The recent molecular cloning of the complementary DNA encoding T cell--replacing factor (TRF) has demonstrated that a single molecule is responsible for B cell growth factor II (BCGF-II) activity and eosinophil differentiation activity. It has been proposed that this molecule be called interleukin 5 (IL-5). We previously reported that purified rIL-5 supports the terminal differentiation and proliferation of eosinophilic precursors. In this study, we examined the effects of IL-5 on functional activities of mature eosinophils. IL-5 maintained the viability of mature eosinophils obtained from peritoneal exudate cells of mice infected with parasites. It also induced superoxide anion production in a dose-dependent manner. The Boyden's chamber Millipore assay revealed that IL-5 had a marked chemokinetic effect on eosinophils in a dose-dependent manner. Moreover, IL-5 was found to be an eosinophil chemotactic factor by the checkerboard assay. In conclusion, IL-5 is suggested to play an important role in increasing the functional activities of eosinophils as well as their production in allergic and parasitic diseases.

No abstract

Murine interleukin‐5 (IL‐5) is known to play an essential role in Ig production of B cells and proliferation and differentiation of eosinophils. Here, we have isolated cDNA clones encoding a murine IL‐5 receptor 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 monoclonal 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 tail (54 amino acids). COS7 cells transfected with the cDNA expressed a 60 kd protein that bound IL‐5 with a single class of affinity (KD = 2–10 nM). FDC‐P1 cells transfected with the cDNA for murine IL‐5 receptor showed the expression of IL‐5 binding sites with both low (KD = 6 nM) and high affinity (KD = 30 pM) and acquired responsiveness to IL‐5 for proliferation, although parental FDC‐P1 cells did not show any detectable IL‐5 binding. In addition, several cDNA clones encoding soluble forms of the IL‐5 receptor were isolated. 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.

TRF has originally been defined as a T-cell-derived lymphokine that triggers activated B cells for a terminal differentiation into Ig-secreting cells. HPLC-purified TRF from Sup of a murine TRF-producing B151 cell is an acidic glycoprotein, exerts BCGF II activity and induces expression of IL-2 receptors. It does not show IL-1, IL-2, IL-3, BSF-1/IL-4, or IFN gamma activity. We prepared monoclonal TB13 and NC17 antibodies against HPLC-purified B151-TRF which are specific for and can inhibit TRF as well as BCFG II activity of B151-TRF. Moreover, TB13 as well as NC17 antibody can immunoprecipitate the 46 Kd molecule from B151 Sup which exerts TRF as well as BCGF II activity. Complementary DNA (pSP6K-mTRF23) encoding for murine TRF/IL-5 was cloned and its entire nucleotide sequences were determined. The murine TRF/IL-5 cDNA encodes 133 amino acids including N-terminal strongly hydrophobic regions. Secreted recombinant TRF/IL-5 (apparent m.w. of 46 Kd) has 113 amino acid residues and also comprises homodimers of a molecule with an apparent m.w. of 25 to 30 Kd. TRF/IL-5 mRNA is constitutively expressed in constitutively TRF-producing B151 and is inducible in some T cell lines upon stimulation with PMA or Con A. TRF/IL-5 mRNA is also expressed in Tbc-primed T cells upon the stimulation with PPD, whereas its expression is not effectively induced in non-primed spleen cells by stimulation with Con A or PMA plus calcium ionophore. The translation product of murine TRF/IL-5 cDNA triggers resting as well as activated (DNP-primed or LPS-stimulated) murine B cells for terminal differentiation into Ig-secreting cells (IgM, IgG1, or IgA) accompanied by increased mRNA expression for secreted forms of relevant Ig heavy chain (mu, gamma, or alpha). Among these, increases in the level of mu, and alpha-specific mRNA for the secreted form of IgM and IgA, respectively, are prominent. Moreover, TRF/IL-5 induces maturation of resting B cells into IgM-secreting cells. TRF/IL-5 promotes growth of activated B cells as well as BCL1 cells. TRF/IL-5 is, therefore, a growth as well as a differentiation inducing factor for B cells. Moreover, it induces functional IL-2 receptors on resting as well as activated B cells, besides TRF and BCGF II activities.(ABSTRACT TRUNCATED AT 400 WORDS)

Murine interleukin‐5 (IL‐5) binds to its receptor with high and low affinity. It has been shown that the high affinity IL‐5 receptor (IL‐5‐R) is composed of at least two membrane protein subunits and is responsible for IL‐5‐mediated signal transduction. One subunit of the high affinity IL‐5‐R is a 60 kDa membrane protein (p60 IL‐5‐R) whose cDNA was isolated using the anti‐IL‐5‐R monoclonal antibody (mAb), H7. This subunit alone binds IL‐5 with low affinity. The second subunit does not bind IL‐5 by itself, and is expressed not only on IL‐5‐dependent cell lines but also on an IL‐3‐dependent cell line, FDC‐P1. Expression of the p60 IL‐5‐R cDNA in FDC‐P1 cells, which do not bind IL‐5, reconstituted the high affinity IL‐5‐R. We have characterized the second subunit of the IL‐5‐R by using another anti‐IL‐5‐R mAb, R52.120, and the anti‐IL‐3‐R mAb, anti‐Aic‐2. The anti‐Aic‐2 mAb down‐regulated binding of IL‐5 to an IL‐5‐dependent cell line, Y16. Both R52.120 and anti‐Aic‐2 mAbs recognized membrane proteins of 130–140 kDa expressed on FDC‐P1 and Y16 cells. The R52.120 mAb recognized both murine IL‐3‐R (AIC2A) and its homologue (AIC2B) expressed on L cells transfected with suitable cDNAs. The high affinity IL‐5‐R was reconstituted on an L cell transfectant co‐expressing AIC2B and p60 IL‐5‐R, whereas only the low affinity IL‐5‐R was detected on a transfectant co‐expressing AIC2A and p60 IL‐5‐R.(ABSTRACT TRUNCATED AT 250 WORDS)