Pharmacologic inhibition of IκB kinase activates immediate hypersensitivity reactions in mice.

Pharmacologic inhibitors of IκB kinase (IKK), especially IKK-ß, have been developed to treat inflammatory diseases. However, their interactions with components of the NF-κB pathways are not fully known in allergic diseases. To examine whether IKK is involved in immediate hypersensitivity reactions and to determine whether counterregulatory mechanisms in the NF-κB activation system were active, we examined the role played by IKK components on mast cell degranulation using a murine ocular immediate hypersensitivity reaction model. Pharmacologic inhibition of IKK in mice caused paradoxical aggravation of the mast cell-mediated immediate hypersensitivity reaction and up-regulation in the expression of inflammatory cytokines. Downstream analyses showed that B-cell deficiency or treatment by IL-1 receptor antagonist corrected the aberrant activation of tissue-resident mast cells, which would indicate contribution by activated B cells. Analyses of co-cultures of tissue-resident mast cells showed the contribution of activated B cells to activation of mast cells and secretion of inflammatory cytokines. Aberrant activation of the NF-κB promoter in isolated B cells was induced exclusively by IKK-ß inhibition and was negated by ablating IKK-α. Aggravated mast cell degranulation by pharmacologic IKK inhibition in the murine immediate hypersensitivity reaction was corrected by B-cell-targeted inhibition of IKK-α. Thus, IKK-ß limits B-cell-mediated mast cell activation and inflammatory cytokine induction in immediate hypersensitivity by counterbalancing the activity of IKK-α.

Involvement of chymase in allergic conjunctivitis of guinea pigs.

It has been reported that chymase activity was increased in allergic conjunctivitis patients and this activity was correlated with the severity of the disease. However, the precise roles of chymase in allergic conjunctivitis are unclear, and whether chymase inhibitors are effective for allergic conjunctivitis has not been reported even in experimental animal models. In this study, the roles of chymase in the pathogenesis were evaluated using a selective chymase inhibitor, ONO-WH-236, in a guinea pig model of allergic conjunctivitis induced by cedar pollen. Sensitized guinea pigs were challenged by the pollen, followed by assessing redness and edema in the conjuntiva, and counting the frequency of eye scratching as an itch-associated response. Treatment with the ONO-WH-236 (40 and 80 mg/kg, p.o.) dose-dependently inhibited the induction of redness, edema and scratching behavior. An anti-histaminic drug, ketotifen (3 mg/kg, p.o.), also significantly inhibited conjunctivitis symptoms. Chymase activity was increased in ophthalmic lavage fluid immediately after the pollen challenge. The increase in chymase activity was inhibited by in vivo treatment with ONO-WH-236. Interestingly, increased histamine in the ophthalmic lavage fluid immediately after the challenge was also inhibited by the chymase inhibitor. Administration of human recombinant chymase by eye dropping (0.09 and 0.9 µg/eye) dose-dependently induced scratching behavior, which was inhibited by not only ONO-WH-236 but also ketotifen; however, chymase administration induced only weak redness in the conjunctiva, which was resistant to treatment with anti-histaminic drugs. In conclusion, it was suggested that chymase was released from mast cells after antigen challenge, followed by the induction of conjunctivitis symptoms through histamine release from mast cells. Thus, chymase could be a potential target for pharmacotherapy for allergic conjunctivitis.

Quantification of tear proteins and sPLA2-IIa alteration in patients with allergic conjunctivitis.

PURPOSE: Allergic conjunctivitis (AC) has been reported to induce the instability of the tear film. The tear protein and the lipid layer play important roles in maintaining the tear film. The aim of this study was to quantify the alteration of the major tear protein components and a lipid related protein secretory type IIa phospholipase A2 (sPLA2-IIa) in tears of seasonal allergic conjunctivitis (SAC) and perennial allergic conjunctivitis (PAC) patients. METHODS: Twenty-one SAC and PAC patients and thirteen normal controls completed a symptom questionnaire and underwent regular ocular examination. SAC and PAC patients were diagnosed based on the clinical presentation and elevated serum IgE levels. Schirmer test paper was used to collect tear samples from SAC and PAC patients and normal controls. Soybean trypsin inhibitor (SBTI) was used as an internal standard to analyze tear samples in 15% SDS-PAGE gel. Total tear protein and its major components from the SAC and PAC patients and normal controls were quantified by band densitometry. The major tear protein bands were determined by MALDI-TOF/TOF spectrum analysis. Western blot was used to detect the content of sPLA2-IIa in tears of allergic conjunctivitis patients and normal controls. RESULTS: Schirmer test scores were more than 10 mm in all the SAC and PAC patients and control subjects. The tear film breakup time of SAC and PAC patients was much shorter than that of the normal controls. We obtained 15 bands of tear protein by one dimensional SDS-PAGE, in which 14 bands were determined by mass-spectrum analysis. The band densitometry analysis revealed that the total tear protein concentration was much higher in SAC and PAC patients than in normal controls (p<0.05). The quantity of tear protein band 4 (serum albumin precursor), band 6 (Ig gamma-2), band 9 (leukocyte elastase inhibitor) were also significantly higher in AC patients (p<0.05). Content of sPLA2-IIa, as shown by western blot, was much higher in AC patients than in controls. CONCLUSIONS: The total tear protein concentration and some of the major tear protein components was increased in tears of SAC and PAC patients. In addition, the content of sPLA2-IIa in tears of SAC and PAC patients was elevated. The tear protein changes in SAC and PAC patients may contribute to instability of tear film.

Elevated expression and activation of matrix metalloproteinase 8 in tear fluid in atopic blepharoconjunctivitis.

PURPOSE: Matrix metalloproteinase 8 (MMP-8) is an effective collagenolytic enzyme that is associated with many ocular inflammatory diseases, such as uveitis, keratitis, and ocular rosacea. We studied the tear fluid concentration and activation of MMP-8 in atopic blepharoconjunctivitis (ABC) and the presence of the enzyme in conjunctival inflammatory cells in vivo. METHODS: Tear fluid samples were collected from 26 patients with ABC and 26 healthy controls. MMP-8 concentrations were determined by immunofluorometric assay, and its molecular forms and degrees of activation were studied by Western blotting. Conjunctival brush cytology samples from patients with ABC were used for MMP-8 immunocytochemistry. RESULTS: : The mean MMP-8 concentration was statistically significantly higher among the patients with ABC (545.6 +/- 879.3 microg/L) than among the healthy controls (50.4 +/- 62.3 microg/L, P = 0.0001). There was a statistically significant correlation between neutrophils detected in brush cytology and tear fluid MMP-8 (P = 0.032, r = 0.47). Both the control and ABC tear fluid samples contained predominantly the larger (60-80 kDa), highly glycosylated polymorphonuclear leukocyte-type MMP-8 isoform, as identified by Western blotting, but neither was found to contain the mesenchymal-type isoform. The active enzyme was in practice present only in the ABC samples. Immunostainings show the MMP-8 protein to be present in all the main inflammatory cell types within the conjunctiva. CONCLUSIONS: : A higher mean concentration and activation of MMP-8 is present in tear fluid in ABC. This finding probably reflects persistent inflammatory and collagenolytic activity associated with the disease.

Chitinase levels in the tears of subjects with ocular allergies.

PURPOSE: Chitin is abundant in the structural coatings of fungi, insects, and parasitic nematodes. The host defense against chitin-containing pathogens includes production of chitinases. An acidic mammalian chitinase (AMCase) is produced in human epithelial cells of lower airways through a TH2-specific, interleukin-13-dependent pathway and appears to be associated with allergic asthma. The role of AMCase in allergic ocular pathologies has never been studied previously. METHODS: Six patients with vernal keratoconjunctivitis (VKC), 7 patients with season allergic conjunctivitis (SAC), and 8 healthy controls (4 children and 4 adults) were enrolled in this study. AMCase activity was measured in tears, RNA was extracted from epithelial cells of the conjunctiva, and AMCase mRNA expression was evaluated by real-time polymerase chain reaction. RESULTS: AMCase activity was increased in patients affected by VKC (33.7 +/- 10.8 nmol/mL/h) and SAC (7.3 +/- 4.1 nmol/mL/h) compared with healthy controls (1.6 +/- 0.2 nmol/mL/h), and AMCase activity was higher in subjects with VKC (P = 0.0001). Receiver operating characteristic analysis showed that the sensitivity and specificity were 100%, addressing the use of AMCase assay in the biochemical diagnosis of VKC and SAC. AMCase mRNA was detected in epithelial cells of the conjunctiva, and the expression was significantly higher in VKC and SAC. CONCLUSIONS: AMCase may be an important mediator in the pathogenesis of TH2 inflammation eye diseases, suggesting a potential diagnostic and therapeutic target in these pathologies.

Trypsin inhibitory capacity in vernal keratoconjunctivitis.

PURPOSE: To study tear trypsin inhibitory capacity (T-TIC), serum trypsin inhibitory capacity (S-TIC) and their relationship to matrix metalloproteinases (MMP)-1 and -9 in patients with vernal keratoconjunctivitis (VKC). METHODS: In the first phase of the study, inactivation of alpha-1 antitrypsin (AAT) by MMP-1 and -9 was investigated in vitro. Subsequently, tear samples were collected after clinical evaluation from 14 patients with active VKC and 15 normal control subjects. Tear cytology was performed on all samples. Levels of T-TIC and S-TIC were determined by spectrophotometry, whereas levels of tear pro-MMP-1, pro-MMP-9, and active MMP-1 and -9 were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: MMP-1 and -9 inactivated AAT in vitro. S-TIC was significantly higher (P < 0.0001), and T-TIC (P < 0.0001) significantly lower in VKC samples. Tear levels of pro-MMP-1 and pro-MMP-9, and the activity of MMP-1 and -9 was significantly greater in patients with VKC than in healthy subjects (P < 0.0001). There was no significant correlation between T-TIC, MMP-1, and MMP-9 activity. In addition, T-TIC was not correlated to the total clinical score or to single clinical sign scores. CONCLUSIONS: In this study, tear trypsin inhibitory capacity was shown to be reduced and tear MMP-1 and -9 activity increased in patients with VKC. Although T-TIC did not correlate with VKC severity, a local reduced inhibitory capacity of ATT may facilitate or prolong conjunctival inflammation in VKC.

Tear levels and activity of matrix metalloproteinase (MMP)-1 and MMP-9 in vernal keratoconjunctivitis.

PURPOSE: To study levels and activity of matrix metalloproteinase (MMP)-1 and -9 and their tissue inhibitor (TIMP-1) in tears of patients with vernal keratoconjunctivitis (VKC), with and without severe corneal damage. METHODS: Tear samples were obtained from 16 patients with active VKC and 10 normal control subjects, after clinical evaluation and tear cytology. Tear levels of pro-MMP-1, pro-MMP-9, and TIMP-1 were measured by enzyme-linked immunosorbent assay (ELISA). Collagenase and gelatinase activity were measured in tears by MMP activity assays. Immunohistochemistry was performed on a fragment of superficial keratectomy from two vernal corneal ulcers. RESULTS: Tear levels of pro-MMP-1 and pro-MMP-9 were significantly increased in patients with VKC compared with control subjects (P < 0.001). MMP-1/TIMP-1 and MMP-9/TIMP-1 molar ratios were significantly increased (P < 0.001) in VKC. MMP-1 and MMP-9 activities were significantly increased in VKC tears compared with control samples (P < 0.005). MMP-9 activity correlated significantly with corneal involvement and giant papillae formation. Immunohistochemistry showed positive staining for MMP-9, fibronectin, and eosinophil cationic protein (ECP) on the superficial corneal stroma of the ulcer bed, but no inflammatory cells. CONCLUSIONS: Increased levels and activity of MMP-1 and -9 and an imbalance between MMPs and TIMP may be involved in the pathogenesis of VKC.

Gelatinase B in vernal keratoconjunctivitis.

OBJECTIVES: To investigate the expression of gelatinase B in the conjunctiva of patients with vernal keratoconjunctivitis (VKC) and the cellular source of this enzyme. METHODS: Conjunctival biopsy specimens from 12 patients with active VKC and 12 control subjects were studied using immunohistochemical techniques and a monoclonal antibody against gelatinase B. The phenotype of gelatinase B(+) inflammatory cells was examined using double immunohistochemical analysis and monoclonal antibodies against eosinophil peroxidase or macrophage CD68. Quantitative zymography was used to compare the activity of gelatinase B in conjunctival biopsy specimens from 10 patients with active VKC and 7 control subjects. RESULTS: Gelatinase B was detected in a few polymorphonuclear cells in 8 control specimens. All VKC specimens showed gelatinase B immunoreactivity in the epithelial and stromal inflammatory infiltrate. Compared with control specimens, VKC specimens showed significantly more gelatinase B-positive cells (mean +/- SD, 40.8 +/- 29.9 vs 10.3 +/- 2.4; P<.02). Most gelatinase B-positive cells were eosinophils (90.2% +/- 3.6%). Zymography revealed that gelatinase B levels in VKC specimens were significantly higher than the levels found in normal conjunctiva (3780.3 +/- 3541.0 vs 610.1 +/- 397.1 scanning units; P<.03). CONCLUSIONS: These findings suggest overexpression of gelatinase B by eosinophils in VKC specimens and participation of gelatinase B in the pathologic changes in VKC. CLINICAL RELEVANCE: Control of the release and/or activation of gelatinase B in eosinophils may provide a new therapeutic strategy for treating VKC.

Tear tryptase in vernal keratoconjunctivitis.

OBJECTIVES: To determine the tear level of tryptase (a marker of mast cell activation) in vernal keratoconjunctivitis (VKC) before and after treatment. In addition, eosinophil counts in conjunctival scrapings and ocular surface temperature before and after treatment were studied. PATIENTS AND METHODS: A total of 20 patients, 7 years or older with VKC, were included in this study. Tear samples for tryptase determination were collected before and 2 weeks after treatment with 4% disodium cromoglycate eyedrops and 0.1% fluorometholone eyedrops. In addition, conjunctival scrapings were obtained for microscopic evaluation, and measurement of the ocular surface temperature was performed before and 2 weeks after treatment. One patient was excluded because the patient did not receive topical treatment. Control tear samples were collected from 20 normal control patients for tryptase determination. RESULTS: There were 19 patients with VKC (17 males, 2 females). The age range was 7 to 17 years with a mean age of 9 years. The mean number of eosinophils prior to initiation of therapy was 11.37 eosinophils with a range of 1 to 34 per high-power field. Following treatment, the mean number of eosinophils was 3.42 eosinophils per high-power field with a range of 0 to 11 (P<.01). The mean ocular surface temperature for the right eye before treatment was 35.56 degrees C (range, 34.46 degrees C-36.50 degrees C) and after treatment was 33.53 degrees C (range, 31.13 degrees C-35.40 degrees C). For the left eye, the mean ocular surface temperature before treatment was 35.49 degrees C (range, 34.86 degrees C-36.16 degrees C) and after treatment was 33.88 degrees C (range, 32.40 degrees C-35.53 degrees C). The ocular surface temperature was found to decrease significantly following treatment (P<.001). The levels of tryptase in tears of patients with VKC were determined before and after treatment. The mean level was 16.77 ng/mL (range, <5-115 ng/mL). Following treatment with topical 4% disodium cromoglycate and 0.1% fluorometholone eyedrops, the mean level of tryptase decreased to 7.29 ng/mL (range, <5-44.1 ng/mL) (P<.05). CONCLUSIONS: Patients with severe VKC had high levels of tryptase in tears. Following treatment, the level of tryptase in tears decreased significantly.

The role of histamine H(1) receptors in late-phase reaction of allergic conjunctivitis.

The role of histamine H(1) receptors in the late-phase reaction of allergic conjunctivitis was studied using histamine H(1) receptor-deficient mice. To clarify the eosinophil infiltration, which is a reliable indicator of late-phase reaction, eosinophil peroxidase activity in the conjunctiva was measured. Mice were actively immunized with ovalbumin, and conjunctivitis was induced by topical instillation of ovalbumin. A significantly high eosinophil peroxidase level in the conjunctiva was observed in sensitized wild-type mice, whereas sensitized histamine H(1) receptor-deficient mice showed no significant increase in the conjunctival eosinophil peroxidase level. In addition, the elevation of eosinophil peroxidase level observed in sensitized wild-type mice was significantly antagonized by pretreatment with anti-P-selectin antibody. From these findings, it was concluded that eosinophil infiltration into the conjunctival tissue in late-phase reaction of allergic conjunctivitis is mediated by P-selectin stored in endothelial cells via histamine H(1) receptors.

Tear chymase in vernal keratoconjunctivitis.

PURPOSE: To determine the levels of mast cell chymase and tryptase activity in the tears of patients with vernal keratoconjunctivitis (VKC). METHODS: Subjects were 38 VKC patients and 18 healthy controls whose chymase and tryptase activity in tears was measured by enzyme assay. VKC severity was quantified based on the following clinical signs: papillary hypertrophy, conjunctival hyperemia, edema, punctate keratitis, Trantas dots, and mucus production. Of the 38 VKC patients, the degree of disease severity was mild in 13, moderate in 18, and severe in 7. RESULTS: Mean chymase activity and standard deviation in tears was 0.23+/-0.07mU in mild VKC, 0.68+/-0.22mU in moderate VKC, 1.91+/-0.71 mU in severe VKC, and 0.11+/-0.05 mU in healthy controls. The increase in all VKC stages was statistically significant compared to that in healthy control. The degree of chymase activity in tears correlated significantly with VKC severity (r = 0.9245, p < 0.001). High tryptase activity was also detected in the tears of VKC patients, although increased tryptase activity in tears did not correlate with disease severity (r = 0.1999). CONCLUSIONS: Chymase activity in tears may thus be a sensitive marker for determining the severity of VKC.

Suppressive effect of antiflammin-2 on compound 48/80-induced conjunctivitis. Role of phospholipase A2s and inducible nitric oxide synthase.

Phospholipase A2s (PLA2s) are a family of esterases that initiate the arachidonic acid cascade, which results in the production of numerous inflammatory mediators. We investigated the expression of Group I and II PLA2 proteins and Group II mRNA in normal conjunctivae and in the conjunctivae of mice with compound 48/80-induced conjunctivitis. Conjunctivitis was induced in C57BL/6 mice by topical instillation of compound 48/80 (C48/80). Mice were then treated with corticosteroid (Pred Forte), antiflammin-2 (AF2, a synthetic peptide that inhibits PLA2), or a placebo (Dacriose, an isotonic, buffered, sterile eye irrigating solution). Low levels of PLA2s were detected on the epithelium of normal conjunctivae. One hr after C48/80 instillation, the expression of PLA2s appeared and increased in the substantia propria, peaked at 6 hr, and returned to baseline 72 hr later. Compared to the placebo, the conjunctivitis was moderate in the AF2-treated group and mild in Pred Forte-treated group. The expression of PLA2s was suppressed in mice treated with Pred Forte and AF2. iNOS mRNA was also diminished in the AF2- and Pred Forte-treated groups. The mechanisms by which anti-allergic medications suppress conjunctivitis may involve the inhibition of PLA2s and iNOS.

The practical detection of mmp-9 diagnoses ocular surface disease and may help prevent its complications.

PURPOSE: To evaluate the importance and practicality of testing for matrix metalloproteinase 9 (MMP-9) in dry eye and ocular surface disease. This enzyme, which can cause tissue damage, seems also to be the most reliable diagnostic indicator of ocular surface disease. METHODS: Enzyme-linked immunosorbent assay, polymerase chain reaction, diffusion, and InflammaDry, a new rapid immunoassay by RPS (Rapid Pathogen Screening Inc). RESULTS: MMP-9 measurement is sensitive and accurate for diagnosing dry eye and ocular surface disease and compares favorably in both sensitivity and specificity against the existing methods of dry eye diagnosis. Abnormal elevations of MMP-9 may predict post-laser in situ keratomileusis complications and refractive complications such as epithelial ingrowth and corneal ulceration. The presence of elevated MMP-9 on the ocular surface will identify those patients who should receive antiinflammatory therapy, such as cyclosporine, and may predict those patients who will respond to this therapy. CONCLUSIONS: A rapid in-office test that is sensitive for identifying inflammatory dry eye and ocular surface disease may facilitate better preoperative management of the ocular surface. Optimization of the ocular surface perioperatively would be expected to reduce complications from laser in situ keratomileusis and other surgeries that often make the underlying disease worse. This test may also indicate the need for antiinflammatory therapies, such as cyclosporine or steroids, and also may predict those patients who are more likely to respond.

Increased conjunctival expression of protease activated receptor 2 (PAR-2) in seasonal allergic conjunctivitis: a role for abnormal conjunctival epithelial permeability in disease pathogenesis?

AIMS: Aeroallergen exposure to the conjunctival epithelium in seasonal allergic conjunctivitis (SAC) may induce a cellular stress response that disrupts the barrier properties of the conjunctival epithelium, resulting in allergic disease. Whether such changes occur in SAC is unknown. Epithelial permeability is known to be increased when protease activated receptor 2 (PAR-2) is activated. We evaluated the expression of PAR-2 in patients with SAC-in-season (SACS) and compared it with control non-atopic subjects or those with out-of-season allergic conjunctivitis (OSAC). METHODS: Six SACS, eight normal and four OSAC specimens were examined immunohistochemically for PAR-2 and quantified in a masked fashion for the percentage of epithelia stained for each marker using Image-J software. Conjunctival epithelial heights were measured in all groups to confirm the presence of allergic eye disease. RESULTS: Mean percentage staining of PAR-2 was significantly greater in SACS that in normal specimens (73.4 ± 15.4% vs 32.8 ± 30.0%, p=0.038) or in OSAC (73.4 ± 15.4% vs 1.4 ± 2.2%, p=0.01). Mean conjunctival epithelial height was significantly raised in SACS (63.8 ± 9.0 µm) versus controls (44.7 ± 11.2 µm) (p=0.003, unpaired t test). CONCLUSIONS: Conjunctival epithelial PAR-2 is significantly upregulated in SAC. This supports the view that disruption of the barrier properties of the conjunctival epithelium is an important event in SAC pathogenesis.

Transfer of a T-helper type 2 clone into conjunctiva induces corneal damage in mice.

PURPOSE: We established a T-helper Type 2 (Th2) clone-induced conjunctival eosinophilia model by injecting D10.G.4.1 (D10) cells, a murine Th2 clone, and conalbumin, its specific antigen, into conjunctiva of AKR/J mice. Using this model, we investigated the effect of a coinjection of D10 cells and conalbumin into conjunctiva on corneal damage. METHODS: Corneal fluorescein staining scores and eosinophil peroxidase (EPO) activity in conjunctiva were measured after coinjection of D10 and conalbumin into conjunctiva, and the effects of cyclosporine A, betamethasone, and anti-interleukin-5 antibody on staining scores and EPO activity were examined. RESULTS: Coinjection of D10 and conalbumin induced an increase of the corneal fluorescein staining score after 24, 48, and 96 hours and 10 days. EPO activity in conjunctiva increased time-dependently until 24 hours after coinjection. The increase in the staining score followed the time dependent increase in EPO activity. The instillation of cyclosporine A, an inhibitor of cytokine production from T-cells, and betamethasone significantly inhibited the increase in corneal fluorescein score and EPO activity. Intraperitoneal administration of anti-interleukin-5 monoclonal antibody, which inhibits the infiltration of eosinophils into the conjunctiva, completely inhibited the increase in staining score. CONCLUSION: The transfer of the Th2 clone into the murine conjunctiva induced corneal damage, which may have been caused by Th2 cell-produced interleukin-5 that mediated the activation of eosinophils.

Matrix metalloproteases in vernal keratoconjunctivitis, nasal polyps and allergic asthma.

BACKGROUND: Allergic conditions in different organs share many similarities in their inflammatory response. Vernal keratoconjunctivitis (VKC), asthma and nasal polyps exhibit several similar, but site-specific mucosal structural changes. The aim of the study was to investigate whether matrix metalloproteases contribute to different tissue remodelling aspects in different organs. METHODS: Mucosal biopsies were obtained from conjunctiva of healthy donors, tarsal conjunctiva of vernal patients, bronchi of non-asthmatic subjects, bronchi of mild stable asthmatic patients, nasal mucosa of non-allergic donors and nasal polyps of allergic patients. Distribution of metalloprotease-1, -3, -9, -13, tissue inhibitor of metalloproteases-1, collagens I and III and the presence of eosinophils and CD4+ cells were evaluated by immunohistochemistry. RESULTS: Collagens were highly diffuse in the giant papillae of VKC and in nasal polyps, and yet less increased in the subepithelium of asthmatic patients. Immunostaining for metalloprotease-1, -3, -9 and -13 was significantly higher in VKC compared with normal conjunctiva. Metalloprotease-9 staining was higher in the stroma of polyps vs. normal nasal mucosa, and only metalloprotease-13 was significantly more expressed in asthmatic vs. non-asthmatic subjects. Metalloprotease-9 immunostaining was more intense in vernal compared with other tissues. In all pathological tissues, metalloprotease-9-positive staining was in association with eosinophils and CD4+ cells. CONCLUSIONS: Expression of metalloproteases may play an important role in inducing the structural changes seen in VKC, nasal polyps and asthma. Tissue remodelling and gelatinase immunoexpression was more dramatic in giant papillae of vernal patients compared with other tissue sites of chronic allergic inflammation.

Histaminase activity in patients with vernal keratoconjunctivitis.

PURPOSE: To investigate the activity of histamine-degradating enzymes in tears and plasma of patients with vernal keratoconjunctivitis (VKC). METHOD: Tear and plasma samples were collected from patients with VKC and from age-matched control subjects. Histamine was measured by enzyme-linked immunosorbent assay in acid samples treated with perchloric to deactivate histaminase and in untreated samples. Tear cytology, skin test reactivity to histamine, and the sum clinical score of allergic signs and symptoms in patients with VKC also were evaluated. Nineteen patients with active VKC and six age-matched control subjects participated in this study. RESULTS: In untreated samples, tear histamine (mean +/- standard error of the mean) was 11.15 +/- 2.16 ng/ml in patients with VKC and 0.855 +/- 0.225 ng/ml in control tears (P < 0.001). In treated samples, mean tear histamine was 22.25 +/- 4.17 ng/ml in patients with VKC versus 10.64 +/- 2.85 ng/ml in control subjects (not statistically different). The ratio of histamine in treated to untreated samples (indicating histaminase activity) was significantly lower in patients with VKC (2.30 +/- 0.263) than in control subjects (17.57 +/- 5.97; P = 0.0001). Plasma histamine levels in untreated and treated samples were significantly higher in patients with VKC (untreated, 2.23 +/- 0.334 ng/ml; treated, 4.37 +/- 0.357 ng/ml) than in control subjects (untreated, 0.254 +/- 0.068, P = 0.0002; treated, 2.96 +/- 0.171 ng/ml, P = 0.0082). The enzymatic breakdown of histamine (treated/ untreated) in plasma was significantly decreased in patients with VKC (2.54 +/- 0.447) compared with control subjects (14.78 +/- 4.86; P = 0.0012). Skin reactivity to histamine was not increased in VKC. Tear histamine levels were significantly correlated to tear lymphocyte content in the general population and to tear basophils in the patients with tarsal-vernal VKC only. An increased number of tear eosinophils were correlated with elevated enzyme activity only in patients with tarsal-vernal VKC and to the clinical score only in limbal-vernal patients. CONCLUSION: The enzymatic degradation of histamine was significantly decreased in patients with VKC compared with control subjects in both tears and plasma, suggesting that this dysfunction may be a primary factor in the pathophysiology of VKC.

Tear tryptase levels and allergic conjunctivitis.

We measured tryptase, a neutral protease stored in the secretory granules of mast cells, by solid-phase radioimmunoassay in tears of 12 subjects with vernal keratoconjunctivitis (VKC) during remission phases, nine subjects with seasonal or perennial allergic conjunctivitis, and eight healthy controls. Mean values of tear tryptase levels were significantly (P < 0.02) increased in VKC patients (14.5 +/- 13 micrograms/l) when compared to those measured in patients with seasonal or perennial allergic conjunctivitis (0.6 +/- 0.1 microgram/l) and in controls (3.3 +/- 3.2 micrograms/l). In subjects with allergic conjunctivitis, the levels of tryptase, almost undetectable before allergen conjunctival challenge, showed a significant increase in the challenged eye 20 min-but not 6 h-after provocation in 5/9 cases. Our results indicate that VKC a severe ocular disease characterized by an increased number and abnormal distribution of mast cells in the conjunctiva, also shows elevated levels of tryptase in tears even during remission phases. Evidence of mast-cell activation, as revealed by a significant increase of tryptase levels in tears, in documented during the early-phase reaction, but not during the late-phase reaction, of allergic conjunctivitis patients challenged topically by specific allergen.

Group IIA phospholipase A2 content of tears in patients with atopic blepharoconjunctivitis.

BACKGROUND: To determine the concentration of group IIA phospholipase A(2) (GIIAPLA(2)) in tears of patients with atopic blepharoconjunctivitis (ABC), and to compare it with the GIIAPLA(2) concentration of tears in age-matched healthy controls. METHODS: The diagnosis of ABC was confirmed with a positive skin prick test and the presence of atopic dermatitis in lids. Conjunctival brush cytology was taken, and the cells including eosinophils, neutrophils, lymphocytes, squamous epithelial cells, columnar epithelial cells, metaplastic changes and the goblet cells were calculated separately. The GIIAPLA(2) concentration of tears was measured with a time-resolved fluoroimmunoassay in 29 patients with ABC (mean age 36.3+/-12.7 years) and 29 normal subjects (mean age 37.0+/-12.0 years). RESULTS: The GIIAPLA(2) concentration of tears in patients with ABC was 43.8+/-33.0 microg/ml, and in normal subjects it was 67.1+/-23.3 microg/ml. The difference was statistically significant (p=0.0018). The concentration of GIIAPLA(2) of tears was lowest in the subgroup of patients with ABC and dry eye (25.8()+/-23.6 microg/ml), whereas it was only slightly decreased in patients with ABC and normal tear secretion (56.6+/-33.3 microg/ml). The difference between these two subgroups was statistically significant (p=0.011). There was no statistically significant correlation between the GIIAPLA(2) concentration of tears and the quantity of different conjunctival cells gathered by the brush cytology. However, an almost significant correlation was found between the GIIAPLA(2) concentration in tears and conjunctival eosinophils. CONCLUSIONS: The results indicate that in patients with ABC the GIIAPLA(2) content of tears was decreased, without any dependence on the quantity of different conjunctival cells.

Active matrix metalloproteinases in the tear fluid of individuals with vernal keratoconjunctivitis.

Corneal epithelial lesions distinguish vernal keratoconjunctivitis (VKC) from other ocular allergic diseases. Such lesions result from degradation of the corneal epithelial basement membrane, which comprises mostly type IV collagen and laminin. Matrix metalloproteinase 2 (MMP-2) and MMP-9 catalyze the degradation of these 2 extracellular matrix proteins. The possible role of MMP-2 and MMP-9 in the pathogenesis of corneal lesions associated with VKC was investigated by assaying tear fluid for the presence of these enzymes. Tear fluid was collected from 6 eyes of 6 patients with active VKC, 14 eyes of 14 patients with active allergic conjunctivitis, and 6 eyes of 6 nonallergic healthy volunteers. Gelatin zymography revealed that the tear fluid of healthy volunteers contained inactive proforms of both MMP-2 and MMP-9 but not the active forms of these enzymes. Active forms of MMP-2 or MMP-9 were detected in a minority of patients with allergic conjunctivitis. However, with the exception of one individual for whom active MMP-9 was not detected, tear fluid from all patients with VKC contained both proforms and active forms of MMP-2 and MMP-9. These results implicate MMP-2 and MMP-9 in the pathogenesis of corneal epithelial disorders associated with VKC.