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. 2000 Feb 7;191(3):435-44.
doi: 10.1084/jem.191.3.435.

A discrete subpopulation of dendritic cells transports apoptotic intestinal epithelial cells to T cell areas of mesenteric lymph nodes

F P Huang  1 , N Platt, M Wykes, J R Major, T J Powell, C D Jenkins, G G MacPherson
Affiliations

A discrete subpopulation of dendritic cells transports apoptotic intestinal epithelial cells to T cell areas of mesenteric lymph nodes

F P Huang et al. J Exp Med. .

Abstract

This study identifies a dendritic cell (DC) subset that constitutively transports apoptotic intestinal epithelial cell remnants to T cell areas of mesenteric lymph nodes in vivo. Rat intestinal lymph contains two DC populations. Both populations have typical DC morphology, are major histocompatibility complex class II(hi), and express OX62, CD11c, and B7. CD4(+)/OX41(+) DCs are strong antigen-presenting cells (APCs). CD4(-)/OX41(-) DCs are weak APCs and contain cytoplasmic apoptotic DNA, epithelial cell-restricted cytokeratins, and nonspecific esterase (NSE)(+) inclusions, not seen in OX41(+) DCs. Identical patterns of NSE electrophoretic variants exist in CD4(-)/OX41(-) DCs, intestinal epithelial cells, and mesenteric node DCs but not in other DC populations, macrophages, or tissues. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL)-positive DCs and strongly NSE(+) DCs are present in intestinal lamina propria. Peyer's patches and mesenteric but not other lymph nodes contain many strongly NSE(+) DCs in interfollicular and T cell areas. Similar DCs are seen in the ileum and in T cell areas of mesenteric nodes in gnotobiotic rats. These results show that a distinct DC subset constitutively endocytoses and transports apoptotic cells to T cell areas and suggest a role for these DCs in inducing and maintaining peripheral self-tolerance.

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Figures

Figure 1
Figure 1
OX41− L-DCs contain apoptotic epithelial cell remnants. (A–C) L-DCs, partially enriched over NycoPrep™, were separated into OX41+ and OX41− populations on a MACS column. Cytospin preparations were labeled for apoptotic DNA by the TUNEL method (green) and for MHC class II (red) and examined on a confocal microscope. (A) OX41− DCs. 20–30% of cells contain TUNEL-positive inclusions. (B) OX41+ DCs. No cells contain TUNEL-positive inclusions. (C) TUNEL-positive inclusions are present within the cytoplasm of a DC. There is little overlap between apoptotic DNA and MHC class II (×80 objective). (D) OX41− L-DCs, separated as above, were labeled for the epithelial cell cytokeratins 4 and 21 by mAbs RK4 and RK5. 4–7% of OX41− DCs contain cytokeratin-positive (RK4) inclusions (>, brown). (E) Cytospins of OX41− DCs were stained for NSE using α-naphthyl butyrate as substrate. More than 80% of DCs are very strongly positive. (F) OX41+ DCs reacted as in E. Esterase reactivity is negative or weak. One positive cell probably represents an OX41− contaminant. (G) OX41− DCs reacted for 1 min for NSE. Esterase reactivity is present as large, irregular, cytoplasmic inclusions.
Figure 2
Figure 2
OX41− DCs and IECs display similar NSE variants. Cells and tissues were lysed, and the lysates were electrophoresed in native form (zymograms). The gels were reacted for NSE using α-naphthyl acetate or α-naphthyl butyrate as substrates. (A) Different numbers of cell equivalents from OX41+ and OX41− DCs and purified IECs were loaded onto the gel. IECs and OX41− L-DCs show a similar pattern of bands, although IECs contain larger amounts of enzyme. OX41+ DCs express very little NSE, that seen probably being due to endogenous NSE variants and/or to contamination with small numbers of OX41− DCs. (B) Zymograms were prepared from OX41+ and OX41− L-DCs, IECs, and total (unseparated) MLN or CLN cells and other tissues. OX41− DCs and IECs show similar isoforms (arrowheads). This pattern is not seen in any other tissue tested. Protein extracts equivalent to 8 × 105 OX41− DCs, 104 IECs, 5 × 105 MLN or CLN cells, 1 mg wet weight (spleen, thymus), or 0.1 mg wet weight (lung, liver, kidney) were loaded onto each lane. (C) Zymograms were prepared from OX41− L-DCs, IECs, DCs isolated from MLN and CLN, bone marrow–derived DCs, aMΦs (Alv. MΦ), and resident and thioglycollate (Thio.)-elicited pMΦs (Pe. MΦ). MLN DCs, but not CLN DCs, contain NSE variants similar to those present in OX41− DCs and IECs (arrowheads). These are not seen in zymograms from any other cell type. Material equivalent to 106 cells was loaded onto each lane, except for IECs (104 cells).
Figure 3
Figure 3
DCs containing IEC markers are present in LP and PP. Cryostat sections of small intestine from SPF rats were labeled for IEC markers, apoptotic DNA, or NSE, with or without DC markers. (A) Intestinal villi labeled for epithelial cell cytokeratin 4 with mAb RK4. The IECs react strongly for cytoplasmic cytokeratin. Occasional large irregular cells in the LP contain granular, cytokeratin-positive inclusions (arrowhead). (B) Intestinal villi labeled for OX62 (blue) and NSE (red). The epithelial cells react strongly for NSE. Several cells in the LP are positive for both NSE and OX62. (C) Intestinal villi labeled for MHC class II and apoptotic DNA. Several cells in the LP are positive for MHC class II and apoptotic DNA. (D) As C, but labeled for OX62 and apoptotic DNA. Some cells express both markers. (E) PP; base, NSE. Many positive cells are in close apposition to the muscle layers. (F) PP. Electron micrograph of basal layers labeled for MHC class II. An MHC class II+, irregular cell contains an apoptotic cell within its cytoplasm.
Figure 4
Figure 4
Cells with the characteristics of OX41−NSE+ L-DCs are present in PP and MLNs but not other nodes. (A) Cryosection of CLN reacted for NSE. Weakly positive cells are present, particularly in the follicles. (B) Cryosection of MLN reacted for NSE. Large numbers of large, irregular, strongly positive cells are present in the subcapsular sinus, interfollicular traffic areas, and the paracortical T cell area (T). (C) Cryosection of MLN reacted for NSE and labeled for MHC class II. The paracortex contains large numbers of large, irregular cells positive for both markers. (D) Cryosection of MLN labeled for NSE and OX62. The paracortex contains large numbers of large irregular cells positive for both markers. (E) Electron micrograph of MLN labeled for MHC class II. An MHC class II+ interdigitating cell in the paracortex contains large, dense cytoplasmic inclusions, similar to those seen in L-DCs. (F) Cryosection of PP labeled for NSE (red) and CD11c (blue). Several double-positive cells (<<) and one CD11c+NSE− cell (>) is present in the T cell area of the patch.
Figure 5
Figure 5
Strongly NSE+ DCs are present in the LP and T cell areas of MLNs from germ-free rats. LNs and ileum from germ-free rats were snap frozen. Cryosections of ileum (A) and MLN (B) were fixed in 2% paraformaldehyde, reacted for NSE (red), and labeled for OX62 (blue in A, brown in B). (A) Double-positive cells (<) are present in apposition to the epithelium. One cell (<<, enlarged in inset) appears to be engulfing an epithelial cell. (B) Several large, irregular, double-positive cells are present in the T cell area of the node (<, >).
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