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Two mouse helper T cell clones that proliferate in response to murine interleukin (IL)-9 could also be grown in conditioned medium of stimulated human connective tissue cells. The activity was not due to known T cell growth factors including human IL-9, which is not effective on mouse cells. This growth-stimulatory activity for TS1 cells (GATS) was co-induced with IL-6 on normal fibroblasts and certain sarcoma cell lines stimulated with IL-1, double-stranded RNA, virus or phorbol ester. However, the conditions for optimal induction and the kinetics of production were found to be different for IL-6 and GATS. GATS from phorbol ester-stimulated human hepatosarcoma cells co-purified with IL-6, but could be separated from it by subsequent cation-exchange fast-protein liquid chromatography and reverse-phase high-performance liquid chromatography. Homogeneous tumor cell-derived GATS was a 25-kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas IL-6 produced by these cells appeared in its 23-kDa form. Pure GATS was found to be inactive in the B cell hybridoma growth assay for IL-6. Finally, GATS was identified by NH2-terminal sequence analysis of the mature protein as leukemia inhibitory factor or human interleukin for DA cells (LIF/HILDA). The effect of LIF/HILDA on T cells was not mediated by IL-2, IL-4 or IL-9 production. Since this cytokine has not previously been reported to act on T cells, further investigation of its role in T cell activation should be taken into consideration.

We have previously shown that certain bone marrow-derived mast cell (BMMC) lines proliferate in response to a mast cell growth-enhancing activity (MEA) that is distinct from interleukin (IL) 3 and IL 4. Here we provide evidence that MEA is identical with the recently cloned mouse T cell growth factor P40. The evidence is as follows: (a) recombinant P40 displayed all the biological activities ascribed to MEA: it supported the growth of MEA-sensitive BMMC lines, it induced IL 6 secretion by these cells, and it enhanced survival of primary mast cell cultures; (b) highly purified MEA stimulated the growth of P40-dependent cell lines; (c) a rabbit monospecific antiserum directed against P40 specifically inhibited the action of MEA on BMMC; (d) specific binding sites for P40 were detected on BMMC and (e) MEA competed with P40 for binding to P40-dependent T cells, indicating that the two molecules interact with the same receptor. These observations further extend the range of biological activities ascribed to P40 and warrant its proposed designation as IL9.

Mouse and human cDNA clones encoding the T-cell and mast cell growth factor P40, now designated IL-9, were used to identify DNA restriction fragment length polymorphisms (RFLPs) in sets of somatic cell hybrids and between inbred strains of mice and interspecific backcross progeny. Segregation of mouse and human chromosomes among somatic cell hybrids indicated a location on mouse chromosome 13 and human chromosome 5. RFLPs were identified among inbred strains of mice. Analysis of chromosome 13 alleles for Tcrg, Dhfr, and Il-9 in an interspecific cross between Mus musculus and NFS/N or C58/J mice indicates that IL9 is distal to Tcrg and proximal to Dhfr.

A new murine T-cell growth factor, designated P40, which supports growth of helper T-cells in the absence of interleukin-2, interleukin-4 and antigen has been isolated from helper T-cell lines in sufficient quantities (100 micrograms) to permit its complete amino acid sequence determination. This was achieved by a combination of sensitive peptide mapping using microbore reversed-phase high performance liquid chromatography and automated microsequence analysis. Attempts to obtain N-terminal sequence data on P40 were unsuccessful due to N-terminal blockage of the native molecule. The nature of this N-terminal blocking was established using a combination of amino acid analysis, fast-atom-bombardment mass spectrometry and peptide synthesis. The P40 molecule, a single polypeptide chain comprising 126 amino acid residues, is structurally distinct from other known T-cell growth factors. No similarity was revealed when the amino acid sequence of P40 was compared with other proteins whose biochemical structure is known. The protein sequence data reported here predict four N-linked glycosylation sites in the P40 molecule.

Serum of lipopolysaccharide (LPS)-treated mice was found to support the growth of interleukin-HP1 (HP1)-dependent hybridoma and plasmacytoma cell lines. This growth-promoting activity, which was undetectable in normal serum, rose more than 1000-fold within 2 h after i.v. injection of the toxin and disappeared in less than 1 day. It could be traced to a single component, which behaved like HP1 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse phase high performance liquid chromatography, and was completely inhibited by a rabbit anti-HP1 antiserum. The LPS-induced factor was apparently not of T cell origin, as indicated by the strong activity found in the serum of LPS-treated nude mice. In vitro, LPS also considerably enhanced the production by macrophages of a factor with similar characteristics.

Cultures of normal diploid fibroblasts and of a human osteosarcoma cell line (MG-63) are shown to be able to produce a factor which promotes the growth of B cell hybridomas (hybridoma growth factor, HGF). The induction is stimulated by treatment of the cells with interleukin 1 (IL 1) (alpha or beta) or polyriboinosinic-polyribocytidylic acid [poly(rI).poly(rC)]. Combined treatment with cycloheximide and actinomycin D also stimulates production and enhances production induced by IL 1 or poly(rI).poly(rC). Extremely small doses of IL 1 (0.1 units/ml) are active as inducer of HGF. Also, under optimal conditions the yield of HGF can attain as much as 10(4) units/ml. Tumor necrosis factor (TNF-alpha), which otherwise shares various properties with IL 1, is a weak inducer of HGF. Although there is a superficial resemblance between induction of HGF and that of interferon-beta, the two activities are serologically distinct and conditions for their induction are quite different. In fact, conditions for induction of HGF are indistinguishable from those described for the induction of the mRNA of the so-called 26-kDa protein (also known as interferon-beta 2). Finally, the HGF derived from IL 1- or poly(rI).poly(rC)-treated fibroblasts is serologically not distinguishable from that produced by mitogen-stimulated peripheral blood leukocytes.

Recently, we described a murine helper T cell-derived molecule with T cell growth factor activity that is functionally and structurally distinct from IL-2, IL-4, and other known growth factors. This molecule, designated P40, was identified as a glycoprotein capable of supporting antigen-independent growth of certain helper T cell clones. Here, we report the cloning and expression of a cDNA for this new growth factor. The predicted mature protein is a cationic cysteine-rich polypeptide of 14 kD without significant homology to previously sequenced proteins.

The human "26-kd protein' is a secreted glycoprotein expressed, for example, in (blood) leukocytes, in epithelial cells treated with various inducers, but most strongly in interleukin-1 (IL-1)-treated fibroblasts. After finding it has antiviral and 2-5A synthetase-inducing activity, one group of authors called this protein IFN-beta 2. However, recently the full-length 26-kd cDNA sequence was shown to be identical with that of a B-cell-differentiating lymphokine called BSF-2, and another report suggested that the 26-kd protein could support the growth of some transformed murine B cell lines. To define its biological activities, we expressed the recombinant 26-kd protein by translating in Xenopus laevis oocytes a pure, synthetic chimeric mRNA containing the 26-kd protein coding region surrounded by Xenopus laevis beta-globin untranslated regions. A similar construction, but containing the HuIFN-beta cDNA coding region, was used to produce HuIFN-beta by the same procedure. Both recombinant glycoproteins were secreted, glycosylated, and their amounts were measured by [35S]methionine incorporation by the oocyte. Here we show that the recombinant 26-kd protein exhibits a high growth factor activity when assayed on an IL-HP1-dependent murine B cell hybridoma (sp. act. approximately 2 X 10(8) U/mg) as well as a potent differentiating activity on human CESS cells (sp. act. approximately 5 X 10(7) U/mg). While rHuIFN-beta was inactive in the latter two assays, it had the expected antiviral activity of 1-5 X 10(8) U/mg. The parallel recombinant 26-kd protein preparations had no detectable antiviral activity (i.e. a maximal specific activity of 1-3 X 10(2) U/mg, if any). The 26-kd protein is thus clearly an interleukin, and considering the confusing nomenclature now in use, this factor may better be renamed "interleukin 6'.

It was recently shown that T cells, macrophages and fibroblasts produce growth factors for B cell hybridomas and plasmacytomas. These factors were subsequently identified as members of a new family of cytokines on the basis of NH2-terminal amino acid sequence analyses. Using T cell-derived interleukin-HP1 (HP1), purified to homogeneity, as the prototype of this family, we examined the effects of these molecules in conventional polyclonal B cell activation assays with anti-immunoglobulin antibodies or dextran sulfate as co-stimulators. In the absence of other cytokines, the only significant effect of HP1 was to stimulate moderately the proliferation of anti-immunoglobulin-activated B cells. By contrast, in conjunction with interleukin 1, HP1 became a major growth and differentiation factor not only for B cells activated with anti-immunoglobulin antibodies but also for dextran sulfate-stimulated and even for unstimulated B cells. In fact, with respect to cell proliferation or IgM synthesis, the IL 1-HP1 combination proved to be equivalent to B cell stimulatory factors like IL 4 or IL 5. This B cell stimulatory activity was not due to the presence of a contaminant in the HP1 preparation because it was also observed with purified plasmacytoma growth factors derived from macrophages and fibroblasts, and could be inhibited by a monoclonal anti-HP1 antibody.

Human hybridoma growth factor (HGF) has been purified to homogeneity and identified with the 26kDa protein, interferon-beta 2 (IFN-beta 2), B-cell stimulatory factor-2 (BSF-2) and hepatocyte stimulatory factor (HSF). This factor, renamed interleukin-6 (IL-6), can be induced in fibroblasts by IL-1, while other cytokines are less active or inactive as inducers. The possible use of this IL-6 induction as an alternative indirect assay system for IL-1 is considered. Also, the direct HGF activity as a test IL-6 has been compared with the other biological activities of IL-6. It was concluded that the HGF assay is the most sensitive, specific and convenient test for IL-6.

A T-cell-derived lymphokine was identified by its ability to support the growth of a subset of B-cell hybridomas. Hybrids that failed to survive in the absence of this molecule represented a major proportion of rat-mouse hybridomas but were very rare among mouse-mouse B-cell hybrids. Stable factor-dependent B-cell hybridomas were used to monitor the purification of the growth factor from the supernatant of a clonotypically stimulated mouse helper T-cell clone. Sequential fractionation using gel filtration, anion-exchange chromatography, and reversed-phase HPLC resolved the factor from other B-cell growth factors and yielded a single-chain protein characterized by a major charge (pI = 5-7) and molecular mass (22- to 29-kDa) heterogeneity, probably due to variations in glycosylation. The NH2-terminal amino acid sequence of this protein, which is active on B-cell hybridomas in the 0.1 pM range, showed no significant homology with that of known lymphokines. Because the purified factor also supported the growth and survival in vitro of murine plasmacytomas (to be published elsewhere), it was provisionally designated interleukin-HP1 (where H stands for hybridoma and P stands for plasmacytoma).

We have recently purified and partially sequenced a new T cell-derived lymphokine with growth factor activity for B cell hybridomas and plasmacytomas, which we named interleukin HP1 (HP1). Here we show that, in response to viral infection or after treatment with poly(rI).poly(rC), L cells produce a factor that is capable of supporting the in vitro growth and survival of HP1-dependent cell lines. Serologic and structural evidence is presented in favor of the identity between the fibroblast factor and HP1, demonstrating that non-T cells can make HP1-related molecules.

Mouse plasmacytoma FLOPC21 was adapted to culture in the presence of a mouse Th cell supernatant. A stable factor-dependent cell line was derived from this culture and the factor responsible for its growth was identified as granulocyte-macrophage colony-stimulating factor.

We reported recently that a human protein, previously described as IL-1 inducible 26K factor (26K) or interferon-beta 2, was a potent growth factor for B cell hybridomas in vitro. Subsequently, it appeared that this protein was also identical with the lymphokine B cell stimulatory factor 2. Here we report that the levels of 26K are considerably increased during the early stages of acute infections of the central nervous system. This elevation in 26K titres was not observed in either chronic infections or in non-infectious diseases.

Several antigen-specific T cell lines were found to secrete a lymphokine upon activation by antigen or lectin that was provisionally termed T cell growth factor III (TCGF III) because it induced the proliferation of a CD4+ T cell clone independently from IL2 and IL4. Amino acid sequence analysis (and the functional properties of TCGF III) revealed that TCGF III was identical with a recently identified lymphokine termed P40. TCGF III/P40 was not only produced by long-term cultured T cell lines but also upon stimulation of freshly isolated Mlsa-reactive T cells. In addition, naive CD4+ T cells secreted TCGF III/P40 upon activation by lectin or allo-major histocompatibility complex structures. However, in spite of its growth-promoting activity for a CD4+ T cell clone this lymphokine does not appear to function as a general growth factor for T cells.