Distribution of non-myelinating Schwann cells and their associations with leukocytes in mouse spleen revealed by immunofluorescence staining

Abstract

The nervous system and the immune system communicate extensively with each other in order to maintain homeostasis and to regulate the immune response. The peripheral nervous system (PNS) communicates specifically with the immune system according to local interactions, including the "hardwiring" of sympathetic/parasympathetic (efferent) and sensory nerves (afferent) to lymphoid tissue and organs. To reveal this type of bidirectional neuroimmune interaction at the microscopic level, we used immunofluorescent staining of glial fibrillary acidic protein (GFAP) coupled with confocal microscopy/3D reconstruction to reveal the distribution of non-myelinating Schwann cells (NMSCs) and their interactions with immune cells inside mouse spleen. Our results demonstrate i) the presence of an extensive network of NMSC processes in all splenic compartments including the splenic nodules, periarteriolar lymphoid sheath (PALS), marginal zone, trabecula, and red pulp; ii) the close association of NMSC processes with blood vessels (including central artries and their branches, marginal sinuses, penicillar arterioles and splenic sinuses); iii) the close "synapse-like" interaction/association of NMSC processes with various subsets of dendritic cells (DCs; e.g., CD4+CD11c+ DCs, B220+CD11c+ DCs, and F4/80+ CD11c+ DCs), macrophages (F4/80+), and lymphocytes (B cells, CD4+ T helper cells). Our novel findings concerning the distribution of NMSCs and NMSC-leukocytes interactions inside mouse spleen should improve our understanding of the mechanisms through which the PNS affects cellular- and humoral-mediated immune responses in a variety of health conditions and infectious/non-infectious diseases.

Keywords: Non-myelinating Schwann cells (NMSC); dendritic cell (DC); immunofluorescence staining.; lymphocyte; spleen.

Figure 1.

Distribution of NMSCs, blood vessels, and DCs in the C57BL/6 mouse spleen. Antibodies against GFAP (green), CD31 (red), and CD11c (blue) label mainly NMSCs, blood vessels, and DCs, respectively. The lower right panel is the merged image of the three immunolabellings. MS, marginal sinus; CA, central artery; MZ, marginal zone; SN, splenic nodule; PALS, periarteriolar lymphoid sheath; RP, red pulp. Objective lens: 20x; scanning mode: tile scan. Scale bar: 200 μm.

Figure 2.

Distribution of NMSCs, blood vessels, and DCs in the PALS (a), splenic nodule (b), and red pulp (c) of C57BL/6 mouse spleen. Antibodies against GFAP (green), CD31 (red), and CD11c (blue) label mainly NMSCs, blood vessels, and DCs, respectively. a) The images in the second row are high-resolution views of another maximal intensity projection of the Z-stack shown in the first row; white arrows show the close interactions of NMSC processes and DCs in PALS. CA, central artery; PALS, periarteriolar lymphoid sheath; SN, splenic nodule; MS, marginal sinus; MZ, marginal zone; SS, splenic sinus; SC, splenic cord. Objective lens: 40x. Each image is a maximal intensity projection of a Z-stack. Stack size: 6.0 μm; optical slice interval: 0.50 μm. Scale bar: 20 μm.

Figure 3.

Distribution of NMSCs, B cells, and DCs in the C57BL/6 mouse spleen. Antibodies against GFAP (green), B220 (red), and CD11c (blue) label mainly NMSCs, B cells, and DCs, respectively. The lower right panel is the merged image of the three immunolabellings. CA, central artery; SN, splenic nodule; PALS, periarteriolar lymphoid sheath; CP, capsule; RP, red pulp; T, trabecula; HL, hilum. Objective lens: 20x; scanning mode: tile scan. Scale bar: 200 μm.

Figure 4.

Distribution of NMSCs, B cells, and DCs in the PALS (a, c) and red pulp (b, d) of C57BL/6 mouse spleen. Antibodies against GFAP (green), B220 (red), and CD11c (blue) label mainly NMSCs, B cells, and DCs, respectively. c) High-resolution view of (a) to show the interaction of NMSCs and immune cells in PALS. d) High-resolution view of (b) to show the interaction of NMSCs and immune cells in red pulp; cyan arrows indicate a B220⁺ B cell closely associated with NMSC processes; white arrows show a B220^- CD11c⁺ DC closely apposed to NMSC processes; yellow arrows indicate a B220⁺ CD11c⁺ DC with a close association with NMSC processes. CA, central artery; SN, splenic nodule; PALS, periarteriolar lymphoid sheath. Objective lens: 40x. Each image is a maximal intensity projection of a Z-stack. Stack size: 6.0 μm; optical slice interval: 0.50 μm. Scale bar: 20 μm.

Figure 5.

Distribution of NMSCs, T helper cells, and DCs in the splenic nodule (a), PALS (b), and red pulp (c) of C57BL/6 mouse spleen. Antibodies against GFAP (green), CD4 (red), and CD11c (blue) label mainly NMSCs, T helper cells, and DCs, respectively.The cyan arrows indicate a CD4+ T helper cell that is closely associated with NMSC process; white arrows show a CD4^-CD11c⁺ DC closely apposed to NMSC processes; yellow arrows indicate a CD4⁺ CD11c⁺ DC having a close association with NMSC processes; yellow arrows indicate a CD4⁺ CD11c⁺ DC having a close association with NMSC processes. MZ, marginal zone; CA, central artery; SN, splenic nodule; PALS, periarteriolar lymphoid sheath; CP, capsule; T, trabecula; RP, red pulp. Objective lens: 40x. Each image is a maximal intensity projection of a Z-stack. Stack size: 6.0 μm; optical slice interval: 0.50 μm. Scale bar: 20 μm.

Figure 6.

Interaction of NMSCs and macrophages in red pulp of spleen from C57BL/6 mouse. Antibodies against F4/80 (red), CD11c (blue), and GFAP (green) label mainly macrophages, DCs, and NMSCs in the red pulp, respectively. b) Various combinations of channels in (a) are shown here. In the fourth image from left, the F4/80⁺CD11c⁺ DCs are marked in white after colocalization analysis. T, trabecula. Objective lens: 40x. Each image is a maximal intensity projection of a Z-stack. Stack size: 6.0 μm; optical slice interval: 0.50 μm. Scale bar: 20 μm.