Patients and methods

After ethics committee approval had been obtained, patients in the Yorkshire Region with a diagnosis of sterile corneal melting in association with systemic vasculitis were invited to take part in the study. The anatomical site of the corneal melting and the systemic disease association were determined from case note review, clinical and slit lamp examination, or both.

DNA was extracted from peripheral blood lymphocytes using a previously described method comprising red cell lysis, proteinase K digestion, salt extraction, and ethanol precipitation.22 At a concentration of 0.1 μg/μl, the DNA was subjected to polymerase chain reaction (PCR) amplification in a GeneAMP PCR System 9600 (Perkin Elmer, CA, USA). The products were analysed by electrophoresis on agarose gels and visualised by ultraviolet transillumination with ethidium bromide staining. A molecular weight marker ranging from 100 bp to 600 bp was used to determine the fragment sizes.

PCR ANALYSIS OF TNF-α GENE POLYMORPHISM

PCR amplification of the polymorphic region at position –308 of the TNF-α promoter region was performed using previously described primers and subjected to specific endonuclease digestion with Nco1.23 PCR conditions were as follows: 94°C for 3 minutes, 60°C for 1 minute, 72°C for 1 minute, followed by 35 cycles of 94°C for 1 minute, 59°C for 1 minute, 72°C for 1 minute and a final cycle of 94°C for 1 minute, 60°C for 1 minute, and 72°C for 1 minute. The resultant PCR products were separated by electrophoresis on a 4% agarose gel and visualised under ultraviolet transillumination. When the TNF1 allele is present, the Nco1 restriction site is intact and an 84 bp product is produced. For the TNF2 allele, the PCR product cannot be cleaved by Nco1 and a 108 bp product is produced.23

PCR ANALYSIS OF IL-1RA GENE POLYMORPHISM

Intron 2 of the IL-1Ra gene contains an 86 bp VNTR. Primers flanking this region were used to amplify the polymorphic region by PCR.12 PCR conditions were as follows: 94°C for 1 minute, 60°C for 1 minute, 72°C for 1 minute followed by 35 cycles of 94°C for 1 minute, 59°C for 1 minute, 72°C for 1 minute and a final cycle of 94°C for 1 minute, 60°C for 1 minute, and 72°C for 1 minute. The PCR products were separated by electrophoresis on a 2% agarose gel and visualised under ultraviolet transillumination. The IL-1Ra alleles exist as variable numbers of the tandem repeat and as PCR products of the following sizes: IL-1Ra*1 = 410 bp, IL-1Ra*2 = 240 bp, IL-1Ra*3 = 500 bp, IL-1Ra*4 = 325 bp, and IL-1Ra*5 = 595 bp.

After determining the genotype for each patient, the allele frequencies (number of times the test allele occurs in the population divided by the total number of alleles) for each of the alleles encountered were calculated. The genotype and frequency of the alleles was then compared with that for unrelated white blood donor controls from the Yorkshire area (J Reid, personal communication).

Statistical analysis was performed using 2×2 contingency tables and χ² tests to calculate the relative risk and significance for each allele.

Results

Twenty white patients were recruited into the study, of whom 17 had rheumatoid arthritis and three had Wegener's granulomatosis. Ten of the patients were male. All the patients had peripheral corneal melting, except for one with rheumatoid arthritis in whom the disease was central and bilateral.

The distribution of the genotypes found in the patients with rheumatoid arthritis or Wegener's granulomatosis and in the control groups is given in Table 1. The frequency of each allele in the combined patient group and the control group is given in Table 2. Although there was a trend for the IL-1Ra*2 allele to be more common in the control group, this failed to reach clinical significance. No allele was found to be significantly associated with the patient group: TNF1 p = 0.89; TNF2 p = 0.89; IL-1Ra*1 p = 0.65; IL-1Ra*2 p = 0.68; IL-1Ra*3 p= 0.50.

Discussion

Necrotising keratitis or corneal melting occurs most commonly in association with a systemic vasculitis.14 The condition may occur in either the central or peripheral cornea and is characterised by ulceration and rapidly progressive loss of the corneal stroma. The pathogenesis of the condition is poorly understood but a combination of factors appears to be involved, including chronic ocular surface disease and inflammatory cell infiltrate.8 24 25Although a tendency exists for the inflammatory ulceration to occur more commonly in the peripheral cornea and for the surface disease associated ulceration to occur in the central or paracentral cornea, this distinction is not absolute and most patients have a combination of surface disease and inflammatory cell infiltrate.26 In our experience, the inflammatory process in the cornea of patients with either anatomical type of vasculitis associated corneal melting is quantitatively very different from that in other patients with the same systemic vasculitis and ocular surface disease but no melting. In this study, we planned to investigate whether this difference may be a reflection of an inherent imbalance in cytokine production relative to the corresponding inhibitor in these patients.

In the normal immune response, inflammation produces little or no damage to the host. However, if the response is self directed and/or ineffectively downregulated, then a situation of chronic leucocyte activation arises with continued production of inflammatory mediators and tissue destruction. This may be the case in the corneal melting associated with rheumatoid arthritis and Wegener's granulomatosis.21 In addition to inflammatory cell infiltrate and surface disease, features of the ocular condition include local vasculitis, an excess of matrix metalloproteinases relative to their tissue inhibitors, and rapid loss of corneal stroma often progressing to perforation.21 24 27 In view of the role of TNF-α and IL-1Ra in the pathogenesis of systemic vasculitis and their presence in normal and inflammatory corneal disease, it seemed reasonable to determine the role of functional polymorphisms of these cytokines in the susceptibility to corneal melting.

Given the similar genotypes in the patients with rheumatoid arthritis and Wegener's granulomatosis and the small number of patients with the latter, the allele frequencies were calculated for the whole patient group (Table 2). Similar frequencies of all the TNF-α and IL-1Ra alleles were observed in patients with corneal melting and healthy controls from the same ethnic group and geographic area. Most of these alleles are biologically silent and only the TNF2 and IL-1Ra*2 alleles have functional significance. There was no evidence to suggest that the functional allele TNF2 was more common and the IL-1Ra*2 allele less common in the patient group. Such a finding would have supported our original hypothesis. However, the number of patients is small and so this study is underpowered. As corneal melting in the context of systemic vasculitis is extremely rare, a multicentre project would be needed to show statistical significance given the similar allele frequencies.28 We have not investigated all of the possible ligand, receptor, and antagonist alleles for TNF-α and IL-1. Our impression from this study is that the alleles of TNF-α at position –308 and within intron 2 of the IL-1Ra gene have either no or a limited role in the susceptibility to corneal melting in this patient group.

Controversy exists with regard to the role of TNF-α and IL-1Ra alleles in relation to other immune disease. Some studies have shown an increased susceptibility to autoimmune disease, including rheumatoid arthritis and Wegener's granulomatosis.16-18 However, these associations are modest and the findings were not supported by other studies.29-31 Given the multifactorial pathogenesis of these systemic diseases, it may be unlikely that a single mutation can confer increased susceptibility.18 19 There is other evidence to support a role for individual alleles in determining the severity of a given autoimmune disease.16 32 Alleles of TNF-α may alter the profile of rheumatoid disease.19 30In relation to ocular disease, variant TNF-α alleles may be responsible for altered levels of TNF-α in the tear film and a poor outcome while IL-1 alleles may predispose to chronic iritis in juvenile rheumatoid arthritis.32 33 It proved difficult to try to classify this small group of patients with corneal melting in terms of the severity of the ocular disease. Using the less common alleles TNF2 and IL-1Ra*2 as independent risk factors, we attempted to determine their influence on the disease severity using the following as examples of measures of severity: unilateral or bilateral disease, perforation requiring corneal grafting, number of corneal grafts, and final visual outcome. However, we were unable to devise a useful measure of disease severity which could be reliably applied to such a retrospective review of the patients and case notes. Not only were the systemic associations different, but so too was the local and systemic immunotherapy before and after presentation, the stage of the disease at the initial presentation, and the relative contribution of ocular surface and inflammatory disease. Our overall impression from a retrospective case note review was that the functional alleles studied were not associated with increased severity of ocular disease.

In summary, the genotype and allele frequencies of the TNF-α and IL-1Ra sites studied did not differ significantly between the patient and control groups. As a result of this study, we conclude that these polymorphic alleles play little or no part in either the susceptibility to or the severity of corneal melting in these patients with systemic vasculitis.