Differential regulation of IL-13 and IL-4 production by human CD8+ and CD4+ Th0, Th1 and Th2 T cell clones and EBV-transformed B cells.

In the present study, the requirements and characteristics for the production of IL-13 by human T cells, T cell clones and B cells were determined and compared with those of IL-4. IL-13 was produced by human CD4+ and CD8+ T lymphocyte subsets isolated from peripheral blood mononuclear cells and by CD4+ and CD8+ T cell clones. CD4+ T cell clones belonging to Th0, Th1-like and Th2-like subsets produced IL-13 following antigen-specific or polyclonal activation. In addition, EBV-transformed B cell lines expressed IL-13 mRNA and produced small amounts of IL-13 protein. Expression of IL-13 mRNA and production of IL-13 protein by peripheral blood T cells and T cell clones was induced rapidly and was relatively long lasting, whereas IL-4 production by these cells was transient. In addition, IL-13 mRNA expression was induced by modes of activation that failed to induce IL-4 mRNA expression. IL-13 shares many biological activities with IL-4 which is compatible with the notion that the IL-13 and IL-4 receptors share a common component required for signal transduction. However, IL-13 lacks the T cell-activating properties of IL-4. Here we have shown that this is related to the fact that T cells fail to bind radiolabeled IL-13 and do not express the IL-13-specific receptor component. Taken together, these results indicate that the differences in expression and biological activities of IL-4 and IL-13 on T cells may have consequences for the relative roles of these cytokines in the immune response.

In vivo cytokine expression in the thymus. CD3high human thymocytes are activated and already functionally differentiated in helper and cytotoxic cells.

CD4 and CD8 are expressed on mutually exclusive T cell populations that can recognize peptides bound to class II and class I MHC Ags, respectively. These populations have different functions and are different in their capacity to produce cytokines. In this paper we demonstrate that this functional differentiation occurs at the CD3low- CD3high transitional stage: single positive mature CD4+ thymocytes express IL-2 mRNA in vivo, whereas CD8+ thymocytes primarily express perforin. IL-2, perforin, and IFN-gamma mRNAs were almost absent in CD4+CD8+ CD3low but were clearly detectable in CD4+CD8+ CD3high cells, indicating that these genes are induced at the CD3low- CD3high transitional stage. In contrast, IL-4 mRNA levels were highest in the precursor cells but dropped sharply at the CD3low-CD3high transitional stage. These data are consistent with and link two earlier observations, i.e., that activation occurs at the CD3low-CD3high transition and that functional differentiation in helper and cytotoxic cells is already accomplished in the single positive thymocytes. This activation may reflect positive selection and concomitant functional differentiation into helper and cytotoxic T cells.

Effects of IL-13 on phenotype, cytokine production, and cytotoxic function of human monocytes. Comparison with IL-4 and modulation by IFN-gamma or IL-10.

Recently, we described the cloning and expression of a human cDNA which is the homologue to P600, a gene transcribed by mouse Th2 clones. Based on its activities on human monocytes and B cells this gene was designated IL-13. In the present study we investigated the effects of IL-13 alone or in combination with IL-4, IFN-gamma, or IL-10 on human monocytes. IL-13 induced significant changes in the phenotype of monocytes. Like IL-4, it enhanced the expression of CD11b, CD11c, CD18, CD29, CD49e (VLA-5), class II MHC, CD13, and CD23, whereas it decreased the expression of CD64, CD32, CD16, and CD14 in a dose-dependent manner. IL-13 induced up-regulation of class II MHC Ag and its down-regulatory effects on CD64, CD32, and CD16 expression were prevented by IL-10. IFN-gamma could also partially prevent the IL-13-induced down-regulation of CD64, but not that of CD32 and CD16. However, IL-13 strongly inhibited spontaneous and IL-10- or IFN-gamma-induced ADCC activity of human monocytes toward anti-D coated Rh+ erythrocytes, indicating that the cytotoxic activity of monocytes was inhibited. Furthermore, IL-13 inhibited production of IL-1 alpha, IL-1 beta, IL-6, IL-8, IL-10, IL-12 p35, IL-12 p40, macrophage inflammatory protein-1 alpha, granulocyte/macrophage-CSF, granulocyte-CSF, IFN-alpha, and TNF alpha by monocytes activated with LPS. In contrast, IL-13 enhanced the production of IL-1 ra by these cells. Similar results on cytokine production were observed or have been obtained with IL-4. Thus IL-13 shares most of its activities on human monocytes with IL-4, but no additive or synergistic effects of IL-4 and IL-13 on human monocytes were observed, suggesting that these cytokines may share common receptor components. Taken together, these results indicate that IL-13 has anti-inflammatory and important immunoregulatory activities.

Identification of human pre-T/NK cell-associated genes.

We have used a combination of subtractive cloning and differential screening techniques to identify genes preferentially expressed in early stages of human T/NK cell development compared with mature T and NK cells. A fetal liver-derived cytoplasmic (c) CD3+ membrane (m) CD3- clone, FL508, which expresses markers characteristic of pre-T and pre-NK cells served as a cell source for our cloning experiments. A cDNA library enriched for genes expressed in FL508 was constructed by removal of cDNA that hybridized to mRNA from a B cell line, JY. One-tenth of the resulting library of 5000 clones was screened by differential hybridization with cDNA probes from JY and a mature CD4+ T cell clone, B21. The relative expression levels of six selected clones were analyzed in 17 different cell/tissue types by semiquantitative polymerase chain reaction. Four of these clones are expressed at higher levels in thymocytes than in mature T or NK cells, and three clones are expressed at higher levels in fetal liver cells than in either mature T or NK cells. Partial and complete DNA sequence information suggests that these six cDNA correspond to previously unidentified genes. Genes identified in this study may be useful not only as markers for early stages of T/NK cell ontogeny, but also as tools for understanding novel developmental events.

Regulation of smooth muscle alpha-actin promoter in ras-transformed cells: usefulness for setting up reporter gene-based assay system for drug screening.

Oncogenic activation of ras results in changes in the transcription of several genes leading to uncontrolled cell growth. In this paper, we demonstrate that transformation of fibroblast cells by the ras oncogene leads to transcriptional repression of the smooth muscle alpha-actin promoter. Transient transfection analysis of plasmids containing the 5' upstream region of the human alpha-actin gene fused to human growth hormone or bacterial chloramphenicol acetyltransferase coding sequences into Rat-2 and ras-transformed Rat-2 (HO6) cells indicates that alpha-actin promoter is repressed in ras-transformed cells. In addition, stable rat fibroblast cell lines expressing human growth hormone or beta-galactosidase under the control of alpha-actin promoter exhibit repressed reporter gene activity following transformation by the ras oncogene. alpha-Actin promoter-driven beta-galactosidase activity is derepressed in revertants of ras-transformed stable cell lines. This revertant cell line expresses elevated levels of ras p21 protein and is resistant to retransformation by Ki and Ha-ras oncogenes. The revertant may have either a defective target protein whose activity is essential for the transforming activity of ras or an activated tumor suppressor gene which can suppress the activity of ras. These results indicate that smooth muscle alpha-actin promoter activity is a sensitive marker to follow phenotypic changes following transformation by ras and subsequent reversion. The advantages of this alpha-actin promoter-reporter gene assay system to screen for drugs that inhibit the transforming activity of ras, either directly or indirectly, are discussed.