Immunoprofiling of human uterine mast cells identifies three phenotypes and expression of ERβ and glucocorticoid receptor

Abstract

Background: Human mast cells (MCs) are long-lived tissue-resident immune cells characterised by granules containing the proteases chymase and/or tryptase. Their phenotype is modulated by their tissue microenvironment. The human uterus has an outer muscular layer (the myometrium) surrounding the endometrium, both of which play an important role in supporting a pregnancy. The endometrium is a sex steroid target tissue consisting of epithelial cells (luminal, glandular) surrounded by a multicellular stroma, with the latter containing an extensive vascular compartment as well as fluctuating populations of immune cells that play an important role in regulating tissue function. The role of MCs in the human uterus is poorly understood with little known about their regulation or the impact of steroids on their differentiation status. The current study had two aims: 1) To investigate the spatial and temporal location of uterine MCs and determine their phenotype; 2) To determine whether MCs express receptors for steroids implicated in uterine function, including oestrogen (ERα, ERβ), progesterone (PR) and glucocorticoids (GR). Methods: Tissue samples from women (n=46) were used for RNA extraction (n=26) or fixed (n=20) for immunohistochemistry. Results: Messenger RNAs encoded by TPSAB1 (tryptase) and CMA1 (chymase) were detected in endometrial tissue homogenates. Immunohistochemistry revealed the relative abundance of tryptase MCs was myometrium>basal endometrium>functional endometrium. We show for the first time that uterine MCs are predominantly of the classical MC subtypes: (positive, +; negative, -) tryptase+/chymase- and tryptase+/chymase+, but a third subtype was also identified (tryptase-/chymase+). Tryptase+ MCs were of an ERβ+/ERα-/PR-/GR+ phenotype mirroring other uterine immune cell populations, including natural killer cells. Conclusions: Endometrial tissue resident immune MCs have three protease-specific phenotypes. Expression of both ERβ and GR in MCs mirrors that of other immune cells in the endometrium and suggests that MC function may be altered by the local steroid microenvironment.

Keywords: ERb; GR; chymase; oestrogen; steroid receptor; tryptase.

Figure 1.. Messenger RNAs encoded by genes encoding mast cell proteases were not cycle stage dependent.

( A) TPSAB1 mRNA and ( B) CMA1 mRNA. Single dots represent different patient samples, and data are expressed as the median. Proliferative phase n=14, early secretory phase n=7, mid secretory phase n=6, and menstrual phase n=3.

Figure 2.. Comparison of immunoexpression of chymase and tryptase in “full thickness” (uterine lumen to endometrial/myometrial junction) tissue samples obtained from across the menstrual cycle.

Note that mast cells were less abundant in the functional layer and appeared to be exclusively tryptase+/chymase-. ( A– C) Functional, basal endometrium and myometrium during proliferative phase (P); ( D– F) Early secretory phase (ES); ( G– I) Mid secretory phase (MS); ( J– L) Late Secretory phase (LS); ( M– O) Menstrual phase (M); ( P– R) Negative control (omission of primary antibody). Double immunofluorescence has revealed the presence of three uterine mast cell subtypes, single tryptase, single chymase and double tryptase-chymase positive cells. (P n=4, ES n=4, MS n=2, LS n=3, M n=2): negative controls were included on all sections.

Figure 3.. A population of chymase positive mast cells that did not express tryptase was identified in uterine tissue samples from the mid secretory phase.

The endometrial compartment shows three different mast cell (MC) subtypes: tryptase single positive, chymase single positive and tryptase and chymase double positive. Basal endometrial MCs are chymase+/tryptase- single positive and double positive, instead functional endometrium MCs are fewer in number and show a chymase-/tryptase+ phenotype. MCs during the secretory appeared to be activated in the myometrium, releasing both proteases from the cytoplasm. (n=3) (Arrowheads: MC _TC cells; Vs: MC _T cells; arrows: MC _C cells).

Figure 4.. Samples from the menstrual phase contained chymase positive cells that appeared to be ‘activated’, as well as abundant double positive cells in the basal endometrium.

Uterine mast cells (MCs) appear to be tryptase and chymase double positive, in both myometrial and basal endometrial layers, with a small portion of chymase+ MCs in the myometrium. MCs were not detectable in the functional layer. MCs during the menstrual phase appeared to be activated only in myometrial compartment, releasing both tryptase and chymase from the cytoplasm. In the basal endometrium, MCs showed a steady state instead. (n=2) (Arrowheads: MC _TC cells; Vs: MC _T cells; arrows: MC _C cells).

Figure 5.. Mast cells are immunopositive for ERβ.

Immunohistochemistry showed co-localization of ERβ in uterine mast cells (MCs). Nuclear expression of ERβ receptor (red staining) was detected in MCs across the tissue structures of uterus, myometrium, basal and functional endometrium, and during both the proliferative and secretory phases of the menstrual cycle. (Proliferative n=5, Secretory n=5).

Figure 6.. Uterine mast cells in the endometrium and myometrium are immunopositive for glucocorticoid receptor.

Mast cell nuclear glucocorticoid receptors (GR; red staining) was detected during the proliferative and secretory phase, throughout the myometrium, functional and basal endometrial and layers. The images are representative of results in proliferative (n=5) and secretory (n=5) phase samples.

Figure 7.. Summary immunoexpression of steroid receptors in human uterine mast cells.

Uterine mast cells are immunopositive for ERβ and GR, and immunonegative for ERα and PR. (Red staining: ERα, ERβ, PR and GR; green staining: tryptase). Arrowheads point to nuclei that have immunopositive (red) staining for ERβ and GR.