Materials and methods

PATIENTS

A total of 30 diseased corneas from patients undergoing penetrating keratoplasty were obtained at the 2nd Department of Ophthalmology, Charles University, Prague, after informed consent was given. Investigations were performed according to the guidelines of the Declaration of Helsinki. The clinical details of the corneal recipients (a total of 15 females between the ages of 26–76 years with a median of 53.5 years and 15 males between the ages of 13–74 years with a median of 36.7 years) are given in Tables 1, 2, and 3.

PREPARATION OF TISSUES FOR IMMUNOHISTO- CHEMISTRY AND IMMUNOFLUORESCENCE

Samples of rejected and explanted corneas were removed, transferred to foil cups containing OCT medium (Miles Corp, Elkhart, IN, USA) and snap frozen in liquid nitrogen cooled isopentane. They were stored at −80°C until used.

IMMUNOHISTOCHEMISTRY

We used the StreptABC method as described previously.21 Cryostat sections (5–6 μm) of tissues were taken onto chromalum coated slides at −20°C and air dried. The samples were fixed for 10 minutes in acetone, rehydrated twice for 5 minutes in TRIS buffered saline (TBS), and incubated with the primary monoclonal antibody in a humid chamber for 1 hour at room temperature. The monoclonal antibodies (mAb) used are shown in Table 4. The sections were washed twice with TBS and incubated in biotinylated rabbit anti-mouse antibody (diluted 1:100 in TBS containing 5% normal human serum) for 30 minutes in a humid chamber at room temperature. After washing twice for 5 minutes with TBS, the slides were incubated for 30 minutes with StreptABComplex/AP (Dako, UK) containing streptavidin and biotinylated alkaline phosphatase in TRIS/HCl. After washing, the substrate containing naphthol, levamisol, and fast red (all Sigma, Poole) in veronal acetate buffer was added to the samples. The tissue sections were mounted in Aquamount Improved (BDH, UK).

IMMUNOFLUORESCENCE

Cryostat sections (5–6 μm) of tissues were taken onto chromalum coated slides at −20°C and air dried overnight. The samples were fixed for 10 minutes in acetone and rehydrated twice for 5 minutes in phosphate buffer saline (PBS), then incubated with the primary antibody against CD1a (Becton-Dickinson, UK) in a humid chamber for 30 minutes. The slides were washed three times for 10 minutes with PBS and incubated with a secondary biotinylated rabbit anti-mouse antibody (diluted 1:100 in PBS containing 5% normal human serum), for 30 minutes. After washing three times for 10 minutes with PBS, the slides were incubated for 30 minutes with Streptavidin-Texas Red (Amersham) diluted 1:50 in PBS. After further three washes for 10 minutes in PBS, the slides were incubated with the primary antibody against CD19 (Dako, UK) or mAb anti-MHC class II molecules (Sapu, UK or Becton Dickinson, USA) as before. The slides were then washed three times for 10 minutes with PBS and incubated for 30 minutes with the second secondary antibody, FITC labelled rat anti-mouse immunoglobulin (diluted 1:50 in PBS containing 5% normal human serum). After washing three times for 10 minutes with PBS, the tissue sections were mounted in Vectashield (Vector Laboratories, CA, USA). We used three kinds of negative controls: (1) tissue sections stained without the first primary mAb; (2) sections stained without the second primary mAb, and (3) sections stained without both primary mAb. Instead of primary antibodies we used PBS for the negative control staining.

LIGHT MICROSCOPY

Tissue sections were examined by light microscopy (Olympus-CH-2, Japan) and immunofluorescence microscopy (Olympus, Japan). Samples were examined using a ×200 original magnification.

HISTOLOGICAL SCORING

The number of cells per 20× objective field was graded as follows: 0 = no positive cells observed; 1 = 1–10 positive cells/field; 2 = 11–20 positive cells/field; 3 = 21–30 positive cells/field; and 4 = >30 positive cells/field. The samples were evaluated by two masked observers. Statistical analysis was performed used a Student's unpaired t test.

Results

The results of the immunohistological analysis of test corneal sections are summarised in Table 5. All 10 control corneal buttons (explanted for keratoconus, Fuchs' endothelial dystrophy, and pseudophakic bullous keratopathy) were negative for the set of antibody markers studied (CD3, CD4, CD8, CD14, CD25, CD68) (Fig 1A). No inflammatory cells were seen and no HLA class II molecules were detected on the surface of epithelial, stromal or endothelial cells. There was also no evidence of LC or DC (CD1a/MHC class II double positive cells) in the corneal tissue.

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Figure 1

(A) Corneal specimen obtained from a patient with keratoconus stained mAb anti-CD68 (original magnification ×100). No staining was observed. (B) Strongly positive MHC class II cells (HLA-DR) in the deep layer of the corneal epithelium and in the stroma from a rejected cornea clinically showing no inflammation at the time of surgery (original magnification ×400). (C) MHC class II (HLA-DP) positive cells in the superficial layers of the epithelium from same specimen as (B) (original magnification ×200). (D) Corneal explant obtained from patient after keratoplasty “a chaud”(in severe inflammation at time of surgery). Specimen shows strong staining for macrophages (mAb anti-CD68) especially around the suture (arrow) (original magnification ×400). (E) Patchy MHC class II (HLA-DR) positive staining of endothelial cells from same specimen as (D) (original magnification ×600). (F) Intensive staining for MHC class II (HLA-DR) on dendritic-like cells in stroma of a corneal specimen from patient with epithelial wound healing problems and no inflammation at the time of corneal transplantation (original magnification ×600). Dual immunofluorescent staining of corneal stromal cells with MHC class II (HLA-DR) and CD1a. (G) Widespread MHC class II staining of many cells in corneal stroma. Arrows show dendritic-like cells in corneal stroma. (H) CD1a staining of individual cells from same region as (G). (I) Dual overlay showing co-expression of MHC Class II and CD1a on stromal dendritic cells, same field as (G) and (H) (original magnification ×600).

The rejected, failed grafts from non-inflamed “quiet” eyes showed moderate positivity for CD14, CD68 (average grade 2.14 (SD 1.7) and 2.0 (1.24)), and CD8 (average grade 0.87 (0.83)) and strong positivity for HLA class II molecules HLA-DP and HLA-DR (average grade 2.27 (1.33) and 2.8 (1.33)) (Fig 1B and C). The presence of HLA-DR positivity was found particularly on cells in the basal layers of the corneal epithelium and in the stroma, and on corneal endothelial cells (Fig 1E). Staining for CD3 and CD4 was negative or low in the majority of grafts. CD1a/MHC class II double positive cells were localised in the deep epithelial layer at the level of Bowman's membrane and also in the corneal stroma.

The grafts collected from eyes with active inflammation “melts” (keratoplasty “a chaud”) showed moderate positivity for CD3, CD4, and CD8 (average grade 2.0 (0.63), 0.8 (0.74), and 1.8 (0.74)), a level which was significantly greater than that in the rejected “quiet” explants. The majority of corneal grafts were strongly positive for the macrophage markers CD14 and CD68 (average grade 3.0 (0.89) and 3.8 (0.89)) (Fig 1D) and for HLA-DP and HLA-DR (average grade 3.2 (0.74) and 3.4 (0.8)). CD1a/MHC class II expressing double positive cells were present in significant number in the deep epithelial layer and throughout the corneal stroma (Fig 1G, H, I and Table 5). Staining for CD19 was also performed and only a few cells expressing this marker were detected. Dual staining for CD1a and CD19 was also performed and no co-expression detected (data not shown).

In the control group (n=10) we observed immunological rejection in 10% of the patients. In the group with previous graft rejection, but no inflammation in the eye at the time of regrafting (n=6), 66.7% of subjects had a rejection episode followed by graft failure, while 33.3% achieved long term acceptance of the graft. In the same group with previous rejection, but who were complicated by having surface wound healing problems without inflammation at the time of rekeratoplasty (n=9), rejection occurred in 44.4% of patients, graft melting in 44.4%, and only 11.2% achieved long term acceptance of the graft. In this group 88.8% of patients suffered graft failure and 77% of these failed grafts required regrafting. In the group with active inflammation at the time of grafting and who also had surface wound healing problems with graft melting (n=5), 60% of subjects suffered a further melting of the graft and further 20% rejected their graft. All subjects have achieved graft failure and only one of these cases did not required regrafting.

Discussion

Immunological rejection is the most important cause of graft failure in corneal transplantation.1 Since there are only limited data on cells which might mediate the immune response particularly antigen presentation in the eye, we measured the expression of HLA-DR and HLA-DP molecules and looked for the presence of DC and macrophages in the corneal buttons from rejected and melted corneal grafts and from control corneas. In this report, for the first time dual immunofluorescent staining has been used for detection of DC using positivity with MHC class II and CD1a and negativity with CD19 antigens as criteria. In a previous paper, low numbers of CD1a+ cells were observed in the corneal epithelium, but not in the corneal stroma.15 20 We observed a population of cells which were MHC class II+, CD1a+, CD19−, had a dendritic morphology in corneal epithelial layer but also in the corneal stroma, and which we believe to be definitive dendritic APC. The source of these cells may be from migrating limbal LC, which have become activated, or from conjunctival stromal DC, which have been recruited from bone marrow.

In the group of 15 corneal buttons examined after rejection (from eyes with no inflammation), a significantly less intense infiltration of CD3, CD4, and CD8 positive cells was found in comparison with explants with severe inflammation. However, prominent expression of HLA-DR and HLA-DP antigens on tissue resident cells was seen in a majority of rejected grafts. It is possible that the expression of class II HLA molecules on the non-professional APC might be induced by cytokines produced by inflammatory cells, as has been proposed,15and we have observed extensive MHC class II expression on epithelial, stromal, and even on endothelial cells. However, since antigen presentation requires not only MHC class II antigen upregulation but also expression of co-stimulatory molecules, it is unlikely that MHC class II antigen alone on tissue cells is responsible for alloantigen presentation to cytotoxic T cells.

We suggested therefore that professional DC are responsible for initiating graft rejection. The number of CD1a/MHC class II double positive cells was not significantly higher in a group with severe inflammation at the time of surgery than in the group with no inflammation. The number of macrophages, which were also regularly observed in rejected corneas, was variable. We have found significantly increased numbers of CD68+ cells in the severely inflamed corneas. These cells may also act locally as APC within the corneal graft and thus perpetuate the rejection reaction, but macrophages are 100 times less potent as APC than DC.22 In addition, it is likely that the influx of macrophages may be secondary to the release of a variety of cytokines and chemokines produced by the graft infiltrating lymphocytes. One of these cytokines, migration inhibitory factor (MIF), is produced by many cells including corneal cells.23

The most important clinical question is the long term acceptance of the corneal graft. From previous studies reduced corneal graft survival time was observed in recipients with grafts containing high numbers of APC.17 In this study we have found immunological rejection followed by graft failure in 66.7% and 88.8% respectively of subjects in the group with no inflammation at the time of surgery. All subjects with severe inflammation in the time of surgery developed graft failure and in 80% underwent repeated corneal transplantation.

More extensive studies on the composition of graft infiltrating lymphocytes have been done in rat cornea, which closely resembles human cornea in terms of MHC molecule expression.24 It was shown that transplantation of syngeneic grafts evokes a focal inflammatory reaction in the vicinity of the sutures and in the wound.25 Allogeneic grafts induce diffuse inflammation with CD8+ and CD4+ lymphocytes and macrophages. The ratio of CD8+ to CD4+ lymphocytes changes with time after grafting, being higher immediately after grafting.26

Taken together, the results of this study support the view that early non-specific inflammation such as occurs in association with surface wound healing problems, previous corneal graft “melting”, and even sutures in the cornea may lead to amplification of the inflammatory response and may trigger an immune response in the eye mediated by CD1a+ and MHC class II+ professional APC. This then leads to chemoattraction of antigen specific and non-antigen specific T cell populations with further tissue damaging macrophage infiltration and finally graft rejection.