高木 智 熊本大学, 医学部, 特別研究員
HITOSHI Yasumiti Kumamoto University, Lecturer, 医学部, 助手 (10222241)
TOMINAGA Akira Kumamoto University, Associate Professor, 医学部, 助教授 (50172193)
YAMAGUCHI Naoto Kumamoto University, Lecturer (00166620)
ONOUE Kaoru Kumamoto University, Professor (60037497)
|Budget Amount *help
¥17,800,000 (Direct Cost : ¥17,800,000)
Fiscal Year 1991 : ¥6,600,000 (Direct Cost : ¥6,600,000)
Fiscal Year 1990 : ¥11,200,000 (Direct Cost : ¥11,200,000)
(1) We reported the establishment of IL-5 and stromal cell-dependent Ly-l^+ early B cell line in long-term bone marrow culture system (Tominaga et al. Growth Factors 1 : 135, 1989). In this study, we obtained another 8 different cell lines that showed germ line configuration of the J region segments of the IgH genes. Their surface markers were CD45R^+, Ly-l^+, Lyb-2^+, sIgM^-, Thy-l^-, and Mac-l^-. When we treated two of them (J8 and J10) with 5-azacytidine followed by coculture with stromal cells (ST2) and IL-5, they became sIgM^+ B cells and Mac-l^+ macrophage-like cells, respectively. After other early lymphold lines were maintained by coculture with ST2 and IL-5 for more than a year, they showed a heterogeneous DNA rearrangement profile of the J region segment of the IgH gene. Northern blot analysis revealed that these cell lines expressed Cu-mRNA, and 5-mRNA. consistent with normal pre-B cells. Intriguingly, they expressed c-fms-mRNA constitutively. When J13 cells were cocultured
with ST2 and, GM-CSF ih place of ST2 and IL-5, they acquired Mac-1 expression and retained Ly-1 expression. They were morphologically macrophages, nonspeclfic-esterase positive, and showed phagocytosis of latex beads. The conversion was inhibited by addition of. IL-5. These results support evidence for a close relationship between the myeloid and Ly-1^+ B cell pathways of differentiation, and indicate that our IL-5-dependent clones are multipotential intermediates in differentiation from pre-B cells to B cells and macrophages.
(2) IL-5 has been suggested to be5 lnvolved in in vitro growth and differentiation of B cells and eoginophlls. To envisage the possible engagement of IL-5 in the development of these cells in vivo, transgenic mice carrying the mouse IL-5 gene legated with a metallothionein promoter were generated. The IL-5 transgenic mice exibited elevated levels of IL-5 In the serum and an increase in the levels of serum IgM and IgA. A massive eosinophilia In peripheral blood and spleen and an infiltration of eosinophils In muscle and liver were observed. When cadmium-containing saline was injected i. p. into transgenic mice, a distinctive Ly-l^+ B cell population became apparent in the spleen after 5 days. IL-5 receptors (lL-5R) were detected on those cells by mabs against la-5R. Another interesting finding in these transgenic mice was an increase in polyreactive anti-DNA antibodies of IgM class. It Is suggested, therefore, that aberrant expression of the IL-5 gene may Induce accumulation of Ly-l^+ B cells and eosinophils. Furthermore, this IL-5 transgenic mouse can be a model mouse for eosinophilia and we can determine the role of IL-5 in the differentiation of Ly-l^+ B cells and eosinophils by using this mouse.
(3) We have Isolated CDNA clones encoding a murine IL-5R by expression screening of a library prepared from a murine IL-5-dependent early B cell line. A CDNA library was expressed In COS7 cells and screened by panning with the use of anti-IL-5 receptor monocional antibodies. The deduced amino acid sequence analysis demonstrates that the receptor Is a glycoprotein of 415 amino acids (Mr 45, 284), Including an N-terminal hydrophobic region (17 amino acids), a glycosylated extracellular domain (322 amino acids), a single transmembrane segment (22 amino acids) and a cytoplasmic tall (54 amino acids). COS7 cells transfected with the CDNA expressed a 60-kD protein that bound IL-5 with a single class of affinity (K_D = 2-10 nM). FDC-PL cells transfected with the CDNA for murine IL-5R showed the expression of IL-5 binding sites with both low (K_D=6 nM) and high affinity (K_D=30 pill) and acquired responsiveness to IL-5 for proliferation, although parental FDC-PL cells did not show any detectable IL-5 binding. Northern blot analysis showed that two species of mRNAs (5.0 kb and 5.8 kb) were detected In cell lines that display binding sites for murine IL-5. Homology search for the amino acid sequence of the IL-5 receptor reveals that the IL-5 receptor contains a common motif of a cytokine receptor family that is recently identified.
(4) The murine high-affinity IL-5R consists of at least two membrane polypeptide chains ; a chain of p6O and B chain of P130/Pl4O. We found that the B chain is constitutively expressed in IL-3-dependent early B cell-line. To evaluate whether a component of IL-3R is the B chain of the high affinity IL-5R, effect of anti-IL-3 receptor mAb (anti-Aic-2 mAb) on DNA synthesis of Y16 was examined. Anti-Aic-2 mAb that immunoprecipitated p13O/p14O in the cell lysates of Y16 cells partially inhibited the IL-5- and IL-3-induced proliferation of Y16 cells, whereas it did not react with the a chain of IL-5R. Since anti-Aic-2 mAb is reported to react with. recombinant AIC2A and AIC2B, we examined whether AIC2A or AIC2B is involved in the formation of the high affinity IL-5R. The expression of the murine IL-5R receptor a chain with AIC2B, but not with AIC2A in L cells resulted in the high affinity IL-5 binding sites. The expression of AIC2B alone did not show any IL-5 binding by itself. Cell lysates of these transfectants were crosslinked to form the complex of a and B chains with radiolabeled IL-5. These results clearly indicate that B chain of the murine IL-5 receptor is an indispensable for the formation of high-affinity IL-5 binding sites together with the a chain and that AIC2B is likely the the B chain of the mouse IL-5 receptor. The B chain was found to be identical to the B chain of GM-CSF receptor. Less