Fibroblast heterogeneity: existence of functionally distinct Thy 1(+) and Thy 1(-) human female reproductive tract fibroblasts

Abstract

Little is known about fibroblasts from the female reproductive tract, much less whether or not functional subsets exist. Fibroblasts are key as sentinel cells for recruiting white blood cells and for wound healing. The purpose of this research was to evaluate the possibility that functional subsets of fibroblasts exist in the human female reproductive tract. The strategy used was to define fibroblast subpopulations based on their surface expression of the Thy 1 antigen. In situ staining of human myometrium and endometrium showed heterogeneous staining for Thy 1. Freshly derived strains of fibroblasts from the myometrium and endometrium also demonstrated heterogeneous Thy 1 expression. For the first time, using magnetic beading and fluorescence-activated cell sorting, human myometrial fibroblasts were successfully separated into functionally unique Thy 1(+) and Thy 1(-) subsets. Both subsets produced the proinflammatory cytokines interleukin (IL)-6 and IL-8 after IL-1beta stimulation, but only the Thy 1(+) subset produced MCP-1. Furthermore, only Thy 1(+) fibroblasts up-regulated CD40 surface expression with IL-1beta or interferon-gamma treatment. Engagement of CD40 in the Thy 1(+) subpopulation induced IL-6, IL-8, and MCP-1. The discovery of functional subsets of reproductive tract fibroblasts now permits assessment of their roles in the normal functions of the reproductive tract and in disease states such as adhesions and menorrhagia.

Figure 1.

Immunohistochemical localization of Thy 1 in human myometrium and endometrium. Frozen tissue sections were fixed in formalin and blocked with 5% normal horse serum for 20 minutes at room temperature. Tissue sections were incubated overnight at 4°C with mouse monoclonal anti-human Thy 1 (F15-421-5, 10 μg/ml). A negative control antibody (mIgG1) was included at 10 μg/ml. Sections were incubated with biotinylated horse anti-mouse IgG and the avidin-biotin-peroxidase detection system was used. Sections were counterstained with hematoxylin. a: Myometrium. Immunoreactivity is present in some smooth muscle cells and in some fibroblastic stromal cells. b: Myometrium. Negative control; primary antibody replaced with mouse immunoglobulin. c: Endometrium basalis. Thy 1 is localized mainly to the perivascular region. d: Endometrium basalis. Negative control. e: Endometrium functionalis. Thy 1 immunoreactivity is present in some stromal cells. f: Endometrium functionalis. Negative control. Original magnifications, ×400. Scale bar, 50 μm.

Figure 2.

Thy 1 expression by the M5 myometrial fibroblast strain and the E7 endometrial fibroblast strain. Fibroblasts were stained for Thy 1 using the F15-421-5 antibody and analyzed using confocal microscopy. A: Thy 1 expression by the M5 fibroblast strain. Some fibroblasts stain positive for Thy 1 whereas others adjacent to them fail to stain. B: Thy 1 expression by the E7 fibroblast strain. A majority of fibroblasts stain positive for Thy 1, although there are clearly some Thy 1⁻ fibroblasts. Original magnifications, ×400.

Figure 3.

Separation of the M5 fibroblast strain into Thy 1⁺ and Thy 1⁻ subsets. A–C: Flow cytometry analysis for Thy 1 expression by M5 parental fibroblasts, showing 41% of the cells positive for Thy 1 (A), the M5 Thy 1⁻ subset, where <1% cells were positive for Thy 1 (B), and the M5 Thy 1⁺ subset, where >99% cells were positive for Thy 1 (C). Separation into Thy 1⁺ and Thy 1⁻ subsets was accomplished using fluorescence-activated cell sorting followed by three to four rounds of magnetic beading as described in Material and Methods. D–F: Phase contrast microscopy (original magnification, ×400) indicating morphology of M5 parental (D), M5 Thy 1⁻ (E), and M5 Thy 1⁺ fibroblasts (F). M5 Thy 1⁺ fibroblasts had more elongated processes whereas the M5 Thy 1⁻ fibroblasts were more rounded and spread. Fibroblasts were cultured on chamber slides and viewed under phase contrast on a light/phase microscope connected to a digital camera. Original magnification, ×400.

Figure 4.

M5 Thy 1⁺ and Thy 1⁻ fibroblast subsets synthesize IL-6 and IL-8, but only M5 Thy 1⁺ fibroblasts produce MCP-1. Cells were serum-starved for 72 hours in 0.5% FBS and RPMI 1640 and stimulated with IFN-γ or IL-1β for the final 24 hours in serum-free media. Supernatants were harvested and analyzed by enzyme-linked immunosorbent assay for IL-6 and IL-8 production as described in the Materials and Methods. IL-6 production (A) and IL-8 production (B) by M5 fibroblast subsets. IL-6 and IL-8 were produced by both Thy 1 subsets only after IL-1β stimulation. C: MCP-1 production by M5 fibroblast subpopulations. Only M5 Thy 1⁺ fibroblasts synthesized significant levels of MCP-1 in response to IFN-γ. IL-1β stimulation yielded a further 4.5-fold increase in MCP-1 production. This is a representative of three experiments (*, P < 0.05; **, P < 0.01 indicates value is statistically significant).

Figure 5.

M5 Thy 1⁺ fibroblasts express surface CD40. Fibroblasts were cultured with or without IFN-γ (500 U/ml) for 72 hours or IL-1β (10 ng/ml) for 24 hours. Mouse anti-human CD40 (G28-5) was used as the primary antibody and mouse IgG1 was used as the isotype control. Cells were then incubated with goat anti-mouse Ig-FITC as a secondary antibody. Samples were analyzed on a FACSCalibur. A: CD40 expression by M5 Thy 1⁺ fibroblasts. Stimulation with IFN-γ led to a 28% CD40 surface expression, whereas IL-1β yielded a 26% CD40 expression. Unstimulated cells only expressed minimal CD40 (<2%). B: CD40 expression by M5 Thy 1⁻ fibroblasts. IFN-γ and IL-1β stimulation did not up-regulate CD40 expression in the M5 Thy 1⁻ subset (CD40 expression, <2%). This figure is representative of three experiments.

Figure 6.

CD40-CD40L interaction induces increased IL-6, IL-8, and MCP-1 production by M5 Thy 1⁺ fibroblasts. Fibroblasts were serum-starved for 72 hours in fresh media containing 0.5% FBS with or without IFN-γ (500 U/ml). The treatments used for the final 24 hours were: no stimulation, IFN-γ alone (500 U/ml), CD40L alone, or IFN-γ together with CD40L. The anti-CD40L antibody MK13A4 (2.5 μg/ml) was used the final 24 hours on fibroblasts stimulated with IFN-γ and CD40L. Mouse IgG1 was added at 2.5 μg/ml as a control antibody. Supernatants were harvested and tested for IL-6, IL-8, and MCP-1 production by enzyme-linked immunosorbent assay. A: IL-6 production. IFN-γ and CD40L stimulation gave a 3.5-fold increase in IL-6 production over IFN-γ or CD40L stimulation alone. Addition of MK13A4 (anti-CD40L antibody) to cells treated with IFN-γ plus CD40L, completely blocked IL-6 production. B: IL-8 production. Treatment with IFN-γ together with CD40L induced a sevenfold increase in IL-8 production that is blocked by MK13A4. C: MCP-1 production. Addition of IFN-γ with CD40L yielded a fourfold increase in MCP-1 production, which is inhibited by MK13A4. Addition of control antibody did not affect induced IL-6, IL-8, or MCP-1 levels (*, P < 0.05; **, P < 0.01 indicates value is statistically significant).