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ICSBP is essential for the development of mouse type I interferon-producing cells and for the generation and activation of CD8alpha(+) dendritic cells.

Schiavoni G, Mattei F, Sestili P, Borghi P, Venditti M, Morse HC, Belardelli F, Gabriele L - J. Exp. Med. (2002)

Bottom Line: Moreover, ICSBP(-/-) CD8alpha(+) DCs exhibited a markedly impaired phenotype when compared with WT DCs.In addition, these cells were unable to undergo full phenotypic activation upon in vitro culture in presence of maturation stimuli such as lipopolysaccharide or poly (I:C), which paralleled with lack of Toll-like receptor (TLR)3 mRNA expression.Finally, cytokine expression pattern was also altered in ICSBP(-/-) DCs, as they did not express interleukin (IL)-12p40 or IL-15, but they displayed detectable IL-4 mRNA levels.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Virology, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.

ABSTRACT
Interferon (IFN) consensus sequence-binding protein (ICSBP) is a transcription factor playing a critical role in the regulation of lineage commitment, especially in myeloid cell differentiation. In this study, we have characterized the phenotype and activation pattern of subsets of dendritic cells (DCs) in ICSBP(-/-) mice. Remarkably, the recently identified mouse IFN-producing cells (mIPCs) were absent in all lymphoid organs from ICSBP(-/-) mice, as revealed by lack of CD11c(low)B220(+)Ly6C(+)CD11b(-) cells. In parallel, CD11c(+) cells isolated from ICSBP(-/-) spleens were unable to produce type I IFNs in response to viral stimulation. ICSBP(-/-) mice also displayed a marked reduction of the DC subset expressing the CD8alpha marker (CD8alpha(+) DCs) in spleen, lymph nodes, and thymus. Moreover, ICSBP(-/-) CD8alpha(+) DCs exhibited a markedly impaired phenotype when compared with WT DCs. They expressed very low levels of costimulatory molecules (intercellular adhesion molecule [ICAM]-1, CD40, CD80, CD86) and of the T cell area-homing chemokine receptor CCR7, whereas they showed higher levels of CCR2 and CCR6, as revealed by reverse transcription PCR. In addition, these cells were unable to undergo full phenotypic activation upon in vitro culture in presence of maturation stimuli such as lipopolysaccharide or poly (I:C), which paralleled with lack of Toll-like receptor (TLR)3 mRNA expression. Finally, cytokine expression pattern was also altered in ICSBP(-/-) DCs, as they did not express interleukin (IL)-12p40 or IL-15, but they displayed detectable IL-4 mRNA levels. On the whole, these results indicate that ICSBP is a crucial factor in the regulation of two possibly linked processes: (a) the development and activity of mIPCs, whose lack in ICSBP(-/-) mice may explain their high susceptibility to virus infections; (b) the generation and activation of CD8alpha(+) DCs, whose impairment in ICSBP(-/-) mice can be responsible for the defective generation of a Th1 type of immune response.

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Impaired number and phenotype of CD8α+ DCs in spleens from ICSBP−/− mice. Spleens from ICSBP−/− or WT mice were pooled and enriched for DCs by Nycodenz density-gradient centrifugation, as described in Materials and Methods. (A) The low-density cell fraction was double-stained for CD11c and CD8α expression. The histograms show the percentage of CD8α+ DCs in ICSBP−/− and WT mice, gated for CD11c positivity and by forward side scatter properties. (B) The CD8α− and CD8α+ CD11c+ DC-subsets were additionally stained for ICAM-1, CD40, CD80, CD86, MHC class I, or class II molecules. Histograms show specific staining for the indicated antigens in CD8α+ CD11c+ (left) and CD8α- CD11c+ (right) gated populations, in ICSBP−/− (filled) and WT (open) mice. The broken profiles represent the background fluorescence for control isotype-matched antibodies. (C) Forward scatter profiles of CD8α− (filled histograms) and CD8α+ (open histograms) CD11c+ DCs from ICSBP−/− and WT mice. (D) Density plot analysis showing CD4 and CD8α expression in CD11c-gated DCs from ICSBP−/− and WT mice. Results are representative of at least five independent experiments.
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fig3: Impaired number and phenotype of CD8α+ DCs in spleens from ICSBP−/− mice. Spleens from ICSBP−/− or WT mice were pooled and enriched for DCs by Nycodenz density-gradient centrifugation, as described in Materials and Methods. (A) The low-density cell fraction was double-stained for CD11c and CD8α expression. The histograms show the percentage of CD8α+ DCs in ICSBP−/− and WT mice, gated for CD11c positivity and by forward side scatter properties. (B) The CD8α− and CD8α+ CD11c+ DC-subsets were additionally stained for ICAM-1, CD40, CD80, CD86, MHC class I, or class II molecules. Histograms show specific staining for the indicated antigens in CD8α+ CD11c+ (left) and CD8α- CD11c+ (right) gated populations, in ICSBP−/− (filled) and WT (open) mice. The broken profiles represent the background fluorescence for control isotype-matched antibodies. (C) Forward scatter profiles of CD8α− (filled histograms) and CD8α+ (open histograms) CD11c+ DCs from ICSBP−/− and WT mice. (D) Density plot analysis showing CD4 and CD8α expression in CD11c-gated DCs from ICSBP−/− and WT mice. Results are representative of at least five independent experiments.

Mentions: We therefore examined the distribution of CD8α− and CD8α+ DC subsets in spleens, thymus, mesenteric and skin-draining lymph nodes from ICSBP−/− or WT mice. Gradient-enriched DCs were double-stained for CD11c and CD8α markers and analyzed by flow cytometry. As illustrated in Fig. 3 A, showing the expression of CD8α in spleen DCs gated by CD11c positivity and forward side scatter properties, 32% of splenic DCs were CD8α+ in WT mice, similarly to values previously found in other mouse strains (19). Surprisingly, spleen CD8α+ detected in ICSBP−/− mice only represented 3.6% of the total DC population. To further analyze the maturation and activation phenotype of CD8α− and CD8α+ subsets of splenic DCs from ICSBP−/− and control mice, we performed three-color flow cytometric analysis combining the CD11c and CD8α markers alternatively with the costimulatory antigens CD40, CD80, CD86, intercellular adhesion molecule (ICAM)-1, and the MHC class I and class II molecules. As shown in Fig. 3 B, the CD8α+ DC subpopulation from ICSBP−/− mice expressed significantly lower levels of the costimulatory antigens CD40, CD80, CD86, and ICAM-1 with respect to the WT counterparts. In contrast, in the CD8α− DC-subset all the considered costimulatory antigens and activation markers were expressed at comparable levels in ICSBP−/− and WT mice. The altered phenotype of CD8α+ DCs in ICSBP−/− mice was further confirmed by the morphologic analysis of the forward scatter profile (Fig. 3 C), showing that, consistently with previous reports (36), splenic CD8α+ DCs were bigger than CD8α− DCs in WT mice. Conversely, CD8α+ DCs recovered from the spleens of ICSBP−/− mice proved to be significantly smaller than CD8α− DCs.


ICSBP is essential for the development of mouse type I interferon-producing cells and for the generation and activation of CD8alpha(+) dendritic cells.

Schiavoni G, Mattei F, Sestili P, Borghi P, Venditti M, Morse HC, Belardelli F, Gabriele L - J. Exp. Med. (2002)

Impaired number and phenotype of CD8α+ DCs in spleens from ICSBP−/− mice. Spleens from ICSBP−/− or WT mice were pooled and enriched for DCs by Nycodenz density-gradient centrifugation, as described in Materials and Methods. (A) The low-density cell fraction was double-stained for CD11c and CD8α expression. The histograms show the percentage of CD8α+ DCs in ICSBP−/− and WT mice, gated for CD11c positivity and by forward side scatter properties. (B) The CD8α− and CD8α+ CD11c+ DC-subsets were additionally stained for ICAM-1, CD40, CD80, CD86, MHC class I, or class II molecules. Histograms show specific staining for the indicated antigens in CD8α+ CD11c+ (left) and CD8α- CD11c+ (right) gated populations, in ICSBP−/− (filled) and WT (open) mice. The broken profiles represent the background fluorescence for control isotype-matched antibodies. (C) Forward scatter profiles of CD8α− (filled histograms) and CD8α+ (open histograms) CD11c+ DCs from ICSBP−/− and WT mice. (D) Density plot analysis showing CD4 and CD8α expression in CD11c-gated DCs from ICSBP−/− and WT mice. Results are representative of at least five independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2194263&req=5

fig3: Impaired number and phenotype of CD8α+ DCs in spleens from ICSBP−/− mice. Spleens from ICSBP−/− or WT mice were pooled and enriched for DCs by Nycodenz density-gradient centrifugation, as described in Materials and Methods. (A) The low-density cell fraction was double-stained for CD11c and CD8α expression. The histograms show the percentage of CD8α+ DCs in ICSBP−/− and WT mice, gated for CD11c positivity and by forward side scatter properties. (B) The CD8α− and CD8α+ CD11c+ DC-subsets were additionally stained for ICAM-1, CD40, CD80, CD86, MHC class I, or class II molecules. Histograms show specific staining for the indicated antigens in CD8α+ CD11c+ (left) and CD8α- CD11c+ (right) gated populations, in ICSBP−/− (filled) and WT (open) mice. The broken profiles represent the background fluorescence for control isotype-matched antibodies. (C) Forward scatter profiles of CD8α− (filled histograms) and CD8α+ (open histograms) CD11c+ DCs from ICSBP−/− and WT mice. (D) Density plot analysis showing CD4 and CD8α expression in CD11c-gated DCs from ICSBP−/− and WT mice. Results are representative of at least five independent experiments.
Mentions: We therefore examined the distribution of CD8α− and CD8α+ DC subsets in spleens, thymus, mesenteric and skin-draining lymph nodes from ICSBP−/− or WT mice. Gradient-enriched DCs were double-stained for CD11c and CD8α markers and analyzed by flow cytometry. As illustrated in Fig. 3 A, showing the expression of CD8α in spleen DCs gated by CD11c positivity and forward side scatter properties, 32% of splenic DCs were CD8α+ in WT mice, similarly to values previously found in other mouse strains (19). Surprisingly, spleen CD8α+ detected in ICSBP−/− mice only represented 3.6% of the total DC population. To further analyze the maturation and activation phenotype of CD8α− and CD8α+ subsets of splenic DCs from ICSBP−/− and control mice, we performed three-color flow cytometric analysis combining the CD11c and CD8α markers alternatively with the costimulatory antigens CD40, CD80, CD86, intercellular adhesion molecule (ICAM)-1, and the MHC class I and class II molecules. As shown in Fig. 3 B, the CD8α+ DC subpopulation from ICSBP−/− mice expressed significantly lower levels of the costimulatory antigens CD40, CD80, CD86, and ICAM-1 with respect to the WT counterparts. In contrast, in the CD8α− DC-subset all the considered costimulatory antigens and activation markers were expressed at comparable levels in ICSBP−/− and WT mice. The altered phenotype of CD8α+ DCs in ICSBP−/− mice was further confirmed by the morphologic analysis of the forward scatter profile (Fig. 3 C), showing that, consistently with previous reports (36), splenic CD8α+ DCs were bigger than CD8α− DCs in WT mice. Conversely, CD8α+ DCs recovered from the spleens of ICSBP−/− mice proved to be significantly smaller than CD8α− DCs.

Bottom Line: Moreover, ICSBP(-/-) CD8alpha(+) DCs exhibited a markedly impaired phenotype when compared with WT DCs.In addition, these cells were unable to undergo full phenotypic activation upon in vitro culture in presence of maturation stimuli such as lipopolysaccharide or poly (I:C), which paralleled with lack of Toll-like receptor (TLR)3 mRNA expression.Finally, cytokine expression pattern was also altered in ICSBP(-/-) DCs, as they did not express interleukin (IL)-12p40 or IL-15, but they displayed detectable IL-4 mRNA levels.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Virology, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.

ABSTRACT
Interferon (IFN) consensus sequence-binding protein (ICSBP) is a transcription factor playing a critical role in the regulation of lineage commitment, especially in myeloid cell differentiation. In this study, we have characterized the phenotype and activation pattern of subsets of dendritic cells (DCs) in ICSBP(-/-) mice. Remarkably, the recently identified mouse IFN-producing cells (mIPCs) were absent in all lymphoid organs from ICSBP(-/-) mice, as revealed by lack of CD11c(low)B220(+)Ly6C(+)CD11b(-) cells. In parallel, CD11c(+) cells isolated from ICSBP(-/-) spleens were unable to produce type I IFNs in response to viral stimulation. ICSBP(-/-) mice also displayed a marked reduction of the DC subset expressing the CD8alpha marker (CD8alpha(+) DCs) in spleen, lymph nodes, and thymus. Moreover, ICSBP(-/-) CD8alpha(+) DCs exhibited a markedly impaired phenotype when compared with WT DCs. They expressed very low levels of costimulatory molecules (intercellular adhesion molecule [ICAM]-1, CD40, CD80, CD86) and of the T cell area-homing chemokine receptor CCR7, whereas they showed higher levels of CCR2 and CCR6, as revealed by reverse transcription PCR. In addition, these cells were unable to undergo full phenotypic activation upon in vitro culture in presence of maturation stimuli such as lipopolysaccharide or poly (I:C), which paralleled with lack of Toll-like receptor (TLR)3 mRNA expression. Finally, cytokine expression pattern was also altered in ICSBP(-/-) DCs, as they did not express interleukin (IL)-12p40 or IL-15, but they displayed detectable IL-4 mRNA levels. On the whole, these results indicate that ICSBP is a crucial factor in the regulation of two possibly linked processes: (a) the development and activity of mIPCs, whose lack in ICSBP(-/-) mice may explain their high susceptibility to virus infections; (b) the generation and activation of CD8alpha(+) DCs, whose impairment in ICSBP(-/-) mice can be responsible for the defective generation of a Th1 type of immune response.

Show MeSH
Related in: MedlinePlus