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.

Interleukin 9 (IL-9) is a T-cell-derived lymphokine that induces the proliferation of various lymphold and hemopoietic cells. A cDNA done encoding the murine receptor was isolated by expression cloning in COS cells and screening with 12SI-labeled IL-9. Transient expression of this cDNA produced high-affinity binding sites for IL-9. The predcid 52-kDa protein contains a putative signal peptide and a pical trsembrane domain. A cDNA for the human homoogue was isolated by cross-hybridization. Transfection of this cDNA in a murine T-cell cone conferred responsiveness to human IL-9. Sequence analysis revealed that the IL-9 receptor belongs to the recently described hematopoletin receptor superfamily and is expressed in membrane-bound and soluble forms.Interleukin 9 (IL-9) was originally identified as a growth factor for murine T helper cell clones (1, 2), murine mast cell lines (3, 4), and a human megakaryoblastic leukemia line (5). Subsequently, additional biological targets were discovered including murine fetal thymocytes (6), murine erythroid progenitors (7), human T-cell lines (8), and human erythroid and myeloid precursors (9, 10). Moreover, involvement of IL-9 in tumorigenesis has been recently suggested by the observations that a murine T helper clone becomes tumorigenic after transfection with the IL-9 cDNA (11) and that lymph nodes from patients with Hodgkin disease or large-cell anaplastic lymphoma express IL-9 constitutively (12). Preliminary characterization of the IL-9 receptor on a murine T-cell clone has demonstrated the existence of saturable and specific binding sites with a Kd of -100 pM. Cross-linking analysis showed that the IL-9 receptor consists of a 64-kDa glycoprotein, the molecular mass of which is reduced to 54 kDa on treatment with N-glycosidase F (13).We report here the expression cloning and sequence of a cDNA encoding the murine IL-9 receptor.t This cDNA was further used to identify a human homologue.t MATERIALS AND METHODS Construction of cDNA Libraries. Poly(A)+ RNA extracted from the murine T-cell clone TS1 (1) was converted to double-stranded cDNA by the method of Gubler and Hoffman (14) with random hexanucleotide primers. EcoRI adaptors were attached and cDNA larger than 1.5 kilobases (kb) was isolated by fractionation on a 5-201% (wt/vol) potassium acetate gradient (15). The size-selected cDNA was inserted into the EcoRI site of the pCDSRa expression vector (16). Two additional cDNA libraries were generated in the BstXI site of the pCDM8 vector (15) with oligo(dT) or random primers. A human cDNA library was constructed by the same method in the pRC/RSV plasmid (Invitrogen, San Diego) with RNA from the megakaryoblastic leukemia cell line Mo7E (17).Transient Expression and Biding Assays. Purified recombinant murine IL-9 was radiolabeled using the Bolton and Hunter reagent as described (18) to yield material with a specific activity of 2300 cpm/fmol. Screening of cDNA pools by direct expression in COS cells was performed essentially as described by Gearing et al. (19). The mu...

To test the transforming potential of deregulated P40/Interleukin 9 expression, we transfected a mouse P40-dependent T cell line with P40 cDNA, and examined the tumorigenicity of the resulting transfectants. When the cells, which grew autonomously in vitro, were injected intraperitoneally or subcutaneously into syngeneic mice, a very high tumor incidence was observed with as few as 10(4) cells per inoculum. Animals died as a result of widespread dissemination of lymphomatous tissue to abdominal and thoracic organs. The same P40-dependent cell line transfected with a control construct did not form tumors even after injection of 10(7) cells. These results indicate that uncontrolled expression of P40 can support T cell proliferation in vivo, and may be a transforming event involved in the development of certain T cell tumors.

To better understand the role of free radicals in liver carcinogenesis, endogenous antioxidant defense systems and the susceptibility of membranes to lipid peroxidation were evaluated in early lesions and in malignant tumors induced by the Solt-Farber resistant hepatocyte protocol. These parameters were also measured in the liver surrounding these tumors. In comparison with the normal liver, both nodules and carcinomas show a different biochemical pattern consisting of decreased glutathione peroxidase (GSH peroxidase) and catalase activities plus increased glutathione reductase (GSSG reductase) activity. In contrast, 1 week after the application of the initiation-selection protocol, the liver displays a high level of glutathione (GSH), high GSSG reductase activity, a reduced production of malondialdehyde and no changes in superoxide dismutase and GSH peroxidase activities. These data suggest that the liver is well protected against reactive oxygen species. During the carcinogenic process, the liver parenchyma surrounding the altered foci recovers from most of the modifications induced by the initiation-selection treatment. These results add additional support for the hypothesis that the appearance of early alterations in the liver, after a carcinogenic treatment, might be an adaptive response to a hazardous environment in which selected cell populations are transformed into nodules and/or carcinomas.

The existence of saturable and specific binding sites for mouse P40/IL-9 was demonstrated on a variety of factor-dependent T cell lines derived from Th clones by long term culture in the presence of P40-containing T cell supernatants. Scatchard transformation of the data obtained with one such line was consistent with the existence of a single class of receptors with a Kd of approximately 100 pM and a density of 3000/cell. P40 binding to these cells was followed by rapid internalization of the ligand. P40-receptors (P40-R)3 were also found on certain Th clones maintained in conventional cultures, especially after stimulation with Ag and APC. Only T cell clones that proliferated in response to P40 showed significant levels of binding, suggesting that the regulation of P40-R expression is an important element in the control of P40-responsiveness. In accord with this idea, fresh T cells, cytolytic T cell clones and a wide variety of other cells including B cells and fibroblasts, which do not proliferate in response to P40, showed no significant binding. However, P40-R were not restricted to a few unusual Th clones. They were also detected on several T cell tumors, on macrophages and on mast cell lines. The latter point is of particular interest in view of the mast cell growth factor activity recently ascribed to P40. Cross-linking studies with T-cell lines and mast cells indicated that the P40-R consists of a 64-kDa glycoprotein, the molecular mass of which is reduced to 54 kDa on treatment with N-glycosidase F.