Abstract
purpose. The Th2-biased immune system can promote penetrating keratoplasty survival in mice. A series of experiments were performed to determine whether this system could prolong corneal limbal transplant (LT) survival.
methods. BALB/c (H-2d) mice were immunized with 50 μg of keyhole limpet hemocyanin (KLH) in incomplete Freud’s adjuvant. Four weeks later, the corneal epithelium, including the limbal area, was removed, and the mice received LT from B10.D2 (H-2d), C57BL/10 (H-2b), or enhanced green fluorescence protein (EGFP) transgenic (H-2b) donor mice. Immediately thereafter, recipient mice were immunized with 50 μg of KLH or Hanks’ balanced salt solution (HBSS; control) in complete Freund’s adjuvant. The allograft fates were assessed clinically. Lymphocytes of recipients were examined for donor-specific proliferation and for donor-specific cytokine production in vitro.
result. The regenerated epithelia of all C57BL/10 (n = 14) and B10.D2 (n = 18) grafts were rejected swiftly in control mice, whereas 66.6% of C57BL/10 grafts (8/12, P < 0.001) and 62.8% of B10.D2 grafts (22/35, P < 0.001) in the KLH immune group remained significantly clear for 8 weeks. Moreover, EGFP donor epithelial cells were detected from the healthy corneas of KLH-immunized mice. As for the in vitro assay, at 1 week after B10.D2 grafting, lymphocytes from KLH-immunized groups showed neither proliferation nor increased cytokine secretion.
conclusions. The Th2-biased immune system can support LT prolongation irrespective of donor disparity and can suppress corneal neovascularization. This prolongation is not due to induction of donor-specific regulatory cells, but is presumably at least associated with the suppression of allosensitization.
Limbal transplantation (LT) is universally performed to reconstitute the ocular surface in patients who have no corneal epithelium stem cells, such as after a chemical burn or Stevens-Johnson syndrome.
1 2 3 If ocular surface damage is bilateral, allogeneic LT is the best means of supplying healthy corneal epithelial and limbal cells, including stem cells. The renewed allogeneic epithelium has been shown to remain viable only under attentive postoperative care, with comparatively long-term use of immunosuppressants to avoid intense and frequent rejection. However, the local and/or systemic use of corticosteroids or alternative general immunosuppressants is associated with significant complications.
4 5 Even with the use of immunosuppressants, certain patients exhibit intense postoperative corneal allograft rejection.
6 Moreover, Mills et al.
7 recently reported, using the rat model of LT, that immunosuppression could mediate clinical allograft survival, but not donor cell survival on the ocular surface. It is therefore apparent that development of immunologic strategies that can suppress allograft rejection may contribute to ocular surface reconstruction.
Previously, Yao et al.
8 reported that either major histocompatibility complex (MHC) only disparate or minor histocompatibility antigen (minor H) only incompatible LT showed epithelial rejection at a frequency similar to that of MHC
+ minor H disparate LT, unlike penetrating keratoplasty (PKP).
9 Yao et al. concluded therefore that major and minor histocompatibility antigens are both related to corneal epithelial rejection. The immune mechanism in LT differs from that in PKP for several reasons. In the LT model, the donor grafts involve the limbus, which contains Langerhans’ cells, allowing for greater host recognition of the graft, and the corneal limbus of the recipient possesses blood vessels and lymphatics, as well as Langerhans’ cells, allowing for acute allosensitization and swift rejection.
However, as in PKP,
10 11 allogeneic LT rejection appears to be mediated primarily by a delayed-type hypersensitivity (DTH) response, rather than by a cytotoxic T lymphocyte (CTL) response.
12 Moreover, donor-specific DTH suppression by induction of anterior chamber-associated immune deviation (ACAID) could promote survival of both LT
13 and PKP allografts.
14 15 16 Therefore, a DTH-suppressing strategy must be realized to avoid epithelial rejection.
DTH is typically mediated by T-helper type 1 (Th1) CD4
+ T cells, that secrete interferon (IFN)-γ.
17 Th1 cells can be cross-regulated by a different subset of CD4
+ T cells that secrete IL-4 and -10, but not IFN-γ, which are termed Th2. Thus, the cytokines produced by Th2 cells suppress cytokine activation and release by Th1 cells,
17 thereby limiting the ability of the latter to mediate effector responses such as the allodestructive DTH response. Actually, a Th2-biased immune system, in a PKP mice model, enhanced allogeneic graft survival.
18 We have used this strategy in an effort to modify LT in mice. Our results indicate that mice with immune systems heavily biased toward Th2 responses accept allogeneic LT at a higher rate than do normal mice. Furthermore, this system is equally effective between a MHC-matched and MHC-mismatched donor–recipient combination. Our results indicate the presence of another suppressive mechanism in the Th2-biased immune system.
Seven to 10-week-old male BALB/c (H-2d) and C57BL/10 (H-2b) mice were purchased from SLC (Osaka, Japan). Mice comprising same-aged male B10.D2 (H-2d) and enhanced green fluorescence protein (EGFP) transgenic mice (H-2b, C57BL/6 background) were purchased from The Jackson Laboratory (Bar Harbor, ME). All animals were treated in accordance with the ARVO Statement on the Use of Animals in Ophthalmic and Vision Research. All experiments were approved by the Committee for Animal Research, Kyoto Prefectural University of Medicine. In the following experiments, all transplant recipients were BALB/c mice.
Keyhole limpet hemocyanin (KLH) of Megathura crenulata (Calbiochem, La Jolla, CA), was used for immunization to induce a Th2-type response. BALB/c and C57BL/6 mice received intraperitoneal (IP) injections of 50 μg KLH emulsified in incomplete Freund’s adjuvant (IFA; Difco Laboratories, Detroit, MI) 28 days before transplantation. Control mice received Hanks’ balanced salt solution (HBSS) plus IFA. In a secondary immunization, mice that received an orthotopic LT in one eye also received, in the nape of the neck immediately thereafter, an injection of KLH (50 μg) or HBSS alone in complete Freund’s adjuvant (CFA; Difco Laboratories).
Characteristics of Alloreactive T Cells from KLH-Immune Mice That Accepted Limbal Grafts
Most limbal grafts that fail in both humans and mice do so because of immune-associated rejection, though even the most immunogenetically disparate corneal grafts placed orthotopically can exhibit prolonged, often indefinite, survival. In comparison with LT, the extraordinary success of PKP can be attributed to various features of the normal cornea and anterior segment that in the aggregate account for their immune-privileged state,
20 21 including the avascularity of the stroma, the absence of corneal lymphatics, and the rarity of indigenous professional antigen-presenting Langerhans’ cells or macrophages in the normal graft bed. Because of the factors responsible for ocular immune privilege, it has been found that minor H antigens, rather than antigens encoded within the MHC, are the most important initiators of alloimmunity after PKP.
9 All peptides derived from minor H antigen processing are loaded onto self-MHC molecules on recipient antigen-presenting cells and presented to T cells by the so-called indirect pathway of allorecognition in the PKP model. The Th2-biased immune system was therefore established to induce donor minor H–specific Th2 type response to suppress Th1-mediated immune rejection, because mice that have mounted Th2-type responses to one peptide antigen often display Th2 responses to subsequent immunizations with different antigens.
22 In fact, donor-specific Th2 cells were induced and donor-specific allorejection suppressed in a PKP model mice.
18
In contrast, less immune privilege is exhibited in LT, which involves the presence of Langerhans’ cells in both donor and recipient limbus. Donor Langerhans’ cells can present alloantigens directly to recipient T cells by the so-called direct pathway of allorecognition. In cases of MHC-disparate combination between donor and recipient, MHC alloantigen is presented without the contribution of recipient antigen-presenting cells. In cases of MHC-matched combination, even minor H can be presented directly to recipient T cells by donor Langerhans cells, because both donor and recipient share the same MHC molecule. Theoretically, it is quite natural for minor H antigen presentation in LT to be influenced by the Th2-biased immune system. However, the fact is that neither positive donor-specific Th2 response nor typical donor-specific regulation was detected. Moreover, both MHC disparate and MHC-matched allogeneic LT showed similar graft survival in this system.
The Th2-biased immune system could suppress neovascular invasion at around 5 days after LT, before the initial rejection reaction appeared. Because corneal NV probably plays an important role in facilitating swift antigen presentation and effector elements in the inflamed cornea and is associated with fulminating graft rejection, suppressed initial NV induction should contribute to LT graft survival. Immunologically, alloantigen is presented and alloreactive T cells proliferate at around 3 to 5 days in the case of skin transplantation. NV suppression in the Th2-biased immune system is therefore presumably not mediated by alloreactive T cells, but by anti-inflammatory factors produced in the Th2 response. This NV suppression may be one mechanism to enhance graft survival in this system.
Current prophylactic and therapeutic regimens for LT rejection are associated with significant complications. Moreover, although immunosuppression can mediate clinical allograft survival, it is suggested that donor cells do not survive on the rat ocular surface indefinitely.
7 The Th2-biased immune system unexpectedly promoted donor epithelial cell survival for more than 4 weeks. Hence, the Th2-biased immune system or a modified system can be an effective immune therapy in LT.
Supported in part by a Research Grant 13557145 and 12771043 from the Japanese Ministry of Education, Culture, and Science and research funds from the Kyoto Foundation for the Promotion of Medical Science.
Submitted for publication July 16, 2002; revised December 24, 2002, and March 26 and May 11, 2003; accepted July 11, 2003.
Disclosure:
K. Maruyama, None;
J. Yamada, None;
Y. Sano, None;
S. Kinoshita, None
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “
advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Corresponding author: Jun Yamada, Department of Ophthalmology, Kyoto Prefectural University of Medicine, 465 Kajiicho Hirokoji-agaru Kawaramachi-dori, Kamigyo-Ku, Kyoto 602-0841, Japan;
[email protected].
The authors thank Junji Hamuro for helpful discussions of the experiments.
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