Apoptosis of crypt cells and lymphocytes in gut-associated lymphoid tissues during small intestinal...
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Apoptosis of Crypt Cells and Lymphocytes in Gut-Associated Lymphoid Tissues During Small Intestinal Graft Rejection in Rats Y.J. Park, S. Fujisaki, K. Kimizuka, K. Shugito, R. Tomita, M. Fukuzawa, and K. Matsumoto ABSTRACT Introduction. We investigated the extent of apoptosis in crypt cells and Peyer’s patches (PPs) during small bowel allograft rejection in rats to examine whether the Fas/FasL pathway participates in apoptosis within grafts during rejection. Materials and methods. Orthotopic small bowel transplantation with portocaval drain- age was performed from Brown Norway to Lewis (LEW) rats. Isografted (LEW 3 LEW) and nontransplanted animals served as the controls. Animals were sacrificed on days 3, 5, on 7 after SBT (each n 5). An in situ end-labeling (ISEL) technique was used to detect apoptotic cells. Indirect immunoperoxidase staining was also performed using monoclonal antibodies against rat Fas or Fas-L. Results. The number of ISEL-positive enterocytes in the allografts increased signifi- cantly on days 3, 5, and 7. Similarly, in the PPs of the allografts, the number of ISEL-positive mononuclear cells increased significantly on days 3, 5, and 7. On day 7 the number of Fas- and FasL-positive enterocytes were increased significantly in the allografts compared with the nontransplanted controls. Similarly, in the PPs, Fas- and FasL-positive mononuclear cells also increased significantly on day 7 in the allograft. Conclusion. Although an increase, number of apoptotic enterocytes and lymphocytes were observed in the early phase, activation of Fas/FasL system occurred during the late phase of small bowel graft rejection. These findings suggest that both rejection-associated and sepsis-induced forms of apoptosis may be associated with small bowel graft rejection. T HE ROLE OF APOPTOSIS during rejection of the lymphoid-rich small bowel graft is still unclear. In addition, little is known about Fas and FasL expression in relation to the presence of apoptotic cells in small bowel allografts. We investigated the expression of apoptosis in crypt cells and Peyer’s patches (PPs) during small bowel allograft rejection in rats to examine whether the Fas/FasL pathway participates in the apoptosis during rejection. MATERIALS AND METHODS Ten- to 12-week-old male rats of the inbred strain. Brown Norway (RT1; 250 g) Lewis (LEW; RT1; 300 g) rats of the inbred strain Brown Norway (RT1; 250g) was used for orthotopic small bowel transplantation with portocaval drainage to Lewis (LEW; RT1; 300g) rats. Isografted (LEW 3 LEW) and nontransplanted animals served as controls. Animals were sacrificed on posttrans- plant days 3, 5, or 7 (each n 5; isograft n 5). An in situ end-labeling (ISEL) technique was used to detect apoptotic cells. To quantify the results, sections were evaluated blindly by five pathologists. We calculated the average number of positive cells in 100 fields of crypt tissue chosen at random. In the PP samples, we calculated the average number of positive cells among 10 randomly chosen fields of vision within 100 areas of lymphocytes. Differences between isograft (LEW 3 LEW) and allografted (BN 3 LEW) were compared using the Student t test and considered to be of statistical significance when P .05. Indirect immunoperoxidase staining was performed using monoclonal antibodies against rat Fas and FasL and graded from 1 (faint) to 4 (high levels) among crypt cells and PPs. The degree of staining of each section was ranked blindly by five independent observers; the median rank of each section was determined. Finally the scores of respective animals were defined as the median rank of each section of three pieces taken from the same animal. Results were expressed as From the First Department of Surgery (Y.J.P., S.F., K.K., K.S., R.T., M.F.), Nihon University School of Medicine, and Depart- ment of Pathology (K.M.), Nippon Medical School, Tokyo, Japan. Address reprint requests to Yeong Ji Park, 7-2-1 Chuo Ka- sukabe, Saitama 344-8588, Japan. E-mail: [email protected] u.ac.jp © 2004 by Elsevier Inc. All rights reserved. 0041-1345/04/$–see front matter 360 Park Avenue South, New York, NY 10010-1710 doi:10.1016/j.transproceed.2004.01.097 Transplantation Proceedings, 36, 353–355 (2004) 353
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poptosis of Crypt Cells and Lymphocytes in Gut-Associatedymphoid Tissues During Small Intestinal Graft Rejection in Rats
.J. Park, S. Fujisaki, K. Kimizuka, K. Shugito, R. Tomita, M. Fukuzawa, and K. Matsumoto
ABSTRACT
Introduction. We investigated the extent of apoptosis in crypt cells and Peyer’s patches(PPs) during small bowel allograft rejection in rats to examine whether the Fas/FasLpathway participates in apoptosis within grafts during rejection.Materials and methods. Orthotopic small bowel transplantation with portocaval drain-age was performed from Brown Norway to Lewis (LEW) rats. Isografted (LEW3 LEW)and nontransplanted animals served as the controls. Animals were sacrificed on days 3, 5,on 7 after SBT (each n � 5). An in situ end-labeling (ISEL) technique was used to detectapoptotic cells. Indirect immunoperoxidase staining was also performed using monoclonalantibodies against rat Fas or Fas-L.Results. The number of ISEL-positive enterocytes in the allografts increased signifi-cantly on days 3, 5, and 7. Similarly, in the PPs of the allografts, the number ofISEL-positive mononuclear cells increased significantly on days 3, 5, and 7. On day 7 thenumber of Fas- and FasL-positive enterocytes were increased significantly in the allograftscompared with the nontransplanted controls. Similarly, in the PPs, Fas- and FasL-positivemononuclear cells also increased significantly on day 7 in the allograft.Conclusion. Although an increase, number of apoptotic enterocytes and lymphocyteswere observed in the early phase, activation of Fas/FasL system occurred during the latephase of small bowel graft rejection. These findings suggest that both rejection-associatedand sepsis-induced forms of apoptosis may be associated with small bowel graft rejection.
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HE ROLE OF APOPTOSIS during rejection of thelymphoid-rich small bowel graft is still unclear. In
ddition, little is known about Fas and FasL expression inelation to the presence of apoptotic cells in small bowelllografts. We investigated the expression of apoptosis inrypt cells and Peyer’s patches (PPs) during small bowelllograft rejection in rats to examine whether the Fas/FasLathway participates in the apoptosis during rejection.
ATERIALS AND METHODS
en- to 12-week-old male rats of the inbred strain. Brown NorwayRT1; �250 g) Lewis (LEW; RT1; �300 g) rats of the inbred strainrown Norway (RT1; �250g) was used for orthotopic small bowel
ransplantation with portocaval drainage to Lewis (LEW; RT1;300g) rats. Isografted (LEW 3 LEW) and nontransplanted
nimals served as controls. Animals were sacrificed on posttrans-lant days 3, 5, or 7 (each n � 5; isograft n � 5). An in sitund-labeling (ISEL) technique was used to detect apoptotic cells.o quantify the results, sections were evaluated blindly by five
athologists. We calculated the average number of positive cells in u2004 by Elsevier Inc. All rights reserved.60 Park Avenue South, New York, NY 10010-1710
ransplantation Proceedings, 36, 353–355 (2004)
00 fields of crypt tissue chosen at random. In the PP samples, wealculated the average number of positive cells among 10 randomlyhosen fields of vision within 100 areas of lymphocytes. Differencesetween isograft (LEW 3 LEW) and allografted (BN 3 LEW)ere compared using the Student t test and considered to be of
tatistical significance when P � .05. Indirect immunoperoxidasetaining was performed using monoclonal antibodies against ratas and FasL and graded from 1 (faint) to 4 (high levels) amongrypt cells and PPs. The degree of staining of each section wasanked blindly by five independent observers; the median rank ofach section was determined. Finally the scores of respectivenimals were defined as the median rank of each section of threeieces taken from the same animal. Results were expressed as
From the First Department of Surgery (Y.J.P., S.F., K.K., K.S.,.T., M.F.), Nihon University School of Medicine, and Depart-ent of Pathology (K.M.), Nippon Medical School, Tokyo, Japan.Address reprint requests to Yeong Ji Park, 7-2-1 Chuo Ka-
ukabe, Saitama 344-8588, Japan. E-mail: [email protected]
.ac.jp0041-1345/04/$–see front matterdoi:10.1016/j.transproceed.2004.01.097
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354 PARK, FUJISAKI, KIMIZUKA ET AL
edian scores of five animals for each time. Differences betweenxperimental groups compared using the Mann-Whitney U testere considered to be statistically significant when P � .05.
ESULTSSEL
SEL showed a dramatic increase in the number of apopto-ic enterocytes among small intestine allografts, whereasncreased labeling was not observed among isogeneic grafts.he number of ISEL-positive enterocytes was increased
ignificantly on days 3, 5, and 7 in the allograft comparedith an isograft (P � .001). ISEL showed a dramatically
ncreased number of apoptotic cells in the PPs of al-ografted small intestines, whereas increased labeling wasot observed in isogeneic small intestinal grafts. The num-er of ISEL-positive cells in PPs increased significantly onays 3, 5, and 7 in the allograft compared with isografts (P
.001).
ig 1. (a) On day 7, the expression of Fas-positive enterocytesf Fas-positive enterocytes increased significantly on day 7 inxpression of FasL-positive enterocytes in isograft was gradenterocytes increased significantly on day 7 in the allografts co
ig 2. (a) On day 7, the expression of Fas-positive cells in Peyexpression of Fas-positive cells in Peyer’s patches increased sigb) On day 7, the expression of FasL-positive cells in Peyer’s
xpression of FasL-positive cells in Peyer’s patches increased significas/FasL
Change of Fas/FasL-positive cryptic cells. On day 7,sografts showed grade 2 expression of Fas-positive entero-ytes while it was grade 4 in allografts (Fig 1). Thexpression of Fas-positive enterocytes increased signifi-antly on day 7 in the allografts compared with the isograftsP � .05).
Change of Fas/FasL-positive cells in PPs. On day 7, thexpression of Fas-positive cells in PPs in isografts was grade, but in allografts it was grade 4 (Fig 2). The expression ofas-positive cells in PPs increased significantly on days 7 in
he allografts compared with isografts (P � .05).
ONCLUSIONS
poptosis has been reported to occur in the course ofxperimental and human hepatic, renal, and pancreaticllograft rejection.1–5 Infiltrating immune cells may destroyhe graft by apoptosis resulting in allograft rejection. This
ograft was grade 2 but in allograft was grade 4. The expressionllografts compared with isograft (*P � .05). (b) On day 7, theut in allograft was grade 4. The expression of FasL-positiveed with isograft (*P � .01).
atches in isograft was grade 2 but in allograft was grade 4. Thently on day 7 in the allografts compared with isograft (*P � .05).hes in isograft was grade 2 but in allograft was grade 4. The
in isthe a2, b
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antly on day 7 in the allografts compared with isograft (*P � .01).
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APOPTOSIS OF CRYPT CELLS AND LYMPHOCYTES 355
ype of apoptosis may be mediated by two major cytotoxicathways: (1) the Fas-FasL system and (2) the granzyme/perforin products of CTLs.6,7 In this study, we investi-ated the changes in Fas and FasL expression. Although anncrease of apoptotic enterocytes and lymphocytes wasbserved among the early phase of small bowel graftejection (postoperative days 3 to 5). Activation of theas/FasL system was observed during the late phase (post-perative day 7) of small bowel graft rejection. Thesendings suggest that not only rejection-associated apoptosisut also sepsis-induced apoptosis may be associated withmall bowel graft rejection.
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copic study of the mode of donor cell death in unmodified
ejection of pig liver allografts. Aust J Exp Biol Med Sci 55:401,9772. Nawax S, Fennel RH: Apoptosis of bile duct epithelial cells in
epatic allograft rejection. Histopathology 25:137, 19943. Olsen S, Burdick JF, Keown PA, et al: Primary acute renal
ailure (“acute tubular necrosis”) in the transplanted kidney:orphology and pathologenesis. Medicine 68:173, 19894. Ito H, Kasagi N, Shomori K, et al: Apoptosis in the human
llo-grafted kidney: analysis by terminal deoxynucleotidyl trans-erase-mediated dUTP-biotin nick end labeling. Transplantation0:794, 19955. Knoop M, McMahon RFT, Jones CJP, et al: Apoptosis in
ancreatic allograft rejection. Exp Pathol 41:219, 19916. Lowin B, Hahne M, Mattmann C, et al: Cytolytic T-cell
ytotoxicity is mediated through perforin and Fas lytic pathways.ature 370:650, 19947. Cohen JJ: Apoptosis: mechanisms of life and death in the
mmune system. J Allergy Clin Immunol 103:548, 1999
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