Expression of Siglec-11 by human and chimpanzee ovarian stromal cells, with uniquely human ligands: implications for human ovarian physiology and pathology

Abstract

Siglecs (Sialic acid-binding Immunoglobulin Superfamily Lectins) are cell surface signaling receptors of the I-type lectin group that recognize sialic acid-bearing glycans. CD33-related-Siglecs are a subset with expression primarily in cells of hematopoietic origin and functional relevance to immune reactions. Earlier we reported a human-specific gene conversion event that markedly changed the coding region for the extracellular domain of Siglec-11, associated with human-specific expression in microglia (Hayakawa T, Angata T, Lewis AL, Mikkelsen TS, Varki NM, Varki A. 2005. A human-specific gene in microglia. Science. 309:1693). Analyzing human gene microarrays to define new patterns of expression, we observed high levels of SIGLEC11 transcript in the ovary and adrenal cortex. Thus, we examined human and chimpanzee tissues using a well-characterized anti-Siglec-11 mouse monoclonal antibody. Although adrenal expression was variable and confined to infiltrating macrophages in capillaries, ovarian expression of Siglec-11 in both humans and chimpanzees was on fibroblasts, the first example of Siglec expression on mesenchyme-derived stromal cells. Cytokines from such ovarian stromal fibroblasts play important roles in follicle development and ovulation. Stable transfection of SIGLEC11 into a primary human ovarian stromal fibroblast cell line altered the secretion of growth-regulated oncogene α, interleukin (IL)-10, IL-7, transforming growth factor β1 and tumor necrosis factor-α, cytokines involved in ovarian physiology. Probing for Siglec-11 ligands revealed distinct and strong mast cell expression in human ovaries, contrasting to diffuse stromal ligands in chimpanzee ovaries. Interestingly, there was a trend of increased Siglec-11 expression in post-menopausal ovaries compared with pre-menopausal ones. Siglec-11 expression was also found on human ovarian stromal tumors and in polycystic ovarian syndrome, a human-specific disease. These results indicate potential roles for Siglec-11 in ovarian physiology and human evolution.

Fig. 1.

Expression patterns of human CD33-related Siglec genes. (A) Expression profile of human Siglec-6 mRNA in 84 human tissues and cell lines, as acquired from BioGPS containing the HG_U133A/GNF1H gene atlas data set. Expression values are based on probe set 206520_x_at. (B) Expression profile of Siglec-11 mRNA in 105 human tissues and cell lines as acquired from microarray Affymetrix U133. Expression values are based on probe set 1552910_at.

Fig. 2.

Detection of Siglec-11 protein expression in human and chimpanzee adrenals and ovaries. Mouse anti-Siglec-11 antibody 4C4 was used to detect the presence of Siglec-11 (shown in red) on paraffin-embedded tissue sections. The scale bar indicates 50 μm. (A) Expression of Siglec-11 is found in macrophage-like cells inside capillaries of human adrenal cortex (upper panel). Some adrenals had infiltrating macrophages but no expression of Siglec-11 (lower panel). Mouse IgG was used as the negative reagent control. CD68 is the macrophage marker. (B) Strong expression of Siglec-11 shows on ovarian stromal cells from human and chimpanzee ovaries. Human spleen sections are used as a positive control (macrophage staining only) and mouse IgG was used as the negative reagent control. (C) Comparison of Siglec-11 expression with Vimentin (stromal marker) and CD45 (hematopoietic cell marker) in human spleen, ovary and prostate. The spleen is the positive control for Siglec-11 expression on macrophages, and the prostate sample shows lack of non-specific staining on stromal cells in general.

Fig. 3.

Siglec-11 expression in transfected HFN0402 cells confirmed by western blot. Human ovarian fibroblast primary cell line HFN0402 was transfected with Siglec-11-pcDNA3.1 or vector only. G418 (100 µg/mL) was used to select positively transfected cells for 3 weeks. Transiently transfected 293T cells with Siglec-11-pcDNA3.1 or vector only were used as positive and negative controls, respectively. Cells were lysed with RIPA buffer (Cell Signaling Technology). Western blots of reduced cell lysates were probed with mouse anti-Siglec-11 antibody 4C4, and then goat anti-mouse IgG horseradish peroxidase.

Fig. 4.

Different Siglec-11 ligands in human and chimpanzee ovaries. Paraffin sections of human or chimpanzee ovaries were overlaid with human Siglec-11-FLAG-Fc or chimpanzee Siglec-11-FLAG-Fc, respectively. (A) Binding was detected using rabbit anti-FLAG and peroxidase-conjugated anti-rabbit IgG. Siglec-6-FLAG-Fc was used as a negative control for non-specific binding. Control slides had no added Siglec-Fc. The scale bars indicate 50 μm. Note that the human ovary slides are deliberately shown at a higher magnification, in order to highlight the bright staining of a few cells, which are shown below to be mast cells. (B) Siglec-11-FLAG-Fc binding was detected using rabbit anti-flag and Cy3-conjugated anti-rabbit IgG. Mast cells in human and chimpanzee ovaries were probed using a biotinylated mouse anti-human tryptase antibody, detected by Alexa-Fluor 488 labeled streptavidin. Rabbit IgG or mouse IgG was used as negative controls (not shown). The scale bar indicates 50 μm. (C) Sections were treated with sialidase or periodate before the overlay of Siglec-11-FLAG-Fc. Efficacy of both treatments was confirmed by loss of binding of SNA to the sections (not shown). The human Siglec-11 ligand on mast cells is not sensitive to either treatment. The chimpanzee Siglec-11 ligand on ovarian stroma is partially sensitive to both treatments.

Fig. 5.

Expression of Siglec-11 in pathologic human ovaries. Paraffin sections of ovaries were overlaid with mouse anti-Siglec-11 antibody 4C4 to detect the presence of Siglec-11 (shown in red). Mouse IgG as the negative control. (A) Siglec-11 expression in human PCOS sample. CD68 is the macrophage marker. The scale bar indicates 500 μm. (B) Siglec-11 expression in one of two human ovarian stromal tumors. A spleen section is shown as a positive control. The scale bar indicates 50 μm.