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Allograft acceptance and rejection, mediated by a liver suppressor factor, LSF-1, purified from serum of liver transplanted rats
Transplant Immunology 1996; 4: 287-292
All-raft acceptance and rejection, mediated by a liver suppressor factor, LSF-1, purified from serum of liver transplanted rats Catherine Edwards-Smith”, Shigeru Goto, Roger Lord”, Yoshinori Shimizub, Frank Vari” and Naoshi Kamadaa aJoint Transplantation Biology Programme, Queensland Institute of Medical Research/Department of Surgery, University of Queensland, Brisbane, and ‘Department of Surgery, Showa University, Tokyo Received 1 March 1996; accepted for publication 7 May 1996
Abstract: In certain rat strain combinations liver allografts are spontaneously accepted without immunosuppression and induce donor-specific tolerance to further skin and heart grafts in the recipient. Such an effect is also transferrable using serum from orthotopically liver transplanted rats (OLT serum). In the OLT serum of one such combination, DA (RTl”) donor into PVG (RTl’) recipient, a 40 kDa protein (liver suppressor factor, LSF-1) has been identified and shown to be immunosuppressive in vitro. The aim of the present study is to investigate the immunological effect of LSF-1 and a polyclonal antibody (anti-LSF-I) against this molecule, in a rat heterotopic heart transplant (HHT) model and OLT model, respectively. Intramuscular injection of 300 pg of LSF-1, 1 h postoperatively, into a PVG recipient of either a DA or BN (RTl”) cardiac allograft caused significant prolongation of graft survival. Intravenous injection of polyclonal rabbit sera raised against an N-terminal peptide of LSF-1 (anti-LSF-l), within 1 h postoperatively, had variable effects on the survival of DA liver grafts in PVG recipients. In 5/6 cases injection of between 1 and 2 ml of anti-LSF-1 resulted in death of the recipient. Histological examination of the liver showed severe rejection with lymphoid cell infiltration of the portal tract and sinusoids and extensive damage to the parenchyma. All control rats survived for more than 60 days without any signs of rejection. The anti-LSF-1 polyclonal antibody prevented the induction of tolerance in the normally tolerogenic model (DA into PVG). This, together with the in viva results, suggests a role for LSF-1 in the induction of tolerance.
Introduction The ability of the liver to be accepted as an allograft without immunosuppression has been reported in several species such as mouse, rat and pig. l-3 Rejection reactions against the liver are relatively weak in comparison with those against other organs. In certain rat and pig strain combinations liver allografts provide a protective effect against rejection of other grafts Address for correspondence: Catherine Edwards-Smith, Transplantation Biology, Queensland Institute of Medical Research, PO Royal Brisbane Hospital, Queensland 4029, Australia. 0 Arnold 1996
which are donor type such as skin, heart and kidney and small bowe1.2@ It is also recognized in the clinical setting that the liver possesses unique survival properties. Co-transplantation of the liver in order to protect other organs against graft rejection has become a common strategy; combined liver and kidney transplantation was associated with a reduced incidence of kidney rejection.7*8 We have studied in detail a rat model of orthotopic liver transplantation (OLT). PVG (RTl’) recipients of DA (RTl”) liver allografts survive without immunosuppression and become tolerant of subsequent grafts of other DA organs such as skin, heart and kidney. 43 This immunosuppressive effect of
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the liver was transferrable to other PVG recipients using OLT serum. OLT serum (1 ml), collected from PVG recipients bearing DA liver allografts for 24 months, suppressed the rejection of PVG.RTl” heart grafts.9*‘o Such donor-specific effects of OLT serum have been attributed to the high concentration of soluble (DA) donor class I antigen’ ’ and anti-class II antibodies.‘2*‘3 Knoop et ~1.‘~ have also demonstrated that 1 ml of OLT serum (DA-PVG) induced tolerance in PVG recipients of third party A0 (RTl”) kidney graft and speculated that it was due to the high levels of anticlass II antibodies. OLT serum from liver grafted mice was also immunosuppressive and the effect was attributed to the high concentration of IgG particularly class II antibodies.’ Studies on purified soluble donor class I antigen have shown that, by continuous infusion, antigen-specific protection of allografts can be achieved.15 Administration of extremely high doses of rat MHC class I antigen could also prolong renal allograft survival.‘6~17 However, others have indicated that class I alone cannot be responsible for the observed prolongation by OLT serum.‘*.r9 Further studies have shown that soluble MHC class I antigen and anti-donor MHC class II antibodies alone cannot be responsible for the immunosuppressive activity observed in pooled OLT serum.2o OLT serum from postoperative day (POD) 60 has previously been shown to be the most effective at inhibiting cell proliferation in MLR (mixed lymphocyte reaction)?’ This serum has also been shown to contain lower concentrations of recipient anti-donor class I and class II antibodies than OLT serum from earlier postoperative times.22 We have previously reported on the identification and isolation of LSF- 1, a 40 kDa protein with a unique N-terminal sequence (SLAATHMHGNRVG) from rat POD 60 OLT serum.2o The addition of 10 p,g of LSF- 1 to MLR showed a degree of donorspecific immunosuppression in anti-DA PVG and anti-PVG DA MLR.” In the present study, LSF-1 was tested in vivo using a rat model of heterotopic heart transplantation (HHT). Subsequently, to fully examine the role of LSF-1, a polyclonal antibody was raised in rabbit against the N-terminal sequence. The polyclonal rabbit sera was injected into the normally tolerogenic combination of OLT (DA liver into PVG recipient) and the effect on the establishment of tolerance observed.
Objective The objective of this study was to examine the effect of LSF1 and a polyclonal antibody (anti-LSF-1) against this molecule, on graft acceptance or rejection using a rat HHT (heterotopic heart transplant) model and OLT model, respectively.
Materials and methods Purification of ISF-1 OLT (DA into PVG) day 60-80 serum was used for the purification of LSF-1, a 40 kDa protein with a unique N-terminal sequence as described previously.“*22 Briefly, the protein was purified by separation of OLT serum using 4-20% SDS-PAGE gradient gel (Biorad) separation followed by staining with Coomassie blue G250. The 40kDa band was identified by comparison to prestained broad range molecular weight markers (Biorad), excised and then eluted from the gel, prior to Transplant Immunology 1996; 4: 287-292
final purification by reverse-phase high-performance liquid chromatography (Model 1350, Bio-Rad Richmond, CA, USA). N-terminal amino acid sequencing was performed by electrophoretic separation of the protein on 4-20% gradient TrisTricine gels (BioRad) followed by transfer to PVDF membrane (BioRad) using the method of Choli and Wittman-Liebold.23 The protein was subjected to gas phase sequencing on an Applied Biosystems 437 protein sequencer (Foster City, CA, USA). Synthesis of peptide The peptide (SLAATHMHGN), derived from the previously reported N-terminal sequence of LSF-1,22 was synthesized using the simultaneous multiple peptide technique described employing derivatized (t-Boc) amino acids by Houghte# (Omni Biochemicals, National City, CA, USA) on a benzhydrylamine resin. An N-terminal cysteine was added to the peptide to facilitate coupling to the carrier protein. The heterobifunctional reagent maleimidocaproyloxysuccinimide (MCS) was employed to add either diphtheria toxoid (DT, Commonwealth Serum Laboratories, Melbourne, Australia) or bovine serum albumin (BSA; Sigma, St Louis, USA). The procedure of Lee et a1.25was the method used for derivatization of DT and BSA. Preparation of rabbit polyclonal antibodies Rabbits were immunized subcutaneously every 2 weeks for 14 weeks with 200 p,g of peptide-DT conjugate in Freund’s complete adjuvant (Sigma) for the first immunization and Freund’s incomplete adjuvant (Sigma) for subsequent boosts. Blood was collected at weeks 6, 8, 10 and 14 after the first immunization at week 0. Blood was collected from the rabbit’s ear by venipuncture using a catheter and allowed to clot at room temperature. Serum was collected by centrifugation at 3000 rpm for 15 min and stored at -2O’C. SDS-PAGE and immunoblotting Aliquots (5 ~1) of previously diluted (l/20 with distilled H20) OLT (DA into PVG) and DA serum were each added to 5 pl of Laemmli26 sample buffer, either with or without B-mercapmethanol, then electrophoretically separated on a 4-20% gradient SDS-PAGE, followed by transfer onto nitrocellulose membrane using a Trans-Blot SD semidry transfer blotter (BioRad). Membranes were then blocked by incubation with 5% skim milk powder in Tris-buffered saline (TBS) with gentle agitation for 30 min. The polyclonal sera was then added directly to the blocking solution at l/500 dilution and the membrane incubated overnight with gentle agitation at room temperature. The membrane was then washed for 5 min in TBS and repeated twice. The secondary antibody, sheep anti-rabbit IgG alkaline phosphatase conjugate (Boehringer Mannheim) diluted l/1000 in TBS, was added to the membrane and incubated at room temperature for 1 h. The membrane was then washed again in TBS, as described. Antibody reaction was detected using an Alkaline Phosphatase Conjugate Substrate Kit (BioRad) and used as described by the manufacturer. Experimental surgery The effect of LSF-1 on heart allografs Male rats, each weighing 230-350 g, of the following strains were used: DA (MHC haplotype RTla), PVG (RTl’) and BN
Role of LSF-1 in tolerance induction
Table 1 The effect of a single administration Recipient
PVG PVG PVG PVG PVG
of LSF-1,
Donor
from OLT serum, on fully allogeneic
Treatment
DA DA DA BN BN
LSF-I was administered intramuscularly, MST, mean survival time.
purified
Nil Intralipos 1.0 ml 300 pg LSF-1 Nil 300 pg LSF-1 dissolved
in 1.0 ml of Intralipos
The effect of anti-LSF-I on tolerogenic liver allografrs Between 0.8 and 2.0 ml of polyclonal rabbit serum (anti-LSF1) was injected intravenously into PVG recipients of DA liver grafts within 1 h of surgery. Control OLT rats received 1.0 ml of normal rabbit sera. OLT was performed using a previously established technique.2s Animals were observed daily and, in the event of death, an autopsy was performed to obtain the histological cause of death.
Results Effectof LSF-1 on heart allograft sunkd Administration of a single injection of LSF-1 (300 Fg, i.m., in 1 ml of Intralipos) into PVG recipients, led to significant prolongation of DA (>lOO days, n = 3) and BN heart grafts (54 f 21 days, n = 3) compared to untreated controls [DA: 8.1 + 0.7; BN: 8.0 f 0.8 days) or Intralipos controls (DA: 8.4 f 0.7 days) (Table 1). The administration of LSF-1, in Intralipos solution intramuscularly, showed no detrimental effects on liver and kidney functions (Table 2). Serum levels
POD
Serum source
heart grafts
Survival of allograft
MST f SD
(days)
(days)
7788888899 .,.,I..,, 7889999 .,..o 103, 126, 112 77888899 .,,,,.. 32, 56, 74
8.1 f0.7 8.4 f 0.7 114f12 8.0 f 0.8 54f21
solution.
(RTl”) (Animal Resources Centre, Perth, Australia). The HHT was performed in the cervical area as described elsewhere.*’ LSF-1 was dissolved in 1 ml of Intmlipos solution (Midori Juji Co., Osaka, Japan) and 300 p,g injected as a single shot, intramuscularly, into PVG recipients of either DA or BN hearts within 1 h of HHT. In control groups, either 1 ml of Intralipos solution was injected intramuscularly into HHT rats, or rats received no additional treatment before or after surgery. Serum samples were taken at various time points after HHT and sent to Veterinary Pathology Services (Brisbane, Australia) for analysis of levels of creatinine, urea, alanine aminotransferase (ALT) and alkaline phosphatase.
Table 2 Tests for normal kidney and liver function:
of creatinine, ALT and alkaline phosphatase all remained within normal levels in both untreated and treated recipients. Untreated PVG animals all showed high levels of serum urea (7.2, 7.7, 8.4 nmol/l) compared to the normal range (2.13.6 nmol/l); however, all the treated PVG recipients showed similar high levels, further indicating that there were no severe effects on kidney function due to administration of LSF-1. All animals followed a standard growth curve for weight increase following surgery (data not shown). Western blotting with anti-LSF-1 Polyclonal rabbit sera was shown by immunoblotting to react with two proteins of approx. 40 kDa in OLT serum as a doublet, after denaturation with p-mercaptoethanol (Figure 1, lane 5). These bands are LSF-1 (the 40 kDa protein) and a protein with an identical N-terminal sequence at 37 kDa which is not immunosuppressive in vifro.*O The 37 kDa protein is believed to be a degraded or modified form of the 40kDa (LSF-1) as described previously.2o The broad bands seen above the 45 kDa marker in all lanes, although slightly higher in fully denatured samples, are due to non-specific cross-reactivity with rat serum albumin. The strong cross-reaction that is observed with a high molecular mass protein (>lOO kDa, lanes 3 and 5) as yet remains unresolved; however, it is not a cross-reaction of either the primary or secondary antibodies with rat IgG (data not shown). We speculate that this is either a multimeric form of LSF-I disrupted by the addition of pmercaptoethanol, or it is the LSF-1 molecule bound to, or held by, a carrier molecule(s) for transport in serum. The difference in intensities between the LSF-1 doublet and the > 100 kDa molecule may be due to a conformational difference between the two resulting in lower antibody affinity under Western blotting conditions. The reactivity of anti-LSF-1 with the N-
serum levels of urea, creatinine,
ALT (alanine
aminotransferase)
and alkaline
Urea (nmol/l) (2.1-3.6)
Creatinine (nmol/l) (0.03-0.33)
ALT (U/ml) (11-61)
(Ufl) (<260)
-
7.2 7.7 8.4
0.04 0.06 0.06
40 40 40
128 1.52 119
PVG recipient of DA HHT, treated with 300 pg of LSF-1
30 30 43 70 80 87
7.0 7.6 8.0 9.0 7.2 7.0
0.05 0.06 0.05 0.05 0.04 0.06
40 40 35 40 40 40
160 180 160 165 126 128
by Veterinary
Pathology
Transplant Immunology 1996; 4: 287-292
Services (Brisbane).
Normal ranges are also indicated
phosphatase
Alkaline phosphatase
PVG serum
Testing was performed
289
in the table. POD, postoperative
day.
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C Edwards-Smith
et al.
79 KDa-
45-
36-
26.9
Figure 1 A Western blot showing the cross-reactivity of anti-LSF-1 with DA serum and OLT POD 60 serum. All serum was diluted l/20 with distilled Hz0 and 5 ~1 added to 5 p,l of Laemmli sample buffer prior to loading onto the gel. Laemmli sample buffer was either with or without @mercaptoethanol (E-Me) added to assist in fully denaturing serum proteins. Serum proteins were separated on a 4-208 SDSPAGE gradient gel (BioRad) prior to transfer onto nitrocellulose and immunoblotting. Lane 1: BioRad broad range prestained molecular mass markers. Lane 2: DA serum in non-denaturing Laemmli sample buffer. Lane 3: DA serum in denaturing Laemmli sample buffer. Lane4 OLT (DA into PVG) POD 60 serum in non-denaturing Laemmli sample buffer. Lane 5: OLT (DA into PVG) POD 60 serum in denaturing Laemmli sample buffer. The 40 kDa doublet, seen running just below the 45 kDa marker in lane 5, is marked with an arrow
peptide was confirmed using a peptide-BSA gate by Western blotting (data not shown). terminal
conju-
Effect of antHSF-1 on a normally tolerogenic liver ahgraft model Injection of between 1.0 and 2.0 ml of anti-LSF-1 sera into PVG recipients of DA livers resulted in the death of the recipient in S/6 cases, as shown in Table 3. The animals died between days 10 and 15 after OLT due to rejection based upon histological findings. One of the recipients of > 1.O ml of antiLSF-1 polyclonal rabbit sera became ill and showed signs of severe weight loss consistent with rejection; however, the animal eventually recovered and survived (>40 days). The administration of less than 1.0 ml of anti-LSF-1 did not prevent the induction of tolerance and the rats survived (n = 3). Controls showed no significant weight loss and followed normal patterns of recovery following OLT. . . H&ologd examination of the effect of anti-LSF-1 Figure 2A, the untreated DA into PVG POD 11 liver, shows mild cellular infiltration, mainly of the portal tract (top), with scattered Transplant Immunology 1996; 4: 287-292
Figure2 HE staining of liver tissue samples after OLT (DA into PVG) treated with or without anti-LSF-1. (A) OLT (DA into PVG) POD 11 liver (magnification x250). There is mild cellular infiltration, mainly of the portal tract (top), with scattered focal necrosis of liver cells and mononuclear cells throughout the sinusoidal spaces. (B) OLT (DA into PVG) anti-LSF-1 treated liver biopsy POD 10 (magnification x200). Histological analysis indicates widespread focal necrosis of the liver cells which is associated with an extensive cellular infiltrate in the sinusoids. (NB: this animal died at POD 14.) (C) OLT (DA into PVG) anti-LSF-1 treated liver POD 10 autopsy sample (magnification x200). Severe rejection with oedema and extensive loss of parenchymal cells, necrosis of remaining liver cells and massive infiltration of the sinusoids
focal necrosis of liver cells and mononuclear cells throughout the sinusoidal spaces. Figure 2B, from POD 10, shows a liver biopsy sample tiom a DA into PVG anti-LSF-1 treated rat. Histological analysis indicates widespread focal necrosis of the liver cells
Role of LSF-1 in tolerance induction
Histology of liver graft biopsy and autopsy samples, following anti-LSF-1 treatment, shows extensive cellular infiltration into the tissue followed by widespread necrosis consistent with an acute rejection episode (Figure 2). These results, taken together with the in vivo testing of LSF-1, clearly implicate a role for LSF-1 in the induction of tolerance. The mechanism underlying such long-term allograft survival by administration of LSF-1 is unknown. The process of tolerance induction appears to be disrupted by administration of anti-LSF-1, suggesting that LSF-1 acts early in the process of tolerance induction. Two possible scenarios are envisaged for the action of LSF-1: 1) LSF- 1 may be regulating cell-cell interaction during the process of alloantigen recognition on the pathway to clonal deletion of alloreactive T cells;31*322) LSF1 may be interfering in ligand-receptor interactions responsible for T cell activation,33 leading to graft acceptance. LSF1 may also be aiding in the formation of T suppressor cells, which appear in the spleen after week 20 in the DA into PVG model,” or it could be involved in the inactivation of donor specific T cell clones.34 It is not clear at this stage whether LSF- 1 is a T cell product or is derived from other cells involved in the allograft response, or from the liver itself. The relatively high serum concentration of LSF-lm is more typical of a protein secreted from the liver than a T cell derived protein, although, under certain conditions of disease and powerful immune activation, cytokines can be clearly detected in serum. In conclusion, LSF-1 clearly has a potent immunosuppressive effect in a rat heart transplant model. Clinical post-transplant therapy using the immunosuppressive compounds CsA or FK506 must usually be maintained throughout the life of a transplant patient. These drugs have a number of possible undesirable side-effects which include nephrotoxicity, neurotoxicity and hepatotoxicity. 35*36The identification of the cell type responsible for the production of LSF- 1, the cellular target and studies on the mechanism of action of LSF-1 and further characterization of the molecule are underway. A better understanding of the mechanisms involved in liver graft induced tolerance without immunosuppression, in certain mammalian combinations, may facilitate new strategies for the prevention of graft rejection. The identification of a human homologue of LSF-1 may provide applications for clinical use in the maintenance and treatment of graft recipients.
which is associated with an extensive cellular infiltrate in the sinusoids. Figure 2C shows an autopsy sample of liver OLT following anti-LSF-I treatment, with severe rejection, oedema and extensive loss of parenchymal cells, necrosis of remaining liver cells and massive infiltration of the sinusoids.
Discussion The immunosuppressive activity of OLT serum has been atttibuted to a number of factors, including MHC restricted factors such as soluble class I antigen and anti-class II antibody.2’ Treatments with MHC antigen (cellular, soluble or membrane) or specific MHC peptides combined with CsA (cyclosporine A)2g or FK506 can result in donor-specific tolerance.30 However, administration of soluble or membrane forms of class I only produces a small degree of prolongation of allograft survival.i5 A single administration (300 pg i.m.) of LSF-1, purified from POD 60 OLT serum, prolonged heart allograft survival in a donor-specific (DA to PVG; graft survival >lOO days) and also in a non-donor-specific (BN to PVG; graft survival 54 f 21 days) manner (see Table 1) without any detrimental side-effects. Although administration of a single dose of LSF-1 does not produce tolerance, it has a very powerful in viva suppressive effect, prolonging survival of DA and BN allografts. There does appear to be some degree of strain combination sensitivity to the effect as prolongation of DA grafts is greater than BN (114 vs 54 days). There are no other reports of a single injection, at therapeutic doses, of an immunosuppressive agent with such powerful effects on fully allogeneic HHT without deleterious side-effects. Lower concentrations of LSF-1 (100 pg/ml) administered intravenously had no effect on prolongation of heart grafts (unpublished A higher concentration observations). (300 pg/ml) was then chosen; however, at this concentration the protein began to precipitate, indicating hydrophobic domains within LSF-1. This necessitated the use of the Intralipos solution and intramuscular administration of LSF- 1. This further raises the possibility that the high molecular weight protein observed in Figure 1 is involved in the stabilization of LSF-1 in serum (i.e. a carrier). Western blot analysis confirmed that the polyclonal antisera was reacting with I-SF-1 in OLT POD 60 samples treated with p-mercaptoethanol (Figure 1). The administration of between 1.O and 2.0 ml of anti-LSF- 1 sera abrogated the tolerance normally seen in this tolerogenic DA into PVG model (Table 3).
Table 3 The effect of anti-LSF-1 on liver transplantation in a normally
Recipient
Donor
tolerogenic
combination
Treatment
(DA into PVG) Survival of individual
rats
(days)
PVG
DA
Nil
>60,>60,>60,>60,>60,>fxl
PVG
DA
1.0 ml normal rabbit sera, i.v.
>60, >60,
>60,
PVG
DA
0.8-0.9
>30,
X0
PVG
DA
1.0 ml anti-LSF-1,
PVG
DA
1.5-2.0 ml anti-LSF-1,
ml anti&SF-l,
i.v.
i.v.
Transplant Immunology 19%; 4: 287-292
X0,
~60
8, 11, 10 i.v.
“A biopsy sample was taken from this animal for histological examination POD 10. ms animal showed severe weight loss at POD 10-14 consistent with the rejecting animals; however,
POD, postoperative day; i.v., intravenous.
291
148, 12, >4Ob
it recovered
and survived (MO days).
292
C Edwards-Smith et al.
Acknowledgements This work was supported with grants from the Sasakawa Foundation Tokyo, Japan, the University of Queensland (External Support Enabling Grant) and the Combined Liver Transplant Trust Funds, Princess Alexandra Hospital and the Royal Children’s Hospital Brisbane, Australia.
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All-raft acceptance and rejection, mediated by a liver suppressor factor, LSF-1, purified from serum of liver transplanted rats Catherine Edwards-Smith”, Shigeru Goto, Roger Lord”, Yoshinori Shimizub, Frank Vari” and Naoshi Kamadaa aJoint Transplantation Biology Programme, Queensland Institute of Medical Research/Department of Surgery, University of Queensland, Brisbane, and ‘Department of Surgery, Showa University, Tokyo Received 1 March 1996; accepted for publication 7 May 1996
Abstract: In certain rat strain combinations liver allografts are spontaneously accepted without immunosuppression and induce donor-specific tolerance to further skin and heart grafts in the recipient. Such an effect is also transferrable using serum from orthotopically liver transplanted rats (OLT serum). In the OLT serum of one such combination, DA (RTl”) donor into PVG (RTl’) recipient, a 40 kDa protein (liver suppressor factor, LSF-1) has been identified and shown to be immunosuppressive in vitro. The aim of the present study is to investigate the immunological effect of LSF-1 and a polyclonal antibody (anti-LSF-I) against this molecule, in a rat heterotopic heart transplant (HHT) model and OLT model, respectively. Intramuscular injection of 300 pg of LSF-1, 1 h postoperatively, into a PVG recipient of either a DA or BN (RTl”) cardiac allograft caused significant prolongation of graft survival. Intravenous injection of polyclonal rabbit sera raised against an N-terminal peptide of LSF-1 (anti-LSF-l), within 1 h postoperatively, had variable effects on the survival of DA liver grafts in PVG recipients. In 5/6 cases injection of between 1 and 2 ml of anti-LSF-1 resulted in death of the recipient. Histological examination of the liver showed severe rejection with lymphoid cell infiltration of the portal tract and sinusoids and extensive damage to the parenchyma. All control rats survived for more than 60 days without any signs of rejection. The anti-LSF-1 polyclonal antibody prevented the induction of tolerance in the normally tolerogenic model (DA into PVG). This, together with the in viva results, suggests a role for LSF-1 in the induction of tolerance.
Introduction The ability of the liver to be accepted as an allograft without immunosuppression has been reported in several species such as mouse, rat and pig. l-3 Rejection reactions against the liver are relatively weak in comparison with those against other organs. In certain rat and pig strain combinations liver allografts provide a protective effect against rejection of other grafts Address for correspondence: Catherine Edwards-Smith, Transplantation Biology, Queensland Institute of Medical Research, PO Royal Brisbane Hospital, Queensland 4029, Australia. 0 Arnold 1996
which are donor type such as skin, heart and kidney and small bowe1.2@ It is also recognized in the clinical setting that the liver possesses unique survival properties. Co-transplantation of the liver in order to protect other organs against graft rejection has become a common strategy; combined liver and kidney transplantation was associated with a reduced incidence of kidney rejection.7*8 We have studied in detail a rat model of orthotopic liver transplantation (OLT). PVG (RTl’) recipients of DA (RTl”) liver allografts survive without immunosuppression and become tolerant of subsequent grafts of other DA organs such as skin, heart and kidney. 43 This immunosuppressive effect of
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the liver was transferrable to other PVG recipients using OLT serum. OLT serum (1 ml), collected from PVG recipients bearing DA liver allografts for 24 months, suppressed the rejection of PVG.RTl” heart grafts.9*‘o Such donor-specific effects of OLT serum have been attributed to the high concentration of soluble (DA) donor class I antigen’ ’ and anti-class II antibodies.‘2*‘3 Knoop et ~1.‘~ have also demonstrated that 1 ml of OLT serum (DA-PVG) induced tolerance in PVG recipients of third party A0 (RTl”) kidney graft and speculated that it was due to the high levels of anticlass II antibodies. OLT serum from liver grafted mice was also immunosuppressive and the effect was attributed to the high concentration of IgG particularly class II antibodies.’ Studies on purified soluble donor class I antigen have shown that, by continuous infusion, antigen-specific protection of allografts can be achieved.15 Administration of extremely high doses of rat MHC class I antigen could also prolong renal allograft survival.‘6~17 However, others have indicated that class I alone cannot be responsible for the observed prolongation by OLT serum.‘*.r9 Further studies have shown that soluble MHC class I antigen and anti-donor MHC class II antibodies alone cannot be responsible for the immunosuppressive activity observed in pooled OLT serum.2o OLT serum from postoperative day (POD) 60 has previously been shown to be the most effective at inhibiting cell proliferation in MLR (mixed lymphocyte reaction)?’ This serum has also been shown to contain lower concentrations of recipient anti-donor class I and class II antibodies than OLT serum from earlier postoperative times.22 We have previously reported on the identification and isolation of LSF- 1, a 40 kDa protein with a unique N-terminal sequence (SLAATHMHGNRVG) from rat POD 60 OLT serum.2o The addition of 10 p,g of LSF- 1 to MLR showed a degree of donorspecific immunosuppression in anti-DA PVG and anti-PVG DA MLR.” In the present study, LSF-1 was tested in vivo using a rat model of heterotopic heart transplantation (HHT). Subsequently, to fully examine the role of LSF-1, a polyclonal antibody was raised in rabbit against the N-terminal sequence. The polyclonal rabbit sera was injected into the normally tolerogenic combination of OLT (DA liver into PVG recipient) and the effect on the establishment of tolerance observed.
Objective The objective of this study was to examine the effect of LSF1 and a polyclonal antibody (anti-LSF-1) against this molecule, on graft acceptance or rejection using a rat HHT (heterotopic heart transplant) model and OLT model, respectively.
Materials and methods Purification of ISF-1 OLT (DA into PVG) day 60-80 serum was used for the purification of LSF-1, a 40 kDa protein with a unique N-terminal sequence as described previously.“*22 Briefly, the protein was purified by separation of OLT serum using 4-20% SDS-PAGE gradient gel (Biorad) separation followed by staining with Coomassie blue G250. The 40kDa band was identified by comparison to prestained broad range molecular weight markers (Biorad), excised and then eluted from the gel, prior to Transplant Immunology 1996; 4: 287-292
final purification by reverse-phase high-performance liquid chromatography (Model 1350, Bio-Rad Richmond, CA, USA). N-terminal amino acid sequencing was performed by electrophoretic separation of the protein on 4-20% gradient TrisTricine gels (BioRad) followed by transfer to PVDF membrane (BioRad) using the method of Choli and Wittman-Liebold.23 The protein was subjected to gas phase sequencing on an Applied Biosystems 437 protein sequencer (Foster City, CA, USA). Synthesis of peptide The peptide (SLAATHMHGN), derived from the previously reported N-terminal sequence of LSF-1,22 was synthesized using the simultaneous multiple peptide technique described employing derivatized (t-Boc) amino acids by Houghte# (Omni Biochemicals, National City, CA, USA) on a benzhydrylamine resin. An N-terminal cysteine was added to the peptide to facilitate coupling to the carrier protein. The heterobifunctional reagent maleimidocaproyloxysuccinimide (MCS) was employed to add either diphtheria toxoid (DT, Commonwealth Serum Laboratories, Melbourne, Australia) or bovine serum albumin (BSA; Sigma, St Louis, USA). The procedure of Lee et a1.25was the method used for derivatization of DT and BSA. Preparation of rabbit polyclonal antibodies Rabbits were immunized subcutaneously every 2 weeks for 14 weeks with 200 p,g of peptide-DT conjugate in Freund’s complete adjuvant (Sigma) for the first immunization and Freund’s incomplete adjuvant (Sigma) for subsequent boosts. Blood was collected at weeks 6, 8, 10 and 14 after the first immunization at week 0. Blood was collected from the rabbit’s ear by venipuncture using a catheter and allowed to clot at room temperature. Serum was collected by centrifugation at 3000 rpm for 15 min and stored at -2O’C. SDS-PAGE and immunoblotting Aliquots (5 ~1) of previously diluted (l/20 with distilled H20) OLT (DA into PVG) and DA serum were each added to 5 pl of Laemmli26 sample buffer, either with or without B-mercapmethanol, then electrophoretically separated on a 4-20% gradient SDS-PAGE, followed by transfer onto nitrocellulose membrane using a Trans-Blot SD semidry transfer blotter (BioRad). Membranes were then blocked by incubation with 5% skim milk powder in Tris-buffered saline (TBS) with gentle agitation for 30 min. The polyclonal sera was then added directly to the blocking solution at l/500 dilution and the membrane incubated overnight with gentle agitation at room temperature. The membrane was then washed for 5 min in TBS and repeated twice. The secondary antibody, sheep anti-rabbit IgG alkaline phosphatase conjugate (Boehringer Mannheim) diluted l/1000 in TBS, was added to the membrane and incubated at room temperature for 1 h. The membrane was then washed again in TBS, as described. Antibody reaction was detected using an Alkaline Phosphatase Conjugate Substrate Kit (BioRad) and used as described by the manufacturer. Experimental surgery The effect of LSF-1 on heart allografs Male rats, each weighing 230-350 g, of the following strains were used: DA (MHC haplotype RTla), PVG (RTl’) and BN
Role of LSF-1 in tolerance induction
Table 1 The effect of a single administration Recipient
PVG PVG PVG PVG PVG
of LSF-1,
Donor
from OLT serum, on fully allogeneic
Treatment
DA DA DA BN BN
LSF-I was administered intramuscularly, MST, mean survival time.
purified
Nil Intralipos 1.0 ml 300 pg LSF-1 Nil 300 pg LSF-1 dissolved
in 1.0 ml of Intralipos
The effect of anti-LSF-I on tolerogenic liver allografrs Between 0.8 and 2.0 ml of polyclonal rabbit serum (anti-LSF1) was injected intravenously into PVG recipients of DA liver grafts within 1 h of surgery. Control OLT rats received 1.0 ml of normal rabbit sera. OLT was performed using a previously established technique.2s Animals were observed daily and, in the event of death, an autopsy was performed to obtain the histological cause of death.
Results Effectof LSF-1 on heart allograft sunkd Administration of a single injection of LSF-1 (300 Fg, i.m., in 1 ml of Intralipos) into PVG recipients, led to significant prolongation of DA (>lOO days, n = 3) and BN heart grafts (54 f 21 days, n = 3) compared to untreated controls [DA: 8.1 + 0.7; BN: 8.0 f 0.8 days) or Intralipos controls (DA: 8.4 f 0.7 days) (Table 1). The administration of LSF-1, in Intralipos solution intramuscularly, showed no detrimental effects on liver and kidney functions (Table 2). Serum levels
POD
Serum source
heart grafts
Survival of allograft
MST f SD
(days)
(days)
7788888899 .,.,I..,, 7889999 .,..o 103, 126, 112 77888899 .,,,,.. 32, 56, 74
8.1 f0.7 8.4 f 0.7 114f12 8.0 f 0.8 54f21
solution.
(RTl”) (Animal Resources Centre, Perth, Australia). The HHT was performed in the cervical area as described elsewhere.*’ LSF-1 was dissolved in 1 ml of Intmlipos solution (Midori Juji Co., Osaka, Japan) and 300 p,g injected as a single shot, intramuscularly, into PVG recipients of either DA or BN hearts within 1 h of HHT. In control groups, either 1 ml of Intralipos solution was injected intramuscularly into HHT rats, or rats received no additional treatment before or after surgery. Serum samples were taken at various time points after HHT and sent to Veterinary Pathology Services (Brisbane, Australia) for analysis of levels of creatinine, urea, alanine aminotransferase (ALT) and alkaline phosphatase.
Table 2 Tests for normal kidney and liver function:
of creatinine, ALT and alkaline phosphatase all remained within normal levels in both untreated and treated recipients. Untreated PVG animals all showed high levels of serum urea (7.2, 7.7, 8.4 nmol/l) compared to the normal range (2.13.6 nmol/l); however, all the treated PVG recipients showed similar high levels, further indicating that there were no severe effects on kidney function due to administration of LSF-1. All animals followed a standard growth curve for weight increase following surgery (data not shown). Western blotting with anti-LSF-1 Polyclonal rabbit sera was shown by immunoblotting to react with two proteins of approx. 40 kDa in OLT serum as a doublet, after denaturation with p-mercaptoethanol (Figure 1, lane 5). These bands are LSF-1 (the 40 kDa protein) and a protein with an identical N-terminal sequence at 37 kDa which is not immunosuppressive in vifro.*O The 37 kDa protein is believed to be a degraded or modified form of the 40kDa (LSF-1) as described previously.2o The broad bands seen above the 45 kDa marker in all lanes, although slightly higher in fully denatured samples, are due to non-specific cross-reactivity with rat serum albumin. The strong cross-reaction that is observed with a high molecular mass protein (>lOO kDa, lanes 3 and 5) as yet remains unresolved; however, it is not a cross-reaction of either the primary or secondary antibodies with rat IgG (data not shown). We speculate that this is either a multimeric form of LSF-I disrupted by the addition of pmercaptoethanol, or it is the LSF-1 molecule bound to, or held by, a carrier molecule(s) for transport in serum. The difference in intensities between the LSF-1 doublet and the > 100 kDa molecule may be due to a conformational difference between the two resulting in lower antibody affinity under Western blotting conditions. The reactivity of anti-LSF-1 with the N-
serum levels of urea, creatinine,
ALT (alanine
aminotransferase)
and alkaline
Urea (nmol/l) (2.1-3.6)
Creatinine (nmol/l) (0.03-0.33)
ALT (U/ml) (11-61)
(Ufl) (<260)
-
7.2 7.7 8.4
0.04 0.06 0.06
40 40 40
128 1.52 119
PVG recipient of DA HHT, treated with 300 pg of LSF-1
30 30 43 70 80 87
7.0 7.6 8.0 9.0 7.2 7.0
0.05 0.06 0.05 0.05 0.04 0.06
40 40 35 40 40 40
160 180 160 165 126 128
by Veterinary
Pathology
Transplant Immunology 1996; 4: 287-292
Services (Brisbane).
Normal ranges are also indicated
phosphatase
Alkaline phosphatase
PVG serum
Testing was performed
289
in the table. POD, postoperative
day.
290
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et al.
79 KDa-
45-
36-
26.9
Figure 1 A Western blot showing the cross-reactivity of anti-LSF-1 with DA serum and OLT POD 60 serum. All serum was diluted l/20 with distilled Hz0 and 5 ~1 added to 5 p,l of Laemmli sample buffer prior to loading onto the gel. Laemmli sample buffer was either with or without @mercaptoethanol (E-Me) added to assist in fully denaturing serum proteins. Serum proteins were separated on a 4-208 SDSPAGE gradient gel (BioRad) prior to transfer onto nitrocellulose and immunoblotting. Lane 1: BioRad broad range prestained molecular mass markers. Lane 2: DA serum in non-denaturing Laemmli sample buffer. Lane 3: DA serum in denaturing Laemmli sample buffer. Lane4 OLT (DA into PVG) POD 60 serum in non-denaturing Laemmli sample buffer. Lane 5: OLT (DA into PVG) POD 60 serum in denaturing Laemmli sample buffer. The 40 kDa doublet, seen running just below the 45 kDa marker in lane 5, is marked with an arrow
peptide was confirmed using a peptide-BSA gate by Western blotting (data not shown). terminal
conju-
Effect of antHSF-1 on a normally tolerogenic liver ahgraft model Injection of between 1.0 and 2.0 ml of anti-LSF-1 sera into PVG recipients of DA livers resulted in the death of the recipient in S/6 cases, as shown in Table 3. The animals died between days 10 and 15 after OLT due to rejection based upon histological findings. One of the recipients of > 1.O ml of antiLSF-1 polyclonal rabbit sera became ill and showed signs of severe weight loss consistent with rejection; however, the animal eventually recovered and survived (>40 days). The administration of less than 1.0 ml of anti-LSF-1 did not prevent the induction of tolerance and the rats survived (n = 3). Controls showed no significant weight loss and followed normal patterns of recovery following OLT. . . H&ologd examination of the effect of anti-LSF-1 Figure 2A, the untreated DA into PVG POD 11 liver, shows mild cellular infiltration, mainly of the portal tract (top), with scattered Transplant Immunology 1996; 4: 287-292
Figure2 HE staining of liver tissue samples after OLT (DA into PVG) treated with or without anti-LSF-1. (A) OLT (DA into PVG) POD 11 liver (magnification x250). There is mild cellular infiltration, mainly of the portal tract (top), with scattered focal necrosis of liver cells and mononuclear cells throughout the sinusoidal spaces. (B) OLT (DA into PVG) anti-LSF-1 treated liver biopsy POD 10 (magnification x200). Histological analysis indicates widespread focal necrosis of the liver cells which is associated with an extensive cellular infiltrate in the sinusoids. (NB: this animal died at POD 14.) (C) OLT (DA into PVG) anti-LSF-1 treated liver POD 10 autopsy sample (magnification x200). Severe rejection with oedema and extensive loss of parenchymal cells, necrosis of remaining liver cells and massive infiltration of the sinusoids
focal necrosis of liver cells and mononuclear cells throughout the sinusoidal spaces. Figure 2B, from POD 10, shows a liver biopsy sample tiom a DA into PVG anti-LSF-1 treated rat. Histological analysis indicates widespread focal necrosis of the liver cells
Role of LSF-1 in tolerance induction
Histology of liver graft biopsy and autopsy samples, following anti-LSF-1 treatment, shows extensive cellular infiltration into the tissue followed by widespread necrosis consistent with an acute rejection episode (Figure 2). These results, taken together with the in vivo testing of LSF-1, clearly implicate a role for LSF-1 in the induction of tolerance. The mechanism underlying such long-term allograft survival by administration of LSF-1 is unknown. The process of tolerance induction appears to be disrupted by administration of anti-LSF-1, suggesting that LSF-1 acts early in the process of tolerance induction. Two possible scenarios are envisaged for the action of LSF-1: 1) LSF- 1 may be regulating cell-cell interaction during the process of alloantigen recognition on the pathway to clonal deletion of alloreactive T cells;31*322) LSF1 may be interfering in ligand-receptor interactions responsible for T cell activation,33 leading to graft acceptance. LSF1 may also be aiding in the formation of T suppressor cells, which appear in the spleen after week 20 in the DA into PVG model,” or it could be involved in the inactivation of donor specific T cell clones.34 It is not clear at this stage whether LSF- 1 is a T cell product or is derived from other cells involved in the allograft response, or from the liver itself. The relatively high serum concentration of LSF-lm is more typical of a protein secreted from the liver than a T cell derived protein, although, under certain conditions of disease and powerful immune activation, cytokines can be clearly detected in serum. In conclusion, LSF-1 clearly has a potent immunosuppressive effect in a rat heart transplant model. Clinical post-transplant therapy using the immunosuppressive compounds CsA or FK506 must usually be maintained throughout the life of a transplant patient. These drugs have a number of possible undesirable side-effects which include nephrotoxicity, neurotoxicity and hepatotoxicity. 35*36The identification of the cell type responsible for the production of LSF- 1, the cellular target and studies on the mechanism of action of LSF-1 and further characterization of the molecule are underway. A better understanding of the mechanisms involved in liver graft induced tolerance without immunosuppression, in certain mammalian combinations, may facilitate new strategies for the prevention of graft rejection. The identification of a human homologue of LSF-1 may provide applications for clinical use in the maintenance and treatment of graft recipients.
which is associated with an extensive cellular infiltrate in the sinusoids. Figure 2C shows an autopsy sample of liver OLT following anti-LSF-I treatment, with severe rejection, oedema and extensive loss of parenchymal cells, necrosis of remaining liver cells and massive infiltration of the sinusoids.
Discussion The immunosuppressive activity of OLT serum has been atttibuted to a number of factors, including MHC restricted factors such as soluble class I antigen and anti-class II antibody.2’ Treatments with MHC antigen (cellular, soluble or membrane) or specific MHC peptides combined with CsA (cyclosporine A)2g or FK506 can result in donor-specific tolerance.30 However, administration of soluble or membrane forms of class I only produces a small degree of prolongation of allograft survival.i5 A single administration (300 pg i.m.) of LSF-1, purified from POD 60 OLT serum, prolonged heart allograft survival in a donor-specific (DA to PVG; graft survival >lOO days) and also in a non-donor-specific (BN to PVG; graft survival 54 f 21 days) manner (see Table 1) without any detrimental side-effects. Although administration of a single dose of LSF-1 does not produce tolerance, it has a very powerful in viva suppressive effect, prolonging survival of DA and BN allografts. There does appear to be some degree of strain combination sensitivity to the effect as prolongation of DA grafts is greater than BN (114 vs 54 days). There are no other reports of a single injection, at therapeutic doses, of an immunosuppressive agent with such powerful effects on fully allogeneic HHT without deleterious side-effects. Lower concentrations of LSF-1 (100 pg/ml) administered intravenously had no effect on prolongation of heart grafts (unpublished A higher concentration observations). (300 pg/ml) was then chosen; however, at this concentration the protein began to precipitate, indicating hydrophobic domains within LSF-1. This necessitated the use of the Intralipos solution and intramuscular administration of LSF- 1. This further raises the possibility that the high molecular weight protein observed in Figure 1 is involved in the stabilization of LSF-1 in serum (i.e. a carrier). Western blot analysis confirmed that the polyclonal antisera was reacting with I-SF-1 in OLT POD 60 samples treated with p-mercaptoethanol (Figure 1). The administration of between 1.O and 2.0 ml of anti-LSF- 1 sera abrogated the tolerance normally seen in this tolerogenic DA into PVG model (Table 3).
Table 3 The effect of anti-LSF-1 on liver transplantation in a normally
Recipient
Donor
tolerogenic
combination
Treatment
(DA into PVG) Survival of individual
rats
(days)
PVG
DA
Nil
>60,>60,>60,>60,>60,>fxl
PVG
DA
1.0 ml normal rabbit sera, i.v.
>60, >60,
>60,
PVG
DA
0.8-0.9
>30,
X0
PVG
DA
1.0 ml anti-LSF-1,
PVG
DA
1.5-2.0 ml anti-LSF-1,
ml anti&SF-l,
i.v.
i.v.
Transplant Immunology 19%; 4: 287-292
X0,
~60
8, 11, 10 i.v.
“A biopsy sample was taken from this animal for histological examination POD 10. ms animal showed severe weight loss at POD 10-14 consistent with the rejecting animals; however,
POD, postoperative day; i.v., intravenous.
291
148, 12, >4Ob
it recovered
and survived (MO days).
292
C Edwards-Smith et al.
Acknowledgements This work was supported with grants from the Sasakawa Foundation Tokyo, Japan, the University of Queensland (External Support Enabling Grant) and the Combined Liver Transplant Trust Funds, Princess Alexandra Hospital and the Royal Children’s Hospital Brisbane, Australia.
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