Received for publication 11 December 2000 Accepted 3 January Cambridge Medical Publications References 1 Saat RE, Heineman E, de Bruin RW, Marquet RL, Jeekel J: Total orthotopic allogeneic small bowel transplantation in rats. Attempts to ameliorate the graft-versus-host disease by irradiation and transfusions of the donor. Transplantation 1989; 47: Deltz E, Ulrichs K, Schack T, Friedrichs B, Muller-Ruchholtz W, Muller-Hermelink HK, et al: Graft-versus-host reaction in small bowel transplantation and possibilities for its circumvention. Am J Surg 1986; 151: Grant D, Wall W, Mimeault R, Zhong R, Ghent C, Garcia B, et al: Successful small-bowel/liver transplantation. Lancet 1990; 335: Todo S, Tzakis AG, Abu-Elmagd K, Reyes J, Fung JJ, Casavilla A, et al: Cadaveric small bowel and small bowel/liver transplantation in humans. Transplantation 1992; 53: Todo S, Tzakis A, Abu-Elmagd K, Reyes J, Starzl TE: Current status of intestinal transplantation. Adv Surg 1994; 27: Kai K, Tanaka S, Orita K: Donor pretreatment with deoxyspergualin on prevention of graft-vs-host reaction after small bowel transplantation. Transplant Proc 1996; 28: Luck R, Klempnauer J, Steinger B: Transplantation of MHC incompatible vascularized mesenteric lymph nodes: identical pattern of lethal graft-versus-host disease as induced by small bowel grafts. Transplant Proc 1990; 22: Pirenne J, Lardinois F, D Silva M, Fridman V, Boniver J, Mahieu P, et al: Relevance of mesenteric lymph nodes to graft-versus-host disease following small bowel transplantation. Transplantation 1990; 50:

7 9 Clark CL, Price BA, Malcolm P, Lear PA, Wood RF: Graft versus host disease in small bowel transplantation. Br J Surg 1991; 78: Kimura K, Money SR, Jaffe BM: The effects of size and site of origin of intestinal grafts on small-bowel transplantation in the rat. Surgery 1987; 101: Address for correspondence Dr H Iwagaki First Department of Surgery, Okayama University Medical School, Shikata-cho, Okayama , Japan. iwagaki@med.okayama-u.ac.jp 228