Donor-specific tolerance is integral to the development of clinical protocols that eliminate the need for chronic immunosuppression. Recent clinical success with protocols of renal and hepatic transplantation illustrates that human tolerance induction is possible (1 – 4). Major histocompatibility complex (MHC)-inbred miniature swine are unique translational animals that serve as a bridge between research in rodents and primates (see Item 1, SDC 1, http://links.lww.com/TP/A560). Through inbreeding, the swine leukocyte antigen (analogous to the human leukocyte antigen) in MGH-miniature swine is defined and fixed (5). Using MGH-miniature swine, we have extensively studied the induction and mechanism of transplantation tolerance for kidney, thymus, heart, and islet grafts (6 – 13).
We have previously reported that 12 days of FK506 administered through continuous IV infusion facilitates tolerance induction to fully MHC-mismatched kidneys (7). This tolerance induction was highly dependent on the dose of FK506 used and was observed only at blood levels more than 35 ng/mL. Subsequent studies from our laboratory in non-human primates suggested that an even higher dose (>80 ng/mL) and longer duration (28 days) of immunosuppression were required for the induction of tolerance in this species (Yamada K, manuscript in preparation). We have therefore reasoned that the level of drug required for tolerance induction may vary among species and among strains of swine. Consistent with this hypothesis, we found that 12 days of FK506 at the dose, which uniformly facilitates tolerance induction of fully MHC-mismatched renal allografts in MGH-miniature swine, was insufficient to induce such tolerance in the fully allogeneic CLAWN-miniature swine maintained at Kagoshima University in Japan (14 – 16) (see Results; see Item 2, SDC 1, http://links.lww.com/TP/A560).
Hepatocyte growth factor (HGF) is a multifunctional, pleoitropic protein with mitogenic, motogenic, and morphogenic effects in a wide variety of cells (17) (see Item 3, SDC 1, http://links.lww.com/TP/A560). Recent studies using rodents have suggested that HGF has immunomodulatory effects for bone marrow and heart transplantation (18 – 20). We wished to test whether immunomodulation with HGF might inhibit acute rejection responses in a large animal model. Given the results of our data in CLAWN versus MGH miniature swine, we have now evaluated the potential immunomodulatory effects of human-recombinant HGF (h-rHGF) combined with 12 days of FK506 on renal allografts in CLAWN-miniature swine. The data reported here indicate that a short course of h-rHGF, in combination with FK506, inhibited acute rejection in this model. To our knowledge, this is the first demonstration of the purported immunomodulatory effects of HGF in a large animal model.
Effects of High-Dose FK506 on Renal Transplantation in CLAWN-Miniature Swine
12 Days of High-Dose FK506 Did Not Induce Tolerance in CLAWN-Miniature Swine
Clinical course and histology: Six animals received renal allografts followed by 12 days of high-dose FK506 (postoperative day [POD] 0–11). Mean FK506 levels during treatment were more than 35.0 ng/mL; 35.0, 35.4, 41.4, 39.6, 38.2, and 40.5 ng/mL, respectively, that were sufficient blood levels to induce tolerance in MGH-miniature swine (7). In contrast to our previous study using MGH-miniature swine (7), all animals in this group experienced severe rejection crises within 1 month of transplantation (Fig. 1a). Four of six animals rejected their grafts within the first month (PODs 15, 26, 29, and 29) and the other two had severe rejection crises with creatinine peaking at 20 mg/dL (no. 18467, POD 37) and 14 mg/dL (no. 18428, POD 19). The rejection crises then spontaneously resolved, however, creatinine levels fluctuated and these animals were killed at POD 91 (no. 18467) and POD 156 (no. 18428) with severe uremic condition.
Histologic analysis revealed both cellular and humoral rejection. Biopsies from rejected grafts taken within the first month of transplantation demonstrated diffuse interstitial mononuclear cell infiltrates, tubulitis (Fig. 1b), acute glomerulitis with multiple microthrombi, and thrombus in small vessels (Fig. 1c). Fibrinoid necrosis with thrombus formation at was also present in the small arteries 26 days after transplantation (Fig. 1d). Biopsies taken during acute rejection crises in animal no. 18467 on POD 30 and animal no. 18428 on POD 32 revealed similar pathology: diffuse mononuclear cell infiltrates and acute glomerulitis. Histology at later time points showed diffuse interstitial fibrosis (Fig. 1e), chronic transplant glomerulopathy and vasculopathy (Fig. 1f,g), indicating severe chronic rejection.
In Vitro Responses
All animals developed antidonor IgG antibodies: Antidonor antibodies were assessed by fluorescence-activated cell sorter (FACS) and immunohistochemistry. Antidonor IgG was detectable in all animals that rejected their grafts within 1 month of transplantation (Fig. 1h). Immunofluorescence of the excised kidney grafts from animal no. 18455 on POD 29 showed deposition of antidonor IgM and IgG antibodies (Fig. 1i, j). Although antidonor IgG antibodies were not detected by FACS analysis in animals that maintained their grafts longer than a month (animal nos. 18428 and 18467; Fig. 1k), glomerular IgG deposition was observed by immunohistochemistry on PODs 156 and 91 (Fig. 1l, m).
Use of h-rHGF in the Induction Period of Kidney Transplantation With 12 days of FK506
CLAWN-miniature swine experienced complete rejection or severe acute rejection crisis soon after cessation of FK506. We hypothesized that (1) peripheral alloregulatory responses were not sufficiently established with 12 days of FK506 in the fully allogeneic CLAWN model; and (2) h-rHGF, which has previously demonstrated immunomodulatory effects (18, 19), would inhibit the initial acute rejection and change the balance of the immune responses towards long-term acceptance. Based on this hypothesis, h-rHGF therapy was started on POD 11 to cover the period of rejection crisis seen in the FK506 monotherapy.
h-rHGF Level in the Serum and Renal Tissue of a Naive Animal
Before testing the effects of h-rHGF on renal allograft survival, we first confirmed that our drug-delivery system resulted in the selective accumulation of h-rHGF in the renal graft. For this purpose, a naive CLAWN-miniature pig received h-rHGF (0.03 mg/kg/hr) continuously for 2 hr through a catheter located in the renal artery. Before the administration of h-rHGF, the concentration of h-rHGF in the kidney was less than 0.3 ng/mL. Two hours after starting h-rHGF administration, the concentration within the kidney increased to 179.9 ng/mL, which is higher than the “optimal” levels described in small animal models (21). The serum level of h-rHGF was 3 ng/mL after 1 hr and 5 ng/mL after 2 hr. Thus, using this model, we were successful in achieving levels of h-rHGF in the kidney that were commensurate with those required to prevent rejection in published small animal series (22).
Short Course of h-rHGF Inhibited Acute Renal Rejection
Clinical Course and Histology
Three animals received a 14-day course of h-rHGF at a dose of 0.015 mg/kg/day beginning on POD 11, in addition to FK506. The mean FK506 level during treatment was 39.5±1.0 ng/mL which was comparable to that of animals treated with FK506 alone (38.4±1.1 ng/mL). In contrast to animals with a FK506 monotherapy that uniformly developed severe rejection in the first 3 weeks, all animals (nos. 19422, 19424, and 19456) maintained stable renal function for at least 7 weeks (Fig. 2a). During FK506 administration, there was no significant difference in mean creatinine levels between the two groups (POD 8, 1.8±0.3 mg/dL in animals with FK506 monotherapy vs. 2.3±0.6 mg/dL in animals with FK506 followed by h-rHGF, P=0.50). Seven days after cessation of FK506, however, the mean creatinine levels of the two groups were significantly different (POD 18, 8.2±1.5 mg/dL with FK506 monotherapy vs. 1.9±0.3 mg/dL in animals with h-rHGF+FK506, P=0.008) (Fig. 2b). One h-rHGF-treated animal (no. 19424) died from pneumonia on POD 43 with stable renal function. Creatinine levels in the other two animals that received h-rHGF increased after POD 50. They were both killed on POD 85 due to graft rejection (Fig. 2a).
One additional animal (no. 19412) received FK506 in addition to h-rHGF at a dose of 0.03 mg/kg/day for 7 days (total dose of h-rHGF equivalent to the animals that received the lower dose of h-rHGF for 14 days). The trend in creatinine for this animal was similar to the three animals that received the lower dose of h-rHGF. It was killed on POD 81 due to graft rejection (Fig. 2a, dashed line).
The 1- and 2-month renal graft biopsies from the three animals that received the lower dose of h-rHGF showed minimal cell infiltrates, no endotheliitis in the small arteries (Fig. 2c), and mild acute glomerulitis in approximately 20% of glomeruli (Fig. 2d, e). Histopathologically, despite observing diffuse interstitial CD3+ cellular infiltration in animals treated with FK506 monotherapy (Fig. 2f, representative data), CD3+ cellular infiltration of the grafts in the h-rHGF treatment group was markedly lower (Fig. 2g). At time of necropsy on POD 85 (nos. 19422 and 19456), pathology showed increased interstitial cell infiltrates and fibrosis in both grafts. The animal that received high-dose h-rHGF had similar pathological findings to those with the lower dose. No additional animals received the high-dose h-rHGF because it did not seem to confer a survival benefit over the lower dose h-rHGF. The higher dose was also associated with protein casts (Fig. 2h).
In Vitro Responses
Animals treated with h-rHGF did not develop antidonor antibodies and were transiently hyporesponsive to donor in cellular assays: FACS analysis revealed no antidonor IgM and IgG antibodies in the serum of the animals treated with h-rHGF (assessed on PODs 0, 7, 14, 21, 30, 60, and on the day of killing) (Fig. 3a, b). Immunofluorescence of excised kidney grafts showed no anti-swine IgM and IgG antibody deposition (Fig. 3c, d).
Carboxyfluorescein diacetate succinimidyl ester-mixed lymphocyte reaction (MLR) assays showed donor-specific hyporesponsiveness on POD 57 (Fig. 3e). PKH26-cytotoxicity assay (CML) also demonstrated donor-specific hyporesponsiveness on POD 44. The percent specific lysis was 6.7% at an effector-to-target ratio of 100:1 (Fig. 3f). However, antidonor responses in both MLR assays and CML assays were restored at time of graft loss in the animals treated with both low and high-dose h-rHGF (Fig. 3e, g).
HGF treatment was associated with maintenance of the FoxP3+ cells in CD4+CD25+ populations in peripheral blood monocyte cells (PBMC): To assess whether peripheral regulatory mechanisms were involved in the prolongation of graft survival observed with h-rHGF therapy, we examined changes in the expression of FoxP3 within CD4+/CD25+PBMCs in all animals. The percentage of FoxP3- positive cells within the CD4/CD25 double-positive cell population significantly decreased in the third and fourth postoperative weeks in animals that received the FK506 monotherapy (Fig. 4a, filled bars). In contrast, no decrease in this subpopulation was observed after transplantation in animals treated with both h-rHGF+FK506 (Fig. 4a, open bars). Additionally, we analyzed the percent FoxP3+/CD4+/CD25+ triple positive cells in PBMC. The percentages of triple-positive cells during FK506 treatment were similar in animals with FK506 alone and with FK506 plus h-rHGF. However, at time of rejection, this population decreased in the animals treated with FK506 alone while no change was observed in the animals treated with h-rHGF and FK506. These data indicate that among CD4+/CD25+ cells, the rejectors had a relative increase in FoxP3 negative cells while the majority of CD4+/CD25+ cells expressed FoxP3 in h-rHGF-treated animals. Histopathologically, the extent of FoxP3+ cells and CD25+/FoxP3+ cell infiltration diminished in animals treated with FK506 monotherapy during induction. On the other hand, grafts in the h-rHGF-treated group exhibited an increased infiltration of FoxP3+ and CD25+/FoxP3+ cells in renal allografts (Fig. 4c).
c-Met analysis: We examined expression of c-Met in kidneys (23, 24). Few c-Met+ cells were present in the naive renal tissue (Fig. 5a). During rejection, c-Met expression increased in the glomeruli (Fig. 5b), graft infiltrating lymphocytes (Fig. 5c) and capillaries (Fig. 5d) of animals treated with FK506 monotherapy (see Item 4, SDC 1, http://links.lww.com/TP/A560).
We have previously reported that 12 days of high-dose FK506, facilitates the induction of tolerance across a full MHC-mismatch in MGH-miniature swine, younger than 12 months (7, 25). Using the same surgical technique and dosage of FK506 and using swine of the same age that we used for the studies in MGH swine, tolerance was not induced in CLAWN-miniature swine. We suspect that, like non-human primates, CLAWN miniature swine are likely to require higher doses of FK506 than are required by MGH miniature to induce tolerance. Whether this difference is based on sharing of MHC antigens (26, 27), on differences in the metabolism of FK506, on environmental factors (leading to differences in memory vs. naive T cells) or on other strain-related differences between the two herds of swine is unclear at present. Nevertheless, the failure of this dose of FK506 to induce tolerance in CLAWN miniature swine has made these animals an excellent model for testing of other immunomodulatory treatments that might extend graft survival.
Using the CLAWN miniature swine model, we have been able to study the role of HGF in prolonging graft survival. After h-rHGF treatment, we observed no rejection episode in the induction period and greater than 30 days of stable graft function after cessation of all immunosuppression, as well as the maintenance of FoxP3 cells in the maintenance of FoxP3+ cells in CD4+CD25+ populations in PBMC. To our knowledge, this is the first demonstration that HGF may be immunoprotective in a large animal model.
Recent rodent studies have suggested that HGF has immunomodulatory effects in vitro and in vivo. Rutella et al. (18) demonstrated that monocytes cultured with HGF-primed CD4+ T cells selectively inhibited the proliferation of naive CD4+CD25− T cells in a cell-contact-dependent manner and induced the expression of FoxP3. Intravenously administered HGF has demonstrated the ability to ameliorate acute graft-versus-host disease after murine, allogeneic bone-marrow transplantation through reduction of IL-12 serum levels and suppression of IFN-γ and TNF-α mRNA in the target organ (18, 19) and regulation likely requires cell-to-cell contact (18). Furthermore, HGF has also been shown to prolong survival of cardiac allografts by enhancing IL-10 and TGF-β mRNA expression during acute rejection in a rodent model (20). Our preliminary results with co-culture assays have failed to show inhibition of naive swine leukocyte antigen-matched T-cell responses to donor-type or third-party stimulators by T cells from HGF-treated animals during periods of stable renal function (data will be reported elsewhere). This suggests that HGF may not directly inhibit T-cell function. We are currently investigating the effect of HGF on monocyte function and development of CD4+/CD25+/FoxP3+ cell populations in our model.
Recent data demonstrated that HGF induces the NF-kB inhibitory and anti-inflammatory protein A20 in RPTEC and EC (Ferran C, personal communication, 2011), demonstrating that HGF decreases inflammation and subsequent alloreactive signaling. Therefore, the immunomodulatory effect may be predominantly induced in grafts in our model that resulted in inhibition of direct antigen presentation by less efficient antigen presentation by antigen-presenting cells but not sufficiently through indirect responses. Although this study was not designed to interrogate the effect of indefinite HGF administration, continued infusion may promote tolerogenic effects by inhibition of systemic responses through the indirect pathway.
In this study, HGF was administered directly into the renal artery of the donor graft. A previous report by Ido et al. (1) demonstrated that h-rHGF administered intravenously or by the portal vein accumulated in livers and spleen but not in kidneys and (2) a half-life of h-rHGF is only 2.4 min when administered intravenously (21). In addition, a study in miniature swine had shown that intravenous r-hHGF at 0.2 or 1.0 mg/kg caused a rapid decrease in systolic BP in 20 min (28). To maintain a high/constant accumulation of h-rHGF in renal allografts, we developed a new method for HGF delivery allowing HGF to accumulate in the graft selectively.
In conclusion, our results demonstrate that CLAWN-miniature swine represent a valuable model for the preclinical studies of transplantation. Furthermore, our results provide evidence that h-rHGF administration may be associated with avoidance of acute rejection. Because HGF itself is biologically unstable in its native form, one possible therapeutic approach is improvement in bioavailablity by using a stable agonist of the HGF tyrosine kinase receptor (29). Additional studies aimed at increasing the length of HGF administration and improved bioavailability using HGF agonists will help to elucidate further the role of HGF as a potentially tolerogenic agent. Although further investigation is needed to clarify the molecular and cellular mechanisms related to HGF-mediated protection from acute rejection and to determine the optimal duration of HGF or HGF-agonist that would lead to maximal and more prolonged protection, we propose that there may be a role for HGF in clinical transplantation.
MATERIALS AND METHODS
CLAWN-miniature swine, aged less than 12 months, were obtained from the Japan Farm CLAWN Institute (Kagoshima, Japan) (14). CLAWN- miniature swine are genetically typed, MHC-inbred miniature swine (15, 16) (see Item 2, SDC 1, http://links.lww.com/TP/A560). In this study, c1-type swine were used as donors of fully MHC-mismatched renal allografts to c2-type recipients. In vitro immunologic responses to assess MHC class I and class II disparities were also confirmed by MLR and CML assays (see Item 5, SDC 1, http://links.lww.com/TP/A560).
Ten CLAWN-miniature swine received fully MHC-mismatched kidney allografts. Recipients were treated with 12 days (days 0–11) of FK506 with blood levels maintained at 35 to 45 ng/mL. Six recipients received only FK506 (group 1). Four recipients received FK506 in combination with 7 days (0.03 mg/kg/day) or 14 days (0.015 mg/kg/day) of HGF starting on POD 11 (group 2). The HGF was infused through a catheter in the renal graft artery.
Catheter placement and kidney transplantation: Two semipermanent indwelling silastic central venous catheters were placed in the external jugular veins of recipient animals. One catheter was used for the administration of FK506; the other facilitated blood sampling for clinical monitoring and in vitro assays. After bilateral nephrectomies, heterotopic renal transplantation to the right iliac space was performed (7) (see Items 6 and 7, SDC 1, http://links.lww.com/TP/A560).
Insertion of the Catheter to Infuse h-rHGF Selectively to Renal Allografts
We have developed a drug-infusion system that selectively administers h-rHGF to renal grafts in miniature swine (see Item 7, SDC 1, http://links.lww.com/TP/A560). The catheter was removed at the time of the first kidney biopsy. No graft arterial thromboses developed as a result of the catheter.
Administration of FK506
FK506 was generously provided by Astelas Healthcare, Inc. (Osaka, Japan). It was continuously administered through an infusion pump for 12 days starting on the day of kidney transplantation (day 0). The FK506 dose was adjusted to maintain blood levels between 35 and 45 ng/mL. This 12 days of high-dose FK506 was previously found to induce tolerance to fully MHC-mismatched kidneys in MGH-miniature swine (7).
Monitoring of Rejection
Rejection was monitored by serum creatinine levels and by histological examination of renal biopsy tissue. Biopsy specimens were stained using hematoxylin-eosin and periodic acid-Schiff (PAS), and periodic acid silver methenamine (PAM).
- Cellular assays: PBMCs were obtained by gradient centrifugation using HISTOPAQUE (Sigma, St. Louis, MO) as previously reported (30) (see Item 8, SDC 1, http://links.lww.com/TP/A560).
- Assessment of antidonor antibodies: Antidonor IgM and IgG antibodies in the serum and in frozen biopsy samples were assessed by indirect FACS and immunohistochemistry, respectively, as previously described (7).
- Assessment of FoxP3+ CD4+CD25+ lymphocytes: The percentage of FoxP3+CD4+CD25+ positive cells in PBMCs was assessed by FACS analysis using FITC conjugated anti-CD4 (VMRD, Inc., Washington), Bio-conjugated anti-CD25 (Fitzgerald industries, MA), and PE-conjugated FoxP3 (eBioscience, Inc., CA) (see Item 9, SDC 1, http://links.lww.com/TP/A560).
- Measurement of the concentration of h-rHGF: The concentration of h-rHGF in serum and tissue extracts was determined using a commercially available ELISA kit (Otsuka Pharmaceutical Co., Tokushima, Japan) (21) (see Item 10, SDC 1, http://links.lww.com/TP/A560).
- Immunohistochemistry: Frozen samples were used for immunohistochemical analysis with the avidin-biotin horseradish-peroxidase complex technique (31). Anti-swine CD3 for T cells (Fitzgerald industries), anti-FoxP3 (ABcam, Cambridge, UK) and anti-CD25 (Fitzgerald industries) and antibodies for Met (C-12) (Santa Cruz biotechnology, Inc., CA) were used for immunohistochemical and immunofluorescent staining.
The authors thank Astelas Healthcare, Inc. (Osaka, Japan) for FK506 provided for this study. They thank Dr. Isabel Hanekamp for reviewing this manuscript. The study protocol was approved by the Ethical Committee of the Faculty of Medicine at Kagoshima University in Japan.
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