Mast Cell-Derived Tryptase in Geographic Atrophy

Abstract

Purpose: Our previous study demonstrated significantly more degranulating mast cells (MCs) in choroids from subjects with age-related macular degeneration compared to aged controls. This study examined the immunolocalization of tryptase, the most abundant MC secretory granule-derived serine protease, in aged control eyes and eyes with geographic atrophy (GA).

Methods: Postmortem human eyes with and without GA were obtained from the National Disease Research Interchange. Tissue was fixed, cryopreserved, sectioned, and immunostained with a monoclonal antibody against tryptase. Sections were imaged on a Zeiss 710 Confocal Microscope.

Results: In the posterior pole of all aged control eyes, tryptase was confined to choroidal MCs, which were located primarily in Sattler's layer. In eyes with GA, many MCs were located in the inner choroid near choriocapillaris and Bruch's membrane (BM). Tryptase was found not only in MCs but also diffusely around them in stroma, suggesting they had degranulated. In contrast with aged control eyes, eyes with GA also had strong tryptase staining in BM. Tryptase was observed within BM in regions of RPE atrophy, at the border of atrophy, and extending well into the nonatrophic region.

Conclusions: Our results demonstrate that tryptase, released during choroidal MC degranulation, binds to BM in GA in advance of RPE atrophy. Tryptase activates MMPs that can degrade extracellular matrix (ECM) and basement membrane components found in BM. ECM modifications are likely to have a profound effect on the function and health of RPE and choroidal thinning in GA.

Figure 1

Aged control whole mount choroid immunolabeled for (A) UEA lectin (blue), (B) tryptase (red), and (C) chymase (green). The vast majority of human choroidal MCs are of the MC_T phenotype (tryptase⁺/chymase⁻). Only a small percentage of MCs are MC_tc (tryptase⁺/chymase⁺) (arrows). Scale bar: 30 μm.

Figure 2

Aged control (A, B) and GA (C, D) whole mount choroids immunolabeled with UEA lectin (blue) and anti-tryptase (red). MC degranulation near the border region of RPE atrophy demonstrates the association between MC degranulation and choriocapillaris atrophy. The free tryptase⁺ granules were at the level of the inner aspect of the choriocapillaris as determined in z stacks. Scale bar: 30 μm.

Figure 3

(A–D) Section from an 85-year-old aged control subject showing (C) tryptase⁺ MCs (arrows), (B) CD34+ blood vessels (green) and BM (arrowhead). Most MCs in aged control eyes were located in Sattler's and Haller's layer where tryptase⁺ granules were confined to MC cytoplasm. In (A) the colors are merged and (D) is differential interference contrast (DIC) illumination so RPE can be distinguished. Scale bar: 20 μm.

Figure 4

(A–D) Section from an 89-year-old GA subject showing (C) a degranulated tryptase⁺ MC (arrow), (B) CD34⁺ blood vessels and BM (arrowhead) in a nonatrophic region of RPE (*). (D) DIC illumination and (A) has all colors merged. MCs in GA eyes often were located near the choriocapillaris where diffuse tryptase immunoreactivity was observed in the surrounding choroidal stroma. Granular tryptase reaction product was localized in BM (arrowhead), sub RPE deposits and drusen. Scale bar: 20 μm.

Figure 5

(A–D) Section from the same 89-year-old GA subject in shown in Figure 4 near the border of RPE atrophy demonstrates granular tryptase immunoreactivity in BM (arrowhead). (B) The density of CD34⁺ choriocapillaris is reduced in this border region and (C) RPE are multilayered with granular tryptase immunoreactivity around them and within BM. (D) DIC illumination and (A) has all colors merged. Scale bar: 20 μm.

Figure 6

(A–D) Section from the same 89-year-old GA subject shown in Figure 5 in a region with RPE atrophy showing, (B) reduced CD34+ choriocapillaris density and diffuse tryptase staining (C) surrounding a degranulating MC (arrow) and granular tryptase immunoreactivity in BM (arrowhead). (D) DIC and (A) has all colors merged. Scale bar: 20 μm.

Figure 7

(A–D) Section from an 86-year-old aged control subject showing (C) a histamine⁺ MC (arrow), (B) CD34⁺ blood vessels and BM (arrowhead). (D) DIC and (A) has all colors merged. The endothelium of an artery is weakly histamine⁺ (*). BM (arrowhead) is unlabeled D. Scale bar: 20 μm.

Figure 8

(A–D) Section from an 89-year-old GA subject near the border of RPE atrophy showing (C) a histamine⁺ MC (arrow), (B) CD34⁺ blood vessels, and BM (arrowhead). (D) DIC illumination shows a drusen⁺ (*), and (A) has all colors merged. BM (arrowhead), drusen (*) and RPE that are unlabeled. Scale bar: 20 μm.

Figure 9

(A–D) Section from the same 89-year-old GA subject shown in Figure 8 in a region of RPE atrophy showing (C) a histamine⁺ MC (arrow), (B) CD34⁺ blood vessel attenuation, and unstained BM (arrowhead). (D) DIC illumination and (A) has all colors merged. Scale bar: 20 μm.

Figure 10

Sections from an 81-year-old aged control subject (A) and an 89-year-old GA subject (B–D) showing oil red O staining. In aged controls, weak diffuse staining was observed in BM. In the GA eyes, intense staining was observed in BM and drusen. The staining was granular in appearance and was remarkably similar to tryptase immunoreaction product in GA. Scale bar: 10 μm.

Figure 11

Semiserial sections of a 90-year-old GA choroid comparing tryptase localization (A) with oil red O staining (B) in BM and drusen. Note the rounded particulate bodies are labeled in both and reside in the same tissue compartment. Scale bar: 5 μm.