IL-3 derived from CD4+ T cells is essential for the in vitro expansion of mast cells from the normal adult mouse spleen

A null cell line (SCM1) was established by a culture of spleen cells (SC) from normal adult C57Bl/6 mice with complete medium alone for 10 days and followed by weekly cultures with a 25% WEHI-3 cell culture supernatant. Phenotype analysis showed that the SCM1 cells were negative for CD3, Thy1.2, B220, Mac-1, Gr-1, NK1.1 and MHC class II, but were positive for MHC class I, FcγRII/III, FcεRI, c-kit and the receptor against wheat germ agglutinin. These findings suggested that the SCM1 cells were mast cells. In an in vitro proliferation assay, SCM1 cells proliferated in the presence of either IL-3 or stem cell factor (SCF), but not in the presence of IL-4, whereas IL-4 showed an augmenting effect on their proliferation in the presence of either IL-3 or SCF. In analysing the mechanism by which such mast cells could be expanded from normal adult mouse SC, the addition of anti-IL-3 MoAb, but not anti-SCF MoAb, into the initial culture inhibited the subsequent expansion of either IL-3- or SCF-responding cells. The prior depletion of CD4+ T cells abrogated the capacity of the SC to enhance the expansion of SCF-responding cells, and this inability was restored by the addition of IL-3. Moreover, the culture supernatant of normal adult SC alone contained considerable levels of IL-3. Taken together, our findings suggest that, in an in vitro culture, CD4+ T cell-derived IL-3 therefore enhances the expansion of mast cells from the normal adult mouse spleen

The role of the ribosomal protein S19 C-terminus in Gi protein-dependent alternative activation of p38 MAP kinase via the C5a receptor in HMC-1 cells

We have demonstrated that an alternative C5a receptor (C5aR) ligand, the homodimer of ribosomal protein S19 (RP S19), contains a unique C-terminus (I134–H145) that is distinct from the moieties involved in the C5a–C5aR interaction. To examine the role of I134–H145 in the ligand–C5aR interaction, we connected this peptide to the C-terminus of C5a (C5a/RP S19) and found that it endowed the second binding moiety of RP S19 (L131DR) with a relatively higher binding affinity to the C5aR on a human mast cell line, HMC-1. In contrast to the C5aR, the second C5aR C5L2 worked as a decoy receptor. As a result, the mitogen-activated protein kinase (MAPK) downstream of the Gi protein exchanged extracellular-signal regulated kinase for p38MAPK. This alternative p38MAPK activation could be pharmacologically suppressed not only by the downregulation of phosphoinositide 3-kinase (PI3K) by LY294002, but also by the over-activation of protein kinase C by phorbol 12-myristate 13-acetate. The activation was reproduced upon C5a–C5aR interaction by a simultaneous suppression of PI3K and phospholipase C with LY294002 and U73122 at low concentrations. Moreover, p38MAPK phosphorylation upstream of the pertussis toxin-dependent extracellular Ca2+ entry was also suppressed by high concentrations of MgCl2, which blocks melastatin-type transient receptor potential Ca2+ channels (TRPMs). The active conformation of C5aR upon the ligation by C5a, at least on HMC-1 cells, is changed by the additional interaction of the I134–H145 peptide, which seems to guide the alternative activation of p38MAPK. This activation is then amplified by a novel positive feedback loop between p38MAPK and TRPM.Electronic supplementary material The online version of this article (doi:10.1007/s10495-010-0511-y) contains supplementary material, which is available to authorized users