Profiling of Cytokines Secreted by Conventional Aqueous Outflow Pathway Endothelial Cells Activated In Vitro and Ex Vivo With Laser Irradiation

Abstract

Purpose: To profile which cytokine genes are differentially expressed (DE) as up- or downregulated by cultured human trabecular meshwork (TMEs) and Schlemm's canal endothelial cells (SCEs) after three experimental treatments consisting of selective laser trabeculoplasty (SLT) irradiation, exposure to media conditioned either by SLT-irradiated TMEs (TME-cm) or by SCEs (SCE-cm). Also, to profile which cytokines are upregulated ex vivo in SLT-irradiated human conventional aqueous outflow pathway (CAOP) tissues.

Methods: After each treatment, Affymetrix microarray assays were used to detect upregulated and downregulated genes for cytokines and their receptors in TMEs and SCEs. ELISA and protein antibody arrays were used to detect upregulated cytokines secreted in SLT-irradiated CAOP tissues ex vivo.

Results: The SLT irradiation upregulated numerous cytokine genes in TMEs, but only a few in SCEs. Exposure to TME- and SCE-cm induced SCEs to upregulate many more cytokine genes than TMEs. Selective laser trabeculoplasty irradiation and exposure to TME-cm downregulated several cytokine genes in TMEs but none in SCEs. Selective laser trabeculoplasty irradiation induced one upregulated and three downregulated cytokine-receptor genes in TMEs but none in SCEs. Exposure to TME-cm induced upregulation of one and downregulation of another receptor gene in TMEs, whereas two unique cytokine-receptor genes were upregulated in SCEs. Cytokine protein expression analysis showed that at least eight cytokines were upregulated in SLT-irradiated human CAOP tissues in situ/ex vivo.

Conclusions: This study has helped us identify a cytokine signaling pathway and to consider newly identified mechanisms regulating aqueous outflow that may lay the foundation for the future development of cytokine-based glaucoma therapies.

Figure 1

Box plots showing the expression for three cytokines in cultured TMEs and SCEs treated with SLT-irradiation (L) and compared with untreated controls (C). The expression level was determined for IL-1α, IL-1β, and IL-8 (CXCL8) using ELISA with one-sided Wilcoxon P value for each treatment-control comparison measured less than 0.05.

Figure 2

For in situ/ex vivo cytokine expression, the data consisted of intensity measurements taken from between 5 and 22 specimen pairs for each cytokine, depending on the particular assay. Using antibody array assay (A) and ELISA (E), the cytokine expression levels were assayed from an equal amount of supernatants of 48-hour cultured human CAOP tissues after SLT irradiation. Separate antibody array assays could not be carried out for CXCL1, 2, and 3 due to the GRO antibody reacting with CXCL1, CXCL2, and CXCL3. Fold values = mean treated intensity/mean control intensity. A cytokine having both a calculated fold value ≥ 1.5 and a one-sided FDR adjusted Wilcoxon P value less than 0.1 was considered upregulated. Cytokine-specific Wilcoxon FDR P values: (A) < 0.005; (B) < 0.05; and (C): ≥ 0.1. TLTD, too low to detect using ELISA.

Figure 3

Proposed unidirectional intercellular signaling pathway and proposed positive feedback loop. (A) Pre-SLT irradiation: cytokine receptor binding prior to laser treatment. (B) Post-SLT irradiation: cytokine receptor binding after laser treatment.