Molecular basis for hematopoietic/mesenchymal interaction during initiation of Peyer's patch organogenesis

Abstract

Mice deficient in lymphotoxin beta receptor (LTbetaR) or interleukin 7 receptor alpha (IL-7Ralpha) lack Peyer's patches (PPs). Deficiency in CXC chemokine receptor 5 (CXCR5) also severely affects the development of PPs. A molecular network involving these three signaling pathways has been implicated in PP organogenesis, but it remains unclear how they are connected during this process. We have shown that PP organogenesis is initiated at sites containing IL-7Ralpha(+) lymphoid cells and vascular cell adhesion molecule (VCAM)-1/intercellular adhesion molecule (ICAM)-1 expressing nonlymphoid elements. Here we characterize these lymphoid and nonlymphoid components in terms of chemokine signals. The lymphoid population expresses CXCR5 and has a strong chemotactic response to B lymphocyte chemoattractant (BLC). Importantly, chemokines produced by VCAM-1(+)ICAM-1(+) nonlymphoid cells mediate the recruitment of lymphoid cells. Furthermore, we show that these VCAM-1(+)ICAM-1(+) cells are mesenchymal cells that are activated by lymphoid cells through the LTbetaR to express adhesion molecules and chemokines. Thus, promotion of PP development relies on mutual interaction between mesenchymal and lymphoid cells.

Figure 1

Chemotactic activity of BLC, ELC, and SLC on CD4⁺CD3⁻IL-7Rα¹ cells. (A) Whole-mount immunostaining analysis of E15.5, E16.5, and E17.5 gut with anti-CD4 mAb. (B) RT-PCR analysis on CXCR5 and CCR7 in FACS^®-sorted CD4⁺CD3⁻IL-7Rα¹ cells from intestine at E17.5. The numbers of cells subjected to RT-PCR are 1,000, 200, 40, and 8 cells from left to right in each panel. Specific primers for CD4, IL-7Rα, and CD3-ε were used as positive and negative controls. (C) CD4⁺ cells were prepared with Dynabeads and placed in the chemotaxis chamber in duplicates. ELC and SLC maximally attracted CD4⁺ cells at 100–300 ng/ml. Results are expressed as the percentage of input cells migrating to the lower chamber containing BLC (1 μg/ml), ELC (300 ng/ml), or SLC (300 ng/ml). Data from individual wells are shown as filled diamonds and means as bars. The results are representative of three independent experiments. (D) Chemotactic response to BLC by CD4⁺ cells from intestine at E17.5 (filled circles) or B cells from Spl (open squares). Lines represent the averages of duplicated Transwells. The results are representative of at least three independent experiments.

Figure 2

Expression of BLC, ELC, VCAM-1, and ICAM-1 in PP anlagen. Whole-mount in situ hybridization analysis of BLC (A) and ELC (B) expression in E17.5 intestines. Whole-mount immunostaining analysis of E16.5 (C and D) and E17.5 (E and F) gut with mAbs against VCAM-1 (C and E) and ICAM-1 (D and F). Cryostat sections of intestines at E17.5 were double labeled with Abs against VCAM-1 (Alexa™ 594; red) in combination with (G) anti–ICAM-1 (FITC; green) or (H) anti-CD4 (FITC; green) (original magnification: ×200) and subjected to confocal microscopic analysis. The insets are included to show the morphology of the V⁺I⁺ cells and CD4⁺ cells (original magnification: ×630).

Figure 3

Chemokine expression by V⁺I⁺ cells. FACS^® analysis of the intestinal cells from at E15.5 (A) and at E17.5 (B–D) with anti–VCAM-1 and anti–ICAM-1. V⁺I⁺ cells are absent in IL-7Rα^−/− embryonic intestine (D) but present in the heterozygous littermate (C). The numbers indicate the percentage of the V⁺I⁺ population. (E) RT-PCR analysis of transcripts encoding ELC and BLC in FACS^®-sorted V⁺I⁺ population (a) and other populations (b to d) from E17.5, as indicated in B. Reverse-transcribed mRNA from Spl tissue was analyzed as positive control. PCR with GAPDH primers is shown as control for the cDNA content of individual samples. Similar results were obtained from three independent experiments.

Figure 3

Chemokine expression by V⁺I⁺ cells. FACS^® analysis of the intestinal cells from at E15.5 (A) and at E17.5 (B–D) with anti–VCAM-1 and anti–ICAM-1. V⁺I⁺ cells are absent in IL-7Rα^−/− embryonic intestine (D) but present in the heterozygous littermate (C). The numbers indicate the percentage of the V⁺I⁺ population. (E) RT-PCR analysis of transcripts encoding ELC and BLC in FACS^®-sorted V⁺I⁺ population (a) and other populations (b to d) from E17.5, as indicated in B. Reverse-transcribed mRNA from Spl tissue was analyzed as positive control. PCR with GAPDH primers is shown as control for the cDNA content of individual samples. Similar results were obtained from three independent experiments.

Figure 4

Signaling through IL-7Rα and LTβR are required for induction of V⁺I⁺ cells. (A) Either A7R34 (3 mg), LTβR-Ig (250 μg), or TNFp55-Ig (250 μg) was injected (inj.) to pregnant mice at E15.5, and cells were prepared from embryonic intestines at E17.5 and analyzed by FACS^®. Control mice were treated with equal amounts of human-IgG Fc fragment or polyclonal rat IgG. The numbers indicate the percentage of the cell population positive for both VCAM-1 and ICAM-1. (B) Expression of LTβR in gated V⁺I⁺ cells was measured by flow cytometry. Cells were incubated with or without labeled anti-LTβR mAb (AFH6). To confirm signal specificity, blocking with unlabeled AFH6 was performed (right).

Figure 5

IL-7 induces LTα/β upregulation in PP inducers. (A–C) Histograms show LTβR-Ig staining in CD4⁺ cells. (A) CD4⁺ cells from E17.5 intestine after in utero treatment with A7R34 or control rat IgG at E15.5 were analyzed. inj., injected. (B) CD4⁺ cells from E15.5 intestine were analyzed after in vitro cultures in the absence or presence of rIL-7 at indicated amounts for 6 h. (C) Cells were preincubated with 3 μg/ml A7R34 or equal amounts of polyclonal rat IgG and then incubated with rIL-7 (40 U/ml) for 6 h. (D–G) E17.5 intestines after in utero treatment with either A7R34 or control rat IgG at E15.5 were analyzed. (D) Whole-mount in situ hybridization analysis of LTβ expression. Arrows indicate positive spots. (E) Whole-mount immunostaining with anti-CD4 mAb. Arrows indicate accumulation of CD4⁺ cells. (F) The number of CD4⁺CD3⁻ cells in the intestine was measured by FACS^®. (G) Chemotactic response of CD4⁺ cells from intestines after treatment with either control rat IgG (white bars) or A7R34 (black bars) to BLC or ELC.

Figure 6

Signal pathway from IL-7R to LTα/β regulates expression of BLC and ELC. (A) Northern blot analysis of BLC and ELC mRNA from E17.5 intestines after treatment with either A7R34 or polyclonal rat IgG at E15.5. GAPDH was used as control to quantitate amount of RNA loaded. Total RNA isolated from Spl tissue was loaded as positive control. inj., injected. (B) Whole-mount in situ hybridization analysis of BLC and ELC expression in E17.5 intestines after treatment with A7R34 or control rat IgG at E15.5. Several spots (arrows) positive for expression of BLC and ELC are visualized in the control intestine.

Figure 7

V⁺I⁺ cells are of a mesenchymal lineage. (A) Three-color FACS^® analysis of various cell surface molecules (CD45, CD11c, VE-cadherin, PDGFRα, PDGFRβ, and MAdCAM-1; filled histograms) or isotype-matched mAbs (open histograms) on gated V⁺I⁺ cells. (B) Representative transmission electron microscopic appearance of cells in the populations indicated in Fig. 3 B obtained from the gut at E17.5. FACS^®-sorted cells were fixed and embedded for sectioning. Arrowheads and arrows indicate well-developed endoplasmic reticulum and lipid droplets, respectively.