Neutrophil collagenase, gelatinase, and myeloperoxidase in tears of patients with stevens-johnson syndrome and ocular cicatricial pemphigoid

Abstract

Objective: To investigate the levels of matrix metalloproteinases (MMPs), myeloperoxidase (MPO), and tissue inhibitor of metalloproteinase-1 (TIMP-1) in tears of patients with Stevens-Johnson syndrome (SJS) and ocular cicatricial pemphigoid (OCP).

Design: Prospective, noninterventional cohort study.

Participants: Four SJS patients (7 eyes), 19 OCP patients (37 eyes), and 20 healthy controls who underwent phacoemulsification (40 eyes).

Methods: Tear washes were collected from all patients and were analyzed for levels of MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, MPO, and TIMP-1 using multianalyte bead-based enzyme-linked immunosorbent assays. Total MMP activity was determined using a fluorometric assay. Correlation studies were performed between the various analytes within study groups.

Main outcome measures: Levels of MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, MPO, and TIMP-1 (in nanograms per microgram of protein) and total MMP activity (in relative fluorescent units per minute per microgram of protein) in tears; MMP-8-to-TIMP-1 ratio; MMP-9-to-TIMP-1 ratio; and the correlations between MMP-8 and MMP-9 and both MMP and MPO.

Results: MMP-8, MMP-9, and MPO levels were elevated significantly in SJS and OCP tears (SJS>OCP) when compared with controls. The MMP activity was highest in SJS patients, whereas OCP patients and controls showed lower and similar activities. The TIMP-1 levels were decreased in SJS and OCP patients when compared with those in controls, with levels in OCP patients reaching significance. The MMP-8-to-TIMP-1 and MMP-9-to-TIMP-1 ratios were markedly elevated in SJS and OCP tears (SJS>OCP) when compared with those of controls. Across all study groups, MMP-9 levels correlated strongly with MMP-8 and MPO levels, and MMP-8 correlated with MPO, but it did not reach significance in SJS patients. There was no relationship between MMP-7 and MPO.

Conclusions: Because MMP-8 and MPO are produced by inflammatory cells, particularly neutrophils, the correlation data indicate that they may be the common source of elevated enzymes, including MMP-9, in SJS and OCP tears. Elevated MMP-to-TIMP ratios and MMP activity suggest an imbalance in tear MMP regulation that may explain the predisposition of these patients to demonstrate corneal melting and chronic complications associated with persistent inflammation. Myeloperoxidase in tears may be a sensitive and specific marker for the quantification of ocular inflammation.

Figure 1

Bar graph showing the mean levels of neutrophil collagenase (MMP-8), gelatinase B (MMP-9) and myeloperoxidase (MPO) in Stevens-Johnson syndrome (SJS) (n = 7 eyes), ocular cicatricial pemphigoid (OCP) (n = 37 eyes) and healthy control (controls) (n = 38 eyes) tears using a fluorimetric bead-based enzyme-linked immunosorbent assay. All values were standardized to µg of protein loaded (ng/µg protein) and the MMP-8, MMP-9 and MPO levels in SJS and OCP tears were compared to controls and between disease groups (*P<0.05, **P<0.01, ***P<0.001, Kruskal-Wallis test). The error bars indicate the standard errors of the mean.

Figure 2

Bar graph showing the mean total matrix metalloproteinase (MMP) activity in Stevens-Johnson syndrome (SJS) (n = 7 eyes), ocular cicatricial pemphigoid (OCP) (n = 27 eyes) and healthy control (controls) (n = 37 eyes) tears using an MMP fluorimetric activity assay. All values were standardized to µg of protein loaded (RFU/min/µg protein). The P values are shown on the graph (* statistically significant, Kruskal-Wallis test). The error bars indicate the standard errors of the mean.

Figure 3

Bar graphs showing (A) mean levels of tissue inhibitor of metalloproteinases-1 (TIMP-1) and (B) the ratios of the amounts of neutrophil collagenase (MMP-8) and gelatinase B (MMP-9) to TIMP-1 in tears of Stevens-Johnson syndrome (SJS), ocular cicatricial pemphigoid (OCP) and healthy control (controls) eyes. (A) SJS (7 eyes), OCP (24 eyes) and controls (33 eyes) tears were assayed using a fluorimetric bead-based enzyme-linked immunosorbent assay where all values were standardized to µg of protein loaded (ng/µg protein). The P values are shown on the graph (* statistically significant, Kruskal-Wallis test). (B) MMP/TIMP-1 ratios in SJS (7 eyes), OCP (23 eyes) and controls (32 eyes) tears were calculated and compared to their controls and between disease groups (*P<0.05, **P<0.01, ***P<0.001, Kruskal-Wallis test). The error bars indicate the standard errors of the mean.

Figure 4

Scatter graphs illustrating the correlations between the levels (in logarithmic values) of (A) neutrophil collagenase (MMP-8) and gelatinase B (MMP-9), (B) MMP-8 and myeloperoxidase (MPO), (C) MMP-9 and MPO and (D) matrilysin (MMP-7) and MPO in Stevens-Johnson syndrome (SJS), ocular cicatricial pemphigoid (OCP) and healthy control (controls) tears using Spearman rank correlation tests. The best fit lines: SJS (-.-), OCP (---) and controls (…). (A) In SJS (7 eyes), OCP (36 eyes) and controls (38 eyes) tears, significant correlation was found in all study groups: SJS (r = 0.96, P<0.01), OCP (r = 0.87, P<0.0001) and controls (r = 0.78, P<0.0001). (B) In SJS (7 eyes), OCP (22 eyes) and controls (32 eyes) tears, correlation was found in all study groups. Significance was achieved in OCP (r = 0.84, P<0.0001) and controls (r = 0.68, P<0.0001) but not in SJS (r = 0.75, P = 0.067). (C) In SJS (7 eyes), OCP (21 eyes) and controls (32 eyes) tears, significant correlation was found in all study groups: SJS (r = 0.85, P<0.05), OCP (r = 0.93, P<0.0001) and controls (r = 0.91, P<0.0001). (D) In SJS (7 eyes), OCP (22 eyes) and controls (30 eyes) tears, no significant correlation was demonstrated in all study groups: SJS (r = 0.68, P = 0.11), OCP (0.26, P = 0.24) and controls (r = 0.089, P = 0.64).