Effects of IL-3 and SCF on Histamine Production Kinetics and Cell Phenotype in Rat Bone Marrow-derived Mast Cells

Abstract

Background: Rat mast cells were regarded as a good model for mast cell function in immune response.

Methods: Rat bone marrow mast cells (BMMC) were prepared both by recombinant rat IL-3 (rrIL-3) and by recombinant mouse stem cell factor (rmSCF), and investigated for both proliferation and differentiation in time course. Rat BMMC was induced by culture of rat bone marrow cells (BMCs) in the presence of both rrIL-3 (5 ng/ml) and rmSCF (5 ng/ml). Culture media were changed 2 times per week with the cell number condition of 5x10(4)/ml in 6 well plate. Proliferation was analyzed by cell number and cell counting kit-8 (CCK-8) and differentiation was by rat mast cell protease (RMCP) II and histamine.

Results: Cell proliferation rates reached a maximum at 8 or 11 days of culture and decreased thereafter. However, both RMCP II production and histamine synthesis peaked after 11 days of culture. By real time RT-PCR, the level of histidine decarboxylase mRNA was more than 500 times higher on culture day 11 than on culture day 5. By transmission electron microscopy, the cells were heterogeneous in size and contained cytoplasmic granules. Using gated flow cytometry, we showed that cultured BMCs expressed high levels of FcepsilonRI and the mast cell antigen, ganglioside, on culture day 11.

Conclusion: These results indicate that rat BMMCs were generated by culturing BMCs in the presence of rrIL-3 and rmSCF and that the BMMCs have the characteristics of mucosal mast cells.

Keywords: Bone marrow; IL-3; Mast cell; Rat; Stem cell factor.

Figure 1

Light microscopic view (A) and Toluidine blue staining (B~D) of rat bone marrow cells. Cultured rat bone marrow cells were harvested at (B) 4 days, (C) 11 days, or (D) 18 days and were stained by Toluidine blue.

Figure 2

TEM of rat bone marrow cells. Rat bone marrow cells cultured in the presence of rrIL-3 and rmSCF were analyzed by TEM at day 0 (A), day 4 (B), day 11 (C), and day 15 (D).

Figure 3

Flowcytometry analysis of cell populations and phenotype of cultured rat bone marrow cells. Cell were analyzed by flow cytometry for cell size by both FSC and SSC (A), and for surface expression of the BC4 (B, C) and AA4 (D, E) antigens. Control with dotted line in (B~E) were only treated with the secondary antibody, PE-goat anti mouse IgG. The solid lines in (B and C) were for the membrane protein for the primary antibody, BC4, and the 2nd antibody. The solid lines in (D and E) were for the membrane protein for the primary antibody, AA4, and the 2nd antibody. (B and D) for gated as the R1, (C and E) gated as the R2.

Figure 4

Cell proliferation, RMCP II production, and histamine synthesis in rat bone marrow cells treated with rrIL-3 and rmSCF. Rat BM cells were incubated under specified culture condition. Cells numbers were counted using a hemocytometer (A~C). RMCP II expression was analyzed with 200 ng/ml cell lysate of cultured rat bome marrow cells (D~F). Histamine levels were analyzed using a Histamine EIA kit (G~I). Culture conditions were controlled in in terms of serum (FBS vs. HS), media (IMDM vs. RPMI) or percoll concentration (50% vs. 70%). Open/white symbol or bar, FBS; and closed/gray symbol or bar, HS. In the line graph: circle, cells separated by 50% percoll; and triangle, cells by 70% percoll. Solid line, IMDM; and dash-dot line, RPMI-1640. : 50 IM FBS, : 50 IM HS, : 50 RP HS, : 50 RP HS, : 70 IM FBS, : 70 IM HS, : 70 RP FBS, : 70 RP HS. In the bar graph, bar with horizontal line, 50% percoll and IMDM; bar with oblique line, 50% percoll and RPMI-1640; bar with vertical line, 70% percoll and IMDM; bar with net cross, 70% percoll and RPMI-1640. : 50 IM FBS, : 50 IM HS, : 50 RP FBS, : 50 RP HS, : 70 IM FBS, : 70 IM HS, : 70 RP FBS, : 70 RP HS. IM for IMDM, RP for RPMI-1640, NA for not applied, ND for not determined. Y axis for both H and I in log scale.

Figure 5

Exocytosis of cultured rat bone marrow cells by IR162 and B5. Cultled rat bone marrow cells were applied to the processing of exocytosis by IR162 and B5, a mouse anti rat IgE mAb.

Figure 6

The effects of cytokine treatment on rat bone marrow cell proliferation and differentiation. The specified cytokines were added to activeted rat bone marrow cells at culture day 8, which were incubated for 3 (black bar) or 7 (white bar) days more. At the indicated time point, cell proliferation was analyzed CCK-8 (A and C) and cell differentiation was analyzed by RMCP II production (B and D). Cells harvested on a 70% Percoll cushion (A and B). Cells harvested on a 50% Percoll cushion. (C and D). rrIL-3, recombinant rat interleukin 3; rmSCF, recombinant rat stem cell factor; RMCP II, rat mast cell protease II; rrIL-4, recombinant rat interleukin-4; rrIL-6, recombinant rat interleukn-6; rmIL-3, recombinant mouse interleukin-3; LPS, lipopolysaccharide; Con A, concanavalin A. Cell number is represented as times of the given condition described in material and methods.

Figure 7

Real time RT-PCR assessment of rat histidine decarboxylase mRNA levels. The ratio was analyzed in log scale on the Y axis. Histidine decarboxylase mRNA levels were normalized against the house keeping gene GAPDH. Black bar, 0.06µg RNA. White bar, 0.6µg RNA. Representative data.