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Effect of 15-Deoxyspergualin (DSG) on Rat Kidney Allograft: Immunological Mechanisms Implicated in Prolonged Survival
~22-5347/95/1546-2197$03.00/0 THE J, IURNAL OF UR0UW;U Copyright 0 1995 by AMERICAN UROLOGICAL ASS~CIATION, INC.
VoL 164,2187-2202. December 19B6 Printed in
u.sA.
EFFECT OF 15-DEOXYSPERGUALIN (DSG) ON RAT KIDNEY ALLOGRAFT: IMMUNOLOGICAL MECHANISMS IMPLICATED IN PROLONGED SURVIVAL TATSUYA CHIKARAISHI, HIROSHI * S I
T0SHIMOR.I SEKI,TOMOHIKO KOYANAGI AND TAKASHI YOSHIKI
From the Departments of Urnlogy and Path&,
Hokkaido University School of Medicine, Sapporn, Japan
ABSTRACT
Purpose and Methods: The effect of short-term administrationof 15-deoxyspergualin(DSG), 5 mg.lkg./day from postoperative days 4 to 7, on rat renal transplantation was studied. Results: Although allografb treated with DSG survived longer than nontreatd ones, cellular infiltration in both grafts did not differ. However, renal tubular cells of DSG-treated grafta proliferated well and escaped apoptotic cell death. A donor-specific tolerance 2 weeks after transplantation was developed, and cells with in vitro Suppressor function were induced in such animals. Conclusions:Treatment with DSG appears to prevent lethal attack of effector cells on tubular cells in situ and to generate suppressor cells in the maintenance phase of graft enhancement. KEYWORDS: kidney transplantation,rate
allow reeipients to survive. Recipients dying within 7 days poetoperatively were regarded ae technical ''errors" and were excluded from the data analysis. To examine the effect of short-term administration of DSG, TO or LEJ rat kidneys were transplanted into WKAH rats. The recipients were divided into 2 groups, a control group and a DSG-treated group. In the former group, no DSG was administered after transplantation. In the latter group, DSG was given in a dose of 5 mgJkgJday intramuscularly, from days 4 to 7, postoperatively. Graft survival was compared between the 2 group. Skin tmnsplantation. Tail skin,10-mm. in diameter, of the donor rats was transplanted to the back of the recipients. A single recipient received skin grafts from 5 separate donors at the same time. On postoperative day 7, the graRe were examined. If the ekin grafts had already become necrotic by this time, they were considered technicalfailures. The graffs were examined every day, and when they had become necrotic by 50% or more, they were considered as rejections. MATERIALS AND METHODS DSG treated WKAH rats with TO kidney transplants re15-Deoxyspergualin (DSG). Nippon lbyaku Co. Ltd., TO- ceived additional skin grafb from WKAH, TO, SDJ, LEJ and kyo,Japan kindly supplied DSG which was dissolved in 0.9% BUF rats. In group A, skin graRs were performed 1week NaCl at a concentration of 2 mg./ml., sterilized with a Milli- after renal transplantation; in groups B and C, the skin pore filter (Kanto Chemical Co., Inc., Tokyo, Japan). and grafts were performed 2 weeks and more than 100 days aRer renal transplantation, respectively. Naive WKAH rats and Stored at -2OC until use. (RT-lk, RT- DSGtreated WKAH rats without TO kidney transplants also Animals. The inbred rat strains, lAkBkDk),TO/Hkm (RT-l", RT-lA"B"D"), S D J h (RT-l", received skin grafts (groups D and El. Monoclonal antibodies. Monoclonal antibodies (Mab) RT-lA"B"D"), LEJ/Hkm (RT-lj, RT-lA"BbDb), BUF/Hkm (RT-lb,RT-lAbBbDb),maintained and fed at the Center for against rat major histocompatibili~class (MHC) class II Biological Experiments, Hokkaido University, were used. (0x6,Biosys S.A.), rat intemllular adhesion molecule1 Male rats weighing more than 250 g. were used for the (ICAM-1, lA29),6 rat lymphocyte function-associated antiexperiment. gen-1 (LFA-1, WT.1)7 and proliferating cell nuclear antigen Renal transpiantation. Renal transplantation was per- (PCNA, Dakopatts, Glostrup, Denmark) were used in this formed according to the method described by Lees with some study. The HA58 Mab (anti-human ICAM-1),6 which has no modifications. To evaluate the enhancing effect of DSG on cross-reaction with rat tissues, was used as a negative conrenal allografts, both kidneys of the recipient were removed trol antibody. The lA29and WT.l were kindly supplied by Dre. SO that graft survival could be estimated as recipient survival M. Myasaka and T. Tamatani of the Department of Immunoldays. When the graft histology was examined chronologi- ogy,Tokyo Metmpolitan Institute of Medical Science. cally, the recipient's leR kidney was not removed, so as to Histologic and immunohistochemical examinations. Serial kidney biopsy was done on days 4,6,8 and 14, under general -pted for publication January 23,1995. Requests for reprints: Department of Pathology, Hokkaido Uni- anesthesia with ether. The biopsy materials were stained versity School of Medicine,N-16,W-7, Kita-ku,Sapporo 060, Japan. with hematoxylin and eosin for histologic examination. For
A guanidinic-like structure, 15-deoxyspergualin (DSG), is an analog of spergualin,which was isolated from the culture filtrates of Bacillus laterosporue.1 In experimental models of transplantation, DSG has been shown to prolong survival of rat skin, heart, liver, pancreas and kidney all0grafb.S' The mechanism of prolonged graft survival by DSG remains unclear. In previous studies, the duration of DSG administration ranged from 7 to 14 days, beginning on the day of transplantation. In the present study, the effect of DSG, administered for a shorter period, 4 days, beginning on an earlier post-transplant day, was investigated in the rat renal transplantation model. The shorter period of treatment was selected to minimize a possible untoward effect by longer administration. These studies also focused on the mechanism of grafk enhancement in this short-term DSG administration model, separately in both the induction and maintenance phases.
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15-DEOXYSPERGUALIN AND RAT KIDNEY ALLOGRAFT
2198
TABLE1. Effect of DSG on renal allograft in the rat the immunohistochemical study using OX6,1A29, WT.l, and HA58, the specimens were rapidly frozen, sliced a t 5 pm Strain combination Experimental group p value thickness with a cryostat and fixed in cold acetone for 20 (donor recipient) contml group DSG group minutes. For PCNA staining, the samples were fixed in 10% TO -+WKAH 8, 8, 9, 9, 11, 16 19, 105*, 114*, 176*, p = 0.0007 buffered formalin for 2 days, embedded in paraffin, cut and 187, 210, 211*, 231 (10.2 -t 3.1) deparaffinized by xylene before being labeled with PCNA. 1156.6 2 71.8) 0x6, LA29,WT.1, PCNA and HA58 were used as primary LEJ -WKAH 9,9, 10, 10, 12 10,69, 115". 271*, 316' p = 0.0317 antibodies in the avidin-biotin-peroxydasecomplex method. (156.2 Z 131.7) (10.0 5 1.2) Nuclei were counterstained with hematoxylin for 0 x 6 , lA29, In the DSG group, DSG was given in a dose of 5 mg./kg./day intramuscularly WT.1 and HA58. The periodate acid-Schiff (PAS)stain was from days 4 to 7 postoperatively. In the control group, DSG was not given. * These animals were sacrificed on the indicated days in order that they used in the PCNA-stained sections in order to distinguish whether labeled cells were graft infiltrating lymphocytes might be employed for other experiments. (GIL) or renal tubular cells (TC). PCNA-positive and negative GIL or TC were counted within 10 randomly selected Mixed lymphocyte culture reaction (MLR). One way MLRs high power fields (the microscopic field at x40 objective lens), followed by calculation of PCNA-positive ratio by the for- were also performed. Lymphocytes from mesenteric lymph nodes, 1 x lo5, were used as responders, and 2 x lo5 of mula: mitomycin C (25 pg./ml.)-treated lymph node cells were used PCNA-positive GIL or TC X 100 as stimulators. These cells were cocultured in RPMI-1640 medium, including 10% fetal calf serum (GIBCO, Grand PCNA-positive ratio (%) = Total GIL or TC Island, New York) and 5 X lop5 MA. 2-mercaptoethanol There were 3 experimental groups. In the isograft group, (Wako Pure Chemical Industries, Osaka, Japan). The MLRs WKAH kidneys were transplanted into WKAH recipients. In were then performed for 96 hours in a humidified 5% CO, the allograft group, TO kidneys were transplanted into atmosphere at 37C. [3Hlthymidine, 0.5 pCi (Japan RadioisoWKAH recipients. In the DSG group, TO kidneys were trans- tope Association, Tokyo, Japan), was added to each well 16 hours before the cells were harvested. The cultured cells were planted into WKAH with short-term DSG administration. Deoxyribonucleic acid nick end labeling. DNA nick end harvested by means of an automatic cell harvester, and the labeling of tissue sections was performed by a method previ- radioactivity was measured with a liquid scintillation ously described.9 In brief, deparaffinized sections were incu- counter. In some experiments, 1 x lo5 of spleen or lymph bated with 20 pg/ml. proteinase K (Sigma Chemical Co., St. node cells from recipient WKAH rats with DSG-treated, longLouis, Missouri) for 20 minutes at room temperature, fol- surviving grafts or similar cells from naive WKAH rats were lowed by multiple washes with distilled water. After blocking added to MLRs as modulator cells to evaluate for the possiof endogenous peroxidase with 2% H,O, for 7 minutes, the bility of tolerance. Results of MLR were expressed as means sections were rinsed with distilled water and immersed in and standard deviations of triplicated wells (96 round-bottom terminal deoxy transferase (TdT) buffer (30 mM. Trizma well, Petray 96U, Terumo, Japan). Three independent experbase, pH 7.2, 140 mM. sodium cacodylate, 1 mM. cobalt iments were performed, each using a long-surviving animal chloride); TdT (0.3 e.u./l) and biotinylated dUTP in TdT (each more than 100 days post-transplant). Statistical analysis. All data values were presented as buffer were then added a t 37C for 90 minutes. The reaction means 2 standard deviation. Statistical analyses were done was stopped by application of TB buffer (300 mM. sodium chloride, 30 mM. sodium citrate) for 30 minutes. ARer mul- with the unpaired Wilcoxon test. A probability of p c0.05 was tiple washes with distilled water, peroxidase-labeled strep- required for significance. toavidin (Dakopatts) was added for 30 minutes at 37C. The RESULTS preparations were then washed with distilled water and stained with diaminobenzidine for 30 minutes at 37C. The Effects of short-term administration of DSG on renal alPAS stain was performed in nick end labeled sections, to lografts. In the DSG group, 7 of 8 allografted kidneys in the distinguish whether the labeled cells were GIL or TC. The TO-to-WKAH combination and 3 of 5 in the LEJ-to-WKAH numbers of labeled nuclei of GIL or TC were counted in 10 combination survived for more than 100 days, whereas all of randomly selected high power fields. Samples that were ob- the untreated controls were rejected in 10.2 3.1 and 10.0 5 tained by partial graft excision on days 4, 6 and 8 were 1.2 days, respectively (table 1). studied. Experimental groups were divided into two: namely, Tolerance induction in DSG-treated long-surviving recipithe allograft group that was not treated with DSG and the ent ruts. In group A, all of the syngeneic WKAH skin grafts DSG group into which allografts were transplanted and survived indefinitely, and 4 of 5 TO grafts survived indefishort-term DSG administration was carried out. nitely. Two of 5 SDJ skin grafts showed prolonged survival, _f
TABLE2 Imniunologrcul tolerance by short-term DSG adminlstratlon ~~
Expenmental
__
POUP
~
WKAH (RT-1'1
TO I RT 1 ' I I ~
A
0
E
C)
. . . . . . . . . . . . . . ................ Skin Graft Donor IRT-1)
-
LEJ (RT-1')
SDJ (RT-1")
... ~ ~ _ _ _ _ .
BUF iRT-lh)
0
( 1
12
C
D
0
.
E
12 0
11 .
0
10 .
.
10
17 0 14 0 13 0
12
.
12
14
14
11
10
9
17 0 10 0 12
10 9 . . in 9 . 0
0
.
10 0
12 .
13
0
10 .
14
12 .
.
11
11
11
11 .
10
8
16
12
8
7
12
10 10 . . 11 9
0 16 .
0
9
i
12
.
n
0
9
11
8
9
8
.
9
8
15-DEOXYSPERGUALINAND RAT KIDNEY m
FIG.1. Histology of allograRa on day 14 after transplantation. In untreated, acute rejection up, renal parenchyma,including tubular e ithelial cells, is largeTdestroyed by cellular in6ltrab, intermin& with hemorrhage and degeneration (A). In DSG-treated group, although prominent cellular infiltrates are observed, r e d parenchyma is well preserved (B).(4, B; X200). whereas all of the LEJ and BUF skin grafh were rejected. In groups B and C,all of the WKAH and TO skin grafts survived indefinitely,but all of the SDJ skin gr& were rejected. In groups D and E,only the WKAH skin grafb survived indef-
W
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initely. All of the skin grafta from other strains were rejected. The evidence indicates that donor-specific tolerance wae induced in DSG-treated recipients mta (table 2). Histologv. In the ieograft group, focal and mild cellular infiltrates, composed chiefly of small lymphocytes, were evident from days 4 to 14. The cellular infiltrates never became intense or diffuee.In the allograft group, cellular infiltrates around the interlobular arteries and veins were seen on day 4; they spread to the peritubular areas on day 6.On day 8, intense cellular infiltrates were observed and interstitial hemorrhage, edema, and thrombosis developed. By day 14, the histologichallmark of =aft loss was noted in which most of the r e d tubules were-destroyed (6g. 1,A). In the DSG group, histologic changes similar to thoae seen in the unhated allograft group were obeerveed on days 4 and 6.Diffuse cellular intiltrates were evidenced in the pentubular areas on days 8 and 14, but interstitial hemorrhage, edema and tubular damage were minimal (fig. 1,B). The histologic appearance of all0grafh-I kidneys from long-term survivors was also investigated. Only small patchy foci of lymphocytic infiltration were observed, and blastic lymphocyteswere very rarely seen. There was no interstitial edema and bleeding, and there was no evidence of tiesue iqjury. Immunohistochemistry.In the isogratt group, no subatantial expression of MHC clam 11 and ICAM-1 antigens was observed on the renal constitutive cells. Major histmompat ibility class class 11and LFA-1 antiens were preaent on the infiltrating cells,some of which aIe0 expressed ICAM-1. In the untreated allograft group, some tubular epithelial cells expressed MHC class II and ICAM-1 a n t i g e ~after day 4. Major hietooompatibility class II and LF'A-1 positive cells infiltrated the graft after day 4. The expression of MHC clasa 11and ICAM-1 antigens on renal tubular cells was gradually augmented by days 6,8 and 14. The number of LFA-1positive infiltrating cells aLS0 gradually increased. In the DSG group, there was essentially the same ICAM-1 and MHC class 11 antigen expression on renal tubular cells as was observedin the untreated allograftgroup. The number of LFA-1-positive cells also gradually increased. Table 3 shows the PCNA-positive rate in GIL and TC. Figure 2 shows a representative FCNA staining in allografts. Although the FCNA-positive rate of GIL became slightly higher in the allograft group than in the DSG group, no statistid significance was noted. The PCNA-positive rate of TC was -&I nificantly higher in the DSG group than in the allograft group (p <0.05).
TABLE3. Rolifemting cell nuclear antigen-positivemte of gmfi infiltmting lymphocytes (GIL)and tubular ceUs fN.3 F'CNA-poaitive rete (96)
cell type
DSG treatment
GlL
(-1
17.6 2 9.91
DSG
11.8 2 3.1
n:
(-)
DSG
Day 4
Day 6 n.S.t
18.82 9.7
9.0 -+ 11.77 n,8.* 12.2 -+ 13.0
Day 8
21.0 f 5.77
13.3 2 12.8
1
37.3
n.8.+
2
]
12.8
n,8.*
23.0 2 11.2 n.8.*
23.7 -+ 20.6
33.2 t 19.6
* n.8.: not significant. TABLE4. Number of DNA-fmgmented nuclei detected by the DNA nick end-hbeling no msitive celldl0 HF'P
Cell type
DSG treatment
GIL
(-)
DSG
n:
(-)
DSG
Dav 4
Day 6
0.3 ? 0.671 n.8. 0.3 2 0.41 0.05 2 0.22 0.06
1
0.3
= 1.321
Day 8 n.s,
0.65 ? 0.691
0.76 2 0.91
0.45 2 0.69
0.6 f 0.82
0.55 2 0.82
n.8,
= 0.22
HPF: High power field repregents the field observed with a 4OX objectiw lens.
n.B.
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15-DEOXYSPERGUALINAND RAT KIDNEY ALLOGRAFT
FIG. 2. Proliferating cell nuclear antigen distribution in renal tubular cells on day 8 after transplantation. Nuclei of renal tubular cells in DSG group allografts ( B )are labeled to greater degree with PCNA than those in untreated allograft group (A). (A, B ; X400). Deoxyribonucleic acid nick end labeling. The number of the labeled TC in the allograft group demonstrated a 30-fold increase from days 4 to 8 (table 4). On day 8, the number of labeled TC in the DSG group was significantly smaller than that in the allograft group (p = 0.0245). The number of labeled GIL in the DSG group did not differ significantly from that in the allograft group. A representative nick end labeling feature in the allografts is shown in figure 3. Mixed lymphocyte culture reaction. By adding spleen cells from DSG-treated WKAH rats with long-surviving TO kidney grafts, TO-to-WKAH MLR was significantly suppressed (as much as 92%). Spleen cells of naive WKAH also suppressed TO-to-WKAH MLR to a lesser extent (p <0.0001), whereas a n addition of lymph node cells from the DSGtreated WKAH rats with long-surviving TO kidney grafts did not suppress TO-to-WKAHMLR but augmented it as did lymph node cells from naive rat. Lymph node cells from DSG-treated WKAH rats responded to TO as strongly as lymph node cells from naive WKAH did (table 5). DISCUSSION
In the present study, the administration of DSG for 4 days, beginning on postoperative day 4,prolonged renal allograft survival significantly. DSG appears to be able to rescue allografts, even from ongoing rejection, in keeping with the observation by Suzuki et al.1° that the initiation of DSG treatment on the day of diagnosis of graft rejection could successfully reverse the rejection.
FIG. 3. Fragmentation of DNA in tubular epithelial cells as evidenced by nick end-labeling. In untreated allograft group, 30-fold increase in DNA-fragmented nuclei is evident on day 8 after transplantation ( B ) ,as compared with post-transplant day 4 (A). In DSG group on day 8 post-transplantation(0, DNA-fragmented nuclei are less evident than those in control, allograft group (B).(A, B, C; x400).
Skin transplants to the DSG-treated recipients with the long-surviving kidney graft demonstrated that donor-specific tolerance was induced in the host beginning 2 weeks after renal transplantation, and continued indefinitely. Tolerance a t 1 week after renal transplantation could be RT- 1-specific, because 2 of 5 SDJ skin grafts that had the same RT-1 A, B and D a s TO also showed prolonged graft survival. It appears that tolerance in the early phase under the DSG treatment may be RT-1-specific, followed by donor-specific tolerance.
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15-DEOXYSPERGUALIN AND RAT MDNEY -RAFT
TABLE5. Suppressive effect of eells from long-surviving DSG-tmated allomoff reciDie& on M U Responder n a n e WKAH nai\e WKAH n a n e WKAH naire WKAH n a n e WKAH
Stimulator To To To
To TO
Modulator mne spleen eella from naive WKAH spleen celln from WKAH (TO,DSG, >100F* lymph node celln from naive WKAH lymph node celln from WKAH 0, DSG, 2100) none
MLR (epm) 93217.7 13620.6 2 666.8 13879.9 2657.4 2 111.1 62808.6 2 528.3
66467.0 2 2902.4 54402.9 2 1973.1
WKAH (TO. DSG. >loo) To * p c 0 0001. ** WKAH ("0,DSG, >loo) repreeenta DSG-treated WKAH with TO kidney allograft, surviving more than 100 days.
SbrdLR 100 .lOS,-J 8.0 189.1 200.1 103.6
In the rat renal allografts, histologic evidence of acute Function for MLR but not for antibody production was invesrejection began to start 3 to 4 days postoperatively with the tigated in this study, and suppression of B cell function appearance of cellular infiltrates around the interlobular appears to be another issue that should be addressed in vessels.11.12 In a previous study, it was shown that the cel- further studies. lular infiltrates were less pronounced in DSGtreated recip Acknowledgement. We wish to thank K Kawai, M. ients as compared with those in acutely rejected kidney al- Hamada and M. Kanda for their technical and secretarial lografts, when the administration of DSG was started assistance. We also wish to thank Dr.Nicholas J. Feduska for immediately &r tran~plantation.1sSince DSG was admin- assisting us with the preparation of this manuscript. istered from day 4 in the present study, diffuse cellular infiltrates similar to those in allografts from untreated recipREFERENCES ients were observed in the grafts of DSG-treated recipients. The expression pattern of MHC class 11,ICAM-1 and LFA-1 1. Umezawa, I€, Kondo, S.,Iimrma,H, Kunimoto, S., Ikeda, Y., in the acutely rejected graft514 was quite similar to that in I ~ a S t i ~H., a , Iheda,D.and T & E ~ IT.: ~ ,StrUduR of an antitumor antibiotic, spergualin. J. Antibiotiica, 34:1622.1981. the grafts of DSGtreated recipients. However, tubular dam2. Suzuki, S.,Kanashiro, M. and Amemiya. H.:Effect of a new age, interstitial edema and hemorrhage were virtually abimmunosuppressant, 15deoxyspergualin,on heterntopic rat sent in the DSGtreated grafts. Proliferating cell nuclear heart transplantation, in comparison with cyclosporine. antigen recognizes the presence of a polypeptide within the Transplantation, 44:483,1987. nucleus in the synthesis phase.16 The PCNA-positive rates on 3. Dickneite, G., Schorlemmer, H. U., Walter, P. and Wacek, GIL in the DSG and the untreated groups were similar and H. H.: Graft survivalin experimentaltranaplantetion could be were not statistically significant. However, the PCNA-poaprolonged by the action ofthe antitumoral drug l!kleoryspertive rate on TC was signiscantly higher in the DSGtreated gualin. Tramplant. Proc., 1& 1295,1986. group on day 8 compared with that in the untreated p u p . 4. Walter, P., Dickneite, G., Fife, G. and Thies,S.:Deoxyspergualin induces tolerance in allogeneic kidney transplantation. TransThe DNA nick end-labeling method identi6es DNA fragmenplant. Proc., 16.3980,1987. tation as a marker of apoptotic cell death.9 The number of 5. Lee, S.:An improved technique of renal transplantation in the apoptotic TC in DSG-treated grafts was signi6cantly smaller rat. Surgery,81: 771,1967. than that in untreated grafk on day 8 &r transplantation 6. Tamatani, T. and Miyamka, M.: Identification of monoclonal Lidicating an apparent "escape" of TC from the "lethal hit" by antibodies reactive with the rat homologue of ICAM-1 in the GIL. The number of apoptotic GIL did not significantlydiffer adherence of resting versus activated lymphocytes to high in DSG-treated and nontreated grafts. These data suggest endothelial cells. Int. Immunol., 8: 166,1990. that GIL in the DSGtreated grafts may not function ade7. T m t a n i , T., Kotani, M., Tanaka, T.and M i , M.: Moleular meehaniems underlying lymphocyte recirculation. Differquately as effector cells during the induction phase of @ ential regulation of LFA-1 in the interaction between lymphoenhancement. The histology of long-surviving graRs indicytes and high endothelial cells. Em. J. Immunol., 21: 855, cated that there was no evidence of tissue Wury or lymph* 1991. cytic activation in these grafts, suggesting a graaual r e p s 8. Tsujieaki, M., Imai, K., Hirata, H.,Hanzawa, Y.. Masuya, J., sion of lymphocytic activation in DSGtreated rats. It haa Nakano, T.,Matsui, M.,Hinoda, Y.and Yachi,Y.:Detectionof been suggested that the major, if not the entire, effect of DSG circulating intercellular adhesion molecule-1 antigen in mamight be inhibition of cyt~toxicT cell precursor differentialignant diseaeee.clin. Exp. Immunol., w 3,1991. tion into cytotoxic effector cells.16-18 out results appear to 9. Gavrieli,Y..Sherman,Y.and BensaesOn, S.A:Identification of support this concept. programmed cell death in eitu via specific labeling of nuclear DNA fragmentation.J. Cell Biol., 116.493, 1992. Suzuki et d.10.1S reported that suppressor cells were induced in DSGtreated rat heart recipients. In line with this 10. Suzuki, S., Kanashiro, M., Watanabe. H. and Amemiya, H.: Therapeutic effect of 15deoxyspergualin on acute rejection observation, cells that can intensely suppress the TO-todetected by "P nuclear magnetic resonance 8p&mgraphy, WKAH MLR were generated in the spleens of DSGtreated, and its effect on rat heart transplantation. Transplantation, long-surviving WKAH recipients, although spleen cells from 48:669,1988. naive rats also suppressed the MLR slightly. In contrast, the 11. Feldman, J. D. and Lee,S.:Renal homotransplantation in rats. lymph node cells of the long-surviving rats responded to TO Allogeneic recipients. J. Exp. Med., 1uI:783,1967. as strongly as lymph node cells of naive WKAH. Based upon 12. Guttman, R. D., Lindquist, R. R., Parker, R. M., Carpenter, C.B. these observations, graft enhancement appears to be mainand Merrill, J. P.: Renal transplantation in the inbred rat. Transplantation, 6 668,1967. tained by suppressor cells in this model and not by the deletion or anergy of donor-specific lymphocyte clones. Thus, 13. Kerr,P. G., Marshall, V. C. and Atkine, R. C.: I m m m o h i s t o l ~ of non-rejected deoxyspergualin-hated rat renal allografts. suppressor cells appear to be responsiblefor the maintenance Transplant. Proc., 21: 3767, 1989. of DSG-induced tolerance. In this study, graft survival in the 14. Kanagawa, K, Ishihura, H.,Takahash', C. and Yoshiki, T.: untreated, rejection p u p was about 10 days, indicating a Identification of ICAM-1-positive cells in the non-grafted and milder alloimmune response as compared with the ACI-totransplanted rat kidney: an immunohiatochemical and ultraLewis combination. The Suppressor mechanism shown in this structural study. Transplantation, 68: 1057, 1991. study should be further evaluated in such stronger strain 15. Bravo, R., Frank,R., Blundell, P.A. and MacDonald-Bravo,H.: combinations. Suppression of B cell-mediated transplantaCyclin/PCNA is the auxiliary protein of DNA polymerase delta. Nature, 328:515,1987. tion immunity has bean shown to be an important aspect Of the effect of 6SG.m The presence of cells with a suppressivf! 16. Nishimura. K and Tokunaga, T.:Mechanism of action of 15
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15-DEOXYSPERGUALINAND RAT KIDNEY ALLOGRAFT
deoxyspergualin. I. Suppressive effect on the induction of alloreactive secondary cytotoxic T lymphocytes in vivo and in vitro. Immunology, 68: 66,1989. 17. Falk, W., Ulrichs, K. and Muller-Ruchholtz, W.: 15-deoxyspergualin (a new guanidine-like drug) blocks T lymphocyte proliferation. Transplant. Roc.,1 9 4239, 1987. 18. Fujii, H., Takada, T.. Nemoto, K, Abe, F., Fujii, A., Talmadge, J. E. and Takeuchi, T.: Deoxyspergualin, a novel immunosuppressant, markedly inhibits human mixed lymphocyte reaction and cytotoxic T-lymphocyte activity in vitro. Int. J. Im-
munopharmacol., 1 4 731, 1992. 19. Suzuki, S., Kanashiro, M., Watanabe. H., Sakakibara, I. and Amemiya, H.: Effect of 15-deoxyspergualin on rejection detected by 31Pnuclear magnetic resonance spectroscopy and in vivo mechanisms of action in rat heart transplantation. Transplant. Proc., 21: 1094, 1989. 20. Fujii, H., Takada, T., Nemoto, K., Yamashita, T., Abe, F., Fujii, A. and Takeuchi, T.: Deoxyspergualin directly suppresses antibody formation in vivo and in vitro. J. Antibiot., 43: 213, 1990.
VoL 164,2187-2202. December 19B6 Printed in
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EFFECT OF 15-DEOXYSPERGUALIN (DSG) ON RAT KIDNEY ALLOGRAFT: IMMUNOLOGICAL MECHANISMS IMPLICATED IN PROLONGED SURVIVAL TATSUYA CHIKARAISHI, HIROSHI * S I
T0SHIMOR.I SEKI,TOMOHIKO KOYANAGI AND TAKASHI YOSHIKI
From the Departments of Urnlogy and Path&,
Hokkaido University School of Medicine, Sapporn, Japan
ABSTRACT
Purpose and Methods: The effect of short-term administrationof 15-deoxyspergualin(DSG), 5 mg.lkg./day from postoperative days 4 to 7, on rat renal transplantation was studied. Results: Although allografb treated with DSG survived longer than nontreatd ones, cellular infiltration in both grafts did not differ. However, renal tubular cells of DSG-treated grafta proliferated well and escaped apoptotic cell death. A donor-specific tolerance 2 weeks after transplantation was developed, and cells with in vitro Suppressor function were induced in such animals. Conclusions:Treatment with DSG appears to prevent lethal attack of effector cells on tubular cells in situ and to generate suppressor cells in the maintenance phase of graft enhancement. KEYWORDS: kidney transplantation,rate
allow reeipients to survive. Recipients dying within 7 days poetoperatively were regarded ae technical ''errors" and were excluded from the data analysis. To examine the effect of short-term administration of DSG, TO or LEJ rat kidneys were transplanted into WKAH rats. The recipients were divided into 2 groups, a control group and a DSG-treated group. In the former group, no DSG was administered after transplantation. In the latter group, DSG was given in a dose of 5 mgJkgJday intramuscularly, from days 4 to 7, postoperatively. Graft survival was compared between the 2 group. Skin tmnsplantation. Tail skin,10-mm. in diameter, of the donor rats was transplanted to the back of the recipients. A single recipient received skin grafts from 5 separate donors at the same time. On postoperative day 7, the graRe were examined. If the ekin grafts had already become necrotic by this time, they were considered technicalfailures. The graffs were examined every day, and when they had become necrotic by 50% or more, they were considered as rejections. MATERIALS AND METHODS DSG treated WKAH rats with TO kidney transplants re15-Deoxyspergualin (DSG). Nippon lbyaku Co. Ltd., TO- ceived additional skin grafb from WKAH, TO, SDJ, LEJ and kyo,Japan kindly supplied DSG which was dissolved in 0.9% BUF rats. In group A, skin graRs were performed 1week NaCl at a concentration of 2 mg./ml., sterilized with a Milli- after renal transplantation; in groups B and C, the skin pore filter (Kanto Chemical Co., Inc., Tokyo, Japan). and grafts were performed 2 weeks and more than 100 days aRer renal transplantation, respectively. Naive WKAH rats and Stored at -2OC until use. (RT-lk, RT- DSGtreated WKAH rats without TO kidney transplants also Animals. The inbred rat strains, lAkBkDk),TO/Hkm (RT-l", RT-lA"B"D"), S D J h (RT-l", received skin grafts (groups D and El. Monoclonal antibodies. Monoclonal antibodies (Mab) RT-lA"B"D"), LEJ/Hkm (RT-lj, RT-lA"BbDb), BUF/Hkm (RT-lb,RT-lAbBbDb),maintained and fed at the Center for against rat major histocompatibili~class (MHC) class II Biological Experiments, Hokkaido University, were used. (0x6,Biosys S.A.), rat intemllular adhesion molecule1 Male rats weighing more than 250 g. were used for the (ICAM-1, lA29),6 rat lymphocyte function-associated antiexperiment. gen-1 (LFA-1, WT.1)7 and proliferating cell nuclear antigen Renal transpiantation. Renal transplantation was per- (PCNA, Dakopatts, Glostrup, Denmark) were used in this formed according to the method described by Lees with some study. The HA58 Mab (anti-human ICAM-1),6 which has no modifications. To evaluate the enhancing effect of DSG on cross-reaction with rat tissues, was used as a negative conrenal allografts, both kidneys of the recipient were removed trol antibody. The lA29and WT.l were kindly supplied by Dre. SO that graft survival could be estimated as recipient survival M. Myasaka and T. Tamatani of the Department of Immunoldays. When the graft histology was examined chronologi- ogy,Tokyo Metmpolitan Institute of Medical Science. cally, the recipient's leR kidney was not removed, so as to Histologic and immunohistochemical examinations. Serial kidney biopsy was done on days 4,6,8 and 14, under general -pted for publication January 23,1995. Requests for reprints: Department of Pathology, Hokkaido Uni- anesthesia with ether. The biopsy materials were stained versity School of Medicine,N-16,W-7, Kita-ku,Sapporo 060, Japan. with hematoxylin and eosin for histologic examination. For
A guanidinic-like structure, 15-deoxyspergualin (DSG), is an analog of spergualin,which was isolated from the culture filtrates of Bacillus laterosporue.1 In experimental models of transplantation, DSG has been shown to prolong survival of rat skin, heart, liver, pancreas and kidney all0grafb.S' The mechanism of prolonged graft survival by DSG remains unclear. In previous studies, the duration of DSG administration ranged from 7 to 14 days, beginning on the day of transplantation. In the present study, the effect of DSG, administered for a shorter period, 4 days, beginning on an earlier post-transplant day, was investigated in the rat renal transplantation model. The shorter period of treatment was selected to minimize a possible untoward effect by longer administration. These studies also focused on the mechanism of grafk enhancement in this short-term DSG administration model, separately in both the induction and maintenance phases.
2197
15-DEOXYSPERGUALIN AND RAT KIDNEY ALLOGRAFT
2198
TABLE1. Effect of DSG on renal allograft in the rat the immunohistochemical study using OX6,1A29, WT.l, and HA58, the specimens were rapidly frozen, sliced a t 5 pm Strain combination Experimental group p value thickness with a cryostat and fixed in cold acetone for 20 (donor recipient) contml group DSG group minutes. For PCNA staining, the samples were fixed in 10% TO -+WKAH 8, 8, 9, 9, 11, 16 19, 105*, 114*, 176*, p = 0.0007 buffered formalin for 2 days, embedded in paraffin, cut and 187, 210, 211*, 231 (10.2 -t 3.1) deparaffinized by xylene before being labeled with PCNA. 1156.6 2 71.8) 0x6, LA29,WT.1, PCNA and HA58 were used as primary LEJ -WKAH 9,9, 10, 10, 12 10,69, 115". 271*, 316' p = 0.0317 antibodies in the avidin-biotin-peroxydasecomplex method. (156.2 Z 131.7) (10.0 5 1.2) Nuclei were counterstained with hematoxylin for 0 x 6 , lA29, In the DSG group, DSG was given in a dose of 5 mg./kg./day intramuscularly WT.1 and HA58. The periodate acid-Schiff (PAS)stain was from days 4 to 7 postoperatively. In the control group, DSG was not given. * These animals were sacrificed on the indicated days in order that they used in the PCNA-stained sections in order to distinguish whether labeled cells were graft infiltrating lymphocytes might be employed for other experiments. (GIL) or renal tubular cells (TC). PCNA-positive and negative GIL or TC were counted within 10 randomly selected Mixed lymphocyte culture reaction (MLR). One way MLRs high power fields (the microscopic field at x40 objective lens), followed by calculation of PCNA-positive ratio by the for- were also performed. Lymphocytes from mesenteric lymph nodes, 1 x lo5, were used as responders, and 2 x lo5 of mula: mitomycin C (25 pg./ml.)-treated lymph node cells were used PCNA-positive GIL or TC X 100 as stimulators. These cells were cocultured in RPMI-1640 medium, including 10% fetal calf serum (GIBCO, Grand PCNA-positive ratio (%) = Total GIL or TC Island, New York) and 5 X lop5 MA. 2-mercaptoethanol There were 3 experimental groups. In the isograft group, (Wako Pure Chemical Industries, Osaka, Japan). The MLRs WKAH kidneys were transplanted into WKAH recipients. In were then performed for 96 hours in a humidified 5% CO, the allograft group, TO kidneys were transplanted into atmosphere at 37C. [3Hlthymidine, 0.5 pCi (Japan RadioisoWKAH recipients. In the DSG group, TO kidneys were trans- tope Association, Tokyo, Japan), was added to each well 16 hours before the cells were harvested. The cultured cells were planted into WKAH with short-term DSG administration. Deoxyribonucleic acid nick end labeling. DNA nick end harvested by means of an automatic cell harvester, and the labeling of tissue sections was performed by a method previ- radioactivity was measured with a liquid scintillation ously described.9 In brief, deparaffinized sections were incu- counter. In some experiments, 1 x lo5 of spleen or lymph bated with 20 pg/ml. proteinase K (Sigma Chemical Co., St. node cells from recipient WKAH rats with DSG-treated, longLouis, Missouri) for 20 minutes at room temperature, fol- surviving grafts or similar cells from naive WKAH rats were lowed by multiple washes with distilled water. After blocking added to MLRs as modulator cells to evaluate for the possiof endogenous peroxidase with 2% H,O, for 7 minutes, the bility of tolerance. Results of MLR were expressed as means sections were rinsed with distilled water and immersed in and standard deviations of triplicated wells (96 round-bottom terminal deoxy transferase (TdT) buffer (30 mM. Trizma well, Petray 96U, Terumo, Japan). Three independent experbase, pH 7.2, 140 mM. sodium cacodylate, 1 mM. cobalt iments were performed, each using a long-surviving animal chloride); TdT (0.3 e.u./l) and biotinylated dUTP in TdT (each more than 100 days post-transplant). Statistical analysis. All data values were presented as buffer were then added a t 37C for 90 minutes. The reaction means 2 standard deviation. Statistical analyses were done was stopped by application of TB buffer (300 mM. sodium chloride, 30 mM. sodium citrate) for 30 minutes. ARer mul- with the unpaired Wilcoxon test. A probability of p c0.05 was tiple washes with distilled water, peroxidase-labeled strep- required for significance. toavidin (Dakopatts) was added for 30 minutes at 37C. The RESULTS preparations were then washed with distilled water and stained with diaminobenzidine for 30 minutes at 37C. The Effects of short-term administration of DSG on renal alPAS stain was performed in nick end labeled sections, to lografts. In the DSG group, 7 of 8 allografted kidneys in the distinguish whether the labeled cells were GIL or TC. The TO-to-WKAH combination and 3 of 5 in the LEJ-to-WKAH numbers of labeled nuclei of GIL or TC were counted in 10 combination survived for more than 100 days, whereas all of randomly selected high power fields. Samples that were ob- the untreated controls were rejected in 10.2 3.1 and 10.0 5 tained by partial graft excision on days 4, 6 and 8 were 1.2 days, respectively (table 1). studied. Experimental groups were divided into two: namely, Tolerance induction in DSG-treated long-surviving recipithe allograft group that was not treated with DSG and the ent ruts. In group A, all of the syngeneic WKAH skin grafts DSG group into which allografts were transplanted and survived indefinitely, and 4 of 5 TO grafts survived indefishort-term DSG administration was carried out. nitely. Two of 5 SDJ skin grafts showed prolonged survival, _f
TABLE2 Imniunologrcul tolerance by short-term DSG adminlstratlon ~~
Expenmental
__
POUP
~
WKAH (RT-1'1
TO I RT 1 ' I I ~
A
0
E
C)
. . . . . . . . . . . . . . ................ Skin Graft Donor IRT-1)
-
LEJ (RT-1')
SDJ (RT-1")
... ~ ~ _ _ _ _ .
BUF iRT-lh)
0
( 1
12
C
D
0
.
E
12 0
11 .
0
10 .
.
10
17 0 14 0 13 0
12
.
12
14
14
11
10
9
17 0 10 0 12
10 9 . . in 9 . 0
0
.
10 0
12 .
13
0
10 .
14
12 .
.
11
11
11
11 .
10
8
16
12
8
7
12
10 10 . . 11 9
0 16 .
0
9
i
12
.
n
0
9
11
8
9
8
.
9
8
15-DEOXYSPERGUALINAND RAT KIDNEY m
FIG.1. Histology of allograRa on day 14 after transplantation. In untreated, acute rejection up, renal parenchyma,including tubular e ithelial cells, is largeTdestroyed by cellular in6ltrab, intermin& with hemorrhage and degeneration (A). In DSG-treated group, although prominent cellular infiltrates are observed, r e d parenchyma is well preserved (B).(4, B; X200). whereas all of the LEJ and BUF skin grafh were rejected. In groups B and C,all of the WKAH and TO skin grafts survived indefinitely,but all of the SDJ skin gr& were rejected. In groups D and E,only the WKAH skin grafb survived indef-
W
2199
initely. All of the skin grafta from other strains were rejected. The evidence indicates that donor-specific tolerance wae induced in DSG-treated recipients mta (table 2). Histologv. In the ieograft group, focal and mild cellular infiltrates, composed chiefly of small lymphocytes, were evident from days 4 to 14. The cellular infiltrates never became intense or diffuee.In the allograft group, cellular infiltrates around the interlobular arteries and veins were seen on day 4; they spread to the peritubular areas on day 6.On day 8, intense cellular infiltrates were observed and interstitial hemorrhage, edema, and thrombosis developed. By day 14, the histologichallmark of =aft loss was noted in which most of the r e d tubules were-destroyed (6g. 1,A). In the DSG group, histologic changes similar to thoae seen in the unhated allograft group were obeerveed on days 4 and 6.Diffuse cellular intiltrates were evidenced in the pentubular areas on days 8 and 14, but interstitial hemorrhage, edema and tubular damage were minimal (fig. 1,B). The histologic appearance of all0grafh-I kidneys from long-term survivors was also investigated. Only small patchy foci of lymphocytic infiltration were observed, and blastic lymphocyteswere very rarely seen. There was no interstitial edema and bleeding, and there was no evidence of tiesue iqjury. Immunohistochemistry.In the isogratt group, no subatantial expression of MHC clam 11 and ICAM-1 antigens was observed on the renal constitutive cells. Major histmompat ibility class class 11and LFA-1 antiens were preaent on the infiltrating cells,some of which aIe0 expressed ICAM-1. In the untreated allograft group, some tubular epithelial cells expressed MHC class II and ICAM-1 a n t i g e ~after day 4. Major hietooompatibility class II and LF'A-1 positive cells infiltrated the graft after day 4. The expression of MHC clasa 11and ICAM-1 antigens on renal tubular cells was gradually augmented by days 6,8 and 14. The number of LFA-1positive infiltrating cells aLS0 gradually increased. In the DSG group, there was essentially the same ICAM-1 and MHC class 11 antigen expression on renal tubular cells as was observedin the untreated allograftgroup. The number of LFA-1-positive cells also gradually increased. Table 3 shows the PCNA-positive rate in GIL and TC. Figure 2 shows a representative FCNA staining in allografts. Although the FCNA-positive rate of GIL became slightly higher in the allograft group than in the DSG group, no statistid significance was noted. The PCNA-positive rate of TC was -&I nificantly higher in the DSG group than in the allograft group (p <0.05).
TABLE3. Rolifemting cell nuclear antigen-positivemte of gmfi infiltmting lymphocytes (GIL)and tubular ceUs fN.3 F'CNA-poaitive rete (96)
cell type
DSG treatment
GlL
(-1
17.6 2 9.91
DSG
11.8 2 3.1
n:
(-)
DSG
Day 4
Day 6 n.S.t
18.82 9.7
9.0 -+ 11.77 n,8.* 12.2 -+ 13.0
Day 8
21.0 f 5.77
13.3 2 12.8
1
37.3
n.8.+
2
]
12.8
n,8.*
23.0 2 11.2 n.8.*
23.7 -+ 20.6
33.2 t 19.6
* n.8.: not significant. TABLE4. Number of DNA-fmgmented nuclei detected by the DNA nick end-hbeling no msitive celldl0 HF'P
Cell type
DSG treatment
GIL
(-)
DSG
n:
(-)
DSG
Dav 4
Day 6
0.3 ? 0.671 n.8. 0.3 2 0.41 0.05 2 0.22 0.06
1
0.3
= 1.321
Day 8 n.s,
0.65 ? 0.691
0.76 2 0.91
0.45 2 0.69
0.6 f 0.82
0.55 2 0.82
n.8,
= 0.22
HPF: High power field repregents the field observed with a 4OX objectiw lens.
n.B.
2200
15-DEOXYSPERGUALINAND RAT KIDNEY ALLOGRAFT
FIG. 2. Proliferating cell nuclear antigen distribution in renal tubular cells on day 8 after transplantation. Nuclei of renal tubular cells in DSG group allografts ( B )are labeled to greater degree with PCNA than those in untreated allograft group (A). (A, B ; X400). Deoxyribonucleic acid nick end labeling. The number of the labeled TC in the allograft group demonstrated a 30-fold increase from days 4 to 8 (table 4). On day 8, the number of labeled TC in the DSG group was significantly smaller than that in the allograft group (p = 0.0245). The number of labeled GIL in the DSG group did not differ significantly from that in the allograft group. A representative nick end labeling feature in the allografts is shown in figure 3. Mixed lymphocyte culture reaction. By adding spleen cells from DSG-treated WKAH rats with long-surviving TO kidney grafts, TO-to-WKAH MLR was significantly suppressed (as much as 92%). Spleen cells of naive WKAH also suppressed TO-to-WKAH MLR to a lesser extent (p <0.0001), whereas a n addition of lymph node cells from the DSGtreated WKAH rats with long-surviving TO kidney grafts did not suppress TO-to-WKAHMLR but augmented it as did lymph node cells from naive rat. Lymph node cells from DSG-treated WKAH rats responded to TO as strongly as lymph node cells from naive WKAH did (table 5). DISCUSSION
In the present study, the administration of DSG for 4 days, beginning on postoperative day 4,prolonged renal allograft survival significantly. DSG appears to be able to rescue allografts, even from ongoing rejection, in keeping with the observation by Suzuki et al.1° that the initiation of DSG treatment on the day of diagnosis of graft rejection could successfully reverse the rejection.
FIG. 3. Fragmentation of DNA in tubular epithelial cells as evidenced by nick end-labeling. In untreated allograft group, 30-fold increase in DNA-fragmented nuclei is evident on day 8 after transplantation ( B ) ,as compared with post-transplant day 4 (A). In DSG group on day 8 post-transplantation(0, DNA-fragmented nuclei are less evident than those in control, allograft group (B).(A, B, C; x400).
Skin transplants to the DSG-treated recipients with the long-surviving kidney graft demonstrated that donor-specific tolerance was induced in the host beginning 2 weeks after renal transplantation, and continued indefinitely. Tolerance a t 1 week after renal transplantation could be RT- 1-specific, because 2 of 5 SDJ skin grafts that had the same RT-1 A, B and D a s TO also showed prolonged graft survival. It appears that tolerance in the early phase under the DSG treatment may be RT-1-specific, followed by donor-specific tolerance.
2201
15-DEOXYSPERGUALIN AND RAT MDNEY -RAFT
TABLE5. Suppressive effect of eells from long-surviving DSG-tmated allomoff reciDie& on M U Responder n a n e WKAH nai\e WKAH n a n e WKAH naire WKAH n a n e WKAH
Stimulator To To To
To TO
Modulator mne spleen eella from naive WKAH spleen celln from WKAH (TO,DSG, >100F* lymph node celln from naive WKAH lymph node celln from WKAH 0, DSG, 2100) none
MLR (epm) 93217.7 13620.6 2 666.8 13879.9 2657.4 2 111.1 62808.6 2 528.3
66467.0 2 2902.4 54402.9 2 1973.1
WKAH (TO. DSG. >loo) To * p c 0 0001. ** WKAH ("0,DSG, >loo) repreeenta DSG-treated WKAH with TO kidney allograft, surviving more than 100 days.
SbrdLR 100 .lOS,-J 8.0 189.1 200.1 103.6
In the rat renal allografts, histologic evidence of acute Function for MLR but not for antibody production was invesrejection began to start 3 to 4 days postoperatively with the tigated in this study, and suppression of B cell function appearance of cellular infiltrates around the interlobular appears to be another issue that should be addressed in vessels.11.12 In a previous study, it was shown that the cel- further studies. lular infiltrates were less pronounced in DSGtreated recip Acknowledgement. We wish to thank K Kawai, M. ients as compared with those in acutely rejected kidney al- Hamada and M. Kanda for their technical and secretarial lografts, when the administration of DSG was started assistance. We also wish to thank Dr.Nicholas J. Feduska for immediately &r tran~plantation.1sSince DSG was admin- assisting us with the preparation of this manuscript. istered from day 4 in the present study, diffuse cellular infiltrates similar to those in allografts from untreated recipREFERENCES ients were observed in the grafts of DSG-treated recipients. The expression pattern of MHC class 11,ICAM-1 and LFA-1 1. Umezawa, I€, Kondo, S.,Iimrma,H, Kunimoto, S., Ikeda, Y., in the acutely rejected graft514 was quite similar to that in I ~ a S t i ~H., a , Iheda,D.and T & E ~ IT.: ~ ,StrUduR of an antitumor antibiotic, spergualin. J. Antibiotiica, 34:1622.1981. the grafts of DSGtreated recipients. However, tubular dam2. Suzuki, S.,Kanashiro, M. and Amemiya. H.:Effect of a new age, interstitial edema and hemorrhage were virtually abimmunosuppressant, 15deoxyspergualin,on heterntopic rat sent in the DSGtreated grafts. Proliferating cell nuclear heart transplantation, in comparison with cyclosporine. antigen recognizes the presence of a polypeptide within the Transplantation, 44:483,1987. nucleus in the synthesis phase.16 The PCNA-positive rates on 3. Dickneite, G., Schorlemmer, H. U., Walter, P. and Wacek, GIL in the DSG and the untreated groups were similar and H. H.: Graft survivalin experimentaltranaplantetion could be were not statistically significant. However, the PCNA-poaprolonged by the action ofthe antitumoral drug l!kleoryspertive rate on TC was signiscantly higher in the DSGtreated gualin. Tramplant. Proc., 1& 1295,1986. group on day 8 compared with that in the untreated p u p . 4. Walter, P., Dickneite, G., Fife, G. and Thies,S.:Deoxyspergualin induces tolerance in allogeneic kidney transplantation. TransThe DNA nick end-labeling method identi6es DNA fragmenplant. Proc., 16.3980,1987. tation as a marker of apoptotic cell death.9 The number of 5. Lee, S.:An improved technique of renal transplantation in the apoptotic TC in DSG-treated grafts was signi6cantly smaller rat. Surgery,81: 771,1967. than that in untreated grafk on day 8 &r transplantation 6. Tamatani, T. and Miyamka, M.: Identification of monoclonal Lidicating an apparent "escape" of TC from the "lethal hit" by antibodies reactive with the rat homologue of ICAM-1 in the GIL. The number of apoptotic GIL did not significantlydiffer adherence of resting versus activated lymphocytes to high in DSG-treated and nontreated grafts. These data suggest endothelial cells. Int. Immunol., 8: 166,1990. that GIL in the DSGtreated grafts may not function ade7. T m t a n i , T., Kotani, M., Tanaka, T.and M i , M.: Moleular meehaniems underlying lymphocyte recirculation. Differquately as effector cells during the induction phase of @ ential regulation of LFA-1 in the interaction between lymphoenhancement. The histology of long-surviving graRs indicytes and high endothelial cells. Em. J. Immunol., 21: 855, cated that there was no evidence of tissue Wury or lymph* 1991. cytic activation in these grafts, suggesting a graaual r e p s 8. Tsujieaki, M., Imai, K., Hirata, H.,Hanzawa, Y.. Masuya, J., sion of lymphocytic activation in DSGtreated rats. It haa Nakano, T.,Matsui, M.,Hinoda, Y.and Yachi,Y.:Detectionof been suggested that the major, if not the entire, effect of DSG circulating intercellular adhesion molecule-1 antigen in mamight be inhibition of cyt~toxicT cell precursor differentialignant diseaeee.clin. Exp. Immunol., w 3,1991. tion into cytotoxic effector cells.16-18 out results appear to 9. Gavrieli,Y..Sherman,Y.and BensaesOn, S.A:Identification of support this concept. programmed cell death in eitu via specific labeling of nuclear DNA fragmentation.J. Cell Biol., 116.493, 1992. Suzuki et d.10.1S reported that suppressor cells were induced in DSGtreated rat heart recipients. In line with this 10. Suzuki, S., Kanashiro, M., Watanabe. H. and Amemiya, H.: Therapeutic effect of 15deoxyspergualin on acute rejection observation, cells that can intensely suppress the TO-todetected by "P nuclear magnetic resonance 8p&mgraphy, WKAH MLR were generated in the spleens of DSGtreated, and its effect on rat heart transplantation. Transplantation, long-surviving WKAH recipients, although spleen cells from 48:669,1988. naive rats also suppressed the MLR slightly. In contrast, the 11. Feldman, J. D. and Lee,S.:Renal homotransplantation in rats. lymph node cells of the long-surviving rats responded to TO Allogeneic recipients. J. Exp. Med., 1uI:783,1967. as strongly as lymph node cells of naive WKAH. Based upon 12. Guttman, R. D., Lindquist, R. R., Parker, R. M., Carpenter, C.B. these observations, graft enhancement appears to be mainand Merrill, J. P.: Renal transplantation in the inbred rat. Transplantation, 6 668,1967. tained by suppressor cells in this model and not by the deletion or anergy of donor-specific lymphocyte clones. Thus, 13. Kerr,P. G., Marshall, V. C. and Atkine, R. C.: I m m m o h i s t o l ~ of non-rejected deoxyspergualin-hated rat renal allografts. suppressor cells appear to be responsiblefor the maintenance Transplant. Proc., 21: 3767, 1989. of DSG-induced tolerance. In this study, graft survival in the 14. Kanagawa, K, Ishihura, H.,Takahash', C. and Yoshiki, T.: untreated, rejection p u p was about 10 days, indicating a Identification of ICAM-1-positive cells in the non-grafted and milder alloimmune response as compared with the ACI-totransplanted rat kidney: an immunohiatochemical and ultraLewis combination. The Suppressor mechanism shown in this structural study. Transplantation, 68: 1057, 1991. study should be further evaluated in such stronger strain 15. Bravo, R., Frank,R., Blundell, P.A. and MacDonald-Bravo,H.: combinations. Suppression of B cell-mediated transplantaCyclin/PCNA is the auxiliary protein of DNA polymerase delta. Nature, 328:515,1987. tion immunity has bean shown to be an important aspect Of the effect of 6SG.m The presence of cells with a suppressivf! 16. Nishimura. K and Tokunaga, T.:Mechanism of action of 15
2202
15-DEOXYSPERGUALINAND RAT KIDNEY ALLOGRAFT
deoxyspergualin. I. Suppressive effect on the induction of alloreactive secondary cytotoxic T lymphocytes in vivo and in vitro. Immunology, 68: 66,1989. 17. Falk, W., Ulrichs, K. and Muller-Ruchholtz, W.: 15-deoxyspergualin (a new guanidine-like drug) blocks T lymphocyte proliferation. Transplant. Roc.,1 9 4239, 1987. 18. Fujii, H., Takada, T.. Nemoto, K, Abe, F., Fujii, A., Talmadge, J. E. and Takeuchi, T.: Deoxyspergualin, a novel immunosuppressant, markedly inhibits human mixed lymphocyte reaction and cytotoxic T-lymphocyte activity in vitro. Int. J. Im-
munopharmacol., 1 4 731, 1992. 19. Suzuki, S., Kanashiro, M., Watanabe. H., Sakakibara, I. and Amemiya, H.: Effect of 15-deoxyspergualin on rejection detected by 31Pnuclear magnetic resonance spectroscopy and in vivo mechanisms of action in rat heart transplantation. Transplant. Proc., 21: 1094, 1989. 20. Fujii, H., Takada, T., Nemoto, K., Yamashita, T., Abe, F., Fujii, A. and Takeuchi, T.: Deoxyspergualin directly suppresses antibody formation in vivo and in vitro. J. Antibiot., 43: 213, 1990.