FK 506 and aminoguanidine suppress iNOS induction in orthotopic corneal allografts and prolong graft survival in mice

doi:10.1016/j.niox.2003.08.003

Nitric Oxide

Volume 9, Issue 2, September 2003, Pages 111-117

https://doi.org/10.1016/j.niox.2003.08.003 Get rights and content

Abstract

The aim of this study was to compare the effectiveness of immunosuppressant FK 506 and the specific inhibitor of inducible nitric oxide synthase (iNOS) aminoguanidine (AG) in prevention of corneal graft rejection and to investigate the iNOS expression in the rejection process. Orthotopic corneal allografting in mice was performed (C57BL/10; H-2^b to BALB/c; H-2^d). FK 506 (0.3 mg/kg per day) or AG (100 mg/kg per day) was injected intraperitoneally for 4 weeks. Grafted mice without therapy served as controls. Immunohistological evaluation of iNOS-positive cells and macrophage infiltration in grafts 27th day after grafting was performed. Within 4 weeks FK 506 prevented graft rejection in 71% and AG in 57% of animals compared to 29% of clear grafts in controls. A significant proportion of iNOS-positive cells was detected in the rejected grafts of the control and AG-treated groups. The treatment with FK 506 resulted in the inhibition of iNOS expression to a high degree in the rejected corneas. Non-rejected corneas of all groups and non-transplanted corneas exhibited no iNOS-positive cells. A massive infiltration of macrophages was detected in the rejected grafts, whereas non-rejected grafts exhibited only slight infiltration of macrophages. The presented data suggest that overexpression of iNOS and/or activation of iNOS is one of the several influential factors that contribute to the rejection process and that iNOS suppression delays corneal allograft rejection. FK 506 and AG are effective drugs in preventing corneal allograft rejection. Higher beneficial effect of FK 506 on graft survival could be explained by its well-known selective T-cell immunosuppression.

Section snippets

Animals

Mice of the inbred strains, which differ in major and minor histocompatibility antigens, were used. Female mice of C57BL/10 (B10; H-2^b) strain (Charles River, Germany) served as corneal graft donors. Male and female mice of BALB/c (H-2^d) strain (Institute of Molecular Genetics, Prague), 6–8 weeks old, were used as recipients. Animals were housed five per cage in plastic cages. Mice were fed ad libitum and maintained in a 12-h light/dark cycle. All animals were treated according to the ARVO

Results

Within 27 days FK 506 prevented graft rejection in 71% and AG in 57% of animals compared to 29% of non-rejected grafts in controls (Fig. 1).

Non-transplanted corneas (Fig. 2A) and non-rejected corneas of all groups (Fig. 4, Fig. 5) were iNOS-negative. A significant proportion of iNOS-positive cells was detected in the rejected grafts of the control group (Figs. 3A, a, B, and C). Non-rejected grafts of the FK 506-treated group exhibited no iNOS-positive cells (Fig. 4A) and the treatment with FK

Discussion

The mechanisms contributing to the rejection process of corneal allograft remain incompletely understood. It has been shown that not CD8+ lymphocytes, activated by CD4+ T-cells, but activated macrophages are the main effector cells in the process of skin allograft rejection in mice [9]. Macrophages are heterogeneous cell population that may exert multiple functions. Besides their phagocytic activity they produce a number of highly active mediators such as tumor necrosis factor (TNF-α) and NO

Acknowledgements

This study was supported by Grants IGA MZ NL/7418-3 and NI/7531-3, GACR 305/00/1129, and Research Programs MSM 111100002 and 111100005. FK 506 was kindly supplied by Fujisawa GmbH (Munich, Germany). We thank Mrs. Jana Bucková for her skillful technical assistance.

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In the present research, the doses of AG which improved anxiety- and depression-like behaviors were not the same as those which affected neuroinflammation and oxidative stress indicators; therefore, it seems that there are other mechanism(s) linking the effects of AG on anxiety- and depression-like behaviors induced by LPS; a point which needs to be investigated in the future studies. The dose of AG used here was based on the previous studies reporting that AG was able to inhibit iNOS [60]. In the present research, the adverse effects of AG were not evaluated, but no mortality was observed.
The effects of aminoguanidine (AG) were investigated in a rat model of lipopolysaccharide (LPS)-induced anxiety- and depression-like behaviors.
The animals were allocated to five groups (n = 10 in each) and treated by: (1) saline as a control group, (2) LPS 1 mg/kg injected two hours before behavioral tests, (3–5) AG 50, 100 or 150 mg/kg before LPS. The open-field test (OFT), elevated plus maze test (EPT), and forced swimming (FS) tests were performed. The brains and blood were then collected to examine oxidative stress and inflammation criteria.
LPS increased the immobility while decreased the active time in the FS test. In EPT, LPS decreased the time spent in the open arms, whereas it increased the time spent in the closed arms. In OFT, LPS decreased the time spent in the central zone compared with the controls. A higher dose of selenium improved the performances of the rats in behavioral tests. LPS injection also increased malondialdehyde (MDA) while it decreased thiol, superoxide dismutase (SOD), and catalase. LPS also increased interleukin (IL)-6 and tumor necrosis factor-alpha (TNF-α), but decreased IL-10 in the LPS group. AG protected the brain from inflammation and oxidative damage.
It was demonstrated that AG improves the behaviors of depression and anxiety in a rat model of LPS-induced anxiety- and depression-like behaviors. Moreover, the effects of AG were accompanied by improved inflammation and oxidative damage biomarkers in brain tissues.
Graft survival and cytokine production profile after limbal transplantation in the experimental mouse model
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The effect of anti-CD4 therapy may be due to elimination of CD4+ T cells which mediate the DTH reaction and are the important source of IFN-γ for iNOS expression [21]. In addition, anti-CD4 antibody can inactivate CD4+ macrophages which play a role in both afferent phase of transplantation reaction as antigen-presenting cells and in the effector phase as cytotoxic macrophages [27–29]. It has been shown that a subpopulation of macrophages expresses CD4 molecules, and these CD4+ macrophages have been shown to be involved in graft rejection [30].
Limbal transplantation or limbal stem cell (LSC) transfer represents the only way to treat severe ocular surface damage or LSC deficiency. However, limbal allografts are promptly rejected in spite of extensive immunosuppressive therapy. To characterize immune response after limbal transplantation, we established an experimental model of limbal transplantation in the mouse. Syngeneic, allogeneic and xenogeneic (rat) limbal grafts were grafted orthotopically in BALB/c mice and graft survival was evaluated. The presence of graft donor cells and the expression of IL-2, IL-4, IL-10, IFN-γ and inducible nitric oxide synthase (iNOS) mRNA in the grafts were detected by real-time PCR. While syngeneic grafts survived permanently, allografts were rejected in 9.0 ± 1.8 days and xenografts in 6.5 ± 1.1 days. The manifestation of clinical symptoms of rejection correlated with the disappearance of donor cells in the graft and in the recipient cornea. Intragraft expression of iNOS mRNA and distinct expression patterns of Th1 (IL-2, IFN-γ) and Th2 (IL-4, IL-10) cytokines were detected during rejection of limbal allografts and xenografts. The limbal graft rejection was prevented with anti-CD4, but not anti-CD8 monoclonal antibody therapy. The results indicate that limbal grafts do not enjoy immune privilege of the eye and are promptly rejected by Th1 (allografts) or by a combined Th1 and Th2 (xenografts) type of immune response involving CD4⁺ cells and iNOS expression. Targeting this pathway may be an effective way to prevent and treat limbal graft rejection.
The complex role of iNOS in acutely rejecting cardiac transplants
2008, Free Radical Biology and Medicine
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The last is produced after up-regulation of inducible NO synthase (iNOS), which occurs initially predominately in macrophage cell infiltrates and later within graft parenchymal cells [8]. NO arising from iNOS is believed to play a significant detrimental role in a variety of allotransplanted cells or tissues, including lung [9,10], kidney [11], pancreatic islets [12,13], cornea [14], and aorta [15]. There are disparate findings regarding the role of iNOS in solid organ transplant rejection in general (see below).
This review summarizes the evidence for a detrimental role of nitric oxide (NO) derived from inducible NO synthase (iNOS) and/or reactive nitrogen species such as peroxynitrite in acutely rejecting cardiac transplants. In chronic cardiac transplant rejection, iNOS may have an opposing beneficial component. The purpose of this review is primarily to address issues related to acute rejection, which is a recognized risk factor for chronic rejection. The evidence for a detrimental role is based upon strategies involving nonselective NOS inhibitors, NO neutralizers, selective iNOS inhibitors, and iNOS gene deletion in rodent models of cardiac rejection. The review is presented in the context of the impact on various components, including graft survival, histological rejection, and cardiac function, which may contribute to the process of graft rejection in toto. Possible limitations of each strategy are discussed in order to understand better the variance in published findings, including issues related to the potential importance of cell localization of iNOS expression. Finally, the concept of a dual role for NO and its downstream product, peroxynitrite, in rejection vs immune regulation is discussed.
Increased iNOS-expressing macrophage in long-term surviving rat small-bowel grafts
2007, American Journal of Surgery
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iNOS may play distinct roles in AR and in long-term survival grafts. Activated macrophages produce NO, and upregulation of iNOS activity has already been reported in AR in animal and human transplantation studies [15–17]. Reactive nitric species, such as proxynitrite, which is a side product of NO, are cytotoxic and cause tissue damage [11].
Inducible nitric oxide synthase (iNOS) produces nitric oxide and modulates many biologic processes critical in the development of rejection; however, its role in chronic rejection (CR) in small-bowel transplantation (SBT) is largely unknown.
FK506 prevented acute rejection (AR); however, recipients eventually lost their bowel grafts to CR. Combined FK506 and rapamycin treatment prevented CR, thus leading to long-term graft survival. We investigated iNOS expression in our rat orthotopic SBT CR model.
Histologically, mesentery vascular occlusion and fibrosis, which are hallmarks of CR, were apparent in bowel grafts in an FK506 single-treatment group. In contrast, patients with long-term surviving grafts receiving FK506 and rapamycin developed mild vascular occlusion and fibrosis. Unlike in AR, low iNOS expression, which is associated with decreased macrophage infiltration, was observed in CR grafts. However, iNOS expression and macrophage infiltration was higher in long-term–surviving grafts than CR grafts. Immunofluorescence staining revealed that the majority of macrophages expressed iNOS in long-term surviving grafts.
Sequential treatment combining FK506 and rapamycin prolonged survival of SBT animals with decreased vasculopathy and collagen deposition of the intestinal grafts. iNOS may play opposing roles in AR and CR in SBT.
Corneal Graft Rejection
2007, Survey of Ophthalmology
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Experimental animal studies also uphold the efficacy of topical steroids and cyclosporine A in the management of corneal graft rejection.200 Derived from fungus streptomycetes tsukybaensis, FK-506,13,14,58,110,111,127,161,167,177,183 which is highly lipophilic in nature, seems to be 10 to 100 times more potent than CsA with similar mechanism of action.7 Whereas CsA binds to the immunophilin, cyclophilin (CyP), FK-506 binds to a separate immunophilin, FK-506 binding protein (FKBP).7,14
Penetrating keratoplasty is the most widely practiced type of transplantation in humans. Irreversible immune rejection of the transplanted cornea is the major cause of human allograft failure in the intermediate and late postoperative period. This immunological process causes reversible or irreversible damage to the grafted cornea in several cases despite the use of intensive immunosuppressive therapy. Corneal graft rejection comprises a sequence of complex immune responses that involves the recognition of the foreign histocompatibility antigens of the corneal graft by the host's immune system, leading to the initiation of the immune response cascade. An efferent immune response is mounted by the host immune system against these foreign antigens culminating in rejection and graft decompensation in irreversible cases. A variety of donor- and host-related risk factors contribute to the corneal rejection episode. Epithelial rejection, chronic stromal rejection, hyperacute rejection, and endothelial rejection constitute the several different types of corneal graft rejection that might occur in isolation or in conjunction. Corneal graft failure subsequent to graft rejection remains an important cause of blindness and hence the need for developing new strategies for suppressing graft rejection is colossal. New systemic pharmacological interventions recommended in corneal transplantation need further evaluation and detailed guidelines. Two factors, prevention and management, are of significant importance among all aspects of immunological graft rejection. Preventive aspects begin with the recipient selection, spread through donor antigenic activity, and end with meticulous surgery. Prevention of corneal graft rejection lies with reduction of the donor antigenic tissue load, minimizing host and donor incompatibility by tissue matching and suppressing the host immune response. Management of corneal graft rejection consists of early detection and aggressive therapy with corticosteroids. Corticosteroid therapy, both topical and systemic, is the mainstay of management. Addition of immunosuppressive to the treatment regimen helps in quick and long term recovery. Knowledge of the immunopathogenesis of graft rejection may allow a better understanding of the immunological process thus helping in its prevention, early detection and management.
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¹: The authors contributed equally to the presented study.

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FK 506 and aminoguanidine suppress iNOS induction in orthotopic corneal allografts and prolong graft survival in mice

Abstract

Section snippets

Animals

Results

Discussion

Acknowledgements

J. Biol. Chem.

Eur. J. Pharmacol.

Eur. J. Pharmacol.

Immunol. Today

J. Neuroimmunol.

The immune privilege of corneal allografts

Transplantation

How successful is corneal transplantation? A report from the Australian corneal graft register

Eye

The collaborative corneal transplantation studies corneal transplantation studies (CCTS): effectiveness of histocompatibility matching of donors and recipients in high-risk corneal transplantation

Arch. Ophthalmol.

The high-risk keratoplasty patient—Quo vadis?

Cornea

Cell subpopulation in failed human corneal grafts

Br. J. Ophthalmol.

Kinetics of corneal transplant rejection in the rat penetrating keratoplasty model

Cornea

T-cell mediated responses in a murine model of orthotopic corneal transplants in mice

Invest. Ophthalmol. Vis. Sci.

Cellular basis of skin allograft rejection in mice: specific lysis of allogeneic skin components by non-T cells

Transplantation

Nitric oxide. A novel signal transduction mechanism for transcellular communication

Hypertension

Nitric oxide IV. Determinants of nitric oxide protection and toxicity in liver

Am. J. Physiol.

Macrophage oxidation of l-arginine to nitrite and nitrate: nitric oxide is an intermediate

Biochemistry