TOPICAL INTERLEUKIN 1 RECEPTOR ANTAGONIST PROMOTES CORNEAL TRANSPLANT SURVIVAL¹

Abstract

Background. 

Interleukin (IL)-1 is a potent proinflammatory cytokine that plays a critical role in initiating and maintaining immunogenic inflammation. We performed a series of experiments to determine whether the topical application of IL-1 receptor antagonist (IL-1ra) can prolong corneal transplant survival in the murine model of orthotopic allotransplantation.

Methods. 

For all experiments, C57BL/6 corneas were transplanted into BALB/c (major histocompatibility and minor histocompatibility-disparate) eyes. “High-risk” transplants were transplants that had been sutured into BALB/c recipient beds with corneal neovascularization induced by placement of three interrupted sutures in the host cornea 2 weeks earlier. Both risk groups were divided in a masked fashion into treatment subgroups that received either 20 mg/ml of IL-1ra mixed in 0.2% sodium hyaluronate vehicle (n=28) or placebo alone (n=25). All transplants were evaluated for 8 weeks after surgery for signs of rejection. At the end of follow-up, corneal specimens were processed for enumeration of Langerhans cells and histopathological evaluation.

Results. 

Survival rates of both normal-risk and high-risk transplants increased significantly among the IL-1ra-treated animals compared with untreated controls by both stratified Mantel-Haenszel (P=0.02) and Kaplan-Meier survival (P=0.03) analyses. Furthermore, both normal- and high-risk IL-1ra-treated grafts had significantly less inflammation and Langerhans cells infiltration compared with untreated controls.

Conclusions. 

Topical treatment with IL-1ra has a significantly positive effect in promoting corneal allograft survival.

Corneal transplantation has emerged as the most common and successful form of solid tissue transplantation. Over 40,000 cases have been performed in the United States alone ⁽¹⁾. In uncomplicated first allografts performed in avascular beds, the 2-year survival rate is over 90% ⁽²⁾. The extraordinary success of penetrating keratoplasty can be attributed to various features of the normal cornea and anterior segment that in the aggregate account for their “immune-privileged” state ⁽³⁾, including: (a) the avascularity of the stroma, (b) the absence of corneal lymphatics, (c) the rarity of indigenous professional antigen-presenting Langerhans cells (LC^*) or macrophages in the normal graft bed, (d) a unique spectrum of locally produced immunomodulatory cytokines that suppress immunogenic inflammation and complement activation (to which the cornea itself contributes), and (e) expression of Fas ligand by these ocular tissues that can directly suppress immunogenic inflammation ⁽⁴⁾.

In spite of the overall success with corneal transplantation, a significant percentage of corneal grafts experience at least one rejection episode. This is significant, since of all the technical and tissue parameters that can affect final graft outcome, immunologic rejection represents the principal threat to allograft longevity, regardless of the degree of allodisparity ^(5-9). This immunological threat to graft survival is nowhere more evident than in vascularized recipient beds, which tend to suffer from earlier and more fulminant rejection episodes that are more resistant to therapy ^{(1, 5, 7, 8, 10)}.

The advent of corticosteroids and their use in the prophylaxis and treatment of corneal transplant rejections have represented the most significant contribution to the prolongation of corneal transplant survival over the last several decades ^{(11, 12)}. However, the local and/or systemic use of corticosteroids, or alternative general immunosuppressants, is associated with significant complications, such as infection, cataracts, glaucoma, and corneal thinning ^(13-16). Therefore, it is apparent that development of molecular strategies that can specifically target a critical cytokine may prove to be an effective modality for circumventing the problems inherent in nonspecific immune suppression.

Interleukin (IL)-1 is a potent proinflammatory cytokine that has a wide range of activities, including the critical mediation of the acute phase response, chemotaxis and activation of inflammatory and antigen-presenting cells, up-regulation of adhesion molecules/costimulatory factors on cells, and stimulation of neovascularization ^(17-20). IL-1 has been implicated as an important cytokine in host immunological reactions to a variety of nonocular allografts ^(21-23). In the eye, IL-1 activity has been correlated with corneal neovascularization ⁽²⁴⁾, endotoxin-mediated uveitis ⁽²⁵⁾, corneal collagenase and metalloprotease expression ^{(26, 27)}, corneal injury in vitamin A deficiency ⁽²⁸⁾, and herpetic stromal keratitis ⁽²⁹⁾. Importantly, Niederkorn and co-workers have shown that IL-1-mediated Langerhans cell migration can play a critical role in host allosensitization in the setting of corneal transplantation ^{(17, 30)}. For all these reasons, IL-1 is an attractive target for therapeutic intervention in immunogenic inflammatory diseases.

IL-1 receptor antagonist (IL-1ra) is a naturally occurring IL-1 isoform with high-affinity binding to both IL-1 receptor subtypes, but it has no agonist activity ^{(31, 32)}. There is a 77% homology between the human and murine IL-1ra, and systemic administration of recombinant human IL-1ra has been shown to have a profound down-regulatory effect on the acute phase cytokine cascade in both man and mouse ^{(33, 34)}. In this series of experiments, we hypothesized that topical application of IL-1ra to corneal allograft recipients would lead to a significant prolongation of transplant survival. Our results demonstrate that IL-1ra has a significant suppressive effect on immune rejection rates in both normal-risk and high-risk recipient beds.

MATERIALS AND METHODS

Mice and anesthesia. Eight- to 10-week-old BALB/c (H2^d) and C57BL/6 (H2^b) mice were purchased from Taconic Farms, Inc. (Germantown, NY). All animals were treated according to the Statement for the Use of Animals in Ophthalmic and Vision Research by the Association for Research in Vision and Ophthalmology. Each animal was deeply anesthetized with an intramuscular injection of 3-4 mg of ketamine and 0.1 mg of xylazine before all surgical procedures.

Corneal transplantation. All 55 transplants involved combined major histocompatibility complex (MHC)- and minor alloantigen-disparate corneas that were grafted from C57BL/6 donors into BALB/c eyes, as follows. On day 0, in each case, the central 2 mm of the donor cornea was marked with a microcurette, and the donor button was excised with Vannas scissors and placed in phosphate-buffered saline (PBS). The recipient graft bed was prepared by excision of the central 2 mm of the cornea. The donor button was then secured in place with eight interrupted 11-0 nylon sutures (Sharpoint, Vanguard, Houston, TX). Antibiotic ointment was applied to the corneal surface, and the lids were shut for 24 hr with an 8-0 nylon tarsorrhaphy for the next day, after which treatment would start. Animals were divided in a masked fashion into cases that would receive active IL-1ra and controls that would receive vehicle/placebo alone, as detailed below. High-risk corneal transplant recipients were treated with IL-1ra or placebo on day -1. Grafted eyes that had technical difficulties (hyphema, infection, or loss of anterior chamber) were excluded from the study. Transplant sutures were removed in all cases on day 7.

Induction and grading of corneal neovascularization. Intrastromal sutures induce robust neovascularization (NV) growth in the normally avascular corneal stroma from the limbus that can be appreciated as early as 3 days after suture placement ⁽³⁵⁾, and untreated allografts in these high-risk beds are rejected swiftly ⁽³⁶⁾. Two parallel protocols were devised to study normal- and high-risk corneal transplantation. In the former case, animals were left unmanipulated until the day of surgery. High-risk beds were developed as described previously ⁽³⁶⁾. Briefly, three interrupted 11-0 sutures were placed in the central cornea of one eye of a normal BALB/c mouse on day -14 using an aseptic microsurgical technique and an operating microscope. The neovascularized beds then served as high-risk graft beds for orthotopic corneal transplants on day 0, as described above (NV-inducing sutures were removed at the time of transplantation). NV was graded between 0 and 8 as described previously based on the degree of centripetal ingrowth and quadrantic involvement of the neovessels ⁽³⁷⁾.

Evaluation and scoring of orthotopic corneal transplants. Grafts were evaluated by slitlamp biomicroscopy twice a week. At each time point, grafts were scored for opacification. A previously described scoring system ⁽³⁷⁾ was used to measure the degree of opacification between 0 and 5+ (0 = clear and compact graft, 1+ = minimal superficial opacity, 2+ = mild deep [stromal] opacity with pupil margin and iris vessels visible, 3+ = moderate stromal opacity with only pupil margin visible, 4+ = intense stromal opacity with the anterior chamber [AC] visible, 5+ = maximal corneal opacity with total obscuration of the AC). Grafts with an opacity score of 2+ or higher after 3 weeks were considered to be rejected (immunological failure); grafts with an opacity score of 3+ or higher at 2 weeks that never cleared were also regarded as rejected. Grafts with an opacity score of 2+ or higher at any time point after 2 weeks were considered to have a rejection reaction, regardless of the opacity score at 8 weeks ⁽³⁷⁾ (since some grafts had only transient opacification).

Pharmacological strategy. One drop (40 μl) of each topical preparation was applied to BALB/c recipient mice three times a day for the 8 weeks of the study. The study medication was composed of 20 mg/ml of human recombinant IL-1ra in 0.2% sodium hyaluronate in PBS (supplied by Amgen, Boulder, CO). Placebo-treated animals received the vehicle 0.2% sodium hyaluronate only.

LC enumeration and histopathological evaluation. The LC were assessed with an immunofluorescence assay performed on whole corneal epithelial mounts, as described previously ⁽³⁸⁾. Briefly, each eye was enucleated and the anterior segment was dissected under the operating microscope. The cornea was placed in 20 mM EDTA buffer and incubated for 30 min at 37°C; then, the epithelium was removed in toto and washed in PBS at room temperature. The cornea was then fixed with 95% ethanol before it was washed and incubated with 1:20 diluted primary anti-murine Ia^d antibody for 45 min at 37°C. The tissue was then washed in PBS and incubated with a fluorescein isothiocyanate-labeled goat anti-mouse secondary antibody for 30 min at 37°C. Negative controls either bypassed this step or were incubated with antibody specific for an unrelated MHC epitope. Sections were then mounted on slides and examined under the fluorescent microscope with a square ocular grid where LC were enumerated. Corneal specimens that were not processed for LC enumeration were fixed, sectioned, and stained with hematoxylin and eosin for light microscopic evaluation.

Statistical techniques. We compared the rates of rejected allografts in the IL-1ra-treated and vehicle-only groups using two methods. First, we obtained the Mantel-Haenszel summary chisquare statistic, stratified by (adjusted for) degree of preoperative risk (i.e., normal versus vascularized stromal bed), to compare the proportion of rejected transplants in the two groups. Second, we constructed Kaplan-Meier survival curves to compare the probability of graft survival over the follow-up period, both overall and separately for normal- and high-risk eyes, in the IL-1ra-treated and untreated controls. This method allowed us to account for the variability in the time-to-graft rejection in addition to the variation in follow-up time (four mice in the normal-risk group had follow-up terminated before the end of the 8-week period, as described below). Comparison of LC population means among IL-1ra-treated eyes and untreated controls was made with Student's t test.

RESULTS

Corneal transplant survival. Graft failure was established based on opacity scores (detailed above). A total of 55 corneal transplants were performed in 55 BALB/c mice, of which 53 were deemed technically acceptable for long-term follow-up (i.e., anterior segment integrity was maintained with no signs of wound leak, infection, or hyphema). These 53 allografts were subdivided based on degree of immunological risk, as described above, into normal- (n=28) and high-risk (n=25) groups.

Statistical analysis of cumulative rejection rates, after stratification for degree of risk based on recipient bed vascularity, revealed a strong association between IL-1ra treatment and graft survival (Mantel-Haenszel test, P=0.02). The 8-week incidences of transplant rejection were lowest in the normal-risk grafts that had been treated with IL-1ra (7%) and highest in the high-risk grafts that were treated with vehicle only (73%) (Table 1).

Four of the animals were censored from further evaluation before completion of the 8-week follow-up course due to development of dystrophic/degenerative corneal calcific deposits ⁽³⁹⁾. To prevent generation of bias by artificial lowering of the denominator size in 8-week rate calculations, Kaplan-Meier survival curves were developed (Fig. 1). Survival analysis revealed that IL-1ra treatment was associated with significant graft longevity in both normal-risk (P=0.1) and high-risk (P<0.05) recipient beds, with an overall reduction in rejection rate of 56% (P=0.03).

Corneal neovascularization. In addition to the graft survival/opacification criteria detailed above, transplants were also followed biomicroscopically for the degree of corneal NV. All high-risk beds had been specially prepared for the previous 2 weeks to develop two or more quadrants of stromal NV, as described previously ⁽³⁶⁾, and all normal-risk corneal beds were avascular.

It has been shown previously in both man ⁽⁴⁰⁾ and mouse ⁽³⁷⁾ that corneal transplantation alone can induce NV. Since postkeratoplasty corneal NV likely plays an important role in facilitating effector elements in the inflamed cornea, we were interested to see whether treatment with IL-1ra had an appreciable effect on this parameter. We observed an angiostatic effect of IL-1ra treatment in the normal-risk, but not high-risk, transplants. Among the normal-risk grafts (Fig. 2), 38% of the untreated corneas had an NV score of ≥3 at 4 weeks compared with none of the IL-1ra-treated cases, and respective rates were 31% for untreated controls and 18% for treated cases at 8 weeks. In contrast, no significant association was apparent with high-risk eyes that had been induced to have corneal NV 2 weeks earlier (Fig. 3). The proportions of corneas with an NV score of ≥3 at 4- and 8-week follow-up were very comparable between untreated controls (91% at both time points) and treated cases (100% and 86% at respective time points).

Furthermore, there was a significant correlation between corneal angiogenesis and rejection in both untreated normal- and high-risk control groups. Among untreated normal-risk transplants (Fig. 2), four of four corneas with an NV score of ≥3 had rejected at 8 weeks. Similarly, among high-risk recipients (Fig. 3), 7 of 10 grafts with an NV score of ≥3 had rejected at 4 weeks, and 8 of 10 grafts with an NV score ≥3 had rejected at 8-week follow-up. In contrast, there was a distinct divergence between corneal angiogenesis and graft survival among both normal- and high-risk IL-1ra-treated cases. For example, among the normal-risk eyes that had received treatment with IL-1ra, the one allograft that rejected had minimal NV, and two treated grafts with significant NV never rejected (Fig. 2).

LC population. The presence of LC in the cornea has been associated with immunogenic inflammation, the host's ability to be allosensitized, and loss of immune privilege ^{(39, 41, 42)}. To explore this point further, naive age-matched BALB/c corneas and allografts were excised at the completion of the follow-up period to assay their LC populations with fluorescence microscopy, as described above.

Consistent with previous findings, the central and paracentral areas of normal naive and avascular corneas had very few LC. Even syngeneic corneal transplantation can induce some degree of LC migration toward the central cornea, as do other corneal insults ⁽⁴³⁾, likely as a result of the nonspecific inflammatory wound-healing response. Interestingly, however, treatment with IL-1ra had a significant dampening effect on LC migration, regardless of the degree of pre- or postoperative corneal NV. Among the normal-risk allografts, the average number of central corneal LC in the IL-1ra-treated cases was 13/mm², compared with 41/mm² (32%) in the untreated controls (P=0.03). A similar reduction in the number of LC was observed after IL-1ra treatment in the vascularized high-risk beds, where the number of LC was 27/mm² in the treated corneas compared with 89/mm² in the untreated eyes (P=0.02).

Corneal inflammation. Histopathological evaluation of IL-1ra-treated and untreated corneas at 8 weeks in both normal- and high-risk eyes demonstrated a significant decrease in the number of leukocytes (particularly neutrophils) infiltrating grafts that had undergone treatment, with an associated decrease in the degree of stromal edema (Fig. 4, A and B). The decreased corneal inflammation in the IL-1ra-treated allografts was reflected by a generally lower opacity score (irrespective of final rejection status) in the IL-1ra-treated transplants. For example, all but one of the untreated normal-risk grafts that eventually failed developed opacity scores ≥3, whereas the single IL-1ra-treated normal-risk graft that failed had an opacity score of 2.

DISCUSSION

Corneal transplantation represents the only recourse available for restoring sight for millions of people with blindness caused by corneal opacification. Corneal transplantation represents the most common form of solid organ allotransplantation in the United States. However, the generally good graft outcomes have tended to overshadow the significant numbers of graft recipients whose transplants reject ^{(2, 5, 6)}. In addition to the significant personal and economic costs associated with transplant rejection, host immune reactions frequently lead to more rejection episodes against the same or a future graft ^{(5, 7, 8)}. The cumulative problem of corneal transplant rejections is reflected in the fact that regrafting is increasingly becoming a leading indication for corneal transplantation in large eye centers. This problem is felt nowhere more acutely than in high-risk keratoplasty, where the diseased corneal bed receiving the graft is further compromised by inflammation and corneal NV, which can abrogate the eye's normal immune-privileged state ⁽³⁵⁾ and lead to fulminant graft rejections ^{(5, 7, 9, 10, 36)}.

The currently available pharmaceutic armamentarium for corneal transplant survival is primarily composed of corticosteroids. Their introduction into ophthalmology is arguably the single most significant factor in the last four decades' advances in corneal transplant surgery ⁽¹³⁾. Nevertheless, beyond their well-known serious complications ⁽¹³⁾, corticosteroids show widely variable efficacy in preventing ultimate immunogenic graft failure, and this is particularly the case in high-risk keratoplasty ^{(1, 7)}. We conducted this series of experiments to test whether the specific inhibition of IL-1 activity by application of high-dose IL-1ra can be successful in prolonging either normal- or high-risk orthotopic corneal allografts in the mouse. In this article, we report that IL-1ra has a significant positive effect in corneal transplantation, irrespective of host bed vascularization, but is associated with a decrease in the degree of host LC migration into the graft bed.

There is little doubt that the presence of corneal NV is a significant risk factor for corneal allograft survival ^{(1, 6, 7, 10, 36)}. The endothelialized channels, in a milieu that is normally devoid of vessels, serve as a conduit for host immune effector elements ⁽³⁶⁾. Furthermore, the vascular endothelial cells can play a critical role in the recruitment and activation of cellular effectors by up-regulating adhesion/costimulatory molecules ^{(44, 45)}. In addition to facilitating expression of the efferent limb of immunity, inflammatory corneal NV is associated with the new development of corneal lymphatics that can provide for a fast and direct route for large molecules and antigen-presenting cells to the highly immunogenic milieu of the draining lymph nodes ⁽⁴⁶⁾.

In our laboratory, as well as in the clinic, high-risk keratoplasty is defined by the presence of corneal NV. We were therefore interested in examining the relationship between IL-1ra treatment and NV scores. Our data suggest that in the normal-risk setting, but not in the high-risk setting, where NV had been induced 2 weeks earlier, IL-1ra treatment is associated with a blunted postkeratoplasty NV response. We have evidence in the laboratory (M.R. Dana, unpublished observations) that IL-1ra can significantly blunt the early-phase, but not late-phase, corneal NV development in response to standard angiogenic stimuli. This suggests that there are non-IL-1-mediated factors that can overshadow IL-1 suppression in corneal angiogenesis. We hypothesize, therefore, that the failure of IL-1ra to lead to significant NV regression in the high-risk beds, as opposed to its demonstrated capacity for angiostasis in the normal-risk beds, is due to the dominance of non-IL-1-driven angiogenic factors in the former.

In the aggregate, development of corneal NV causes sufficient perturbation of the ocular microenvironment to lead to a loss of immune privilege as measured by the ability to induce AC-associated immune deviation ⁽³⁵⁾. We were initially surprised at the efficacy with which IL-1ra could blunt rejection in the high-risk corneas without an equal degree of suppression in corneal NV in the high-risk setting, but this could mirror what has been described previously in neovascularized corneas, where therapeutic measures that have been shown to restore immune privilege are associated with highly variable degrees of angiostasis ⁽³⁵⁾. For example, it may be hypothesized that IL-1ra blunts the effector role of corneal NV in immunity by suppressing the IL-1-mediated up-regulation of intercellular adhesion molecule-1 or other co-stimulatory signals ^{(47, 48)}.

We are intrigued by the degree to which the migration of LC into the central cornea can be blunted by application of IL-1ra. Because the healthy and unoperated cornea is essentially devoid of these constitutively antigen-presenting cells as well as other MHC class II-bearing “passenger leukocytes,” the presence of LC in the central cornea has been implicated in the loss of local immune privilege by virtue of their critical role in immune surveillance, and allosensitization is the “indirect pathway” ^{(2, 17, 36)}. Niederkorn has shown that IL-1 is a critical regulator of LC migration in the cornea ⁽¹⁷⁾, and a number of laboratories have shown that the activity of epidermal LC is at least partially controlled by IL-1 ^{(30, 49)}. Hence, the demonstrated constitutive expression by normal corneal cells of IL-1ra ⁽⁵⁰⁾ likely plays an important immune regulatory role in the avascular/nontraumatized cornea by keeping the microenvironment an inhospitable site for sensitization. We had hypothesized that the induction of allosensitization in the corneal allograft could be tilted in favor of unresponsiveness by the application of high doses of IL-1ra, and that this would be reflected in greater longevity of these allografts. Our data on LC numbers in IL-1ra-treated and untreated controls, although no direct proof of suppressed allosensitization, offer circumstantial evidence to support this hypothesis.

The specific regimen utilized in our study for the delivery of IL-1ra to the cornea was based on the observation that ocular bioavailability of topical medications is enhanced in viscous formulations ^{(51, 52)}. Traditional formulations that rely on aqueous drops for topical treatment often provide low bioavailability because of efficient elimination processes active on the ocular surface that typically lead to a very short drug residence time. Our choice of sodium hyaluronate as the vehicle of choice in this series of experiments was based on previous observations that 0.2% sodium hyaluronate is a vehicle with a very long contact (residence) time on the ocular surface that is also well-tolerated due to its pseudoplastic biophysical properties, which offer little resistance to high shear rates ^{(53, 54)}. We cannot, at this point, comment on the transcorneal penetration of this drug formulation, but the fact that resident corneal epithelial cells can produce significant levels of IL-1 ^{(17, 55)} would mean that an effective delivery system of IL-1ra to the ocular (epithelial) surface, even in the absence of efficient transcorneal penetration, can still play an effective IL-1-antagonizing role.

The therapeutic use of IL-1ra has generally met with mixed success. Several investigators have demonstrated that systemic administration of IL-1ra can have a profound down-regulatory effect on IL-1-mediated responses, such as mortality from endotoxin shock ⁽³⁴⁾ or from severe graft-versushost disease ⁽³³⁾. However, success with local administration of IL-1ra for treatment of inflammatory disease has been more guarded. Kondo and colleagues have shown that intradermal injections of IL-1ra can suppress contact hypersensitivity ⁽⁵⁶⁾, but intraocular treatment of IL-1ra for suppression of lipopolysaccharide-mediated uveitis has met with little success ⁽⁵⁷⁾. In any case, it is apparent that a very significant excess of IL-1ra (over IL-1 or constitutive IL-1ra levels) is required to effectively quench IL-1-mediated inflammation in vivo ⁽⁵⁸⁾.

We are not aware of any reports on the topical use of IL-1ra to suppress immunogenic inflammation in the eye, or elsewhere. Based on the observations of Dinarello and colleagues, that high concentrations of IL-1ra need to be used to suppress IL-1 activity in vivo ⁽⁵⁸⁾, and because we had developed an empirical approach to bioavailability of the compound in the sodium hyaluronate vehicle, we selected a high dose for our treatment protocol. The work of Kondo and colleagues in suppressing contact hypersensitivity has shown that IL-1ra can be effective over a wide range of concentrations ⁽⁵⁶⁾; hence, it is not unlikely that lower doses of IL-1ra would show similar efficacy in prolonging graft survival.

Current prophylactic and therapeutic regimens for corneal transplant rejection are associated with significant complications. Hence, it is critical to devise intervention strategies that can prolong graft survival by specifically targeting molecules that mediate and facilitate the immunogenicity of the allotransplant. Our data indicate that the topical application of IL-1ra holds promise as an effective modality for suppressing IL-1-mediated processes in the context of corneal transplantation.

Acknowledgments. The authors thank Dr. Debra A. Schaumberg, Associate Epidemiologist, Department of Medicine, Brigham & Women's Hospital, Harvard Medical School, for her biostatistical assistance.

Figure 1: Kaplan-Meier survival curves for normal-risk (n=28) and high-risk (n=25) corneal allograft recipients stratified by treatment with IL-1ra active agent or placebo. In both cases, there is an association between IL-1ra treatment and transplant outcome. Survival rates of high-risk grafts treated with the active agent closely mirror those of normal-risk transplants that received placebo.

Figure 2: Association between corneal allograft survival and NV score in normal-risk recipients based on IL-1ra treatment. Corneal NV is associated with graft rejection in untreated controls (A) but not in treated cases (B), and IL-1ra treatment alone has a dampening effect on postkeratoplasty corneal NV (B compared with A).

Figure 3: Association between corneal allograft survival and NV score in high-risk recipients based on IL-1ra treatment. Corneal NV is associated with graft rejection in untreated controls (A) but not in treated cases (B), and IL-1ra treatment alone does not appear to have a dampening effect on postkeratoplasty corneal NV (B compared with A) in contrast to normal-risk cases.

Figure 4: (A) Photomicrograph (×250) of failed high-risk transplant 8 weeks after keratoplasty, treated with placebo alone, shows abundant stromal inflammation, edema, and full-thickness disorganization of the collagen lamellae. (B) Photomicrograph (×50) shows a high-risk transplant 8 weeks after keratoplasty that had been treated with topical IL-1ra, as described in Materials and Methods. In contrast to panel A, only minimal anterior stromal edema and few leukocytes are present. The cornea has maintained its normal architecture, which accounts for its clarity on biomicroscopic examination.