Expression of ESE-3 isoforms in immunogenic and tolerogenic human monocyte-derived dendritic cells

Abstract

Dendritic cells (DC) are the only hematopoietic cells expressing the epithelial specific Ets transcription factor ESE-3. Here we analyzed presence and quantity of isoforms ESE-3a, ESE-3b and ESE-3j in various immunogenic and tolerogenic human monocyte-derived DC (moDC) and blood DC populations using quantitative real time PCR and immunoblot analyses. ESE-3a and ESE-3b were detectable in all moDC populations with ESE-3b being the main transcript. ESE-3b expression was upregulated in immunogenic moDC and downregulated in tolerogenic moDC compared to immature moDC. ESE-3a had similar transcript levels in immature and immunogenic moDC and had very low levels in tolerogenic moDC. In blood DC populations only splice variant ESE-3b was detectable. ESE-3j was not detectable in any of the DC populations. These findings suggest that ESE-3b is the functionally most important ESE-3 isoform in DC.

Figure 1. Exon composition of ESE-3 transcript variants.

Exons are marked with numbers 1–9. Critical exons for distinction between the splice variants are color-coded. The different domains and translational start (→) and stop (|<$>\vskip 1pt\raster="rg1"<$>) are marked. An alternative exon 1a distinguishes ESE-3j from ESE-3a and ESE-3b. Translation of ESE-3j is initiated in exon 1a, whereas translation of ESE-3a and ESE-3b starts in exon 2. ESE-3a is the shortest transcript variant owing to the lack of exon 6.

Figure 2. Overexpressed ESE-3a and ESE-3b are located in the nucleus of HeLa cells.

HeLa cells were transfected with expression vectors coding for ESE-3a-EGFP and ESE-3b-EGFP. After 24 h, cells were fixed and subjected to immunocytochemistry using a monoclonal antibody specific for human ESE-3. The ESE-3a- and ESE-3b-constructs localized exclusively to the nucleus as revealed by both the intrinsic green fluorescence and the ESE-3 immunoreactivity. One representative experiment out of three is shown. Scale bar: 50 µM.

Figure 3. Phenotypic characterization of immunogenic and tolerogenic moDC populations by flow cytometry.

Monocytes were negatively selected from PBMC using magnetic beads. Immature moDC were generated with IL-4 and GM-CSF for 6 days. 15d-PGJ₂ (PGJ2 DC) and dexamethasone plus 1α,25-dihydroxyvitamin were added to generate tolerogenic moDC, respectively (PGJ2 DC and Dex/VD3 DC). To generate immunogenic moDC, immature moDC were stimulated for 24 h with LPS, polyI:C and a cytokine cocktail containing TNF-α, IL-1β, IL-6 and PGE₂, respectively. The phenotypes of the cells were analyzed by flow cytometry. Live cells were gated according to FSC/SSC. One representative experiment out of three is shown.

Figure 4. ESE-3b is the main ESE-3 isoform in moDC.

Monocytes were negatively selected from PBMC using magnetic beads. Immature moDC were generated with IL-4 and GM-CSF for 6 days. 15d-PGJ₂ (PGJ2 DC) and dexamethasone plus 1α,25-dihydroxyvitamin were added to generate tolerogenic moDC, respectively (PGJ2 DC and Dex/VD3 DC). To generate immunogenic moDC, immature moDC were stimulated for 24 h with LPS, polyI:C and a cytokine cocktail containing TNF-α, IL-1β, IL-6 and PGE₂, respectively. ESE-3a and ESE-3b mRNA levels were determined by quantitative RT-PCR analyses. (A) Primers and probes used for quantitative real-time RT PCR are marked. Exons are color-coded as in Figure 1. Positions of the base pairs of the open reading frames are indicated by numbers. (B) Relative amounts of ESE-3a and ESE-3b cDNA in various moDC populations. Transcript levels of GAPDH were used to calculate delta Ct values. Lower delta Ct values indicate higher amounts of target mRNA. N = 4–6. Significant differences between delta Ct values of ESE-3a and ESE-3b within the same moDC population are indicated. ***p<0.001.

Figure 5. ESE-3b expression is increased in immunogenic moDC.

Monocytes were negatively selected from PBMC using magnetic beads. Immature moDC were generated with IL-4 and GM-CSF for 6 days. 15d-PGJ₂ (PGJ2 DC) and dexamethasone plus 1α, 25-dihydroxyvitamin were added to generate tolerogenic moDC, respectively (PGJ2 DC and Dex/VD3 DC). To generate immunogenic moDC, immature moDC were stimulated for 24 h with LPS, polyI:C and a cytokine cocktail containing TNF-α, IL-1β, IL-6 and PGE₂, respectively. (A) ESE3a and (B) ESE-3b mRNA levels were analyzed by quantitative real-time RT PCR using isoform specific primer/probes. GAPDH was used for normalization, immature moDC were used as reference. N = 4–6, *p<0.05. (C) Protein levels of ESE-3 in nuclear extracts were analyzed by Western blotting using a monoclonal antibody specific for human ESE-3. Histone H3 was included to ensure equal loading of the gel. One representative experiment out of three is shown.

Figure 6. ESE-3b is the only ESE-3 isoform transcribed in blood DC.

MDC1, mDC2 and pDC were isolated from PBMC by positive selection with magnetic beads. Quantitative real-time RT-PCR using isoform specific primer/probes was used to analyze mRNA levels of ESE-3a and ESE-3b. GAPDH was used for normalization. No signal for ESE-3a was detected. N = 5.