Secondary Logo

Prolongation of Survival of Fully Allogeneic Cardiac Grafts and Generation of Regulatory Cells by a Histamine Receptor 2 Antagonist

Inoue, Fumihiko1; Zhang, Qi2; Akiyoshi, Takurin1; Aramaki, Osamu2,3; Iwami, Daiki2; Matsumoto, Kenji1; Kitagawa, Yuko1; Shirasugi, Nozomu2,4; Niimi, Masanori2

doi: 10.1097/01.tp.0000287641.69795.cc
Original Articles: Experimental Transplantation
Free

Background. The effects of histamine on immunologic responses via the histamine receptor 2 (HR2) have been studied, but few investigations explored the immunomodulatory role of histamine in vivo. We examined whether the HR2 antagonist ranitidine affects the alloimmune response in a murine model of cardiac transplantation.

Methods. CBA (H-2k) recipients were given no treatment or one intravenous injection of ranitidine on the day of transplantation of a heart from C57BL/10 (H-2b) donors. Survival of the allografts was recorded. The effect of the ranitidine treatment on cell proliferation and cytokine production was assessed by mixed leukocyte culture and enzyme-linked immunosorbent assays. An adoptive transfer study was conducted to determine whether regulatory cells were generated. The effect on graft survival of adding FK506 to the ranitidine treatment was also examined.

Results. CBA recipients given ranitidine (60 mg/kg) had prolonged graft survival (median survival time [MST], 87 days). Ranitidine treatment also suppressed the proliferation of splenocytes and production of interleukin (IL)-2 and up-regulated IL-10 production. Adoptive transfer of splenocytes and CD4+ cells from ranitidine-treated allograft recipients induced significant prolongation of allograft survival in naive secondary recipients (MST, 71 and >100 days, respectively). CBA recipients given both ranitidine and FK506 (0.1 mg/kg/day for 14 days) had indefinite survival of cardiac allografts (MST, >100 days). CBA recipients treated with FK506 alone rejected the allografts (MST, 27 days).

Conclusion. In our model, ranitidine treatment induced significantly prolonged survival of fully allogeneic cardiac grafts, generated CD4+ regulatory cells, and indefinite survival when combined with FK506 (0.1 mg/kg/day).

1 Department of Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, Japan.

2 Department of Surgery, Teikyo University, 2-11-1 Kaga, Itabashi-ku, Tokyo, Japan.

3 Department of Surgery, Nihon University School of Medicine, 30-1 Ooyaguchi, Kamimachi Itabashi-ku, Tokyo, Japan.

4 Address correspondence to: Nozomu Shirasugi, M.D., Ph.D., Department of Surgery, Teikyo University, 2-11-1 Kaga, Itabashi-ku, Tokyo, Japan 173-8605.

E-mail: nozomu_joseph_shirasugi@1988.jukuin.keio.ac.jp

Received 8 June 2007. Revision requested 1 August 2007.

Accepted 22 August 2007.

Histamine is one of the most important mediators in several physiologic and pathologic conditions, including inflammatory and immediate hypersensitivity reactions. Recent studies have indicated that histamine can modulate T-cell immune responses through histamine receptors (HRs). HR1 and HR2 have been observed on T cells (1), but there is controversy about whether the effects produced by histamine through these receptors are stimulatory or inhibitory. Moreover, the immunomodulatory effect of histamine is likely to vary according to the type of experimental model or conditions. Jutel et al. (2) reported that HR2-deficient mice had up-regulation of both T-helper (Th) 1 and Th2 cytokines. Other investigations showed histamine to be a negative regulator of T cells (3, 4). Histamine also was found to decrease interleukin (IL)-18-induced production of interferon (IFN)-γ and IL-12 in mixed lymphocyte cultures of human cells, although it had no such effects in the absence of IL-18 (5).

In contrast, mice deficient in HR2 had decreased levels of tumor necrosis factor (TNF)-α and IFN-γ after antigen-specific stimulation in an experimental model of allergic encephalomyelitis (6). Kohka et al. (7) reported that histamine up-regulated production of IFN-γ and down-regulated production of IL-10 by human peripheral blood mononuclear cells and that administration of an HR2 antagonist reversed these changes. Xu et al. (8) recently reported that histamine produced by neutrophils contributes to lung inflammation and that HR2 antagonist inhibits this inflammation.

Since the 1960s, transplantation studies have shown that histamine is induced during allograft rejection (9, 10). In 1995, Minami et al. (11) demonstrated that histamine release in the microenvironment of allografts was involved in the rejection process after lung transplantation in rats. Li et al. (12) found that an increase in the numbers of degranulated mast cells in allografts with the release of inflammatory mediators such as histamine was well correlated with the severity of rejection. However, little research has been done on the effect of HR antagonists on the immunologic response to organ allografts in vivo. One brief report described an experiment in which an HR2 antagonist slightly shortened allograft survival in a rat model of cardiac transplantation (median survival time [MST], 6 days in treated recipients compared with 8 days in untreated recipients) (13).

We therefore studied the effects of an HR2 antagonist, ranitidine, on the alloimmune response to fully allogeneic cardiac grafts in a murine model. We found that a single dose of ranitidine given on the day of transplantation induced marked prolongation of the survival of cardiac allografts through the generation of regulatory cells. When the conventional immunosuppressant FK506 was given with ranitidine, allografts survived indefinitely. HR2 antagonists are used routinely to prevent stress-induced gastroduodenal ulcers and are often given to organ transplant recipients for this purpose (14). Our findings suggest that HR2 antagonists may have potential as immunomodulatory agents to inhibit rejection of allografts. Administration of an HR2 antagonist to patients who have undergone transplantation may be found to permit a reduction in the use of conventional immunosuppressants, thereby decreasing the drug-related toxicity associated with those agents.

Back to Top | Article Outline

MATERIALS AND METHODS

Mice

Inbred male C57BL/10 (H2b), CBA (H2k), and BALB/c (H2d) mice were purchased from Sankyo Ltd (Tokyo, Japan), housed in conventional facilities at the Biomedical Service Unit of Teikyo University, and used when between eight and 12 weeks of age in accordance with the guidelines of the Animal Use and Care Committee of Teikyo University.

Back to Top | Article Outline

Heart Transplantation

All transplant procedures were performed with the mice under general anesthesia. Fully vascularized heterotopic hearts from C57BL/10 or BALB/c donors were transplanted into CBA recipients by using microsurgical techniques (15). Postoperatively, graft function was assessed by palpation for evidence of contraction. Rejection was defined as complete cessation of contraction and confirmed by direct visualization and histologic examination of the graft.

Back to Top | Article Outline

Treatment With Ranitidine

On the day of cardiac transplantation, transplant recipients were given either no treatment, one intravenous injection of saline (300 μL), or one intravenous injection of 0.6, 6, or 60 mg/kg of ranitidine (Glaxo SmithKline, KK, Tokyo, Japan). The ranitidine was diluted in normal saline before administration.

Back to Top | Article Outline

Histologic Studies of Cardiac Grafts

Morphologic analyses using hematoxylin and eosin staining were conducted on specimens of functioning cardiac grafts obtained 100 days after transplantation from mice that had been given ranitidine. Specimens of cardiac grafts obtained seven days after transplantation from control recipients (saline group) and ranitidine-treated recipients were also examined histologically.

Back to Top | Article Outline

Adoptive Transfer Study

Adoptive transfer studies were conducted to assess the development of regulatory cells after ranitidine treatment. Thirty days after CBA mice (primary recipients) underwent transplantation of C57BL/10 hearts and treatment with ranitidine (60 mg/kg), splenocytes (5×107) from primary recipients with functioning cardiac allografts were adoptively transferred into naive CBA mice (secondary recipients). Cardiac grafts from C57BL/10 donors were transplanted into the secondary recipients immediately after the adoptive transfer. Control secondary recipients received adoptive transfer of splenocytes from naïve CBA mice. In some experiments, CD4+ cells were purified from the spleens of primary recipients given ranitidine by positive selection using a magnetically activated cell sorter and CD4 microbeads (Miltenyi Biotec, Auburn, CA; purity >96%) and 2×107 CD4+ cells were then adoptively transferred into naïve secondary recipients, which then immediately underwent transplantation of a C57BL/10 (donor-specific) or BALB/c (third-party) heart. The CD4+ cells purified for the adoptive transfer were also stained with fluorochrome-conjugated antibodies (anti-CD4, anti-CD25 monoclonal antibody (mAb, BD Biosciences Pharmingen, San Diego, CA) and anti-mouse FOXP3 antibody (eBioscience, San Diego, CA) as well as their isotype controls).

In another experiments, CD4+ CD25+ cells also were purified from the spleens of primary recipients given ranitidine using a magnetically activated cell sorter and mouse CD4+ CD25+ Regulatory T cell Isolation Kit (Miltenyi Biotec) and 106 CD4+ CD25+ cells were adoptively transferred into naïve secondary recipients, which then immediately underwent transplantation of a C57BL/10 heart.

Back to Top | Article Outline

Combined Treatment With a Conventional Immunosuppressant

To investigate whether ranitidine treatment is compatible with a conventional immunosuppressant currently used in clinical transplantation, we gave some transplant recipients FK506. We previously found that a 0.1 mg/kg/day of FK506 did not interfere with the generation of regulatory cells in our murine cardiac transplantation model, whereas 0.3, 0.5 or 1.0 mg/kg/day of FK506 and cyclosporine abrogated it (16); therefore, 0.1 mg/kg/day dose was used in the current study. The FK506 (a gift of Astellas Pharma Inc, Tokyo, Japan) was sustained in normal saline. Intraperitoneal injections of 0.1 mg/kg of FK506 were given daily for two weeks after transplantation of C57BL/10 hearts in naïve CBA mice treated with ranitidine and mice not given ranitidine. All mice survived the drug administration, without any adverse events.

Back to Top | Article Outline

Mixed Leukocyte Cultures

Cellular alloproliferation was assessed in mixed leukocyte cultures (MLCs). The responder cells were splenocytes from naïve CBA mice or ranitidine-treated CBA mice that had undergone transplantation of C57BL/10 hearts seven days earlier. The stimulator cells were C57BL/10 (allogeneic) or CBA (syngeneic) splenocytes treated with 100 μg/ml of mitomycin C (MMC; Kyowa Hakko, Osaka, Japan) for 30 min at 37°C. After the MMC treatment, the cell suspension was washed three times. Cell viability after this treatment, as assessed by a trypan blue (Cosmo Bio, Tokyo, Japan) exclusion test, was more than 90%.

The responder cells (2.5×106 cells/mL) were co-cultured with the stimulator cells (5×106 cells/mL) in complete medium in a humidified 5% CO2 atmosphere (CH-16M; Hitachi, Tokyo) at 37°C in 96-well, flat-bottomed tissue-culture plates (Iwaki Scitech Division, Tokyo) for 3 to 6 days (17). The complete medium was RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with HEPES (2 mM/L; Sigma, St Louis, MO), penicillin (100 μg/mL; Life Technologies), streptomycin (100 μg/mL; Life Technologies), 2-mercaptoethanol (50 μM/L; Sigma), and 10% fetal-calf serum (Life Technologies). Proliferation was assessed by using an enzyme-linked immunosorbent assay (ELISA) for bromodeoxyuridine incorporation (Biotrak, version 2; Amersham, Little Chalfont, United Kingdom [UK]) (17, 18) according to the manufacturer’s instructions.

Back to Top | Article Outline

Production of Cytokines

An ELISA was performed to detect IL-2, IL-4, IL-10, and IFN-γ in the supernatant of the MLC on day 4. The IL-10 capture mAb (JES5-2A5), detection mAb (JES5-16E3), and recombinant standard were from BD Biosciences Pharmingen. The capture and detection mAbs 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 Pepro Tech (London, UK). Absorbance was read at 405 nm by using an ELISA reader (EL ×800 Universal Microplate Reader, Bio-Tek Instruments, Winooski, VT).

Back to Top | Article Outline

Immunohistochemistry Studies

Syngeneic grafts (n=5), allografts in untreated mice (n=8), and allografts in mice treated with 60 mg/kg of ranitidine (n=7) were removed 8 days after transplantation and examined by immunostaining for intercellular adhesion molecule 1 (ICAM-1) to assess activation of endothelial cells in the grafts. The immunolabeling was performed on 4-μm thick, frozen coronal sections of cardiac grafts fixed in periodate-lysine-paraformaldehyde solution. Nonspecific binding of the antibody was blocked with Nonspecific Staining Blocking Reagent (DakoCytomation, Kyoto, Japan) for 10 min. This was followed by incubation with hamster anti-ICAM-1 mAb (5 μg/mL; BD Biosciences Pharmingen) at room temperature for 1 hr. Endogenous peroxidase activity was stopped by adding 0.6% H2O2-methanol for 30 min. Afterward, the sections were incubated for 30 min at room temperature with biotinylated goat antihamster immunoglobulin G (Vector Labs, Burlingame, CA). They were then washed with 0.05 M Tris-HCl and incubated with avidin-biotin-peroxidase complex (Vectastain Elite ABC Kit; Vector Labs) for 30 min at room temperature. Peroxidase activity was demonstrated by using 3,3′ diaminobenzidine. Hamster immunoglobulin G was used as the negative control. The sections were counterstained with Meyer’s hematoxylin.

To quantify the extent of ICAM-1 expression on endothelial cells in the grafts, immunostaining for ICAM-1 was graded numerically on a scale of 0 to 5 (19), with grade 0 indicating no staining; grade 1; patchy and weak staining; grade 2, uniform and weak staining; grade 3, patchy and moderate staining; grade 4, uniform and less intense staining; and grade 5, uniform and intense staining. Multiple fields in two or three cross-sections from each cardiac graft were graded for ICAM-1 immunostaining on endothelial cells of coronary arteries and capillaries by a single observer, who was blinded to the origin of the graft. Results for syngeneic grafts, allografts in untreated mice, and allografts in ranitidine-treated mice were expressed as the mean±standard deviation (SD) value for all scores in each of the three groups.

Back to Top | Article Outline

Statistical Analysis

Cardiac allograft survival in groups of mice was compared by using the log-rank test. The results of the MLC, ELISA, and immunohistochemistry assessments were compared by using unpaired Student’s t tests. All statistical analyses were done with Stat View SE + Graphic software (Abacus Concepts, Berkeley, CA). A P value of less than 0.05 was considered to represent statistical significance.

Back to Top | Article Outline

RESULTS

Prolonged Survival of Fully Allogeneic Cardiac Grafts in Mice Treated With Ranitidine

Naive CBA mice rejected C57BL/10 cardiac allografts acutely, as did mice given normal saline alone; the MSTs of the grafts in these groups were 8 and 9 days, respectively (Fig. 1). CBA mice given one injection of 0.6 or 6 mg/kg of ranitidine on the day of transplantation also had acute rejection of the allograft (MSTs, 8 and 12 days, respectively; no significant difference from the saline or untreated controls). In contrast, CBA recipients given 60 mg/kg of ranitidine had significantly prolonged allograft survival (MST, 87 days; P<0.005 compared with controls). These results indicate that a single injection of ranitidine induced hyporesponsiveness to fully allogeneic cardiac grafts in a dose-dependent manner.

FIGURE 1.

FIGURE 1.

Back to Top | Article Outline

Histologic Features of Grafts From Ranitidine-Treated Recipients

Histologic studies showed that C57BL/10 cardiac grafts from CBA mice given 60 mg/kg of ranitidine at transplantation were free of myocardial injury 7 and 100 days after the procedure (Fig. 2A and 2B) and had only rare, sparse leukocyte infiltrations. In contrast, C57BL/10 cardiac grafts from CBA mice given normal saline were found to have extensive leukocyte infiltrates, edema, and myocyte damage and architectural distortion with focal interstitial fibrosis as well as proliferation of fibroblasts seven days after transplantation (Fig. 2C–E).

FIGURE 2.

FIGURE 2.

Back to Top | Article Outline

Prolonged Graft Survival in Secondary Recipients After Adoptive Transfer

In the adoptive transfer studies, the secondary recipients of C57BL/10 cardiac grafts from primary recipients given 60 mg/kg of ranitidine at transplantation had significantly prolonged survival of those grafts (MST, 71 days; P<0.01 compared with controls; Figure 3A). On the other hand, CBA mice given adoptive transfer of splenocytes from naïve CBA mice rejected C57BL/10 cardiac allografts acutely (MST, 14 days). These findings indicate that regulatory cells were generated in the primary recipients after treatment with a single dose of ranitidine.

FIGURE 3.

FIGURE 3.

When CD4+ cells were purified from spleens of ranitidine-treated CBA primary recipients of allografts and adoptively transferred into naïve CBA secondary recipients that underwent transplantation of C57BL/10 hearts immediately afterward, the secondary recipients had markedly prolonged allograft survival (MST, >100 days; P<0.005 compared with controls; Figure 3B). Adoptive transfer of CD4+ cells from naïve mice did not induce prolongation of allograft survival in secondary recipients (MST, 13 days). When CD4+ cells were purified from spleens of ranitidine-treated primary recipients and adoptively transferred into naïve CBA secondary recipients that underwent transplantation of BALB/c (third-party) hearts immediately afterward, survival of the allografts was only moderately prolonged (MST, 18 days). These data indicate that CD4+ cells constitute one of the regulatory populations induced by ranitidine treatment. Histological studies of C57BL/10 allografts, which were harvested 100 days after transplantation from the secondary recipients with the adoptive transfer of CD4+ cells purified from spleens of ranitidine-treated primary recipients, showed preserved graft structure with few sparse interstitial infiltrates (Fig. 3C). Moreover, the allografts did not show any evidences of chronic rejection such as interstitial fibrosis or obliterative vasculopathy (Fig. 3D). These results suggest that the adoptive transfer of the CD4+ regulatory cells inhibited acute and chronic rejection in the secondary recipients in our model.

When 106 CD4+ CD25+ cells, purified from the splenocytes of ranitidine-treated CBA recipients with functioning C57BL/10 allografts 30 days after transplantation, were transferred into naïve CBA mice (secondary recipients) that underwent transplantation of C57BL/10 hearts immediately afterward, all allografts showed significantly prolonged survival in the secondary recipients (graft survival, >20 days ×4, n=4, P<0.05 compared with controls). In contrast, naïve secondary recipients with adoptive transfer of 106 naïve CD4+ CD25+ cells eventually rejected C57BL/10 allografts (graft survival, 7, 7, 8, 9, and 10 days, MST=8 days). Moreover, when CD25 and FOXP3 expressions were analyzed on CD4+ splenocytes purified from ranitidine-treated recipients by flowcytometry, population of CD4+ CD25+ cells increased in the spleen of ranitidine-treated recipients, compared to those in naïve CBA mice or saline-treated CBA recipients. The CD4+ CD25+ cells were also FOXP3 positive (Fig. 3E, F). These data suggest that the CD4+ regulatory cells contained a regulatory population that was CD4+ CD25+ FOXP3+ in our model.

Back to Top | Article Outline

Indefinite Survival of Fully Allogeneic Cardiac Grafts in Mice Treated With Ranitidine and Low-Dose FK506

CBA recipients of C57BL/10 cardiac allografts given both a single dose of 60 mg/kg of ranitidine on the day of transplantation and 0.1 mg/kg/day of FK506 the day of transplantation and for two weeks afterward had indefinite allograft survival (MST, >100 days, Fig. 4). In contrast, CBA recipients given FK506 alone (0.1 mg/kg/day) had rejection of the allografts significantly earlier (MST, 27 days; P<0.05 compared with the group given both ranitidine and FK506). These results suggest that, in our model treatment with ranitidine was compatible with 0.1 mg/kg/day of FK506 to induce indefinite survival of allografts in our model.

FIGURE 4.

FIGURE 4.

Back to Top | Article Outline

Cell-Proliferation Assay

In the MLC assays, maximum proliferation of splenocytes from naive CBA mice (responder cells) against C57BL/10 splenocytes (stimulator cells) treated with MMC occurred on day four. Proliferation of cells from CBA recipients given ranitidine was markedly suppressed compared with alloproliferation of splenocytes from naive CBA mice in response to C57BL/10 stimulator cells (Fig. 5A; P<0.05).

FIGURE 5.

FIGURE 5.

Back to Top | Article Outline

Production of Cytokines

Production of IL-2 by splenocytes from CBA recipients treated with ranitidine was significantly lower compared with that by CBA splenocytes with an alloproliferative response (Fig. 5B; P<0.05 compared with the alloimmune-response group). In contrast, production of IL-10 was up-regulated in splenocytes from recipients given ranitidine (Fig. 5C; P<0.05 compared with the alloimmune-response group). Production of IL-4 was undetectable in all groups (data not shown). There were no differences in the production of IFN-γ between ranitidine-treated group and saline-treated group (46.6±3.4 ng/mL in ranitidine-treated recipients, 54.5±14.7 ng/mL in saline-treated recipients, 53.2±14.8 ng/mL in naïve CBA responders against C57BL/10 stimulators, not significantly different between the groups).

Back to Top | Article Outline

Inhibition of ICAM-1 Expression on Endothelial Cells by Ranitidine

The results of the immunohistochemistry studies of ICAM-1 expression are shown in Figure 6. Eight days after transplantation, syngeneic grafts showed low levels of ICAM-1 expression on endothelial cells constitutively (score, 0.83±0.41, Figure 6A and 6D); these levels are similar to those reported previously (19–21). Cells in grafts from untreated mice that underwent cardiac allograft transplantation had significantly higher levels of ICAM-1 expression (score, 4.68±0.57; P<0.0001 compared with syngeneic grafts; Fig. 6B and 6D). In transplant recipients given 60 mg/kg of ranitidine, ICAM-1 expression on endothelial cells in the allografts was significantly lower than that in allografts from untreated transplant recipients (score, 2.21±0.98; P<0.0001 compared with allogeneic grafts; Figure 6C and 6D). These data suggest that treatment with ranitidine inhibited up-regulation of ICAM-1 expression on endothelial cells triggered by the alloimmune response.

FIGURE 6.

FIGURE 6.

Back to Top | Article Outline

DISCUSSION

In this study in a murine model of cardiac transplantation, we found that treatment with a single dose of an HR2 antagonist, ranitidine, prolonged survival of fully allogeneic cardiac grafts and generated regulatory cells that contained a CD4+ population. These cells showed alloproliferative hyporesponsiveness in an MLC and up-regulation of IL-10 production. We also observed that when ranitidine treatment was given in combination with a conventional immunosuppressant, FK506, the regimen induced indefinite survival of allografts.

Our data suggest several possible mechanisms for the induction of hyporesponsiveness by ranitidine. The first is that the ranitidine treatment induced regulatory cells in the transplant recipients. In addition to their mechanisms of deletion and anergy that eliminate or functionally inactivate alloreactive cells, regulatory cells apparently have an important role in the induction of unresponsiveness to alloantigen (22). In our model, adoptive transfer of CD4+ splenocytes from ranitidine-treated allograft recipients induced indefinite survival of allografts in secondary transplant recipients, whereas third-party allografts were acutely rejected. These data indicate that ranitidine treatment generated CD4+ regulatory cells. The exact mechanisms for the generation of regulatory cells by ranitidine remain unclear. Our MLC finding of up-regulation of IL-10 production by splenocytes in ranitidine-treated recipients suggests that IL-10 contributes to generation of regulatory cells. IL-10 has antiinflammatory and suppressive effects on most hematopoietic cells, and it plays a crucial role not only in the function of regulatory cells but also in their generation (23). We previously demonstrated the importance of IL-10 in generating regulatory cells in our murine cardiac transplantation model (24). Thus, it is probable that in the current study, treatment with ranitidine induced CD4+ regulatory cells, presumably through up-regulation of IL-10. Moreover, Roncarolo et al. (25) showed that a subset of CD4+ regulatory cells was induced by immature dendritic cells (DCs). In addition, histamine was reported to activate immature DCs and to up-regulate costimulatory molecules on immature DCs (26), and DC activation by histamine was prevented by an HR2 antagonist. In studies in our model, a flow cytometric analysis of costimulatory molecules on DCs revealed that splenic DCs purified from ranitidine-treated recipients had significantly lower expression of CD80 than DCs purified from untreated recipients (mean±SD [in three separate experiments] fluorescent intensity of CD80, 55.2±7.6 in ranitidine-treated recipients compared with 170.5±34.6 in untreated recipients; P<0.05). In the light of these observations, we consider it likely that the HR2 antagonist inhibited up-regulation of costimulatory molecules on DCs that remained immature and subsequently induced regulatory cells.

A second possible mechanism for the induction of hyporesponsiveness by ranitidine is suggested by our finding that treatment with ranitidine inhibited up-regulation of ICAM-1 expression on endothelial cells in allografts from untreated mice. Histamine has been found to induce IL-6 production by endothelial cells. In addition, up-regulated IL-6 production by endothelial cells triggered by histamine and TNF-α was inhibited by an HR2 antagonist in a dose-dependent manner (27). Histamine was also observed to enhance ICAM-1 expression induced by TNF-α on HUVECs. Several studies have demonstrated that ICAM-1 is involved in allograft rejection (28–30). Together, these data suggest that ranitidine inhibited allograft rejection in our model by down-regulating ICAM-1 expression and subsequent leukocyte-endothelial cell interaction.

A third possible mechanism for ranitidine-induced hyporesponsiveness is that ranitidine itself has some immunomodulatory effects on alloimmune responses, rather than exerting effects through the HR2. However, the existence of such a mechanism is not supported by studies in our model because another HR2 antagonist, famotidine, also induced significantly prolonged survival of C57BL/10 cardiac allografts in CBA recipients (survival times of 35, 45, 48, and >100× 3 days; n=6; MST, 74 days). These data suggest that HR antagonism was the means by which ranitidine induced hyporesponsiveness in the current study.

A fourth possible mechanism for ranitidine-induced hyporesponsiveness is its effect on other types of immune cells. HR2 was found on CD8+ cells (1) and B cells (31), as well as CD4+ cells. Gantner et al. (32) found that histamine induced IL-16 from CD8+ cells through HR2 and that HR2 antagonist inhibited the IL-16 release. On the basis of these findings, it is possible that also in our model HR2 antagonist ranitidine might inhibit rejection process to cardiac allografts by inhibition of trafficking of several immune cells as the result of down-regulation of IL-16 (33), or attenuating humoral immune responses.

Histamine is produced primarily by mast cells and basophils. Ultrastructural analyses of biopsy specimens obtained from cardiac allografts in patients showed that activated mast cells, with release of granule contents, appeared in the first week after transplantation and that the density of mast cells and their granules correlated with volume of fibrosis in cardiac allografts since the activated mast cells participate in the stimulation of fibroblasts via histamine (12). In the present study, allografts in saline-treated recipients showed focal interstitial fibrosis and proliferation of fibroblasts as well as extensive leukocyte infiltrates and interstitial edema. Taken together, it is possible that HR2 antagonist ranitidine might protect the allograft from alloimmune responses in the rejection process by inhibiting functions of mast cells to induce graft fibrosis.

Nonspecific immunosuppressive agents, including calcineurin inhibitors, are currently used to inhibit allograft rejection in patients. Prolonged acceptance of allografts requires long-term use of these agents, which is associated with such complications as infection and malignancy, as well as direct regimen-related toxic effects. Induction of transplantation tolerance by generation of regulatory cells might be one strategy for achieving permanent acceptance of allografts in patients without incurring serious toxicity. Clinical tolerance-inducing strategies should be compatible with current immunosuppressant regimens (34). In our model, ranitidine induced indefinite survival of fully allogeneic grafts when given with a short course of low-dose FK506, an agent that, used alone, induced only moderate prolongation of allograft survival. We previously showed that a low dose of FK506 does not interfere with induction of regulatory cells (16). Thus, it is conceivable that clinical allograft rejection might be mitigated by administering low-dose FK506 along with a regulatory cell-inducing HR2 antagonist.

Although there are few other in vivo studies examining the effects of HR2 antagonists, our results in the present study are in conflict with the data by Olausson et al. (13). They reported that, an HR2 antagonist, cimetidine, shortened the allograft survival in rat cardiac transplantation model, although ranitidine did not show any effect. There are several possibilities for different results between Olausson’s and ours. First, it is possible that besides its HR2 antagonistic effect, cimetidine might have other different functions to shorten the allograft survival, such as inhibiting cytochrome P450 monooxygenases (CYPs) (35). Some CYPs are predominantly detected in the heart, vasculature, kidney and lung. A large body of recent data demonstrates that the activation of CYPs in endothelial cells is an integral component in nitric oxide (NO) and prostacyclin (PGI2)-independent vasodilation of vascular beds in heart and kidney (36). Thus, it is possible that inhibition of CYPs by cimetidine might cause acceleration of graft rejection through impairment of microcirculation by inhibiting endothelium-dependent relaxation, while ranitidine may not impair the microcirculation of the allografts since ranitidine does not inhibit CYPs. Second, the effects of histamine through HRs still remain controversial about whether the effects are stimulatory or inhibitory. Findings from a number of investigators indicate that the effects of histamine on immune systems vary according to immunologic stimuli or experimental conditions. As shown above, cimetidine was found to shorten the allograft survival in rat cardiac transplantation model. On the other hand, Festen et al. showed that cimetidine did not accelerate rejection of fully allogeneic skin graft in mice (37). Thus, it is possible that in our experiments ranitidine, another HR2 antagonist, could induce hyporesponsiveness to fully allogeneic grafts in mouse cardiac transplantation model.

The dose used in our model to prolonged survival of fully allogeneic grafts is higher than that in human clinical dose. We performed another experiment, in which CBA recipients received treatment with 6 mg/kg of ranitidine from day 0 through day 7 along with transplantation of C57BL/10 cardiac allografts on day 0. In this group, CBA recipients eventually rejected C57BL/10 allografts (survival time, 12, 12, 13, 21, 22, 35, and 38 days, MST=21 days). These data suggest that in mice, at least in our model, the clinically appropriate human dose of ranitidine was not likely to modulate the alloimmune responses.

Although 60 mg/kg dose is larger than that in treatment in humans, we have not experienced any death or drug-related toxicities in more than 200 cases of mouse cardiac transplantation with ranitidine treatment. In the light of clinical application of the strategies using ranitidine, further studies are needed to clarify an optimum therapeutic dose as well as optimum concentration for inducing unresponsiveness to allografts.

In conclusion, we found that the HR2 antagonist ranitidine had immunomodulatory effects that induced prolonged survival of fully allogeneic grafts and generation of regulatory cells in our in vivo model. HR2 antagonists are already used successfully and with few toxic effects to prevent gastrointestinal complications in patients who have undergone transplantation. Our findings suggest that administration of ranitidine may provide additional benefits with respect to immune regulation. Subsequent research on this possibility must include studies in large animal models.

Back to Top | Article Outline

ACKNOWLEDGMENTS

The authors thank Ryuichi Taki, Japan Cytology, and Pathology Laboratory, Inc. (Tokyo, Japan), for technical assistance with the immunohistochemical and histologic studies.

Back to Top | Article Outline

REFERENCES

1. Perry NL, Zola H, Nicholson IC. Histamine receptors on T lymphocytes. J Biol Regul Homeost Agents 2005; 19: 78.
2. Jutel M, Watanabe T, Klunker S, et al. Histamine regulates T-cell and antibody responses by differential expression of H1 and H2 receptors. Nature 2001; 413: 420.
3. Kunzmann S, Mantel PY, Wohlfahrt JG, Akdis M, Blaser K, Schmidt-Weber CB. Histamine enhances TGF-beta1-mediated suppression of Th2 responses. FASEB J 2003; 17: 1089.
4. van der Pouw Kraan TC, Snijders A, Boeije LC, et al. Histamine inhibits the production of interleukin-12 through interaction with H2 receptors. J Clin Invest 1998; 102: 1866.
5. Itoh H, Takahashi HK, Iwagaki H, et al. Effect of histamine on intercellular adhesion molecule-1 expression and production of interferon-gamma and interleukin-12 in mixed lymphocyte reaction stimulated with interleukin-18. Transplantation 2002; 74: 864.
6. Teuscher C, Poynter ME, Offner H, et al. Attenuation of Th1 effector cell responses and susceptibility to experimental allergic encephalomyelitis in histamine H2 receptor knockout mice is due to dysregulation of cytokine production by antigen-presenting cells. Am J Pathol 2004; 164: 883.
7. Kohka H, Nishibori M, Iwagaki H, et al. Histamine is a potent inducer of IL-18 and IFN-gamma in human peripheral blood mononuclear cells. J Immunol 2000; 164: 6640.
8. Xu X, Zhang D, Zhang H, et al. Neutrophil histamine contributes to inflammation in mycoplasma pneumonia. J Exp Med 2006; 203: 2907.
9. Moore TC, Chang JK. Urinary histamine excretion in the rat following skin homografting and autografting. Ann Surg 1967; 167: 232.
10. Dy M, Lebel B, Kamoun P, Hamburger J. Histamine production during the anti-allograft response. Demonstration of a new lymphokine enhancing histamine synthesis. J Exp Med 1981; 153: 293.
11. Minami M, Nakahara K, Matsumura A, et al. Histamine release from pulmonary mast cells after lung transplantation in rats. J Heart Lung Transplant 1995; 14: 505.
12. Li QY, Raza-Ahmad A, MacAulay MA, et al. The relationship of mast cells and their secreted products to the volume of fibrosis in posttransplant hearts. Transplantation 1992; 53: 1047.
13. Olausson M, Wramner L, Blohme I, Mjornstedt L. Heart allograft rejection in rats triggered by H2 inhibitors. Transplant Proc 1997; 29: 3127.
14. Benoit G, Moukarzel M, Verdelli G, et al. Gastrointestinal complications in renal transplantation. Transpl Int 1993; 6: 45.
15. Niimi M. The technique for heterotopic cardiac transplantation in mice: Experience of 3000 operations by one surgeon. J Heart Lung Transplant 2001; 20: 1123.
16. Shibutani S, Inoue F, Aramaki O, et al. Effects of immunosuppressants on induction of regulatory cells after intratracheal delivery of alloantigen. Transplantation 2005; 79: 904.
17. Akiyama Y, Shirasugi N, Uchida N, et al. B7/CTLA4 pathway is essential for generating regulatory cells after intratracheal delivery of alloantigen in mice. Transplantation 2002; 74: 732.
18. Perros P, Weightman DR. Measurement of cell proliferation by enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody to bromodeoxyuridine. Cell Prolif 1991; 24: 517.
19. Tanaka H, Sukhova GK, Swanson SJ, Cybulsky MI, Schoen FJ, Libby P. Endothelial and smooth muscle cells express leukocyte adhesion molecules heterogeneously during acute rejection of rabbit cardiac allografts. Am J Pathol 1994; 144: 938.
20. Lee JR, Huh JH, Seo JW, Suk CJ, Jeong HM, Kim EK. Time-dependent expression of ICAM-1 & VCAM-1 on coronaries of the heterotopically transplanted mouse heart. J Korean Med Sci 1999; 14: 245.
21. Briscoe DM, Yeung AC, Schoen FJ, et al. Predictive value of inducible endothelial cell adhesion molecule expression for acute rejection of human cardiac allografts. Transplantation 1995; 59: 204.
22. Wood KJ, Sakaguchi S. Regulatory T cells in transplantation tolerance. Nat Rev Immunol 2003; 3: 199.
23. Wan YY, Flavell RA. The roles for cytokines in the generation and maintenance of regulatory T cells. Immunol Rev 2006; 212: 114.
24. Aramaki O, Inoue F, Takayama T, et al. Interleukin-10 but not transforming growth factor-beta is essential for generation and suppressor function of regulatory cells induced by intratracheal delivery of alloantigen. Transplantation 2005; 79: 568.
25. Roncarolo MG, Gregori S, Battaglia M, Bacchetta R, Fleischhauer K, Levings MK. Interleukin-10-secreting type 1 regulatory T cells in rodents and humans. Immunol Rev 2006; 212: 28.
26. Caron G, Delneste Y, Roelandts E, et al. Histamine induces CD86 expression and chemokine production by human immature dendritic cells. J Immunol 2001; 166: 6000.
27. Delneste Y, Lassalle P, Jeannin P, Joseph M, Tonnel AB, Gosset P. Histamine induces IL-6 production by human endothelial cells. Clin Exp Immunol 1994; 98: 344.
28. Stepkowski SM, Wang ME, Condon TP, et al. Protection against allograft rejection with intercellular adhesion molecule-1 antisense oligodeoxynucleotides. Transplantation 1998; 66: 699.
29. Lacha J, Bushell A, Smetana K, et al. Intercellular cell adhesion molecule-1 and selectin ligands in acute cardiac allograft rejection: A study on gene-deficient mouse models. J Leukoc Biol 2002; 71: 311.
30. Labarrere CA, Nelson DR, Park JW. Pathologic markers of allograft arteriopathy: Insight into the pathophysiology of cardiac allograft chronic rejection. Curr Opin Cardiol 2001; 16: 110.
31. Akdis CA, Blaser K. Histamine in the immune regulation of allergic inflammation. J Allergy Clin Immunol 2003; 112: 15.
32. Gantner F, Sakai K, Tusche MW, Cruikshank WW, Center DM, Bacon KB. Histamine h(4) and h(2) receptors control histamine-induced interleukin-16 release from human CD8(+) T cells. J Pharmacol Exp Ther 2002; 303: 300.
33. Conti P, Kempuraj D, Kandere K, et al. Interleukin-16 network in inflammation and allergy. Allergy Asthma Proc 2002; 23: 103.
34. Auchincloss H Jr. In search of the elusive Holy Grail: The mechanisms and prospects for achieving clinical transplantation tolerance. Am J Transplant 2001; 1: 6.
35. Rendic S, Di Carlo FJ. Human cytochrome P450 enzymes: A status report summarizing their reactions, substrates, inducers, and inhibitors. Drug Metab Rev 1997; 29: 413.
36. Fleming I. Cytochrome p450 and vascular homeostasis. Circ Res 2001; 89: 753.
37. Festen HP, Berden JH, Koene RA. Cimetidine does not accelerate skin graft rejection in mice. Clin Exp Immunol 1980; 40: 193.
Keywords:

Ranitidine; Regulatory cells; Cardiac transplantation; Mouse

© 2007 Lippincott Williams & Wilkins, Inc.