Immunofluorescent characterization of non-myelinating Schwann cells and their interactions with immune cells in mouse mesenteric lymph node

Abstract

The central nervous system (CNS) influences the immune system in a general fashion by regulating the systemic concentration of humoral substances, whereas the autonomic nervous system communicates specifically with the immune system according to local interactions. Data concerning the mechanisms of this bidirectional crosstalk of the peripheral nervous system (PNS) and immune system remain limited. To gain a better understanding of local interactions of the PNS and immune system, we have used immunofluorescent staining of glial fibrillary acidic protein (GFAP), coupled with confocal microscopy, to investigate the non-myelinating Schwann cell (NMSC)-immune cell interactions in mouse mesenteric lymph nodes. Our results demonstrate i) the presence of extensive NMSC processes and even of cell bodies in each compartment of the mouse mesenteric lymph node; ii) close associations/interactions of NMSC processes with blood vessels (including high endothelial venules) and the lymphatic vessel/sinus; iii) close contacts/associations of NMSC processes with various subsets of dendritic cells (such as CD4+CD11c+, CD8+CD11c+ dendritic cells), macrophages (F4/80+ and CD11b+ macrophages), and lymphocytes. Our novel findings concerning the distribution of NMSCs and NMSC-immune cell interactions inside the mouse lymph node should help to elucidate the mechanisms through which the PNS affects cellular- and humoral-mediated immune responses or vice versa in health and disease.

Figure 1.

Overview of NMSC distribution in the C57BL/6 mouse mesenteric lymph node. Antibodies against B220 (red) and GFAP (green) label mainly B cells and NMSCs inside the lymph node, respectively; objective lens: 20x; laser scanning mode: tile scan. BF, B cell follicle; GC, Germinal center, C, cortex; PC, paracortex; HEV, high endothelial venules; M, Medulla; MS, medullary sinus; MC, medullary cord; HL, Hilum. Scale bar: 100 μm.

Figure 2.

Distribution of NMSCs in the cortex (A), subcapsular sinus (B), paracortex (C), and medulla (D) of mesenteric lymph node from C57BL/6 mouse. Antibodies against B220 (red) and GFAP (green) label mainly B cells and NMSCs inside the lymph node, respectively. BF, B cell follicle; GC, Germinal center; SCS, subcapsular sinus; C, cortex; PC, paracortex; MS, medullary sinus; MC, medullary cord. BV, blood vessel; objective lens: 40x; scale bar: 20 μm. A) The white arrow indicates the soma (cell bodies) of NMSCs. B) Highresolution views of a subcapsular sinus are shown in inserted windows; the yellow lines indicate the border of a subcapsular sinus. The white arrow indicates NMSC processes associated with sinus. C) The white arrows indicate a few B cells that have a close association with NMSC processes. D) The images are maximal intensity projections of Z-Stack images; number 1 indicates a medullary cord that has been sectioned according to its longitudinal axis; number 2 indicates a medullary cord that has been sectioned perpendicular to its longitudinal axis; stack size: 3.5 μm; optical slice interval: 0.25 μm. E) High-resolution views of a medullary cord in (d).

Figure 3.

Distribution of NMSCs and blood vessels in the cortex (A), paracortex (B), and medulla (C,D) of a mesenteric lymph node from a C57BL/6 mouse. Antibodies against CD31 (red) and GFAP (green) label mainly blood vessel endothelial cells and NMSCs inside lymph node, respectively; BV, Blood vessel; HEV, high endothelial venules; MS, medullary sinus; MC, medullary cord; objective lens: 40x; scale bar: 20 μm. C) The images are maximal intensity projections of Z-Stack images acquired from medulla region; the yellow arrow indicates a few cells (macrophages or DCs) that are weakly positive for CD31. Stack size: 5.5 μm; optical slice interval: 0.25 μm. D) The images are maximal intensity projections of Z-Stack images; they show a medullary cord that has been sectioned according to its longitudinal axis; the medullary sinus (lymphatic vessels, green arrow) and sinus macrophages (cyan arrow) are weakly positive for CD31; Stack size: 5.4 μm; optical slice interval: 0.25 μm.

Figure 4.

Interaction of NMSCs and DCs in the paracortex of a C57BL/6 mouse mesenteric lymph node. HEV, high endothelial venules; objective lens: 40x; scale bar: 20 μm. A) The yellow arrows indicate a few B220^– CD11c⁺ DCs that have a close association with the NMSC processes. B) The white arrow indicates a B220⁺ CD11c⁺ DC that has a close association with the NMSC processes. C,E) CD4⁺ CD11c⁺ DCs appear magenta in the CD4⁺ CD11c channel. D,F) The colocalized structures (CD4⁺ CD11c⁺) are shown in white. In the right panels, parts of the images (left merged images) are shown at higher-resolution to demonstrate the interaction of DCs/T cells and NMSC processes. The white arrows show CD4^– CD11c⁺ DCs that have a close association with NMSC processes. The magenta arrows indicate CD4⁺ CD11c⁺ (appearing white) DCs that have a close association with NMSC processes. The yellow arrows indicate CD4⁺ T cells that have a close association with NMSC processes. The red circle shows two DCs (CD4⁺ CD11c⁺)-T cell (CD4⁺) clusters that have a close association with NMSC processes. The yellow circle indicates one DC (CD4^–CD11c⁺)-T cell (CD4⁺) cluster that has a close association with NMSC processes.

Figure 5.

Interaction of NMSCs and DCs in the paracortex of C57BL/6 mouse mesenteric lymph node. HEV: high endothelial venules; objective lens: 40x; scale bar: 20 μm. A) CD8⁺ CD11c⁺ DCs appear magenta in the CD8⁺CD11c channel. B) Colocalized structures (CD8⁺ CD11c⁺) are shown in white. C) Parts of (B) -merged image panel- are shown at a higher resolution to demonstrate the interaction of DCs/T cells and NMSCs. The white arrows show two CD8^– CD11c⁺ DCs that have a close association with the NMSC processes. The magenta arrows indicate CD8⁺ CD11c⁺ (appearing white) DCs that have a close association with the NMSC processes. The yellow arrows indicate a few CD8⁺ T cells that have a close association with the NMSC processes. The red circle shows two DC (CD8⁺ CD11c⁺) and T cells (CD8⁺) clusters that have a close association with NMSC processes. The yellow circle indicates a DC (CD8^– CD11c⁺) and T cell (CD8⁺) cluster that has a close association with NMSC processes.

Figure 6.

Interaction of NMSCs and F4/80⁺ macrophages in the cortex (A), paracortex (B), and medulla (C,D) of mesenteric lymph node from C57BL/6 mouse; BF, B cell follicle; MS, medullary sinus; MC, medullary cord; IF, interfollicular region; objective lens: 40x; scale bar: 20 μm. Antibodies against F4/80 (red), CD11c (blue), and GFAP (green) label mainly macrophages, DCs, and NMSCs inside the lymph node, respectively. The white arrows, yellow arrows, and cyan arrows indicate F4/80⁺ CD11c⁺ DCs, F4/80⁺ CD11c^– macrophages and F4/80^–CD11c⁺ DCs that have a close association with the NMSC processes inside the lymph node, respectively. The sinus macrophages are shown with magenta arrows. The white circle indicates interaction of F4/80^–CD11c⁺ DCs and NMSC processes inside one medullary cord. (E) Part of (D) shown at higher resolution.

Figure 7.

Interaction of NMSCs and Mac1⁺ macrophages in the cortex (A), paracortex (B), and medulla (C) of mesenteric lymph node from C57BL/6 mouse. BF, B cell follicle; MS, medullary sinus; MC, medullary cord; objective lens: 40x; scale bar: 20 μm. The white arrows and yellow arrows indicate Mac1⁺ CD11c⁺ DCs and Mac1⁺ CD11c^– macrophages that have a close association with the NMSC processes inside the lymph node, respectively. A) The white lines indicate the border between cortex and paracortex. B) Almost all the Mac1⁺ cells are also CD11c⁺. C) The yellow arrows indicate a Mac1⁺ macrophage that has a close association with NMSC processes inside a medullary cord.