Systemic and Ocular Long Pentraxin 3 in Patients with Age-Related Macular Degeneration

Abstract

Age-related macular degeneration (AMD) has been associated with both systemic and ocular alterations of the immune system. In particular dysfunction of complement factor H (CFH), a soluble regulator of the alternative pathway of the complement system, has been implicated in AMD pathogenesis. One of the ligands for CFH is long pentraxin 3 (PTX3), which is produced locally in the retinal pigment epithelium (RPE). To test the hypothesis that PTX3 is relevant to retinal immunohomeostasis and may be associated with AMD pathogenesis, we measured plasma PTX3 protein concentration and analyzed the RPE/choroid PTX3 gene expression in patients with AMD. To measure the ability of RPE cells to secrete PTX3 in vitro, polarized ARPE-19 cells were treated with activated T cells or cytokines (interferon (IFN)-gamma and/or tumor necrosis factor (TNF)-alpha) from the basolateral side; then PTX3 protein concentration in supernatants and PTX3 gene expression in tissue lysates were quantified. Plasma levels of PTX3 were generally low and did not significantly differ between patients and controls (P=0.307). No statistically significant difference was observed between dry and exudative AMD nor was there any correlation with hsCRP or CFH genotype. The gene expression of PTX3 increased in RPE/choroid with age (P=0.0098 macular; P=0.003 extramacular), but did not differ between aged controls and AMD patients. In vitro, ARPE-19 cells increased expression of the PTX3 gene as well PTX3 apical secretions after stimulation with TNF-alpha or activated T cells (P<0.01). These findings indicate that PTX3 expressed in the eye cannot be detected systemically and systemic PTX3 may have little or no impact on disease progression, but our findings do not exclude that locally produced PTX3 produced in the posterior segment of the eye may be part of the AMD immunopathogenesis.

Fig 1. Plasma levels of PTX3 do not differ between AMD patients and controls.

PTX3 protein was quantified in plasma samples from patients with AMD (CARMS grade 2–5) and controls (< or >/ = 60 years of age) using ELISA. CARMS grade 2–3, early AMD; 4, geographic AMD; 5, exudative AMD. P = 0.307 in a one-way ANOVA.

Fig 2. RPE/choroid gene expression of PTX3 is increased with age.

Data were extracted from a publicly available gene expression data set in the NCBI GEO database. PTX3 gene expression is shown relative to beta actin (ACTB) gene expression. Values on bars indicate P values in unpaired t tests with Welch’s correction for unequal variances. Ctrl<60y comprise healthy controls aged 59 or younger (macular, n = 15; extramacular, n = 16). Any >/ = 60y comprise controls (macular, n = 35; extramacular, n = 30) and patients with AMD aged 60 or older (macular, n = 40; extramacular, n = 37).

Fig 3. PTX3 gene expression is increased in ARPE-19 cells following co-culture with activated T cells.

Pigmented monolayers of ARPE-19 cells grown on membrane inserts were basolaterally (replicate I-IV) or apically (replicate V-VI) exposed to CD3/CD28-activated human T cells (co-T), recombinant interferon γ (IFNγ) or tumor necrosis factor α (TNFα). RNA was purified and gene expression analyzed using microarrays. **, P<0.01 in one-way ANOVA with Dunnett’s multiple comparison.

Fig 4. PTX3 secretion from ARPE-19 cells is primarily increased in the apical direction following basolateral inflammatory treatment.

Pigmented monolayers of ARPE-19 cells grown on membrane inserts were exposed to CD3/CD28-activated human T cells (co-T) or recombinant cytokines basolaterally. Media was collected from apical and basolateral compartments, and PTX3 was quantified using ELISA. *, P<0.05; **, P<0.01 in repeated measures one-way ANOVA with Dunnett’s multiple comparison. Bars represent mean values from seven independent setups, error bars represent standard error of the mean.