![Fig. 3. CD40L induces FL expression. / Stromal cells (either HUVECs, BMEC line [TrBMEC], LTBMCs, or bone marrow-derived fibroblasts [BMmyofibr]) were cultured 48 hours with medium alone (control) or 10% supernatant of nontransfected COS cells (COS) or with 10% supernatant of CD40L-CD8α–transfected COS cells (CD40L) and the resulting effect on the expression of FL studied. (A) Up-regulation of the production of the soluble form of FL. ELISA measurements showing the stimulation of the production of the soluble form of FL (average ± SD of 7 experiments). The production of soluble FL in LTBMCs or BMmyofibr is below the level of ELISA detection (15 pg/mL). No FL is detected in untransfected or transfected COS cell supernatants. Statistically significant results (*) are indicated. (B) Western blotting showing FL protein in HUVEC and LTBMC culture medium and increased production by CD40L. Track A, recombinant FL protein, track B and D, HUVECs and LTBMCs respectively, cultured in the presence of 10% supernatant of nontransfected COS cells; track C and E, HUVECs and LTBMCs, respectively, cultured with 10% supernatant of CD40L-CD8α–transfected COS cells. (C) Up-regulation of membrane-bound FL expression by CD40L on ECs is shown by flow cytometry: dark gray, isotype control;gray, EC stimulated 48 hours with 10% supernatant of nontransfected COS cells, black, ECs stimulated 48 hours with 10% supernatant of CD40L-CD8α–transfected COS cells. (D, E) Indirect immunofluorescence showing up-regulation of FL by CD40L at the surface of ECs (top panel: ECs stimulated 48 hours with 10% supernatant of nontransfected COS cells; bottom panel: ECs stimulated with 10% supernatant of CD40L-CD8α–transfected COS cells).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/95/12/10.1182_blood.v95.12.3758/8/bloo01244003ax.jpeg?Expires=1724949409&Signature=qXhivIRt3~gowm-nmtZAijzhNJGcQJ5MO78Qbk0G5m3snWphJ0PsqiLQ5LMzL556sevXE9qfogDc31aLk0rvuuG7tg5jSIWnGnk4vMT69UpOhdW3-9EcfAsazFqwrox-R9CfhKIkwnZI77fLPlzwAJ4eJqbdaREPPGdtUQH2tWpR9IaO3Y0-3F45CcSXs-Ijcq~hJpJLZduGm7ce2X2mtLlB2~jGKbNtJCGfDwA02Po7ihBuvQALE6PCFL2Ne1JPhAypcDVl8Jrd6YPe57BBlaoImLkpuWVEuqcNwNq~60lxtEIHi6UA-cv97nOZ0mUsXQmrs09WKCNMblPfZh33bw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
CD40L induces FL expression.
Stromal cells (either HUVECs, BMEC line [TrBMEC], LTBMCs, or bone marrow-derived fibroblasts [BMmyofibr]) were cultured 48 hours with medium alone (control) or 10% supernatant of nontransfected COS cells (COS) or with 10% supernatant of CD40L-CD8α–transfected COS cells (CD40L) and the resulting effect on the expression of FL studied. (A) Up-regulation of the production of the soluble form of FL. ELISA measurements showing the stimulation of the production of the soluble form of FL (average ± SD of 7 experiments). The production of soluble FL in LTBMCs or BMmyofibr is below the level of ELISA detection (15 pg/mL). No FL is detected in untransfected or transfected COS cell supernatants. Statistically significant results (*) are indicated. (B) Western blotting showing FL protein in HUVEC and LTBMC culture medium and increased production by CD40L. Track A, recombinant FL protein, track B and D, HUVECs and LTBMCs respectively, cultured in the presence of 10% supernatant of nontransfected COS cells; track C and E, HUVECs and LTBMCs, respectively, cultured with 10% supernatant of CD40L-CD8α–transfected COS cells. (C) Up-regulation of membrane-bound FL expression by CD40L on ECs is shown by flow cytometry: darkgray, isotype control;gray, EC stimulated 48 hours with 10% supernatant of nontransfected COS cells, black, ECs stimulated 48 hours with 10% supernatant of CD40L-CD8α–transfected COS cells. (D, E) Indirect immunofluorescence showing up-regulation of FL by CD40L at the surface of ECs (top panel: ECs stimulated 48 hours with 10% supernatant of nontransfected COS cells; bottom panel: ECs stimulated with 10% supernatant of CD40L-CD8α–transfected COS cells).
