Interleukin (IL) 4 and IL-13 act on human lung fibroblasts. Implication in asthma.

Airway hyperresponsiveness leading to subepithelial fibrosis is mediated by inflammatory cells activated by T helper (Th) 2-derived cytokines such as IL-4 and IL-5. By analyzing the phenotype and response of human lung fibroblasts derived from either fetal (ICIG7) or adult (CCL202) tissue as well as from a Th2-type stromal reaction (FPA) to IL-4 and IL-13, we provide evidence that human lung fibroblasts may behave as inflammatory cells upon activation by IL-4 and IL-13. We show that the three types of fibroblasts constitute different populations that display a distinct pattern in cell surface molecule expression and proinflammatory cytokine and chemokine release. All fibroblasts express functional but different IL-4/IL-13 receptors. Thus, while IL-4 receptor (R) alpha and IL-13Ralpha1 chains are present in all the cells, CCL202 and FPA fibroblasts coexpress the IL-13Ralpha2 and the IL-2Rgamma chain, respectively, suggesting the existence of a heterotrimeric receptor (IL-4Ralpha/IL-13Ralpha/IL-2Rgamma) able to bind IL-4 and IL-13. Stimulation with IL-4 or IL-13 triggers in the fibroblasts a differential signal transduction and upregulation in the expression of beta1 integrin and vascular cell adhesion molecule 1 and in the production of IL-6 and monocyte chemoattractant protein 1, two inflammatory cytokines important in the pathogenesis of allergic inflammation. Our results suggest that when activated by IL-4 and IL-13, different subsets of lung fibroblasts may act as effector cells not only in the pathogenesis of asthma but also in lung remodeling processes. They may also differentially contribute to trigger and maintain the recruitment, homing, and activation of inflammatory cells.

IL-15 is produced by a subset of human melanomas, and is involved in the regulation of markers of melanoma progression through juxtacrine loops.

IL-15 is a novel cytokine active through the IL-2R/betagamma. Since several human melanoma cell lines display functional IL-2Rs, we studied the IL-15/melanoma cells interactions. Ten out of 17 melanoma cell lines express the IL-15 transcript and four of them express levels of IL-15 mRNA similar to those detected in control activated monocytes. Nine out of ten cell lines also express two transcripts for the IL-15R alpha originated by the alternative splicing of exon'3'. Two melanoma cell lines, MELP and MELREO, derived from patients with rapidly progressive primary melanomas, co-express the two IL-15 transcripts, originated by alternative splicing of exon 'A'. Intracellular IL-15 protein was only detected in these two cells lines and it is mainly retained in the Endoplasmic Reticulum (ER). However, a small amount of IL-15 is also found in the Golgi apparatus and in the early endosomes, suggesting production and intercellular trafficking of endogenous IL-15 protein. Nevertheless, no biologically active IL-15 could be detected in the supernatant of all melanoma cells. The anti IL-15 blocking mAb M111 causes the up regulation of HLA Class I in dense MELP and MELREO cultures. These data suggest that IL-15 is probably active through juxtacrine loops negatively controlling HLA Class I molecules expression. These data offer, for the first time, a likely explanation to the controversial issue of IL-15 secretion and constitute a natural model for understanding IL-15 routing. Moreover, we identify a subset of melanoma cells producing IL-15, possibly involved in tumor escape mechanisms.

IL2 triggers a tumor progression process in a melanoma cell line MELP derived from a patient whose metastasis increased in size during IL2/INFalpha biotherapy.

Human melanomas may express both in vivo and in vitro functional IL-Rs and may be expected to directly respond to injected IL2. This may generate biological situations which may be favourable for the patient, but also for tumor progression. Here, we analyse the latter hypothesis. MELP is a melanoma cell line derived from a patient whose metastasis increased in size during IL2/IFN alpha biotherapy [correction of biotheraphy]. These cells have been characterized in vitro for their phenotype and for their sensitivity to IL2. In vitro MELP cells express an IL2-R alpha(+) beta(+) gamma(-) phenotype and IL2 treatment induces the acquisition of new functional characteristics represented (i) by the increased surface expression of two markers of metastatic evolution (ICAM-1 and CD44); (ii) by the stable induction of the IL2-R gamma with the appearance of functional IL2-R beta complex, which are also recognized by GM-CSF; (iii) by the inhibition of transcription of a regulatory cytokine such as IL6; (iv) by a differential effect of IL6 on CD44 surface expression in MELP cells treated or not with IL2 (MILG cells); (v) by the acquisition of faster growth rates and appearance of piling up and multilayer cellular organization; (vi) by the development of rapidly growing tumors in nude mice. IL2 induces in MELP cells a tumor progression process that could mimic the metastatic evolution observed in vivo during biotherapy. Therefore, MELP phenotype may help to define a subset of patients in which IL2 therapy may trigger unfavourable evolution.

Are interleukin-2 and interleukin-15 tumor promoting factors for human non-hematopoietic cells?

Human normal non hematopoietic cells of mesenchymal and neuroectodermal origin may express functional IL-Rs. For instance, in these cell types, IL-2 can stimulate proliferation (endothelial, intestinal and nervous cells) or modify the expression of adhesion molecules (fibroblasts) or inhibit proliferation (bone marrow stromal cells). Therefore, some cytotoxic effects described during IL-2 biotherapy could be due to a direct interaction between IL-2 and non-hematopoietic tissues. The expression of functional IL-2-R has also been reported in several human cell lines derived from solid tumors. In some instances IL-2 inhibits cell growth (head and neck, gastric and renal carcinomas), but in other tumors, growth stimulation and increased expression of markers of tumor progression have been reported (intestinal, breast, and lung carcinomas, gliomas, fibrosarcomas and melanomas). Additionally, secretion of biologically active IL-2 has been reported in some melanoma and breast cancer cell lines. Transcripts for the novel cytokine IL-15, which utilizes the beta and gamma chains of the IL2-R, have been found in melanoma cells and anti-IL-15 mAbs inhibit HLA class I expression in these cells. Therefore these cytokines may modify, inside a tumor, the behavior of both stromal and neoplastic cells. All these data may have important implications in our understanding of tumor host interactions and in future strategies of immunotherapy.

Role of interleukin (IL)-2 and IL-15 in the tumour progression of a melanoma cell line MELP, derived from an IL-2 progressor patient.

MELP is an interleukin (IL)-2 receptor (IL-2R; alpha+ beta+ gamma-) melanoma cell line that was derived, before the beginning of the immunotherapy, from a patient whose metastasis increased in size during treatment with IL-2/interferon-alpha. In these cells, continuous culture in the presence IL-2 (1000 UI/ml) causes the selection of a cell sub-line (termed MILG) expressing the gamma-chain which is tumorigenic in nude mice. Here, we further analysed the characteristics of MELP and MILG cells as well as clones selected at limiting dilution in the presence of high concentrations of IL-2 or IL-15, or those selected after transfection for the expression of a human IL-2 transgene (MELP-CL1). MELP cells, but not six other melanomas cell lines, shed two soluble immunosuppressive molecules, CD25 and intercellular adhesion molecule-1, whose levels also strongly increase in vivo during immunotherapy. In vitro MELP cells express transcripts for IL-6, transforming growth factor, basic fibroblast growth factor and vascular-endothelial growth factor. Cloning at limiting dilution was obtained in culture fed with IL-2 or IL-15. All these clones, as MILG cells, express the transcript for the IL-2R gamma chain. This could favour improved interactions with cytokines using this chain. By contrast, MELP-CL1 cells, which secrete low amounts of biologically active IL-2 (200 UI/10(6) cells) exhibit a phenotype and growth characteristics similar to those of the parental MELP cells. Indeed, a crosslinking experiment with 125I-IL-2, has showed that MELP and MELP-CL1 cells display a scant IL-2 binding ability that is strongly increased in MELP cells fed for 1 week with 1000 UI/ml IL-2. These cells, as well as MILG cells express a betagamma-complex which can also bind IL-15. IL-2 induces a rapid tyrosine phoshorylation in MILG cells, which is followed by a prolonged induction of c-fos and c-jun genes. By contrast, in MELP cells IL-2 only causes a delayed induction of c-myc gene. All MELP derivatives, but not MILG cells, express the transcripts for IL-15, which is not secreted but is present as an intracellular protein. All MELP cells express the transcript for the IL-15R alpha chain. MELP-CL1 cells are not tumorigenic in nude mice, whereas MILG cells form rapidly growing tumours in 75% of the mice. Coinjection at the same site of MILG and MELP-CL1 cells causes the rapid regression of MILG tumours in 80% of the mice, whereas their bilateral injection causes the rapid development of MILG tumours in 100% of the nude mice. Finally, treatment in nude mice of MILG cells with low amounts of IL-2 (1000 UI per mouse) and IL-15 (50 ng per mouse) induces the development of much more aggressive tumours.The expression of functional IL-2Rs in a subset of human melanomas could be responsible for tumour progression.

Chemokines and CD40 expression in human fibroblasts.

Chemokines are cytokines specialized for recruiting leukocytes in inflammatory responses. Recent data indicate that besides macrophages and leukocytes fibroblasts may also be a source of these important immune molecules. We assayed chemokine expression (mRNA/ protein) in cultured fibroblasts isolated from a variety of human tissues and different pathologic states: normal bone marrow vs. myelometaplastic spleen, normal lung vs. metastasis stroma, and normal breast vs. radiation fibrosis and tumor stroma. In all fibroblasts, transcripts for chemokines IL-8, stromal cell-derived factor-1, monocyte chemotactic protein (MCP)-1 and eotaxin were detected. Although the production of IL-8 was abundant in most of the fibroblasts studied, fibroblasts from lung and pathologic breast tissue produced significantly less. Conversely, eotaxin production was low in most fibroblasts except in those isolated from myelometaplastic tissue where it was highly produced. Moreover, chemokines MCP-4, RANTES and macrophage inflammatory protein-1alpha were found to be expressed only in fibroblasts from select tissues. When the expression of CD40, an activating surface molecule for immune cells, was investigated, we found that most of the fibroblasts expressed this antigen. Overall these results indicate that cultured human fibroblasts from various tissues and pathologic settings produce a distinct panel of chemokines and express CD 40, suggesting a possible fundamental role of fibroblasts in immune responses and disease processes.

IL-4 and IL-13 specifically increase adhesion molecule and inflammatory cytokine expression in human lung fibroblasts.

Subepithelial fibrosis in the bronchi of asthmatics is the result of an irreversible lung fibroblast activation, triggered by cytokines secreted by IL-4- and IL-5-activated inflammatory cells. Here, we provide evidence that human lung fibroblasts (ICIG7 cells) express a single class of high-affinity IL-4 receptor (IL-4R). This receptor is functional and composed of at least the IL-4Ralpha and IL-13Ralpha1 chains in the absence of the IL-2Rgamma chain. The IL-4Ralpha is efficiently internalized at 37 degrees C within 15 min in the presence of IL-4, whereas this process is slower with IL-13. In ICIG7 cells, IL-4 triggers the tyrosine phosphorylation of at least two proteins (110 and 180 kDa), and up-regulates the transcription of c-fos, c-jun and c-myc proto-oncogenes. In addition, the secretion of several cytokines [IL-6, granulocyte colony stimulating factor and granulocyte macrophage colony stimulating factor (GM-CSF)] as well as the expression of beta1 integrin and VCAM-1 adhesion molecules are augmented by IL-4. IL-13 displays similar biological activities, but less effectively than IL-4. On the other hand, ICIG7 cells could constitute a lung fibroblast population defined by the spontaneous release of several pro-inflammatory cytokines (IL-6, IL-11 and GM-CSF) and cell surface phenotype (CD4 and Thy-1). Through this peculiar cytokine pattern and the IL-4/IL-13-dependent activities, these cells could act as effector cells in the pathogenesis of asthma, triggering and maintaining the recruitment, homing and activation of bone marrow-derived inflammatory cells, and playing a role in the remodeling process of the airways.

Non random activation of endogenous interleukin-2, (IL-2), IL-2 receptor alpha and IL-2 receptor beta genes after transfection of mouse fibroblasts with a cDNA for the alpha chain of the human IL-2 receptor.

Mouse fibroblasts do not ordinarily express components for the interleukin-2 receptor (IL-2R alpha, beta, and gamma). An analysis of these cells by reverse transcription followed by polymerase chain reaction, however, indicates the presence of transcripts specific for the IL-2R beta and gamma genes. Transfection of the cDNA for the alpha chain of the human IL-2R into LTK- mouse fibroblast cell line (L3 cells) leads, in long-term cultures, to the formation of transcripts of endogenous mouse IL-2, IL-2R alpha and beta genes, as detected by Northern blotting. Based upon the results of the binding of 125I-labeled IL-2 to the transfected cells, three IL-2-binding proteins of 55 kDa, 65 kDa and 75 kDa were expressed by the transfected cells. The 65-kDa and 75-kDa proteins bound IL-2 in the presence of monoclonal antibodies for the IL-2R alpha chain. These polypeptides assembled to form high-affinity IL-2R, as shown by Scatchard binding analyses. The receptors were functionally active, since the expression of H-2k major histocompatibility complex antigens on the surface membranes of L3 cells was enhanced by exposing the cells to IL-2. Activation of the IL-2 gene was also observed in long-term cultures of L alpha beta cells, another LTK- transfectant expressing the human IL-2R alpha chain. This type of gene activation was not observed in LTK- fibroblasts transfected with cDNA for human IL-2 or IL-2R beta genes. In L3 and L alpha beta cells, transcription of the endogenous IL-2 gene was suppressed by cyclosporin A and enhanced by cycloheximide. These data may have implications for gene therapy of cancer cells.

Effects of human fibroblasts from myelometaplasic and non-myelometaplasic hematopoietic tissues on CD34+ stem cells.

Fibroblasts demonstrate different phenotypes and functions according to the tissue of origin and its physiopathologic state. We previously showed that fibroblasts isolated in culture from myelometaplasic (MM) spleen differed phenotypically from fibroblasts from normal bone marrow (BM). We compared the influence of each type of fibroblasts on the behavior of CD34+ stem cells. Expansion of nucleated cells was observed when blood CD34+ cells were co-cultured for 3 weeks with MM spleen-derived fibroblasts in monolayers. Myeloid cell differentiation was also observed as indicated by a decline in CD34+ cells and increases in CD14+, CD15+ and CD41+ cells. This myeloid differentiation was enhanced in the presence of MM spleen compared with normal BM-derived fibroblasts. Similarly, proliferation and differentiation of BM CD34+ cells was better in the presence of BM rather than MM spleen-derived fibroblasts. In addition, fibroblasts from MM spleen also induced a differentiation of CD56+ natural killer (NK) cells whereas BM-derived fibroblasts did not. Overall, the data indicate that cultured fibroblasts from diseased tissue have distinct growth and differentiation regulatory characteristics. They also suggest a role for these cells in hematopoietic disorders.