Tear fluid concentration of mmp-8 is elevated in non-allergic eosinophilic conjunctivitis and correlates with conjunctival inflammatory cell infiltration.

BACKGROUND: To investigate tear fluid concentration of matrix metalloproteinase 8 (MMP-8) and its relation to conjunctival inflammatory cell infiltration in persistent non-allergic eosinophilic conjunctivitis (NAEC). METHODS: Two groups were included: 26 consecutive adult patients with NAEC (conjunctival eosinophils at least 1+ [1-10 eosinophils/slide], skin prick test [SPT] to common allergens negative), and 26 asymptomatic adult persons (no conjunctival eosinophils, SPT negative). MMP-8 tear fluid concentrations were determined by immunofluorometric assay, and conjunctival brush cytology samples from NAEC patients were used for MMP-8 immunocytochemistry. Gelatin zymography was used to illustrate proteolytic activity within the tear fluid samples. RESULTS: The mean MMP-8 concentration was significantly higher among NAEC patients (214.3 +/- 327.7 microg/l) than among healthy persons (50.4 +/- 62.3 microg/l, P < 0.0001). In the NAEC patients, tear fluid MMP-8 correlated with the numbers of conjunctival neutrophils (r = 0.66, P = 0.0002) as well as with goblet cells and columnar epithelial cells (r = 0.54 for both, P = 0.045), but not with the lymphocyte numbers (r = -0.36, P = 0.0741). By immunocytology, MMP-8 protein could also be detected in vivo in the inflammatory cell population within the conjunctiva. Zymography revealed that proteolysis was significantly higher in the NAEC group, and activated enzymes were practically found only in the NAEC group. CONCLUSIONS: The results showed that NAEC is an inflammatory condition characterized by increased tear fluid MMP-8 levels, probably derived from both inflammatory and structural conjunctival cells. The increased proteolytic activity in NAEC patients may indicate risk of conjunctival structural changes (remodeling).

Group IIA phospholipase A(2) concentration of tears in patients with ocular rosacea.

PURPOSE: To determine the concentration of group IIA phospholipase A(2) (GIIAPLA(2)) in tears of patients with ocular rosacea, and to compare it with GIIAPLA(2) concentration in tears of age-matched healthy controls. METHODS: The GIIAPLA(2) concentration in tears was measured with a time-resolved fluoroimmunoassay in 21 patients with ocular rosacea (mean age 55.6+/-9.2 years) and in 21 normal subjects (mean age 53.4+/-8.2 years). Conjunctival brush cytology was carried out and eosinophils, neutrophils, lymphocytes, squamous epithelial cells, columnar epithelial cells, metaplastic changes and goblet cells were calculated separately. RESULTS: The GIIAPLA (2) concentration in tears was statistically significantly lower in patients with ocular rosacea (31.0+/-18.4 microg/ml, p=0.0099) and, more specifically, in patients who had dry eye (25.8+/-15.1 microg/ml, p=0.0034), compared to that in normal controls. There was no correlation between the GIIAPLA (2) content of tears and the conjunctival cells collected by the brush cytology. CONCLUSION: The tears of patients with dry eye symptoms due to ocular rosacea have decreased GIIAPLA (2) content. The pathogenic importance of this finding is discussed.

Involvement of cyclooxygenase-2 in rat models of conjunctivitis.

PURPOSE: Using two animal models to determine which isoform of cyclooxygenase (COX), constitutive COX-1 or inducible COX-2, is involved in the progression of anterior ocular inflammation. METHODS: Lambda-carrageenan (500 mg/eye) or bacterial lipopolysaccharide (LPS; 3 mg/eye) was injected into rat conjunctiva to induce conjunctivitis. Vascular permeability in inflamed conjunctiva was measured by uptake of systemic Evans blue. Changes in mRNA for COX-1 and COX-2 in conjunctiva were detected by RT-PCR. Changes in COX-2 protein were detected by immunoblotting after immunoprecipitation. To assess involvement of COX-2 in carrageenan and LPS-induced conjunctivitis, NS-398 (a selective COX-2 inhibitor) or indomethacin (non-selective COX inhibitor) was topically administrated at 15 and 30 minutes before inflammatory stimulator-injection. RESULTS: In the carrageenan-injected model, the dye content of conjunctiva (12.4 +/- 2.8 mg/eye) was significantly increased 4 hours after injection compared to saline-injected control rats (3.7 +/-1.1 mg/eye). mRNA for COX-2 was significantly increased by 2 hours and gradually increased until 24 hours; COX-1 mRNA did not show major changes until 24 hours after injection. COX-2 protein was markedly elevated 4 hours after injection of carrageenan. COX-2 protein levels were well correlated with increased mRNA levels. In the LPS-injected model, the dye content of conjunctiva (5.8 +/- 1.2 mg/eye) was significantly increased 4 hours after injection compared to saline-injected control rats (3.1 +/- 0.6 mg/eye). Expression of COX-2 mRNA was increased 1 hour after injection, peaked at 2 hours, and decreased at 4 hours. mRNA for COX-1 did not change by 24 hours. COX- 2 protein increased 2 hours after injection of LPS. COX-2 protein levels were well correlated with increased mRNA. Topical administration of 1% NS-398 exhibited strong inhibition of dye-leakage into conjunctiva 4 hours after injection of carrageenan or LPS, since 59% or 83% of dye-uptake were inhibited, respectively. 1% of indomethacin eye drops showed only a minimal effect. CONCLUSIONS: These results suggest that the mechanism for anterior ocular inflammation may be due to up-regulation of COX-2.

The cell-layer- and cell-type-specific distribution of GalNAc-transferases in the ocular surface epithelia is altered during keratinization.

PURPOSE: It has been hypothesized that the biosynthesis of O-linked glycans on proteins, particularly on the highly O-glycosylated mucins, by the corneal and conjunctival epithelium is necessary for the protection and maintenance of a healthy ocular surface. The initial step in O-glycosylation is the enzymatic addition of N-acetyl galactosamine (GalNAc) to serine and threonine residues by a large family of polypeptide GalNAc-transferases (GalNAc-Ts). The purpose of this study was to determine the cellular distribution of GalNAc-Ts in the normal ocular surface epithelia and to compare their distribution with that in pathologically keratinized conjunctival epithelia. METHODS: Five conjunctival biopsy specimens and 5 corneas from normal individuals, and 14 conjunctival specimens from patients with ocular cicatricial pemphigoid (OCP) were used. Based on the histologic characteristics of their epithelia, OCP specimens were divided into two groups: less advanced, nonkeratinized (n = 6), and late-stage, keratinized (n = 8). Five monoclonal antibodies raised against the GalNAc-T1, -T2, -T3, -T4, and -T6 isoenzymes, were used for immunofluorescence microscopic localization according to standard protocols. RESULTS: Immunohistochemical studies revealed the presence of GalNAc-T2, -T3, and -T4 isoforms within the stratified epithelium of the cornea and the conjunctiva. The GalNAc-T4 isoenzyme was found in the apical cell layers, whereas GalNAc-T2 was found in the supranuclear region of the basal cell layers of both cornea and conjunctiva. GalNAc-T3 was distributed throughout the entire ocular surface epithelium, whereas GalNAc-T1 was found in scattered cells in conjunctiva only. Binding of antibody to GalNAc-T6 was restricted exclusively to conjunctival goblet cells. There were distinct alterations in expression patterns of GalNAc-T2, -T6, and -T1 in nonkeratinized OCP epithelia compared with normal epithelia. Both GalNAc-T2 and -T6 were expressed in the apical stratified epithelia, and T1 was detected in all cell layers in five of six biopsy specimens. By comparison with nonkeratinized OCP epithelia, a marked reduction in the binding of GalNAc-T antibody was observed in the late-stage keratinized conjunctival epithelia of patients with OCP. In all samples, apical GalNAc-T2 was absent, and GalNAc-T6 was entirely absent. Only one of eight samples was positive for GalNAc-T1. CONCLUSIONS: The presence of GalNAc-T isoenzymes in the human corneal and conjunctival epithelia is cell-layer and cell-type specific. The increased distribution of GalNAc-Ts observed in early stages of the keratinization process in patients with OCP suggests a compensatory attempt of the ocular surface epithelium to synthesize mucin-type O-glycans to maintain a wet-surface phenotype. This early increase in isoenzymes in nonkeratinized OCP epithelia is reduced as keratinization proceeds in the disease.

Effective treatment of ligneous conjunctivitis with topical plasminogen.

PURPOSE: The etiology of ligneous conjunctivitis is now known to be due to an underlying type 1 plasminogen deficiency. We hereby report the clinical features of three cases and their response to topically administered plasminogen. DESIGN: Observational case series. METHODS: Two Caucasian females aged 5 years and an 18-month male of north African descent presented with a membranous conjunctivitis, which recurred after surgical excision. Case 1 presented before the association with plasminogen deficiency was known with a bilateral chronic membranous mucopurulent conjunctivitis from the age of 14 months associated with bronchiolitis and gingival hyperplasia. A diagnosis of ligneous conjunctivitis was entertained and a number of drops were instituted. At the age of 4 years plasminogen levels were ordered. Case 2 presented at the age of 4 years with a unilateral chronic membranous conjunctivitis. Plasminogen levels were requested as soon as a diagnosis of ligneous conjunctivitis was suspected. Case 3 was born with congenital hydrocephalus. Conjunctivitis was treated with antibiotics from the age of 1 month. He presented to the eye clinic at the age of 5 months when a clinical diagnosis of ligneous conjunctivitis was entertained and treated with a number of medications. Plasminogen levels were available at 9 months of age. RESULTS: The two female patients returned plasminogen levels of 0.25 U/ml and 0.3 U/ml, well below the normal level of 0.7-1.0 U/ml. Functional plasminogen levels in the male infant were not recordable with plasminogen antigen levels of 0.125 U/ml (normal range, 0.52-1.82). All cases have responded well to excision of the membranes and institution of topical plasminogen drops. There has been no recurrence with more than 12 months' follow-up. CONCLUSIONS: With the knowledge of the etiology of ligneous conjunctivitis, efforts are underway to identify the best method of delivery of plasminogen. Topical plasminogen concentrate from fresh frozen plasma holds promise as the definitive treatment for this chronic membranous conjunctivitis

Homozygous and compound-heterozygous type I plasminogen deficiency is a common cause of ligneous conjunctivitis.

Severe type I plasminogen deficiency has been recently linked to ligneous conjunctivitis, a rare and uncommon form of chronic conjunctivitis. In this study, eight unrelated ligneous conjunctivitis patients living in different parts of the world were examined. All affected subjects from which plasma was available displayed absent or markedly reduced plasminogen antigen and plasminogen functional activity. Molecular genetic studies of seven patients identified a Lys19-->Glu mutation in two boys in a homozygous state, and in two girls in a compound-heterozygous state in which the second plasminogen gene carried a missense (Arg134-->Lys) and a nonsense mutation (Cys133--> Stop), respectively. A fifth patient was shown to be homozygous for a frameshift mutation in plasminogen exon 14 (Gly565ins-G). In two unrelated subjects with ligneous conjunctivitis no mutations in the plasminogen gene were identified. Our results suggest that the Lys19-->Glu mutation is the most prevalent mutation in the plasminogen gene of patients with ligneous conjunctivitis.

Expression of gelatinase B in trachomatous conjunctivitis.

BACKGROUND/AIMS: Gelatinase B is a matrix metalloproteinase involved in extracellular matrix (ECM) breakdown often associated with scarring and other pathological disorders. It was investigated whether gelatinase B is involved in the pathogenesis of ECM degradation associated with trachomatous conjunctivitis. METHODS: Conjunctival biopsy specimens obtained from six patients with active trachoma, six patients with active vernal keratoconjunctivitis (VKC), and seven control subjects were studied. Immunohistochemical techniques and a specific monoclonal antibody against human gelatinase B were used, and a monoclonal antibody against macrophage CD68 to identify mononuclear cells with gelatinase B immunoreactivity. In addition, quantitative zymography was used to compare the activity of gelatinase B in conjunctival biopsy specimens from seven patients with active trachoma and seven control subjects. RESULTS: Gelatinase B was detected by immunohistochemistry only in polymorphonuclear cells located in the vascular lumens in three normal conjunctival biopsy specimens. In all trachoma specimens and in five VKC specimens, gelatinase B was localised in monocyte/macrophage cells, positive for the CD68 marker, and in polymorphonuclear cells. The majority of the latter cell type was located in intravascular spaces. Compared with VKC specimens, trachoma specimens showed significantly more immunoreactive gelatinase B monocyte/macrophage cells (52.3 (21.9) v 8.2 (6.4); p <0.001) and polymorphonuclear cells (23.2 (14.2) v 6.3 (5.4); p = 0. 013). Activated macrophages with giant cell morphology clearly stained with the gelatinase B specific monoclonal antibody were observed in trachoma specimens. Zymography revealed that gelatinase B levels in trachoma specimens were significantly higher than the levels found in normal conjunctiva (1739.6 (1078.3) v 609.3 (395.9) scanning units; p = 0.0127). CONCLUSIONS: The increased activity of gelatinase B and numbers of inflammatory cells containing gelatinase B in trachoma specimens suggest that this enzyme plays a part in the pathogenesis of conjunctival scarring in trachoma.

Polymorphonuclear elastase in patients with homozygous type I plasminogen deficiency and ligneous conjunctivitis.

Laboratory studies were performed on six female patients (ranging in age from 1 to 31 years) with ligneous conjunctivitis, which we regard as a systemic condition consisting of ligneous conjunctivitis and other pseudomembranous lesions. Plasminogen levels were severely reduced in all six patients; five patients were homozygous, and one patient was double heterozygous for type I plasminogen deficiency. Of family members tested, 11 of 12 parents and two of six siblings tested were diagnosed as heterozygous. No thrombotic episodes had occurred in any of the patients. Polymorphonuclear (PMN) elastase protein levels were markedly elevated in all, significantly more so in the homozygous patients (range 88 to 335 ng/mL; normal range, 20+/-10 ng/mL) than in the heterozygous patient (58 ng/mL). Of 11 parents examined, only 1 mother had normal PMN elastase (27 ng/mL, with plasminogen antigen 60% and plasminogen functional activity 86%), whereas values were moderately elevated (range 42 to 110 ng/mL) in the other 10 parents examined. After plasminogen substitution, PMN elastase levels consistently decreased but did not reach normal values. We interpret our findings as indicating that non-plasmin-induced fibrinolytic processes, possibly mediated via elastase, may be intensified in patients with plasminogen deficiency.

The level of tryptase in human tears. An indicator of activation of conjunctival mast cells.

Tryptase, a neutral endoprotease, is secreted by activated mast cells in human tissues. Tryptase levels in various body fluids have been used as an indicator of mast cell activation. The authors determined tryptase levels in unstimulated tears collected from the following groups of patients: (1) normal control, (2) nonallergic ocular inflammation, (3) asymptomatic seasonal allergic conjunctivitis, (4) symptomatic seasonal allergic conjunctivitis, (5) vernal conjunctivitis, and (6) contact lens-associated giant papillary conjunctivitis. They also assessed the release of tryptase into the tear fluid after provoking the conjunctiva with (7) allergens, (8) compound 48/80, and (9) rubbing. Tryptase levels were elevated in tears of patients with active ocular allergy and also increased after provoking the conjunctiva with allergens in atopic subjects and with compound 48/80 and rubbing in nonatopic subjects. Tryptase levels in tear fluid may prove useful as a clinical indicator of mast cell involvement in ocular allergic disorders. In provocation experiments, tryptase levels may be used to evaluate and compare different mast cell stabilizing agents.

Blood histaminase in phlyctenulosis.

Blood Histaminase estimations were done in 42 cases of phlyctenulosis and 25 normal subjects. Significantly higher level of blood Histaminase was found in patients of phlyctenulosis, which may be a consequence of its induction secondary to increased release of histamine in the early phase of disease.

Lysozyme content of tears in normal subjects and in patients with external eye infections.

We studied the lysozyme content of tears in 267 subjects (521 eyes), including 241 healthy subjects, 7 patients (14 eyes) with bilateral blepharitis, 8 patients (12 eyes) with conjunctivitis, and 11 patients (16 eyes) with keratitis. The concentration of lysozyme in the tears rises with age between childhood and maturity. The highest values were seen in the age group of 21-40 years, and a decrease of lysozyme concentration occurred with an increase in age from 30-40 years. The mean lysozyme content of tears was 1,768 micrograms/ml in healthy subjects; no significant differences occurred between the sexes. Patients with blepharitis, conjunctivitis, and keratitis had normal mean lysozyme content of tears. The tears of patients with herpes simplex keratitis had low lysozyme values.

Immunoassay of tear lysozyme in conjunctival diseases.

The tear lysozyme content in 111 normal subjects and in 159 patients with various conjunctival diseases was determined by a single radial immunodiffusion technique. Tear lysozyme level in normal people was 1.33/mg/ml. (SI conversion: mg/ml = g/l.) The mean tear lysozyme levels in patients with chronic irritative conjunctivitis (0.97 mg/ml) and nutritional deficiency with epithelial xerosis (0.76 mg/ml) were significantly lower than in the normal controls. The mean tear lysozyme levels in tears from patients with vernal conjuctivitis (1.20 mg/ml), phlyctenular conjunctivitis (1.10 mg/ml), and acute bacterial conjunctivitis (1.48 mg/ml) were not significantly different from those in the normal controls. Superimposition of acute bacterial conjunctivitis on trachoma did not alter the low tear lysozyme level that existed before in these patients.

Chronic ulcerative conjunctivitis in a patient with alpha-1-antitrypsin deficiency.

A 62-year-old woman had chronic bilateral conjunctival ulceration of the palpebral and bulbar conjunctivae. Conjunctival scrapings for viral, chlamydial, and bacteriologic studies were unrevealing. A conjunctival biopsy specimen was taken and submitted for histopathologic and immunofluorescent studies. Hematoxylin-eosin-stained tissue sections showed lymphocytes, plasma cells, and eosinosphils. Laboratory findings showed serum alpha-1-antitrypsin deficiency. alpha-1-Antitrypsin has a molecular weight of approximately 60,000 and inhibits a number of proteolytic enzymes including cellular trypsin, elastase, collagenase, and proteases. The deficiency of alpha-1-antitrypsin may have caused such enzymes to perpetuate the tissue damage, thus eventuating in chronic ulcerative conjunctivitis. The association of deficient alpha-1-antitrypsin with chronic ulcerative conjunctivitis could thus have been coincidental or a contributing factor to the conjunctival disease.