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CD40 Ligation Alters the Cell Cycle of Differentiating Keratinocytes

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Presentation on theme: "CD40 Ligation Alters the Cell Cycle of Differentiating Keratinocytes"— Presentation transcript:

1 CD40 Ligation Alters the Cell Cycle of Differentiating Keratinocytes
Jérôme Grousson, Daniel Schmitt, Josette Péguet-Navarro  Journal of Investigative Dermatology  Volume 114, Issue 3, Pages (March 2000) DOI: /j x Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 IFN-γ potentiates the CD40-mediated inhibition of keratinocyte proliferation. Keratinocytes (K) were grown for 5 d in microtiter plates with an equal number of irradiated CD40Lc or CD32c. IFN-γ (50 U per ml) either was, or was not, added to keratinocytes at the beginning of culture. Cell proliferation was assessed by [3H]methylthymidine incorporation during the last 18 h of culture. Results are the mean cpm ± SD of triplicate values. As assessed by statistical analysis using the Student’s t test: p <0.001 for K/CD40Lc and K/CD32c + IFN versus K/CD32c, and p <0.001 for K/CD40Lc + IFN versus K/CD32c + IFN at all days, except day 5, of culture. Results are representative of three independent experiments using keratinocytes from various donors. Journal of Investigative Dermatology  , DOI: ( /j x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 CD40 ligation in the presence or absence of IFN-γ does not affect keratinocyte nuclear morphology. Keratinocytes (K) were grown for 4 d on CD32c or on CD40Lc, and treated (right panel) or not (left panel) with IFN-γ (200 U per ml) during the last 2 d of culture. Nuclear staining was then performed using 20 μg bisbenzimide per ml (Hoechst 33342) and observed under fluorescence microscopy. Very few keratinocytes exhibited apoptotic characteristics, i.e., condensed or fragmented nuclei (arrow). The number of apoptotic keratinocytes was not significantly different under the four experimental conditions. Journal of Investigative Dermatology  , DOI: ( /j x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 CD40 ligation in the presence or absence of IFN-γ does not enhance CHX or ultraviolet B-induced keratinocyte apoptosis. Keratinocytes (2 × 104 cells) were cultured for 4 d with an equal number of CD32c (K/CD32c) or CD40Lc (K/CD40Lc) in DMEM supplemented with IFN-γ (100 U per ml), or not. Keratinocyte apoptosis was induced by either addition of CHX (50 μg per ml) during the last day of culture, or ultraviolet B irradiation (2000 J per m2) 24 h before harvesting the cells. Apoptotic cells were enumerated using the Hoechst technique. Results are the mean ± SD number of apoptotic cells under each experimental condition. Journal of Investigative Dermatology  , DOI: ( /j x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 DNA content. Keratinocytes (K) were grown for 4 d on either 3T3, CD32c or CD40Lc. Fluorescence analysis of the DNA content (propidium iodide staining; FL2-A) was carried out, showing the various cell cycle phases (G1, S, G2/M). 104 keratinocytes were analyzed under each experimental condition. These results are representative of 11 experiments, using keratinocytes from various donors. Journal of Investigative Dermatology  , DOI: ( /j x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

6 Figure 5 Each phase of the keratinocyte cell cycle is altered after CD40 ligation. Keratinocytes (K) were cocultured for 4 d on CD40Lc or CD32c in DMEM supplemented (B) or not (A) with IFN-γ (50 U per ml). Cells were then incubated with BrdUrd, fixed, and, after DNA denaturation, a simultaneous fluorescence analysis of DNA content (propidium iodide staining; FL2) and of BrdUrd incorporation (fluorescein isothiocyanate-conjugated anti-BrdUrd monoclonal antibody; FL1) was performed. 104 keratinocytes were analyzed under each experimental condition These results are representative of eight experiments using keratinocytes from various donors. Journal of Investigative Dermatology  , DOI: ( /j x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

7 Figure 6 CD40 ligation inhibits cyclin A and cyclin E expression. Keratinocytes (K) were grown for 4 d on either 3T3, CD32c, or CD40Lc and recovered from culture. Total cellular proteins were isolated and analyzed by western blot (70 μg per lane) for CDK1, CDK2, cyclin A, cyclin E, and p21waf1 expression. Journal of Investigative Dermatology  , DOI: ( /j x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

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