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. 2010 Jun;160(3):440-9.
doi: 10.1111/j.1365-2249.2010.04103.x. Epub 2010 Feb 10.

CCR6 identifies lymphoid tissue inducer cells within cryptopatches

A Lügering  1 , M Ross, M Sieker, J Heidemann, I R Williams, W Domschke, T Kucharzik
Affiliations

CCR6 identifies lymphoid tissue inducer cells within cryptopatches

A Lügering et al. Clin Exp Immunol. 2010 Jun.

Abstract

The chemokine receptor CCR6 is expressed by dendritic cells, B and T cells predominantly within the organized structures of the gut-associated lymphatic tissue. Its ligand CCL20 is synthesized by the follicle-associated epithelium and is crucial for the development of M cells within Peyer's patches. In addition, lineage-negative c-kit positive lymphocytes within cryptopatches (CP) express CCR6. CCR6-deficient mice exhibit an altered intestinal immune system containing increased amounts of intraepithelial lymphocytes and show smaller Peyer's patches, while progression of cryptopatches to mature isolated lymphoid follicles (ILF) is inhibited. In this report, we show that lin(-) c-kit(+) lymphocytes express a variety of different chemokine receptors and that CCR6 identifies those cells located within CP. In contrast, cells found outside CP are positive for CXCR3 and exhibit a different surface marker profile, suggesting that at least two different populations of lin(-) c-kit(+) cells are present. The presence of CCR6 does not influence the expression of Notch molecules on lin(-) c-kit(+) cells, nor does it influence Notch ligand expression on bone marrow-derived dendritic cells. In the human gut, CCR6 identifies clusters of lymphocytes resembling murine CP. CCR6 seems to have an important role for lin(-) c-kit(+) cells inside CP, is expressed in a regulated manner and identifies potential human CP.

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Figures

Fig. 1
Fig. 1
CCR6 characterizes lymphoid tissue inducer (Lti) cells within cryptopatches. Fluorescence activated cell sorter (FACS) analysis of lin- c-kit+ lamina propria cells (a) revealed a significant proportion of CCR6-expressing cells within the lin- c-kit+ cell fraction [approximately 15–20%; analysis of heterozygous enhanced green fluorescent protein (EGFP)–CCR6 knock-in mice, n = 10]. However, when analysed by immunohistochemistry (b), a significantly higher number of cryptopatch cells expressed this receptor (75 ± 8%, n = 10, P < 0001). All control stainings were negative.
Fig. 2
Fig. 2
CCR6 does not affect Notch signalling. RNA was isolated from sorted lin- c-kit+ cells from wild-type and CCR6-deficient mice. Reverse transcription–polymerase chain reaction (PT–PCR) analysis revealed a similar expression of Notch receptors by CCR6-deficient lymphoid tissue inducer (Lti) cells (a). In addition, 24 h stimulation of CCR6 expressing bone marrow dendritic cells (bmDC) by Macrophage inflammatory protein 3α (Mip3α) (10 ng/ml) did not induce Notch-ligand expression as measured by real-time PCR (b; CCR6-deficient bmDC served as controls; n = 5 per group).
Fig. 3
Fig. 3
Chemokine receptor expression of lin- c-kit+ cells. Samples of cDNA prepared from sorted lin- c-kit+ and lin+ c-kit+ cells, Percoll gradient-purified intraepithelial lymphocytes (IELs) and Peyer's patches were amplified with multiplex polymerase chain reaction (PCR) primer sets specific for CCR1 to CCR5 (top panels) or CCR6 to CCR9 and CX3CR1 (bottom panels). The location of the specific PCR amplimers in the positive control samples are indicated. Data are representative for three individual experiments. Lin- c-kit+ and lin+ c-kit+ cells were derived from the lamina propria.
Fig. 4
Fig. 4
Reciprocal expression of CCR6 and CXCR3 by lin- c-kit+ cells. Percoll-purified lamina proria lymphcytes from wild-type mice were stained with allophycocyanin (APC)-anti-c-kit, biotinylated monoclonal antibodies (mAbs) to lineage markers followed by streptavidin–peridinin chlorophyll (PerCP), phycoerythrin (PE)-anti-CCR6 and rabbit anti-mouse CXCR3 followed by fluorescein isothiocyanate (FITC) goat anti-rabbit immunoglobulin. A dot-plot of CCR6 versus CXCR3 staining is shown for the gated lin- c-kit- population (a). Immunofluorescence staining of a cryptopatch with anti-CXCR3 (green), PE-anti-Thy-1 and Topro-3 as a nuclear counterstain (b). Only rare cells within the cryptopatches are found to be CXCR3+. All control stainings were negative. The data are representative for stainings obtained from five individual wild-type mice.
Fig. 5
Fig. 5
Fluorescence activated cell sorter (FACS) analysis of lymphoid tissue inducer (Lti) cells. Lamina propria cells were isolated and lin- c-kit+ cells (LTi cells) characterized by staining for surface markers. While LTi cells outside (CCR6-) and inside (CCR6+) cryptopatches express similar levels of the activation markers CD25 and CD127, as well as CD44, significantly different expression patterns could be found for CD45Rb, CD4 and CD8, suggesting that at least two different subtypes of LTi cells are present in the intestine. The data are representative for stainings obtained from five individual CCR6 knock-in mice.
Fig. 6
Fig. 6
CCR6 and c-kit expression in the gut. Staining of human (a) and murine (b) intestinal tissue specimens showed that in contrast to the cryptopatches-restricted expression in the murine gut, c-kit+ lymphocytes are found diffusely within the human intestine. However, small clusters of lymphocytes partially expressing c-kit (c) are also found in the human intestine containing a significant number of CCR6+ lymphocytes (d). In addition, multiple expanding clusters of CCR6-expressing cells are found in the mucosa of ulcerative colitis patients (e+f). All control stainings were negative.
Fig. 7
Fig. 7
Detection of cryptopatch cells in the human gut. Lamina propria leucocytes were isolated from large intestinal specimens and stained for the expression of CCR6, c-kit and retinoic acid-related orphan receptor gamma (RORγ) in CD3-, CD11c- and CD19- cells. In contrast to the murine intestine, a second population of SSChigh leucocytes expresses high levels of c-kit but does not express RORγ (right panel). In contrast, CD3-CD11c-CD19- c-kitlow cells within the lymphocyte gate express RORγ homogeneously and are partially positive for the chemokine receptor CCR6 (left panel), highly suggestive for the human correlate of murine cryptopatch cells.
Fig. 8
Fig. 8
Intestinal inflammation does not extend the number of cryptopatch cells. Large intestinal inflammation was induced by dextran sulphate sodium (DSS) colitis (a) and Citrobacter rodentium colitis (b). The amount of lin- c-kit+ CCR6+ cryptopatch cells within the large intestinal lamina propria was determined at various time-points by flow cytometry giving no evidence for de novo cryptopatch formation over time.
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