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BRIEF COMMUNICATIONS: Experimental Transplantation

High dose of antithrombin III induces indefinite survival of fully allogeneic cardiac grafts and generates regulatory cells

Aramaki, Osamu1 2; Takayama, Tadatoshi1; Yokoyama, Takeshi1; Takano, Seigo1; Akiyama, Yoshinobu3; Shibutani, Shintaro3; Matsumoto, Kenji3; Shimazu, Motohide3; Kitajima, Masaki3; Ikeda, Yoshifumi2; Shirasugi, Nozomu2; Niimi, Masanori2 4

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doi: 10.1097/01.TP.0000041781.94679.A1


Antithrombin III (AT-III) is a physiologic inhibitor of thrombin and other serine proteases in the clotting cascade (1). Conventional doses of AT-III, such as 50 U/kg of body weight, have been used clinically to treat sepsis and disseminated intravascular coagulation and to prevent further activation of the clotting cascade associated with consumption of AT-III (2). Because the clotting cascade is also activated after organ transplantation, some groups administer AT-III to patients who have undergone liver transplantation (3). Moreover, in both clinical and experimental studies, AT-III was found to inhibit inflammatory reactions independent of its anticoagulant activity when given in doses higher than those conventionally used (4). Anti-inflammatory agents such as steroids, cyclooxygenase-2 inhibitor (5), and aspirin were shown to have beneficial effects after transplantation (6). In contrast, heparin is well known to prevent not only coagulopathy but also rejection of allografts (7).

Therefore, the authors hypothesized that a high dose of AT-III might help prevent rejection of transplanted organs not only because of its antithrombotic function but also because of its anti-inflammatory function. The authors examined whether AT-III could induce unresponsiveness to fully allogeneic cardiac grafts in mice.

The study used inbred male C57BL/6 (H2b) and CBA (H2k) mice (Sankyo Ltd., Tokyo, Japan) between 8 and 12 weeks of age. Mice were housed in conventional facilities at the Biomedical Service Unit of Teikyo University and used in accordance with animal study protocols approved by the university.

Fully vascularized heterotopic hearts from C57BL/6 donors were transplanted into CBA recipients using microsurgical technique. On the day of transplantation, transplant recipients were untreated or given intravenous injection of 50 or 500 U/kg human AT-III (Aventis Behring Japan and Mitsubishi Pharma, Tokyo, Japan) or control human plasma (Japan Red Cross Blood Center, Tokyo, Japan). AT-III concentrate was prepared from heat-treated pooled human plasma by the cold ethanol method. Subsequently, beating of the transplanted hearts was monitored by daily palpation. Cardiac grafts were considered rejected when they ceased to contract; rejection was confirmed by electrocardiography, visual inspection, and histologic studies. Median survival time (MST) of grafts in different groups of mice was compared with the Mann-Whitney U test (StatView SE, Abacus Concepts, Inc., Cary, NC).

Naive CBA mice rejected C57BL/6 cardiac allografts acutely, as did mice given control plasma; the MST of the grafts were 9 days and 7 days, respectively (Fig. 1A). Mice given the 50-U/kg dose of AT-III had moderately increased graft survival (MST, 25 days;P =0.0084 vs. control group), but all grafts were rejected within 30 days. In contrast, mice given the 500-U/kg dose of AT-III had indefinite graft survival (>100 days;P =0.0052 vs. control group). No hemorrhagic complications occurred in mice given AT-III.

Figure 1
Figure 1:
Graft survival studies. (A) CBA mice were untreated or treated with 50 U/kg or 500 U/kg AT-III, or 0.2 mL control plasma the same day as transplantation of a heart from a C57BL/6 mouse. Results shown are representative of five experiments (*P <0.01 compared with control group and #P <0.01 between two groups). (B) In an adoptive transfer study, CBA mice (primary recipients) were treated with 500 U/kg AT-III and given transplants of C57BL/6 cardiac grafts the same day. Thirty days later, the mice were killed and 5×107 splenocytes from them were adoptively transferred into naive CBA mice (secondary recipients). C57BL/6 cardiac grafts were transplanted into the secondary recipients the same day as the adoptive transfer. The control group underwent adoptive transfer of splenocytes from naive CBA mice. Results shown are representative of three experiments (*P <0.01 compared with control group).

Histologically, C57BL/6 cardiac grafts in CBA mice given 500 U/kg AT-III (Fig. 2A) were similar to syngeneic grafts 100 days after transplantation (Fig. 2B), with little leukocyte infiltration. At 7 days, however, grafts in mice given the same dose of AT-III showed moderate leukocyte filtration (Fig. 2C), whereas those in mice given control plasma had severe leukocyte infiltration and necrosis (Fig. 2D).

Figure 2
Figure 2:
Histologic studies of cardiac grafts. Specimens shown were obtained on day 100 after transplantation from a mouse given 500 U/kg AT-III (A), on day 100 after transplantation from a syngeneic graft (B), on day 7 from a mouse given 500 U/kg AT-III (C), and on day 7 from a mouse given control plasma (D). Grafts were immersion fixed in 5% neutrally buffered formalin, embedded in paraffin, and stained with hematoxylin-eosin (magnification ×50).

To determine whether regulatory cells developed after administration of AT-III, the authors performed an adoptive transfer study. Thirty days after transplantation, CBA mice previously given 500 U/kg AT-III (primary recipients) were killed, and splenocytes (5×107) from these mice were adoptively transferred into naive secondary CBA recipients. The secondary recipients underwent transplantation of a C57BL/6 heart the same day as the adoptive transfer. C57BL/6 cardiac grafts had indefinite survival (>100 days) in naive secondary CBA recipients when the 500-U/kg dose of AT-III was given to primary recipients (Fig. 1B). In contrast, the MST of grafts in secondary recipients given splenocytes from naive CBA mice was 9 days. These results showed that regulatory cells were generated after treatment with 500 U/kg AT-III.

For in vitro studies, CBA mice were untreated or treated with 50 or 500 U/kg AT-III, or control plasma, and then given a C57BL/6 cardiac graft. Fourteen days later, these mice were killed and their splenocytes used as responder cells in mixed leukocyte culture (MLC) analyses (8). Production of interleukin (IL)-2, IL-4, IL-10, and interferon (IFN)-γ in MLC was examined by enzyme-linked immunosorbent assay (ELISA) (8). In the MLC, maximum proliferation of splenocytes from naive CBA mice against C57BL/6 splenocytes treated with mitomycin C occurred on day 4. Proliferation of splenocytes from CBA recipients given an intravenous injection of 500 U/kg AT-III was markedly suppressed compared with that of splenocytes from CBA recipients given either no treatment, 50 U/kg AT-III, or control plasma (Fig. 3A). Production of IL-2 by splenocytes from CBA recipients treated with either 500 U/kg or 50 U/kg AT-III was lower than that of splenocytes from CBA recipients given either no treatment or control plasma (Fig. 3B). There were no differences between groups in the production of IL-4, IL-10, or IFN-γ.

Figure 3
Figure 3:
Mixed leukocyte reaction results. (A) CBA mice were untreated or treated with 50 or 500 U/kg AT-III, or control plasma, given transplants of cardiac grafts from C57BL/6 mice, and killed 14 days later. Splenocytes (0.25×106) from these mice (responder cells) were co-cultured with C57BL/6 splenocytes (1×106; stimulator cells) treated with mitomycin C (MMC) (Kyowa Hakko, Osaka, Japan) in complete medium at 37°C in humidified 5% carbon dioxide (CH-16M, Hitachi, Tokyo, Japan) in 96-well, flat-bottomed tissue-culture plates (Iwaki Scitech Division, Tokyo, Japan) for 4 days. The complete medium was RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with HEPES (5 M; Sigma, St. Louis, MO), penicillin (100 μg/mL; Life Technologies), streptomycin (100 μg/mL; Life Technologies), 2-mercaptoethanol (50 μmol/L; Sigma), and 10% fetal-calf serum (Life Technologies). Proliferation activity of responder cells was determined with an ELISA (Biotrak Version 2, Amersham Pharmacia Biotech, Little Chalfont, United Kingdom) and ELISA reader (EL × 800 Universal Microplate Reader, Bio-Tek Instruments, Highland Park, VT) at 450 nm (8). The cell proliferation ELISA is based on the measurement of 5-bromo-2′-deoxyuridine incorporation during DNA synthesis in proliferating cells. For treatment of stimulator cells with MMC, a suspension of 3×106 splenocytes was resuspended in 2 mL complete medium with 100 μg/mL MMC and incubated for 30 min at 37°C. Cells were washed three times before being used as stimulators. Cell viability assessed by trypan blue (Cosmo Bio, Tokyo, Japan) exclusion testing was always more than 90% after MMC treatment. Results were compared by using unpaired Student t tests. Similar results were obtained in three additional experiments (##P <0.01 between two groups). (B) Production of IL-2, IL-4, IL-10, and IFN-γ in MLC on day 4 was examined by ELISA. The IL-10 capture monoclonal antibody (mAb) (JES5-2A5), detection mAb (JES5-16E3), and recombinant standard were from PharMingen (San Diego, CA). Capture and detection mAb for IL-2 (JES6-1A12 and JES6-5H4, respectively), IL-4 (BVD-1D11 and BVD-24G2), and IFN-γ (R4-6A2 and XMG1.2) were from Caltag Laboratories (Burlingame, CA). Recombinant standards for IL-2, IL-4, and IFN-γ were from PeproTech (London, United Kingdom). Absorbance was read at 405 nm with the ELISA reader (8). Results were compared by using unpaired Student t tests. Similar results were obtained in three additional experiments (##P <0.01 and #P <0.05 between the two groups).

Thus, the authors found that a high dose (500 U/kg) but not a low dose (50 U/kg) of AT-III induced indefinite survival of fully mismatched cardiac allografts in mice. Moreover, the high dose of AT-III suppressed the mixed leukocyte reaction.

A conventional dose of AT-III (50 U/kg) can block the coagulation cascade (1), which is frequently activated after organ transplantation. Takeshita et al. (9) demonstrated that administration of the conventional dose of AT-III prevented hyperacute rejection of cardiac grafts from dogs in pig recipients (from 10 min to 18 min in an untreated group) as a result of its anticoagulant effect.

Clinical and experimental studies have shown that high doses of AT-III inhibit inflammatory reactions independent of its anticoagulant activity. Jochum (4) reported that administration of 500 U/kg AT-III in patients with trauma and sepsis markedly reduced production of inflammatory effectors or mediators and thus improved the clinical condition. No bleeding, thromboses, or other complications occurred in these patients. Other investigators demonstrated the usefulness of the anti-inflammatory function of high doses of AT-III in preventing ischemia-reperfusion injury, thermal injury, acute respiratory distress syndrome, and septic shock.

In the mice given 500 U/kg AT-III in the authors’ study, histologic findings in the allogeneic cardiac grafts 100 days after transplantation were similar to those in syngeneic grafts. However, marked leukocyte infiltration was observed 7 days after transplantation in this group, indicating that the anti-inflammatory effect induced by AT-III was not completely operative in the grafts. In fact, the heartbeat in these mice was somewhat weak between 5 and 20 days after transplantation. Moreover, the authors used a single injection of AT-III at the time of transplantation, and the half-life of human AT-III in mice was approximately 2 days in their study (data not shown). Thus, for the anti-inflammatory function to provide maximum benefits in maintaining graft function, the high dose of AT-III would have to have been given continuously.

Therefore, the authors hypothesized that the 500 U/kg dose of AT-III also had an immunomodulating function. Indeed, the generation of regulatory cells with this dose of AT-III in the authors’ adoptive transfer studies suggested the presence of such a function. The prevention of rejection with administration of high-dose AT-III may also have had other mechanisms, such as deletion or anergy, but none of these are mutually exclusive. It may be that until regulatory cells or other mechanisms that prevent rejection were completely operative, the anti-inflammatory function of AT-III helped to increase graft survival.

The authors also found that a high dose of AT-III suppressed proliferation of leukocytes and generation of IL-2 in vitro. These findings are in agreement with those of Zuo et al., who showed that AT-III inhibited T- and B-lymphocyte activation in a rat model of acute rejection of lung allografts (10). The same investigators also concluded that AT-III inhibited messenger RNA expression of IL-2, IFN-γ, and IL-4 (10).

Conventional immunosuppressive regimens predispose patients to numerous complications, including drug toxicity, opportunistic infections, and malignant diseases, that might be reduced if doses of immunosuppressant agents could be decreased. Here, the authors found that high-dose AT-III had not only an antithrombotic effect but also anti-inflammatory and immunomodulating effects, all of which are beneficial after organ transplantation. The agent also induced generation of regulatory cells that prevented graft rejection. Previous clinical and experimental studies found that high doses of AT-III produced no side effects (4). Therefore, administration of high-dose AT-III may be useful as adjuvant therapy used to allow a reduction in the total dose of conventional immunosuppressant agents after organ transplantation.


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