Figure 4. FIZZ1 effects on BM cell recruitment.
(A) Fresh isolated whole BM cells from day 7 BLM (“BLM-BM”) or PBS (“CON-BM”) treated mice were preloaded with fluorescent dye, and then plated into 5 µm-inserts in 24-well transwell plates. After 2 hours of incubation with the indicated doses of FIZZ1 in the lower chambers, the cells that have migrated to the lower chambers were quantitated by measuring the fluorescence with an excitation and emission wavelengths of 494 and 517 nm, respectively. The results were normalized to the controls and expressed as percentages of controls, and shown as mean ± SE (n = 3). In (B) purified BM-derived DC from day 7 BLM (“BLM-BMDC”) or PBS (“CON-BMDC”) treated mice were similarly analyzed as in (A) for migration to media only (“None”) or to 50 ng/ml FIZZ1 in the lower chambers. The results were expressed as in (A). (C) BM from GFP transgenic mice were transplanted into WT or FIZZ1 KO mice to create GFP BM chimera mice of the respective recipient strain. After stable engraftment the mice were treated with BLM and 7 days later were analyzed for GFP expression in the lung cell population by flow cytometry. Three populations were discerned corresponding to the cells with undetectable (R1), low (R2) or high (R3) GFP fluorescence. One representative data was shown from two individual experiments, and the combined lung cells from two mice were used for flow cytometry in each group. Circulating FIZZ1 protein was measured in the sera (1∶50 dilution) by ELISA assay 21 days after BLM or PBS treatment (D). Results were shown as mean ± SE with 5–6 mice in each group. (E) Effect of FIZZ1 on migration of primary cultured mouse lung fibroblasts was also analyzed as described in legend for (A). *P<0.05 compared to control.