Insulin-like growth factor I messenger RNA and protein are expressed in the human lymph node and distinctly confined to subtypes of macrophages, antigen-presenting cells, lymphocytes and endothelial cells

Abstract

Insulin-like growth factor I (IGF-I) is a potent hormone that stimulates growth and differentiation and inhibits apoptosis in numerous tissues. Preliminary evidence suggests that IGF-I exerts differentiating, mitogenic and restoring activities in the immune system but the sites of synthesis of local IGF-I are unknown. Identification of these sites would allow the functional role of local IGF-I to be clarified. The presence of IGF-I in non-immune cells suggests that it acts as a trophic factor, while its occurrence in subtypes of lymphocytes or antigen-presenting cells indicates paracrine/autocrine direct regulatory involvement of IGF-I in the human immune response. The present study investigated the location of IGF-I messenger RNA and protein on archival human lymph node samples by in situ hybridization, immunohistochemistry and double immunofluorescence staining using an IGF-I probe and antisera specific for human IGF-I and CD3 (T lymphocytes), CD20 (B lymphocytes), CD68 (macrophages), CD21 (follicular dendritic cells), S100 (interdigitating dendritic cells) and podoplanin (fibroblastic reticular cells). Numerous cells within the B- and T-cell compartments expressed the IGF-I gene, and the majority of these cells were identified as macrophages. Solitary follicular dendritic cells exhibited IGF-I. A few T lymphocytes, and no B lymphocytes, contained IGF-I immunoreactive material. Furthermore, IGF-I immunoreactive cells outside the follicles that did not react with CD3, CD20, S100 or podoplanin markers were identified as high-endothelial venule (HEV) cells. From this we conclude that the main task of IGF-I in human non-tumoral lymph node may be autocrine and paracrine regulation of the differentiation, stimulation and survival of lymphocytes, antigen-presenting cells and macrophages and the differentiation and maintenance of HEV cells.

Figure 2

(a,b) Localization of insulin-like growth factor I (IGF-I) messenger RNA (mRNA) and protein within the human lymph node T-cell area by (a,c) in situ hybridization with an IGF-I-specific probe and by (b,d) immunohistochemistry with an IGF-I-specific antiserum. IGF-I gene (a,c) and protein (b,d) expressions are localized in numerous high-endothelial venule (HEV) cells (arrows) around the blood vessel lumen (asterisk) and in large solitary cells (arrowheads), presumably macrophages. (a,b) Bar: 50 μm, (c,d) Bar: 25 μm.

Figure 1

(a,b) Localization of insulin-like growth factor I (IGF-I) messenger RNA (mRNA) and protein within the human lymph node B-cell area by (a) in situ hybridization with an IGF-I-specific probe and by (b,c) immunohistochemistry with (b) an IGF-I-specific antiserum and with (c) a mouse monoclonal CD21 surface marker. IGF-I gene (a) and protein (b) expressions are localized in solitary large cells (arrows) and in numerous cytoplasmic branches within the germinal center (arrowheads), forming a meshwork arrangement similar to (c) follicular dendritic cells. Bar: 50 μm.

Figure 3

Localization of insulin-like growth factor I (IGF-I) immunoreactivity (green) by double immunofluorescence staining using a monoclonal antibody directed against (a) follicular dendritic cells (CD21), (b) B lymphocytes (CD20) and (c) macrophages (CD68), visualized in red. (a-c) Numerous cells contain IGF-I immunoreactivity (arrows) within the germinal centre (Gc) and along the mantle zone (Mz). (a,b) No IGF-I imunoreactivity is visible in co-localization with the cytoplasmic branches of the follicular dendritic cells (a, double arrowheads) and with the B cells (b, arrowheads). (c) Co-localization with IGF-I-immunoreactivity (yellow) is found in numerous macrophages within the Gc (double arrowheads), but some macrophages (red) do not contain IGF-I (single arrowheads). Numerous IGF-I immunoreactive cells (green) are not CD68 immunoreactive (arrows) the majority of which are arranged in an epithelial formation along the high-endothelial venule (HEV) lumina (asterisks). Bar: 25 μm.

Figure 6

Localization of insulin-like growth factor I (IGF-I) immunoreactivity (green) by double immunofluorescence staining with a monoclonal antibody against podoplanin/gp36, which identified the fibroblastic reticular cells (red, arrowheads). IGF-I immunoreactivity is present in numerous cells (arrows), probably macrophages and high-endothelial venule (HEV) cells, arranged around the vessel lumen (asterisk), but not co-localized with podoplanin immunoreactivity. Bar: 25 μm.

Figure 5

(a,b) Localization of insulin-like growth factor I (IGF-I) immunoreactivity (green) by double immunofluorescence staining with a monoclonal antibody directed against S100 protein to detect the interdigitating dendritic cells (red) that reveal the typical veil-like appearance (double arrowheads). Numerous solitary cells, especially around the high-endothelial venule (HEV) (asterisk), contain IGF-I immunoreactivity (arrows), but this is not co-localized with S100 immunoreactivity (arrow heads). Bar: 25 μm.

Figure 4

Localization of insulin-like growth factor I (IGF-I) immunoreactivity (green) by double immunofluorescence staining with a monoclonal antibody directed against the CD3 surface marker (red). Only very few T lymphocytes also contain IGF-I-immunoreactive material (arrowheads). Numerous macrophages identified by their wide cytoplasm (double arrows) and high-endothelial venule (HEV) endothelial cells (arrows) exhibit IGF-I immunoreactivity. Bar: 25 μm.