Figure 2.
Figure 2. Analysis of chemokine and chemokine receptor expression and function. (A) Transcription of CCL2, CCL17, and CCL20 was quantified by real-time PCR. To monitor the amplification of the specific cDNA at the indicated points of time, the fluorescence of PCR products caused by intercalated SYBR-Green was determined. The specifically amplified cDNA was quantified in relation to β-actin transcripts from the same cDNA preparation and is shown as x-fold induction of the gene transcripts of activated cells compared with nonactivated cells. At the bottom, an agarose gel separation of the corresponding product is shown. (B) The concentration of CCL2 (left) and CCL20 (right) in the supernatant of stimulated cells was determined at the indicated points of time using a chemokine-specific ELISA. The detection limit of the CCL2 and the CCL20 ELISAs was 4 pg/mL and 150 pg/mL, respectively. (C) The expression of CC receptor transcripts on sorted marginal zone (CD21+ CD23–) and naive follicular B cells (CD21+ CD23+) was analyzed by RT-PCR. (D) The surface expression of CCR6 after ligand binding was analyzed using flow cytometry. Splenocytes were incubated with an anti-CCR6 mAb for 20 minutes at 4°C. Subsequently, the ligand CCL20 was added and the cells were incubated at room temperature. After the times indicated, the cells were placed on ice anda secondary antibody was used to reveal the CCR6 expression on naive (CD21+ CD23+) follicular B cells (bold line indicates CCR6 expression on naive untreated cells; thin line, CCR6 expression of B cells after binding of CCL20; and broken line, isotype). (E) The chemokines CXCL12 (2; 10 ng/mL) and CCL20 (3; 100 ng/mL) were used to induce locomotion in CCR6-positive (▪) and CCR6-negative (□) B cells in a transwell assay. Cells were allowed to migrate for 2 hours at 37°C, harvested from the lower chamber, and analyzed by flow cytometry. As controls, no CCL20 was added to the lower chamber (1, medium control). Furthermore, as desensitization controls B cells were preincubated (10 minutes) with CCL20 (100 ng/mL) before being added to the upper chamber with (4) or without (5) the chemokine. The results of 3 independent experiments are presented as mean ± SEM. Statistical significance was determined using an unpaired Student t test (*P ≤ .001, **P ≤ .001, ***P ≤ .002). (F) The adhesion of B cells to endothelial cells activated for 6 hours was determined after Ptx treatment or desensitization of splenocytes with recombinant CCL20 at a concentration of 10 ng/mL and 1000 ng/mL. The number of B cells was quantified using electronic gating. Adhesion of untreated B lymphocytes to activated endothelial cells was set at 100% (*P ≤ .001, **P ≤ .004, ***P ≤ .005). The results of 4 independent experiments are presented as mean ± SEM. Statistical significance was determined using an unpaired Student t test.

Analysis of chemokine and chemokine receptor expression and function. (A) Transcription of CCL2, CCL17, and CCL20 was quantified by real-time PCR. To monitor the amplification of the specific cDNA at the indicated points of time, the fluorescence of PCR products caused by intercalated SYBR-Green was determined. The specifically amplified cDNA was quantified in relation to β-actin transcripts from the same cDNA preparation and is shown as x-fold induction of the gene transcripts of activated cells compared with nonactivated cells. At the bottom, an agarose gel separation of the corresponding product is shown. (B) The concentration of CCL2 (left) and CCL20 (right) in the supernatant of stimulated cells was determined at the indicated points of time using a chemokine-specific ELISA. The detection limit of the CCL2 and the CCL20 ELISAs was 4 pg/mL and 150 pg/mL, respectively. (C) The expression of CC receptor transcripts on sorted marginal zone (CD21+ CD23–) and naive follicular B cells (CD21+ CD23+) was analyzed by RT-PCR. (D) The surface expression of CCR6 after ligand binding was analyzed using flow cytometry. Splenocytes were incubated with an anti-CCR6 mAb for 20 minutes at 4°C. Subsequently, the ligand CCL20 was added and the cells were incubated at room temperature. After the times indicated, the cells were placed on ice anda secondary antibody was used to reveal the CCR6 expression on naive (CD21+ CD23+) follicular B cells (bold line indicates CCR6 expression on naive untreated cells; thin line, CCR6 expression of B cells after binding of CCL20; and broken line, isotype). (E) The chemokines CXCL12 (2; 10 ng/mL) and CCL20 (3; 100 ng/mL) were used to induce locomotion in CCR6-positive (▪) and CCR6-negative (□) B cells in a transwell assay. Cells were allowed to migrate for 2 hours at 37°C, harvested from the lower chamber, and analyzed by flow cytometry. As controls, no CCL20 was added to the lower chamber (1, medium control). Furthermore, as desensitization controls B cells were preincubated (10 minutes) with CCL20 (100 ng/mL) before being added to the upper chamber with (4) or without (5) the chemokine. The results of 3 independent experiments are presented as mean ± SEM. Statistical significance was determined using an unpaired Student t test (*P ≤ .001, **P ≤ .001, ***P ≤ .002). (F) The adhesion of B cells to endothelial cells activated for 6 hours was determined after Ptx treatment or desensitization of splenocytes with recombinant CCL20 at a concentration of 10 ng/mL and 1000 ng/mL. The number of B cells was quantified using electronic gating. Adhesion of untreated B lymphocytes to activated endothelial cells was set at 100% (*P ≤ .001, **P ≤ .004, ***P ≤ .005). The results of 4 independent experiments are presented as mean ± SEM. Statistical significance was determined using an unpaired Student t test.

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