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Interleukin 4 (IL 4)-induced IgE production by peripheral blood lymphocytes and tonsil cells from normal donors was enhanced in a dose-dependent fashion by IL 5. IL 5 tested alone was not effective. The synergistic effects of IL 5 were most pronounced at suboptimal IL 4 concentrations, whereas at saturating IL 4 concentrations (200-300 U/ml), IL 5 had no effect. Interferon-gamma (IFN-gamma) and F(ab')2 fragments of monoclonal antibody 25 directed against the CD23 antigen, that blocked IL 4-induced IgE synthesis, also inhibited the production of IgE in the presence of combinations of IL 4 and IL 5, indicating that IL 5 potentiates the activation pathway through which IL 4 induces IgE production. In contrast, IL 4 (50 U/ml) blocked IL 5-induced IgA synthesis. IL 5 was ineffective in inducing the release of soluble CD23 (sCD23), but in the presence of IL 4 an enhanced release of sCD23 was observed, provided IL 4 was present at suboptimal concentrations. IFN-gamma completely blocked sCD23 release induced by IL 4 and IL 5. These results demonstrate that there is a strong quantitative correlation between sCD23 release and induction of IgE synthesis. sCD23 fraction-correlation between sCD23 release and induction of IgE synthesis. sCD23 fractionated from the Epstein-Barr virus-transformed B cell line RPMI 8866 was ineffective in inducing IgE production. However, sCD23 acted synergistically with suboptimal concentrations of IL 4. sCD23 did not modulate the IgE response at saturating concentrations of IL 4. Collectively, these data indicate that sCD23 plays an important regulatory role in the modulation of IL 4-induced IgE synthesis mediated by IFN-gamma and IL 5.

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The growth-promoting activities of interleukin-6 (IL-6) in combination with different factors were assessed in bone marrow (BM) cultures prepared from normal mice and from mice treated with 5-fluorouracil (5- FU). Effects on hematopoietic colony formation with respect to number, size, and cellular composition were evaluated. In agreement with previous reports, IL-6 acts synergistically with IL-3 to stimulate increased numbers of granulocyte/macrophage (GM) and multilineage colonies in day-2 and day-4 post-5-FU BM cultures. Furthermore, day 4 but not day 2 post-5-FU BM showed enhanced GM colony formation when stimulated with IL-6 plus interleukin-4 (IL-4) or granulocyte colony- stimulating factor (G-CSF). In contrast, IL-6 did not increase the number of colonies supported by M-CSF or GM-CSF. Nevertheless IL-6 interacted with all factors, including M-CSF and GM-CSF, to stimulate an increase in colony size. Many of these myeloid colonies attained a diameter of greater than or equal to 0.5 mm, suggesting they derive from high proliferative potential cells (HPP-CFC). The response of normal and day-8 post-5-FU BM containing high numbers of more mature progenitors was also assessed. We found IL-6 enhanced colony formation by lineage-restricted megakaryocytic and erythroid progenitors in the presence of IL-3 and IL-4 plus erythropoietin (Epo), respectively. The sum of these results shows that IL-6 interacts with a variety of factors to regulate the growth of progenitor cells at different stages of lineage commitment and maturation.

authors request that the following error be noted on p. 1272, in the second paragraph from the bottom of the right-hand column. The bacteriophage T4 gene identified by Belfort and colleagues (ref. 53) as containing an intron encodes thymidylate synthase, not thymidine kinase. CorrectionsProc. Nail. Acad. Sci. USA Vol. 83, pp. 1242-1245, March 1986 BiochemistryThe primary structure of rat parathymosin ABSTRACTParathymosin has been isolated from rat thymus and from rat liver. Its primary structure is reported 10 as follows:AcSer-Lys-Ser-Glu-Val -Glu-Ala-Ala-Ala-Glu -20 Leu-Ser-Ala-Lys-Asp-Leu-Lys-Glu-Lys-Lys -Asp-Lys- Glu-Glu-Lys-Ala-Gly-Arg-Lys-Glu-Arg-Lys-Lys-Glu- Val-Glu-Glu-Glu -Glu-Asn-Gly-Ala-Glu-Glu-Glu-Glu-Gluso 80Glu -Thr-Ala-Glu-(Gly3,Asx7,Glx14)-Glu-Gly-Pro -Val-Arg- 90Lys-Arg-Thr-Ala-Glu-Glu-Glu-Asp -Glu-Ala-Asp-Pro-Lys- 100Arg-Gln-Lys-Thr-Glu Asn-Gly-Ala-Ser-AlaOH. The blockmg group at the NH2 terminus was identified by mass spectrometry as acetyl. Regions homologous to amino acid sequences in prothymosin a were found to be located between residues 14-20, 23-25, 33-39, 41-43, and 83-87 of parathymosin.Parathymosin was first isolated from rat thymus (1) as a by-product of the procedure used for the isolation of prothymosin a. It was named parathymosin because of its structural similarity to prothymosin a, the putative precursor of thymosin a1 (2, 3 In the present paper, we report a simplified procedure for the isolation of both peptides from rat thymus and the nearly complete amino acid sequence ofparathymosin. A procedure for the isolation of parathymosin from rat liver, a richer source for this peptide, is also described. MATERIALS AND METHODSMaterials. Rat tissues were collected immediately after sacrifice of the animals by decapitation, frozen in liquid N2, and stored at -70'C. Other materials were from sources identified earlier (1, 3, 4).Methods. Chromatography on Sephacryl S-200 was carried out as described (1). For the HPLC, a Waters Associates system with an Altex Ultrasphere ODS C18 column was used.Protein and peptide analyses were carried out fluorometrically following alkaline hydrolysis and reaction with fluorescamine (7). Automated sequence analysis was as described (3). The quantitative radioimmunoassay for prothymosin a was as described by Haritos and Horecker (4 9.0 with K2CO3. After 4 hr at 50°C, the solution was acidified with 26 ul of concentrated HCOOH, diluted with 10 ml of buffer A (1 M HCOOH/0.2 M pyridine, pH 2.8) and pumped through a Sep-Pak C18 cartridge. After the cartridge was washed with 10 ml of buffer A, the peptides were eluted with 3 ml of 40% acetonitrile in buffer A, and the eluate was lyophilized.Digestion with Staphylococcus aureus V8 protease was in 25 ,ul of 0.1 M NH4HCO3 containing 50 ,ug of peptide, 2 mM EDTA, and 1.67 ,ug of protease. After 13 hr at 37°C, the solution was lyophilized.For the isolation of peptides in the proteinase digests and after treatment with H2NOH, the lyophilized solutions were dissolved in buffer A and separated by HPLC using the OD...

In the present study, we investigated the effects of human recombinant interleukin-7 (IL-7) on the proliferation of enriched hematopoietic cells isolated from human adult and fetal bone marrow (BM). In cultures of CD34+ cells, IL-7 was found to induce dose-dependent incorporation of 3H-thymidine (3H-TdR), but had no demonstrable effect on the development of myeloid colony-forming cells. Numbers of B-cell precursors (BCP), initially present within CD34+ populations and which included a CD34+CD20+ subset, were significantly increased when CD34+ BM cells were cultured in the presence of IL-7. This effect was most striking on CD20+ BCP, and resulted at least partly from higher numbers of cycling cells as indicated by Hoechst 33342 fluorescence (Calbiochem, Behring Diagnostics, La Jolla, CA). These results indicate that IL-7 promotes the growth of BCP within the CD34+ compartment. In line with the B-lineage affiliation of CD34+ target cells, committed BCP (CD10+ CD19+ surface IgM-) isolated from BM were also found to proliferate in response to IL-7. Interestingly, this effect of IL-7 was strongly potentiated by the addition of IL-3. Taken together, and in accordance with previous observations on murine cells, our data indicate that IL-7 acts as a growth factor during the ontogeny of human B lymphocytes.

A method utilizing reversed-phase high-performance liquid chromatography has been developed for the purification to homogeneity of interleukin 2 (IL-2) isolated from a human T-cell leukemia. A rmal purification of 500,000-fold was obtained with a specific activity of pure IL-2 of 10' units/mg. The amino acid analysis of natural IL-2 is strikingly similar to the composition deduced from sequence analysis of a cDNA coding for human IL-2. Protein sequence analysis of CNBr-derived peptides yields data consistent with the sequence proposed from cloned cDNA. The availability of homogeneous IL-2 will allow accurate biological studies of its activity free from the contamination of the numerous lymphokine species that are known to be co-produced with IL-2 during the induction procedure.Interleukin 2 (IL-2), formerly known as T-cell growth factor, was first described as a lymphokine capable of modulating lymphocyte reactivity and promoting the long-term in vitro culture of antigen-specific effector T lymphocytes (1, 2). IL-2 partially purified from mouse (3, 4), rat (5, 6), and human (7, 8) normal T lymphocytes has been found to retain different types of biological activity, including: (i) marked enhancement of thymocyte mitogenesis (7, 9); (ii) promotion of long-term in vitro proliferation of antigen-specific helper or killer T-cell lines (3, 10); and (iii) induction of cytotoxic Tlymphocyte reactivity and plaque-forming cell responses in cultures of nude mouse spleen cells (7,9). These activities indicate that IL-2 may be useful in augmenting immune responses and restoring immune-deficient T-cell populations (nude mouse spleen cells) to normal levels of cellular and humoral immunity. Furthermore, these results suggest that IL-2 production and response are important parameters of immunological functions and their measurement may be useful in clinical diagnosis of aberrant immune status.The sources and techniques that have been used to produce IL-2 have yielded low concentrations of IL-2, with large volumes of conditioned media being required to obtain only small quantities of human IL-2 activity. As a consequence, sufficient quantities of concentrated human IL-2 have not been available for in vivo experiments or for its characterization. Several groups have reported purification procedures for both murine (9, 11) and human (7,(12)(13)(14) IL-2, although the purity of these preparations is not clear. The present study reports a procedure for the purification of human IL-2 to homogeneity using several steps of reversed-phase high-performance liquid chromatography (HPLC). Chemical characterization of the purified lymphokine is reported. EXPERIMENTAL PROCEDURESSource of IL-2. Human leukemia T cells designated Jurkat-H33HJ-JAI (a subclone of the parent Jurkat-FHC1C cell line) (15) were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum, 50 units of penicillin per ml, 50 ug of gentamycin per ml, and 300 .g of fresh L-glutamine per ml. The cells were grown in T175-cm2 flasks (volume, 150 ml) in a humi...

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