Effect of alloantibodies on corneal allograft survival.

PURPOSE: The precise role of antibodies in corneal transplantation is ambiguous, with evidence to support as well as repudiate their involvement in graft rejection. Accordingly, this study was undertaken to investigate the direct contribution of donor-specific antibodies to corneal graft rejection. METHODS: Serum samples from CB6F1 rejecters of orthotopically grafted C3H/Hej corneas were tested by ELISA for elevated levels of donor-specific alloantibody. Orthotopic corneal allograft rejection was also examined in B-cell-deficient mice. In a prospective study, naïve BALB/c T-cell-deficient nude mice and complement-depleted nude mice were passively infused with immune donor-specific serum and grafted with fully allogeneic C57BL/6J corneas. The incidence and speed of graft rejection were observed in each case. The susceptibility of corneal cells to antibody-mediated lysis was tested in vitro. RESULTS: Seventy percent of the CB6F1 hosts that rejected the C3H/Hej corneal allografts possessed significantly elevated levels of alloantibody in serum. Although BALB/c corneal allografts were rejected by B-cell-deficient mice at the same incidence as wild-type control mice, their mean survival time (MST) was significantly longer than that of their wild-type counterparts. Serum of BALB/c mice immunized against C57BL/6J alloantigens produced complement-dependent cytolytic activity against C57BL/6J corneal cells in vitro. Passive transfer of this alloantiserum to T-cell-deficient BALB/c nude mice produced complement-dependent corneal lesions, resulting in significantly increased opacity of C57BL/6J corneal grafts, compared with the relatively clear grafts in control hosts. CONCLUSIONS: Alloantibody, although not necessary for corneal graft rejection, can produce extensive injury to corneal allografts in a complement-dependent manner.

Fate of MHC-matched corneal allografts in Th1-deficient hosts.

PURPOSE: To determine whether the Th1 cytokine, interferon (IFN)-gamma, is necessary for corneal graft rejection. METHODS: Full-thickness penetrating keratoplasties were performed in normal mice and in IFN-gamma knockout (KO) mice. RESULTS: Sixty-four percent of the MHC-mismatched corneal allografts were rejected in IFN-gamma KO mice. By contrast, MHC-matched corneal allografts were rejected in 50% to 77% of the wild-type hosts, but were not rejected in any of the IFN-gamma KO mice or the wild-type mice treated with anti-IFN-gamma monoclonal antibody. Corneal graft rejection in IFN-gamma-deficient hosts was characterized by an eosinophilic infiltrate compared with a mononuclear inflammatory infiltrate in normal mice. CONCLUSIONS: IFN-gamma is not necessary for the rejection of MHC-mismatched corneal grafts. However, IFN-gamma and Th1 immune mechanisms are necessary for the rejection of MHC-matched corneal allografts that confront the host with foreign minor histocompatibility antigens. The immune response in atopic patients, as in IFN-gamma KO mice, is characterized by cross-regulation of Th1 cytokines, such as IFN-gamma. The present results indicate that MHC matching dramatically reduces the risk of corneal graft rejection when IFN-gamma is depressed or absent. Thus, MHC matching may reduce the risk of corneal graft rejection in patients with atopic keratoconus.

Are corneal cells susceptible to antibody-mediated killing in corneal allograft rejection?

PURPOSE: The precise role of antibodies in corneal transplantation is controversial. Clinical and experimental evidence both supports and refutes the contribution of donor-derived alloantibody in corneal allograft rejection. Accordingly, we prospectively evaluated the presence of donor-derived alloantibody in two high-risk donor-host combinations. We also evaluated the ability of this alloantibody to kill corneal epithelial, keratocytes, and endothelial cells in complement-dependent and complement-independent fashions. METHODS: C3H/Hej (H-2(k)) and Balb/c (H-2(d)) corneal grafts were transplanted orthotopically to CB6F1 (H-2(b/d)) and C57BL/6 (H-2(b)) recipients, respectively. These two donor-host combinations represent disparity at the entire MHC and multiple minor histocompatibility loci. Four objectives were addressed. First, we wished to determine if there was a correlation between the appearance of donor-specific serum IgG antibody and corneal graft rejection. Second, we evaluated the effect of passive transfer of hyperimmune donor-specific antibody on corneal allograft rejection. Third, we examined the capacity of donor-specific alloantibody to mediate complement-dependent cytolysis of corneal cells. Finally, we determined the capability of donor-specific alloantibody to mediate apoptosis of corneal cells. RESULTS: The presence of donor-specific serum IgG alloantibodies did not correlate with corneal graft rejection. One hundred percent of CB6F1 and C57BL/6 hosts rejected their C3H and Balb/c orthotopic corneal allografts, respectively. However, two of these seven CB6F1 hosts and one C57BL/6 host did not produce donor-specific IgG alloantibody that was significantly different from naive donors. Passive transfer of hyperimmune allo-antiserum prior to corneal transplantation did not increase the incidence, severity, or tempo of corneal allograft rejection in either donor-host combination. Hyperimmune allo-antiserum produced complement-mediated lysis of C3H corneal endothelial but not C3H corneal epithelial cells in the C3H-CB6F1 donor-host combination. Interestingly, all three corneal cell layers were vulnerable to complement-mediated cytolysis in the Balb/c-C57BL/6 donor-host combination. Additionally, Balb/c corneal epithelial, keratocytes, and endothelial cells were vulnerable to complement-independent, antibody induced apoptosis. CONCLUSIONS: Corneal graft rejection does not appear to correlate with the production of IgG alloantibody and can occur in the absence of donor-specific IgG alloantibody. Antibody-mediated killing of the corneal endothelium can occur in a complement-dependent or complement-independent fashion.

Preliminary findings in corneal allograft rejection in patients with keratoconus.

PURPOSE: Classically, corneal allograft rejection is thought to be a T(H)1-mediated phenomenon. However, T(H)2-mediated allograft rejection has been reported in other transplanted organ systems, including the heart and kidney. We previously reported a form of T(H)2-mediated corneal allograft rejection in a murine model with a T(H)2 immune bias. In this study we sought to determine if there was any evidence for this form of corneal allograft rejection in humans. DESIGN: Experimental study with an interventional case series. METHODS: The clinical records of all keratoconus patients undergoing penetrating keratoplasty at the University of Texas, Southwestern Medical Center from 1994 to 1999 were reviewed. Careful attention was paid to a clinical history of atopy. Atopic patients were selected, because these patients have been shown to have a "T(H)2 immune bias." The corneal graft rejection rate in these patients and the number of repeat corneal transplants performed was determined. The experimental group consisted of patients with a clinical history of atopy and keratoconus who had at least one repeat penetrating keratoplasty for an immunologically rejected corneal transplant. Any patient with evidence of primary allograft failure was excluded from this study. Tissue specimens from these patients were embedded in paraffin, serially sectioned, stained with Giemsa stains, and examined histologically. The control group consisted of patients without a clinical history of allergy (and therefore no T(H)2 immune bias) who underwent corneal transplantation for Fuch corneal endothelial dystrophy, or aphakic/pseudophakic bullous keratopathy. Failed grafts from these control patients were also paraffin embedded, serially sectioned, stained, and examined histologically. The human experimental and control corneal specimens were compared with data obtained in a murine model of T(H)2-mediated corneal allograft rejection. Briefly, full-thickness penetrating C57BL/6ByJ corneal allografts were transplanted onto Balb/cByJ and Balb/c-IFN-gamma(tm1Ts) (Balb/c-IFN-gamma knockout) mice. Additionally, full-thickness Balb/cByJ corneal allografts were transplanted onto C57BL/6ByJ and C57BL/6ByJ-IFN-gamma(tm1Ts) mice. Corneal allograft rejection rates and mean rejection times were calculated and compared between wild-type and interferon gamma (IFN-gamma) knockout hosts. The rejected allografts were examined histologically by the same methods used in the human tissue. RESULTS: There were 84 penetrating keratoplasties performed from 1994 to 1999 for keratoconus. Seven of these 84 patients rejected their corneal grafts. Of the 7 patients who rejected their corneal allografts, 4 had repeat penetrating keratoplasty. Of these 4 repeat corneal allografts, 3 showed eosinophilia when compared with rejected grafts in control patients. Atopic keratoconus patients had a mixed inflammatory cellular infiltrate in the rejected corneal tissue specimen with a significantly greater density of eosinophils (P =.001) compared with patients who did not have a pre-existing T(H)2 bias. The inflammatory infiltrate in these patients without a T(H)2 immune bias was mononuclear. In the murine model, corneal allograft rejection did occur in the absence of IFN-gamma, a critical T(H)1 cytokine in both fully allogeneic donor-host combinations. Histologically, rejection in these ("T(H)2 mice") was characterized by a predominant eosinophilic infiltrate in the rejected graft bed when compared with wild-type animals ("T(H)1 mice") that had a predominantly mononuclear infiltrate in the rejected corneal graft bed. CONCLUSIONS: Preliminary findings show that corneal allograft rejection in patients with a pre-existing T(H)2 phenotype is similar to what is seen in the murine model of T(H)2-mediated corneal allograft rejection. Based on this small sample, it appears that eosinophils may play a role in corneal allograft rejection in this group of patients. However, further study is necessary to determine the importance of these cells in allograft rejection.

Possible role of the vitamin E solubilizer in topical diclofenac on matrix metalloproteinase expression in corneal melting: an analysis of postoperative keratolysis.

OBJECTIVE: To analyze tissue matrix metalloproteinase (MMP) expression in three patients who developed postoperative corneal melts after treatment with topical diclofenac sodium 0.1% (Falcon; Fort Worth, TX) ophthalmic solution. DESIGN: Retrospective noncomparative interventional case series with tissue analysis. MAIN OUTCOME MEASURES: Three patients were examined in this study. We report two patients from the same center with acute corneal melts after uncomplicated photorefractive keratectomy (PRK). Prior to these cases, 1500 patients were treated at the Zale Lipshy University Laser Center for Vision with no adverse effects. All 1500 patients were treated with the same postoperative regimen of ciprofloxacin, rimexolone, and suprofen ([Profenal, (CIBA, Duluth, GA]). The next 27 cases were treated postoperatively with ciprofloxacin and rimexolone. However, diclofenac sodium 0.1% was used instead of Profenal. A third case was also discussed. This melt occurred at another center in a postoperative cataract patient who developed cystoid macular edema after cataract extraction with intraocular lens placement. He was initially treated with diclofenac sodium 0.1% (Ciba Vision, Duluth, GA) then with diclofenac sodium 0.1%. He subsequently developed a corneal perforation requiring penetrating keratoplasty. All tissue specimens were examined by light microscopy. Microbiologic cultures and stains were also performed. Immunolocalization and in situ hybridization were performed on all keratoplasty specimens to detect expression and localization of MMPs. All patients had a complete diagnostic evaluation for systemic autoimmune diseases. RESULTS: Postoperatively, all patients developed corneal perforations requiring surgical intervention while being treated with diclofenac sodium 0.1%. Microbiologic cultures and special stains were negative for microorganisms. Induced expression of specific tissue degrading enzymes of the matrix metalloproteinase family was demonstrated within corneal epithelial cells, stromal keratocytes, and at the level of Descemet's membrane. The uniform distribution pattern of expression was not consistent with the localization expected of a repair response, suggesting the involvement of some outside agent. CONCLUSIONS: Whereas MMP expression is a normal component of repair, excessive or inappropriate MMP activity is associated with corneal keratolysis. Our study provides preliminary evidence that topical application of diclofenac sodium 0.1% may be associated with aberrant MMP expression in the cornea.