Volume 127, Issue 1, Pages 213-223 (July 2004) Lymphotoxin β receptor signaling induces the chemokine CCL20 in intestinal epithelium  Martin Rumbo, Frédéric Sierro, Nathalie Debard, Jean-Pierre Kraehenbuhl, Daniela Finke  Gastroenterology  Volume 127, Issue 1, Pages 213-223 (July 2004) DOI: 10.1053/j.gastro.2004.04.018

Figure 1 Identification of hematopoietic precursor cells, lymphotoxin α1β2 and its receptor, and CCL20 in PP anlage during embryonic development. Serial sections of the small intestine at different embryonic days were analyzed. (A-C) Double staining with anti-CD4 (peroxidase, brown) and anti-CD3 antibodies (alkaline phosphatase, blue). Clusters of CD4+ cells were detected at day E17.5 and E18.5. CD3+ cells were rare in the follicles. Epithelium strongly express endogenous alkaline phosphatase and shows a strong blue staining. (D-F) In situ hybridization with LTβ anti-sense probe. At day E17.5 and E18.5, there is a strong labeling of follicles in PP anlage. (G-I) In situ hybridization with LTβR anti-sense probe. Receptor is expressed in the epithelium at all stages. (J-L) In situ hybridization with CCL20 anti-sense probe. A weak epithelial CCL20 signal over the PP anlage was detected as early as day E17.5 and increased at day E18.5. Scale bars represent 50 μm. Gastroenterology 2004 127, 213-223DOI: (10.1053/j.gastro.2004.04.018)

Figure 2 LTβ and CCL20 expression in RAG2 mice. (A) In situ hybridization with LTβ anti-sense probe. A signal is clearly seen in association with the follicle of the rudimentary PPs. (B) In situ hybridization with CCL20 anti-sense probe. CCL20 signal is present on the epithelium over the rudimentary PP’s follicles. Scale bars represent 50 μm. Gastroenterology 2004 127, 213-223DOI: (10.1053/j.gastro.2004.04.018)

Figure 3 LTβ and CCL20 expression in MyD88−/− mice. Tissue before and after laser microdissection of crypt epithelium (parts A and B, respectively) and epithelial villi (parts C and D, respectively) is shown. (E) Laser dissection microscopy was used to prepare samples highly enriched in FAE (1), villus epithelium (2), crypt epithelium (3), or follicular material (4). (F) CCL20 relative expression in the different tissue types of MyD88−/− and C57BL/6 mice. Tissue from 3 different mice from each strain was processed and qPCR performed on the corresponding cDNA. Results show the average and SD among the 3 different mice processed. (G) LTβ relative expression in the different tissue types in MyD88−/− and C57BL/6 mice. Tissue from 3 different mice from each strain was processed and qPCR performed on the corresponding cDNA. Results show the average and SD among the 3 different mice processed. Scale bars represent 50 μm. Gastroenterology 2004 127, 213-223DOI: (10.1053/j.gastro.2004.04.018)

Figure 4 LTβR signaling induces CCL20 expression in human intestinal T84 cells. Results are from at least 3 independent experiments. (A) Dose-response curve of LTα1β2 stimulation. T84 cells were stimulated for 3 hours with different concentrations of recombinant LTα1β2. CCL20-specific mRNA was measured by RT-PCR. (B) TNFR55-Ig fusion protein does not block the effect of recombinant LTα1β2. TNF-α or LTβ was preincubated for 1 hour with a 100-fold molar excess of TNFR55-Ig. The cells were incubated as in part A. The fusion protein abolished TNF-α-induced, but not LTα1β2-induced, CCL20 mRNA accumulation. (C) Agonist LTβR-specific antibodies induce CCL20 expression by T84 cells. Agonist antibodies directed against the LTβR were used to stimulate cell culture as in part A. CCL20 induction was observed. Gastroenterology 2004 127, 213-223DOI: (10.1053/j.gastro.2004.04.018)

Figure 5 (A) Kinetics of CCL20 induction. T84 cells were stimulated for the indicated times with LTα1β2 recombinant protein (1 μg/mL). CCL20 expression after stimulation was measured as described in the Materials and Methods section. A sustained induction of CCL20 was observed at least up to 24 hours of stimulation. (B) Analysis of CCL20 promoter activity on LTβ stimulation. T84 cells were transfected with a normalizer plasmid and a luciferase reporter construct containing the 1.6-kb sequence upstream of the CCL20 putative transcription start. In parallel, construct with mutated NF-κB-binding site sequences was used. Reporter activity was measured 8 hours after LTα1β2 stimulation and normalized to Renilla luciferase. (C) Kinetics of NF-κB activation on LTα1β2 stimulation. T84 cells were stimulated for different times using LTα1β2 recombinant protein (1 μg/mL) preincubated 1 hour with an excess of TNFR55-Ig fusion protein as in Figure 3C. Nuclear fractions from treated cells were analyzed by Western blots using either an anti-NF-κB p65 or anti-NF-κB p52 antibody. Time of treatment (hours) is indicated on top of each lane. As negative control (−), nuclear extract from untreated T84 cells was used. Total protein lysate from Raji B cells was used as positive control. (D) Kinetics of NF-κB activation on flagellin stimulation. T84 cells were stimulated during different times with flagellin (1 μg/mL). Nuclear fractions from treated cells were analyzed by Western blots as in part C. Time of treatment (h) is indicated on top of each lane. Similar controls as in C were used. (E) Quantitative analysis of p52 and p65 induction in nuclear extracts upon flagellin and LTα1β2 stimulation. Blots showed in sections C and D were scanned using the ImageJ software. Relative quantification was done by assigning an arbitrary value of 1 to the lowest signal in each group of experiments. Gastroenterology 2004 127, 213-223DOI: (10.1053/j.gastro.2004.04.018)

Figure 6 CCL20 expression is induced in vivo after anti-LTβR agonist antibody treatment. (A) CCL20 expression on treatment in different mice strains. CCL20-specific expression was determined by real-time PCR using RNA recovered from duodenal segments of mice injected 2 hours before with 50 μg of agonist antibody or control antibody. Wild-type C57BL/6, Myd88−/− mice, and LPS hyporesponsive C3H/HeJ strains were tested. The results shown are the average of 3 independent experiments. (B) CCL20 expression in treated LTα−/− mice. CCL20 specific expression was determined as described in part A from duodenal segments of newborn mice injected 2 hours before with either 5 μg of agonist antibody or control antibody. The results shown are the average of 3 independent experiments. (C) CCL20 mRNA up-regulation is induced at 2-hours postinjection and sustained for up to 8-hours postinjection. Treatment as described in part A was performed. Mice were killed at different times posttreatment, and CCL20 specific expression was monitored. The results shown are the average of 3 independent experiments. (D) CCL20 expression in small intestine epithelial tissue from treated mice. CCL20 specific expression was determined by real-time PCR on RNA from laser-dissected epithelial tissue from anti-LTβR-treated mice or from PBS-treated or isotype matched antibody controls. CCL20 is up-regulated in the epithelium of mice treated with the anti-LTβR antibody. Gastroenterology 2004 127, 213-223DOI: (10.1053/j.gastro.2004.04.018)