Dynamic populations of dendritic cell-specific ICAM-3 grabbing nonintegrin-positive immature dendritic cells and liver/lymph node-specific ICAM-3 grabbing nonintegrin-positive endothelial cells in the outer zones of the paracortex of human lymph nodes

Abstract

In the paracortex of lymph nodes, cellular immune responses are generated against antigens captured in peripheral tissues by dendritic cells (DCs). DC-SIGN (dendritic cell-specific ICAM-3 grabbing nonintegrin), a C-type lectin exclusively expressed by DCs, functions as an antigen receptor as well as an adhesion receptor. A functional homologue of DC-SIGN, L-SIGN (liver/lymph node-SIGN, also called DC-SIGN-related), is expressed by liver sinus endothelial cells. In lymph nodes, both DC-SIGN and L-SIGN are expressed. In this study, we analyzed the distribution of these two SIGN molecules in detail in both normal and immunoreactive lymph nodes. DC-SIGN is expressed by mature DCs in paracortical areas and in addition by DCs with an immature phenotype in the outer zones of the paracortex. L-SIGN expression was also detected in the outer zones on sinus endothelial cells characterized by their expression of the lymphatic endothelial markers LYVE-1 and CLEVER-1. During both cellular and humoral immune responses changes in the amount of DC-SIGN+ immature and mature DCs and L-SIGN+ endothelial cells were observed, indicating that the influx or proliferation of these cells is dynamically regulated.

Figure 1

The polyclonal antibodies CSRD and PTTS recognize DC-SIGN and L-SIGN, respectively. A: Monocyte-derived DCs and K562, mock transfected or transfected with DC-SIGN or L-SIGN, were stained with AZN-D1, AZN-D2, and AZN-D3 (5 μg/ml), followed by anti-mouse FITC antibodies, and analyzed by flow cytometry. An isotype control antibody was included. B: Alternatively, cytospin preparations of cells were fixed and stained with CSRD and PTTS (1:500), anti-rabbit biotin, and the ABC-AP Vectastain kit. Original magnifications, ×20.

Figure 2

DC-SIGN and L-SIGN are expressed on distinct cells in the outer zone of paracortical areas in lymph nodes. A: Tissue cryosections of human cervical lymph nodes (patient 1) were fixed in acetone and stained with CSRD and PTTS (1:500), followed by anti-rabbit biotin and ABC-AP Vectastain kit to detect expression of DC-SIGN and L-SIGN, respectively. B: Paraffin tissue sections (patient 1) were pretreated by boiling in citric acid and subsequently stained to detect DC-SIGN, L-SIGN, and S100 expression, using CSRD, PTTS, and anti-S100 antibodies (1:500), followed by anti-rabbit biotin, ABC-PO, and diaminobenzidine tetrahydrochloride. Arrows point to positive cells. S, sinus; OZ, outer zone of paracortex; P, paracortex; F, B-cell follicle. Original magnifications: ×10 (A and B, left); ×40 (A and B, right).

Figure 3

In the cortical outer zone, DC-SIGN⁺ cells express mannose receptor and CD68 intracellularly and L-SIGN⁺ cells co-express LYVE-1 and CLEVER-1. A: Tissues sections of lymph nodes (patient 1) double stained with AZN-D1 and PTTS, followed by anti-mouse Texas Red and anti-rabbit FITC, and analyzed by confocal microscopy. Arrowheads indicate DC-SIGN⁺L-SIGN⁻ cells in the paracortex. B: Sections (patient 1) were double stained with CSRD, followed by anti-rabbit FITC and anti-CD68 or anti-mannose receptor, followed by anti-mouse Texas Red. C: Sections (patient 1) were double stained with PTTS, followed by anti-rabbit FITC and anti-CD31 and by anti-mouse Texas Red or with PTTS and anti-rabbit Texas Red in combination with anti-LYVE-1 or anti-CLEVER-1 and anti-mouse Alexa 488. Arrowheads indicate single-positive cells (CD68⁺, mannose receptor⁺, LYVE-1⁺, or CLEVER-1⁺), arrows point to double-positive cells. S, sinus; OZ, outer zone of paracortex; P, paracortex. Original magnifications, ×40.

Figure 4

Changes in the SIGN-expressing cell populations during immune responses. Tissue cryosections of human immunoreactive lymph nodes were fixed in acetone and stained with CSRD and PTTS (1:500), followed by anti-rabbit biotin and ABC-AP Vectastain kit to detect expression of DC-SIGN and L-SIGN, respectively. Lymph nodes were classified using H&E-stained paraffin sections and showed hyperplasia either in the cortex, containing numerous follicles (humoral response, patient 5; A), or in the paracortex (cellular response, patients 6 and 3; B and C). An overview is shown, as well as details from the medulla and paracortex. Arrows point to positive cells. S, sinus; F, B-cell follicle; P, paracortex. Similar results were obtained with patients 2 (humoral response) and 4 (cellular response). Original magnifications: ×5 (overview) and ×40 (others).