Network of dendritic cells within the muscular layer of the mouse intestine

Abstract

Dendritic cells (DCs) are located at body surfaces such as the skin, respiratory and genital tracts, and intestine. To further analyze intestinal DCs, we adapted an epidermal sheet separation technique and obtained two intestinal layers, facing the lumen and serosa. Unexpectedly, immunolabeling of the layer toward the serosa revealed a regular, dense, planar network of cells with prominent dendritic morphology within the external muscular layer and with increasing frequency along the length of the intestine. Direct examination of the serosal-disposed layers showed a significant fraction of the DCs to express DEC-205/CD205, CD11c, Langerin/CD207, Fcgamma receptor/CD16/32, CD14, and low levels of activation markers, CD25, CD80, CD86, and CD95. By more sensitive FACS analyses, cells from this layer contained two CD11c(+) populations of CD45(+) CD205(+), CD19(-) leukocytes, MHC II(+) and MHC II(-). When ovalbumin conjugated to an anti-DEC-205 antibody was injected into mice, the conjugate targeted to these DCs, which upon isolation were able to stimulate ovalbumin-specific, CD4(+) and CD8(+) T cell antigen receptor-transgenic T cells. In vivo, these DCs responded to two microbial stimuli, systemic LPS and oral live bacteria, by up-regulating CD80, CD86, DEC-205, and Langerin within 12 h. This network of DCs thus represents a previously unrecognized antigen-presenting cell system in the intestine.

Fig. 1.

A dendritic, strongly MHC-II⁺ population in the intestinal layer toward the serosa. (A) The intestine was incubated in 0.5 M EDTA for 30 min at 37°C, two layers were separated by traction by using fine forceps, and the layers were fixed in cold acetone. (a and b) Upon labeling, MHC-II⁺ cells with clear dendritic morphology and a regular pattern of distribution are observed in only one of the two intestinal layers obtained (toward the serosa). To study the precise histological location of this DC population, transverse semithin sections of each layer were obtained. The layer (c) toward the lumen comprises the lamina propria and submucosa, whereas the layer (d) toward the serosa corresponds to the external muscular layer. (B) The frequency of this MHC-II⁺ population was also analyzed in each of the different anatomical regions of the intestine. (C) The possible association of the MHC-II⁺ (brown) DC to vessels was assessed by double labeling for CD31 endothelial molecule (blue). The MHC-II⁺ DCs do not appear associated with CD31⁺ (blue) vessels (blood or lymphatics).

Fig. 2.

CD11c and DEC-205 are also expressed in this intestinal DC population. (A) Using DC markers such as CD11c and DEC-205, we identified positive cells with dendritic morphology in this intestinal muscular layer. (B) Double labeling for MHC-II (red) and CD11c or DEC-205 (green). (C) CD11c⁺ and DEC-205⁺ cells in the layer have a lower frequency than the MHC-II⁺ cells. (D) Intestinal layers from the CD11c-EYFP^high mice also show strongly EYFP⁺ cells with prominent dendritic morphology. FACS analysis of single-cell suspensions of the muscularis layer stained for MHC-II and CD11c show class II negative and positive subsets. (E) The indicated fractions were costained for other antigens, DEC-205, CD45, and CD19, and the expression on the MHC-II⁺ cells is shown.

Fig. 3.

Phenotype of the intestinal DCs in situ. The intestinal layers were fixed and processed for immunohistochemistry. (A) (Left) Several functionally significant cell surface molecules were analyzed, and in all cases positive cells with clear dendritic morphology were observed. (Right) Quantification of their frequency. (B) Activation markers were also used to characterize these DCs.

Fig. 4.

DCs from the intestinal layer stimulate proliferation of antigen-specific T cells. (A) DEC-205 targeting: 18 h after i.p. inoculation of the rat anti-mouse DEC-205 antibody and a nonreactive isotype control antibody (10 μg), the intestine was processed to obtain intestinal layers to search for bound antibody by using phycoerythrin (PE)-conjugated anti-rat Ig and indirect immunofluorescence. An FITC-conjugated anti-IA/I-E was concomitantly used to confirm that the cells targeted with DEC-205 were also MHC-II⁺.(B) Single-cell suspensions from the mesenteric lymph node were prepared 24 h after i.p. inoculation of DEC-OVA conjugate (10 μg). CD11c⁺ cells were purified by magnetic-activated cell sorting; CD11c^- cells were also kept. Both suspensions were used to stimulate OT-I transgenic CD8 T cells, in a 1:3 (DC:T cell) ratio. T cell proliferation was measured by [³H]thymidine incorporation (filled bars, DEC-OVA-inoculated mice; open bars, PBS-inoculated mice). (C) Single-cell suspensions from the intestinal layer were obtained and tested for a stimulation of transgenic OT-I and OT-II T cells (1:10 ratio) 24 h after i.p. inoculation of the DEC-OVA conjugate (10 μg). (D) Single-cell suspensions from the muscularis layer were depleted with isotype control antibody (Iso) or mAbs to CD11c, CD19, and CD86 before addition to TCR transgenic T cells. Lamina propria cells were evaluated in parallel and spleen CD11c⁺ cells used as control. T cell proliferation was measured by [³H]thymidine incorporation (filled bars, DEC-OVA inoculated mice; open bars, Iso-OVA-inoculated mice). Representative data from four different experiments are shown.

Fig. 5.

DCs from the intestinal layer mature in vivo. The intestine was processed to obtain intestinal layers to analyze the phenotype and frequency of these DCs 12 h after LPS inoculation (30 μg i.v.) (A) or live bacteria (3.5 × 10⁷ bacteria orally) (B). In both cases, an up-regulation of DEC-205⁺ and Langerin⁺ DCs was observed, and the number of cells expressing activation markers such as CD80 and CD86 increased significantly (filled bars, control animals; open bars, stimulated animals).