Microchimerism following vascularized bone allotransplantation

Microchimerism Following Vascularized Bone Allotransplantation K. Muramatsu and A.T. Bishop

F

OLLOWING organ transplantation, cell migration from the graft into the recipient, or microchimerism, may play a role in development of graft acceptance.1 Thymic microchimerism has since been considered by many to be especially important for induction of tolerance. At present, however, it is not clear that microchimerism is the cause, or merely the result of immunotolerance.2 Vascularized bone allotransplantation remains impractical at present. Such non–life-supporting organ allotransplantation requires long-term immunosuppressive therapy, which, using currently available drugs, cannot be justified due to risk of infection, neoplasm, and toxic adverse effects.3 Thus, one strategy that would allow living bone allotransplantation would be the induction of drug-free, or classical, immunotolerance. Understanding the mechanisms underlying tolerance is therefore of great importance in the future development of treatments aimed at establishing classical tolerance. In this study, vascularized tibial allografting was performed in rat gender-mismatched pairs. The high ration patterns of donor cells into the recipient was analyzed semiquantitatively by polymerase chain reaction (PCR) using Y-chromosome–specific primers. The extent of microchimerism and evidence of graft rejection were evaluated following transplantation across minor and major histocompatibility behavior with and without immunosuppression. MATERIALS AND METHODS Graft donors were adult male rats, either Dark-Agouti rats (RT1*), weighing 200 g, or PVG rats (RT-1*), weighing 220 g. All recipients were adult female PVG rats. All transplants were performed from male to female rats. The animals were divided into three groups (Table 1). Group I consisted of PVG rats (n ⫽ 5) that received PVG isografts and long-term FK 506 (FK) immunosuppressive therapy (3 mg/kg per week in three divided intramuscular injections). Group II consisted of DA rats (n ⫽ 5) that received PVG allografts but no immunosuppressive therapy. Group 3

consisted of DA rats (n ⫽ 25) that received PVG allografts and long-term FK therapy, as described earlier. In the donor, the proximal 2.5 cm of the rat tibiofibular complex was raised as a vascularized flap. In the recipient, the distal femur, knee, and proximal tibiofibular complex were removed to create a bone defect of about 2 cm spanning the knee joint. The graft was transplanted into the bone defect of the recipient and vascular anastomoses were performed. At the final evaluation, blood samples and specimens of liver, spleen, thoracic lymph nodes, thymus, contralateral (nonoperated) tibia, and humerus were obtained prior to exposure of the bone graft to avoid contamination by donor cells. Genomic DNAs were prepared from tissue samples by a simplified phenol/chloroform method. The PCR reaction mixture contained 0.5 ␮g of genomic DNA, 1 U of Ampli-Taq (Intermountain Scientific Corp, Kaysville, Utah), 25 pmol of rat Sry-specific primers, 8 pmol of rat GAPDHspecific primers, 4 ␮L of 2.5 mmol/L dNTP, 5 ␮L of 10⫻ PCR extraction buffer (160 mmol/L [NH4]2SO4, 670 mmol/L Tris-HCl [pH 8.8], and 0.1% Tween-20), and 1.5 ␮L of 50 mmol/L MgCl2 in a final volume of 50 ␮L. The PCR reaction was carried out in a programmed thermal cycler (Intermountain) for 30 cycles of denaturation (96°C for 1 minute), annealing (54°C for 1 minute), and extension (72°C for 1 minute).

RESULTS Microchimerism in Blood and Lymphoid Tissue

In group I, donor-derived Y-chromosome genes were detected in peripheral blood, spleen, thymus, and cervical lymph nodes in all recipients at 18 weeks posttransplant. The ratios of donor cells were 0.1% (low level) in blood and 1% (high level) in other tissues in all cases. In immunosupFrom the Department of Orthopedic Surgery and Microvascular Research, Mayo Clinic, Rochester, Minnesota. Address reprint requests to Dr Keiichi Muramatsu, Department of Orthopedic Surgery and Microvascular Research, Mayo Clinic, 200 First Street SW, Rochester, MN 55905.

Table 1. Microchimerism in Blood, Lymphoid, and Bone Tissues Group

Tx ⫹ FK

Post-Tx

N

Blood [n (%)]

Spleen

Thymus

Liver

Lymph node

Tibia

Humerus

I II

Isograft ⫹ FK Allograft ⫺ FK

III

Allograft ⫹ FK

18 wk ⬍7 d 7–14 d 1 wk 12 wk 18 wk

5 2 3 5 8 10

5 1 (50) 2 (67) 2 (40) 4 (50) 8 (80)

5 1 (50) 2 (67) 2 (40) 1 (13) 6 (60)

5 0 0 0 1 (13) 6 (60)

5 0 0 0 1 (13) 0

5 0 0 0 0 0

5 0 0 0 4 (50) 8 (80)

5 0 0 0 4 (50) 8 (80)

0041-1345/02/$–see front matter PII S0041-1345(02)03387-0

© 2002 by Elsevier Science Inc. 360 Park Avenue South, New York, NY 10010-1710

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Transplantation Proceedings, 34, 2722–2724 (2002)

MICROCHIMERISM POST-TX

pressed allograft rats (group II), donor cells were detected in one of two recipients in blood and spleen within 1 week posttransplant and in two of three recipients evaluated at between 1 and 2 weeks. Donor cells were detected in the peripheral blood (0.1%) and spleen (1%) in two of five immunosuppressed recipients (group III) at 1 week posttransplant. At 12 weeks, donor cells were found in the peripheral blood (four of eight recipients), spleen (one), thymus (one), and liver (one). At 18 weeks, eight of ten recipients showed microchimerism in peripheral blood at the ratio of 0.1% and six recipients showed both intrathymic and intrasplenic microchimerism at the ratio of 1%. No donor cells were detected in the cervical lymph nodes in all recipients throughout the posttransplant periods. Detection of Donor Cell Migration Into Bone Tissues

In all group I recipients, donor cells were detected in the bone graft, adjacent femur and tibia, contralateral tibia, and humerus at 18 weeks posttransplant. The ratios of donor cells were 10% in the bone graft and 1% in other bone tissues. Microchimerism was not seen in recipient bone tissues in nonimmunosuppressed allografts by 2 weeks (group II). Donor-derived cells were detected in the bone graft itself (ratio ⬎50%) in all rats. Similarly, immunosuppressed bone allograft rats had no microchimerism in recipient bone at 1 week posttransplant, and the ratios of donor cells were ⬎50% in all recipients. At 12 weeks, donor cell migration was found in all bone tissues examined in four of five recipients, and the ratios of donor cells were 1%. At 18 weeks, donor cells were detected in recipient femur and tibia in eight and ten of ten recipients. The ratios of donor cells were 1% and 10% in six and two femurs and 1% and 10% in seven and three tibias, respectively. Donor cell migration was found in the contralateral tibia and humerus in eight of ten recipients and the ratios of donor cells were all 1%. DISCUSSION

Starzl and coworkers hypothesized that the microchimerism observed in long-term liver transplant survivors results in a state of graft acceptance.1 Several mechanisms by which low-level microchimerism can induce recipient immunotolerance have been demonstrated. However, a cause-andeffect relationship between microchimerism and immunotolerance has not been proven conclusively. Suberbielle et al found microchimerism in only 5 of 15 long-term kidney allograft transplant patients.4 Schlitt et al demonstrated that microchimerism had no relationship to acute or chronic rejection episodes in heart transplants.5 Recently, the kinetics of chimeric cell accumulation and distribution in allograft recipients was investigated using rigorous techniques, but they remain largely unknown. The exact relationship between graft acceptance and microchimerism, cause or consequence, has not been verified in detail. Microchimerism can frequently be detected after successful organ transplantation in animal models. In tolerant

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animals, however, the degree of chimerism as well as tissue distribution is highly variable, due perhaps in part to the type of organ transplanted. In rat allogeneic liver transplantation studies using the PCR technique, Tashiro et al demonstrated that peripheral (blood) microchimerism was detected in 66% of nonimmunosuppressed recipients and 100% of FK-treated rats at 60 days posttransplant.6 Furukawa et al found no microchimerism in heart transplant recipients.7 Our results demonstrate that 78% (14 of 18) of FK-treated recipients evaluated beyond 1 week posttransplant showed peripheral microchimerism. Because vascularized bone grafts have numerous undifferentiated stem cells in the bone marrow, donor cell migration into the recipient circulation may be more frequent than in other organ transplant procedures such as the heart. Although detection of microchimerism is noteworthy, little information exists on the cumulative load in different organs. Tashiro et al6 detected cells of donor liver origin in lymph nodes and spleen, but not in thymus. Suberbielle et al4 demonstrated skin chimerism in kidney transplant recipients. In our results, 39% (7 of 18) of FK-treated recipients showed intrathymic and intrasplenic microchimerism, but not in cervical lymph nodes. Intrathymic microchimerism may be an indicator of immunotolerance. Starzl et al and Charlton et al1 reported that persistence of donor cells in the recipient thymus led to the deletion of recipient alloreactive T cells. Donor-specific tolerance may be achieved by thymic injection of donor cells. Interestingly, 67% of these same FK-treated recipients showed microchimerism in bone tissues at ⬎12 weeks posttransplant. The level of microchimerism changes with time. The initial wave of microchimerism (within a few weeks posttransplant) is due to the release of differentiated hematopoietic cells from the graft into the recipient circulation. A second wave of donor cells derived from pluripotent stem cells follows. The donor cells were detected in recipient spleens at 1 week posttransplant may thus be differentiated hemopoietic cells, whereas the later osseous microchimerism detected beyond 12 weeks may be derived from donor tissue pluripotent cells.9 Our study demonstrated intrasplenic microchimerism in nontreated recipients within 2 weeks posttransplant. At that time, histology revealed severe rejection in the graft (blood extravasation in the marrow and osteocyte death). It is likely that this initial wave of microchimerism has no connection with graft acceptance and is simply a measurement of differentiated marrow hematopoietic cells via the circulation.9 Any such cells would likely accumulate in the spleen, which serves as a filter for the blood. All isograft recipients (n ⫽ 5) treated with FK therapy demonstrated a high level of microchimerism in lymphoid and skeletal tissue. Transplantation from a male rat to a syngeneic female recipient results in a minor histocompatibility disparity due to a weak H–Y antigen–antibody reaction. Any weak rejection potentially caused by the H–Y antigen was likely completely controlled by FK therapy. The recipients showed more frequent and higher levels of microchimerism than allograft recipients. Acceptance of

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the vascularized bone allograft was not always associated with microchimerism. Isograft recipients showed a higher level of microchimerism than the allograft recipients. Nontreated recipients rejected the graft but showed intrasplenic microchimerism. Microchimerism is supposed to reflect the immunosuppressive state of the recipient and may be a consequence, rather than a cause, of tolerance. REFERENCES 1. Kashiwagi N, Porter KA, Penn I, et al: Surg Forum 20:374, 1968

MURAMATSU AND BISHOP 2. Adams DH, Hutchinson IV: Lancet 349:1336, 1997 3. Muramatsu K, Doi K, Kawai S: Clin Orthop 320:194, 1995 4. Suberbielle C, Caillat-Zucman S, Legendre C, et al: Lancet 342:1468, 1994 5. Schlitt HJ: Transplant Proc 29:82, 1997 6. Tashiro H, Fukuda Y, Hoshino S, et al: Cell Transplant 4(suppl):S61, 1995 7. Furukawa M, Fukuda Y, Tashiro H, et al: Cell Transplant 5(suppl):S75, 1996 8. Charlton B, Auchincloss H Jr, Fathman CG: Annu Rev Immunol 12:707, 1994 9. Ueda M, Hundrieser J, Hisanaga M, et al: Clin Transplant 11:193, 1997