Immunohistochemical evidence of cytokine networks during progression of human melanocytic lesions

Melanoma cells in culture express a variety of growth factors and cytokines and some of their autocrine and paracrine roles have been investigated. However, less information is available on the potential dynamic changes in expression of these molecules on cells during melanoma development and progression in situ. Using immunohistochemistry, we tested 40 nevi and primary and metastatic melanoma lesions for the expression of 10 growth factors and cytokines and the respective receptors representing 10 cell surface molecules. Nevi and thin (< 1 mm) primary melanomas showed little expression of ligands except weak reactivity of tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-beta (TGF-beta), interleukin-8 (IL-8) and reactivity of TGF-betaR and c-kit. Marked up-regulation of growth factors, cytokines and receptor expression was observed in thick (> 1 mm) primary melanomas, which were stained with polyclonal or monoclonal antibodies (MAbs) for IL-1alpha, IL-1beta, IL-6, IL-8, TNF-alpha, TGF-beta, granulocyte-macrophage colony-stimulating factor (GMCSF) and stem cell factor (SCF), but not IL-2. Metastases showed similar expression patterns except that SCF was absent. Co-expression of ligand and receptor was observed for TGF-beta, GM-CSF and IL-6, suggesting an autocrine role for these ligands. TNF-alpha appears to be a marker of benign lesions; IL-6 and IL-8 expression is associated with biologically early malignancy; TGF-beta, GM-CSF and IL-1alpha are highly expressed in biologically late lesions; and TNF-beta is an apparent marker of metastatic dissemination. Our results indicate that melanoma cells utilize cascades of growth factors and cytokines for their progression.     

The role of interleukin-1 beta in the pathogenesis of multiple myeloma

Interleukin-1 beta has potent OAF activity, can increase the expression of adhesion molecules, and can induce paracrine IL-6 production (see Fig. 1). These biologic effects of IL-1 beta closely parallel several of the clinical features of human myeloma, such as osteolytic bone lesions, homing of myeloma cells to the bone marrow, and IL-6-induced cell growth. The increased production of adhesion molecules could explain why myeloma cells are found predominantly in the bone marrow. These fixed monoclonal plasma cells could subsequently stimulate osteoclasts through the production of IL-1 beta and paracrine generation of IL-6, resulting in osteolytic disease. Also, IL-6 produced by either a paracrine or autocrine mechanism can support the growth of the myeloma cells that may be manifested clinically by an elevated labeling index. In the future, continued follow-up of IL-1 beta-positive and IL-1 beta-negative MGUS patients should determine whether aberrant expression of IL-1 beta by monoclonal plasma cells is a critical genetic event in the progression of MGUS to myeloma. Because MGUS is relatively common in the general population and myeloma is incurable in almost all cases, identification of MGUS patients who are likely to progress to active myeloma will be important in the development of new therapeutic strategies. For example, an effective chemopreventive agent that prevents or delays the transition from MGUS to myeloma could have a major effect on the treatment of patients with monoclonal gammopathies.     

IL-6/IL-6R as a potential key signaling pathway in prostate cancer development

Interleukin-6 (IL-6) is a pleiotropic cytokine involved in prostate regulation and in prostate cancer (PC) development/progression. IL-6 acts as a paracrine and autocrine growth stimulator in benign and tumor prostate cells. The levels of IL-6 and respective receptors are increased during prostate carcinogenesis and tumor progression. Several studies reported that increased serum and plasma IL-6 and soluble interleukin-6 receptor levels are associated with aggressiveness of the disease and are associated with a poor prognosis in PC patients. In PC treatment, patients diagnosed with advanced stages are frequently submitted to hormonal castration, although most patients will eventually fail this therapy and die from recurrent castration-resistant prostate cancer (CRPC). Therefore, it is important to understand the mechanisms involved in CRPC. Several pathways have been proposed to be involved in CRPC development, and their understanding will improve the way to more effective therapies. In fact, the prostate is known to be dependent, not exclusively, on androgens, but also on growth factors and cytokines. The signaling pathway mediated by IL-6 may be an alternative pathway in the CRPC phenotype acquisition and cancer progression, under androgen deprivation conditions. The principal goal of this review is to evaluate the role of IL-6 pathway signaling in human PC development and progression and discuss the interaction of this pathway with the androgen recepto pathway. Furthermore, we intend to evaluate the inclusion of IL-6 and its receptor levels as a putative new class of tumor biomarkers.The IL-6/IL-6R signaling pathway may be included as a putative molecular marker for aggressiveness in PC and it may be able to maintain tumor growth through the AR pathway under androgen-deprivation conditions. The importance of the IL-6/IL-6R pathway in regulation of PC cells makes it a good candidate for targeted therapy.     

Growth regulation of the AML-193 leukemic cell line: evidence for autocrine production of granulocyte-macrophage colony-stimulating factor (GM-CSF), and inhibition of GM-CSF-dependent cell proliferation by interleukin-1 (IL-1) and tumor necrosis factor (TNF alpha)

The human leukemic cell line AML-193 was tested for its proliferative response to endogenously produced autocrine factors and to a variety of cytokines and colony-stimulating factors. Cells grown in the absence of GM-CSF incorporated tritiated thymidine, and this was partially reversed by adding neutralizing anti-GM-CSF antibodies to the culture medium, suggesting that it was due, at least in part, to autocrine GM-CSF production. This was confirmed by immunopurification of a GM-CSF-like activity from cell supernatant of AML-193 cells grown in serum free medium in the absence of exogenous GM-CSF. When AML-193 cells were cultured with GM-CSF in combination with other cytokines, Interleukin-1 alpha and beta (IL-1 alpha and beta), Interleukin-3 (IL-3), Interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF) and tumor necrosis factor alpha (TNF alpha), none of them affected the concentration of GM-CSF required to induce 50% of maximum proliferation (D50). However, the maximum proliferation induced by GM-CSF alone was drastically decreased by IL-1 alpha, IL-1 beta and TNF alpha. Inhibition caused by exposure of the AML-193 to IL-1 for up to 24 hr was reversible, ruling out a direct cytotoxic effect.     

Production and Effects of Interleukin-6 and Other Cytokines in Patients with Non-Hodgkin's Lymphoma

Cytokines are regulatory molecules that are produced by a variety of cell types and are characterized by numerous biologic functions involved in the regulation of the immune system and hematopoiesis. This review summarizes the functions and regulation of cytokines in lymphomas and discusses the effect of a specific cytokine, interleukin-6 (IL-6), in B-cell lymphomas. IL-6 is a multipotent cytokine that can mediate the differentiation of B-cells into immunoglobulin-secreting cells, stimulate the autocrine or paracrine growth of myeloma cells, induce acute-phase proteins in liver cells, and may influence the pathogenesis of several diseases by autocrine or paracrine mechanisms. Patients with non-Hodgkin's lymphoma (NHL) have increased serum concentrations of IL-6; increased IL-6 levels can be associated with the presence of B-symptoms. Data presented in this review indicate that neoplastic cells from patients with NHL contained high concentrations of IL-6. Thus, elevated serum levels of IL-6 appear to originate from the lymphoma cells in patients with B-cell NHL, suggesting that the neoplastic cells may modulate the general status of patients with B-cell NHL. The inhibition or modification of the production of IL-6 in lymphoma cells may lead to a more effective control of the general status of patients with B-cell NHL.     

Radioprotection with cytokines--learning from nature to cope with radiation damage.

The quest for methods to protect cells from the damaging effects of ionizing radiation led to the observation that cytokines, endogenously produced hormone-like polypeptides, are radioprotective. Interleukin-1 and tumor necrosis factor-alpha, given before irradiation, can protect mice from doses of radiation that would be fatal to untreated animals. At lower doses of radiation, the hemopoietic growth factors, interleukin-1, interleukin-4, interleukin-6, tumor necrosis factor-alpha, interferon, and leukemia inhibitory factor can promote recovery when administered after irradiation. Exposure to ionizing radiation selectively induces expression of some cytokines. Recent work suggests that certain cytokines may initiate autocrine/paracrine regulated recovery and repair pathways. Thus, the radioprotective and therapeutic effects of supplementary pharmacological doses of cytokines may act by amplifying innate defenses to ionizing radiation.     

Intratumoral Production of IL-6 in B Cell Chronic Lymphocytic Leukemia and B Lymphomas

Interleukin-6 is a major B lymphocyte growth factor, and may play a role in the proliferation of malignant B lymphocytes. In order to provide arguments supporting such a role, the intratumoral production of IL-6 was studied by in situ hybridization and immunohistochemistry in 53 neoplastic tissues from B cell chronic lymphocytic leukemia or B lymphomas. IL-6-producing cells were detected in all samples but 5. However, the number of IL-6 producing cells was variable amongst the different cases. Increased density of IL-6-producing cells was highly dependent on the presence of malignant immunoblasts within the neoplastic clone. IL-6 was produced in a paracrine way, macrophages and endothelial cells being the main producers of the cytokine while malignant immunoblasts expressed the IL-6 receptor. Taken together, these results suggest that IL-6 may indeed act as a growth factor for malignant cells in some B lymphoproliferations and that this paracrine loop could be the target of new therapeutic approaches.     

Procathepsin D and cytokines influence the proliferation of lung cancer cells

Enzymatically inactive procathepsin D (pCD) has been established as an important factor in the development of lung cancer. In addition to serving as a growth factor, pCD is also involved in communication between cancer cells and surrounding cells. In the present investigation, a possible combination of autocrine and paracrine actions of pCD was investigated. pCD initiated secretion of interleukin (IL)-4, IL-8, IL-10 and IL-13 from lung cancer cells. These cytokines participated in the proliferation of the cancer cells, as demonstrated both by adding the cytokines and by inhibition using specific anti-cytokine antibodies. Using several clones of lung cancer cells differing in production of activation peptide, the pCD/cytokine cooperation and action was shown to be dependent on pCD secretion. Further proof that pCD is one of the key molecules involved in cancer development is presented.     

Growth factor independence and growth regulatory pathways in human melanoma development

Summary This review concentrates on growth autonomy of tumor cells in relation to tumor progression. Human malignant melanoma serves as an example for progressive growth factor independence at subsequent stages of tumor progression. Mechanisms by which malignant cells acquire growth factor independence are discussed. In melanoma, deregulation of growth regulatory pathways has been described on four levels: 1) aberrant production of autocrine growth factors that substitute for exogenous growth factors (basic fibroblast growth factor [bFGF]); 2) alterations in the response to negative autocrine growth factors (interleukin [IL]-6 and transforming growth factor [TGF]-); 3) overexpression of epidermal growth factor receptors (EGF-R); and 4) alterations of cellular protooncogenes involved in signal transduction (RAS, MYB) and growth suppression (p53). In addition to bFGF and IL-6, multiple other growth factor genes are activated in malignant melanoma cells but not normal melanocytes. These include both chains of platelet-derived growth factor (PDGF), TGF-, IL-1, IL-8, and tumor necrosis factor (TNF)-. Of these, PDGF-B has been investigated in more detail. Melanoma-derived PDGF clearly does not act in a direct autocrine mode, but has important paracrine effects on normal tissue constituents, notably fibroblasts and endothelial cells, that are essential for tumor developmentin vivo. It is speculated that other melanoma-derived growth factors with as yet undefined functions similarly exert such paracrine or indirect autocrine effects that cannot be sufficiently addressed in studies on cultured cells.     

The inflammatory cytokine tumor necrosis factor-alpha generates an autocrine tumor-promoting network in epithelial ovarian cancer cells

Constitutive expression of the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is characteristic of malignant ovarian surface epithelium. We investigated the hypothesis that this autocrine action of TNF-alpha generates and sustains a network of other mediators that promote peritoneal cancer growth and spread. When compared with two ovarian cancer cell lines that did not make TNF-alpha, constitutive production of TNF-alpha was associated with greater release of the chemokines CCL2 and CXCL12, the cytokines interleukin-6 (IL-6) and macrophage migration-inhibitory factor (MIF), and the angiogenic factor vascular endothelial growth factor (VEGF). TNF-alpha production was associated also with increased peritoneal dissemination when the ovarian cancer cells were xenografted. We next used RNA interference to generate stable knockdown of TNF-alpha in ovarian cancer cells. Production of CCL2, CXCL12, VEGF, IL-6, and MIF was decreased significantly in these cells compared with wild-type or mock-transfected cells, but in vitro growth rates were unaltered. Tumor growth and dissemination in vivo were significantly reduced when stable knockdown of TNF-alpha was achieved. Tumors derived from TNF-alpha knockdown cells were noninvasive and well circumscribed and showed high levels of apoptosis, even in the smallest deposits. This was reflected in reduced vascularization of TNF-alpha knockdown tumors. Furthermore, culture supernatants from such cells failed to stimulate endothelial cell growth in vitro. We conclude that autocrine production of TNF-alpha by ovarian cancer cells stimulates a constitutive network of other cytokines, angiogenic factors, and chemokines that may act in an autocrine/paracrine manner to promote colonization of the peritoneum and neovascularization of developing tumor deposits.     

Cytokine network in human multiple myeloma.

In multiple myeloma (MM), an overproduction of IL-6, indicated by increased plasma C-reactive protein levels, is found in 37% of MM patients at diagnosis and is associated with disease aggressiveness, myeloma-cell proliferation, and poor prognosis. IL-6 is produced by the tumoral environment mainly and not by myeloma cells themselves. IL-6 is a major growth factor for malignant plasmablastic cells in vitro, and it is possible to reproducibly obtain IL-6-dependent myeloma-cell lines. Moreover, anti-IL-6 therapies in patients with terminal disease block myeloma-cell proliferation in vivo. The myeloma-cell growth factor activity of IL-6 is probably the consequence of IL-6 being a growth factor for normal plasmablastic cells. Hematopoietic cytokines (GM-CSF, IL-3, IL-5, G-CSF) synergize with IL-6 to support myeloma-cell proliferation. IFN-alpha and TNF induce an autocrine production of IL-6 in myeloma-cell lines and make possible the autonomous growth of these cell lines. On the contrary, IFN-gamma completely inhibits the IL-6-mediated myeloma-cell proliferation. The identification of some major cytokines involved in the control of the myeloma clone has immediate therapeutic implications, because some of these cytokines are, or might be, used in the treatment of patients with MM.     

Tumor necrosis factor, but not other hematopoietic growth factors, prolongs the survival of hairy cell leukemia cells.

In order to determine the growth factor requirements of hairy cell leukemia (HCL) cells, we studied the in vitro effects of tumor necrosis factor (TNF), interleukin (IL) 1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, B-cell growth factor (BCGF), GM-CSF, PHA-stimulated lymphocyte-conditioned media (CM), and 5637 bladder carcinoma CM on HCL cells obtained from spleens of patients with HCL. Mononuclear cells from a normal donor, obtained at post-traumatic splenectomy, served as a control. TNF prolonged the survival of HCL cells obtained from five different HCL patients when compared to cells cultured in control media alone, although cell proliferation could be demonstrated in only two of the five. HCL cells stained negative for the Epstein-Barr nuclear antigen (EBNA) both before and after 4 weeks in culture. BCGF, 5637 CM, and PHA-stimulated lymphocyte CM also prolonged the survival of HC25 and HC56 cells, although not to the same degree as TNF. Cells cultured in BCGF, however, stained positive for EBNA. None of the other recombinantly produced or purified cytokines prolonged the survival of the leukemic cells. With the exception of IL-2, none of the growth factors studied prolonged the survival of purified normal spleen (NS) cells over a 4-week period of time when compared to NS cells incubated in media alone. TNF prolonged the survival of HC25 cells in a dose-dependent manner, and a highly purified antibody to TNF abrogated the effects of TNF. HC25 cells incubated in the presence of control media alone did not constitutively produce TNF mRNA; however, incubation of the cells in the presence of TNF for 48 h induced the cells to express TNF message. We conclude that TNF is important in prolonging the survival of HCL cells, and thus may be important in the pathogenesis of this disease.     

The Use of a Commercially Available Immunoassay to Determine the Level of Interleukin-6 in the Serum of Patients with Myeloma

Interleukin-6 (IL-6) is the B cell growth factor which stimulates the final differentiation of B cells to plasma cells and has been suggested to be an autocrine growth factor in patients with multiple myeloma. We have compared the concentration of IL-6 in the serum of patients with myeloma at diagnosis (n = 11), in plateau phase (n = 10) and with progressive disease (n = 13) using Intertest-6, a commercially available enzyme-linked immunoassay (ELISA). Longitudinal studies were performed with 6 patients. IL-6 levels were normal in all myeloma sera studied and did not change with disease progression. Serum IL-6 levels did not correlate with disease activity as determined by serum thymidine kinase (r = 0.07) nor did serum IL-6 provide any useful prognostic data. These results contradict previous studies using bioassays which reported that IL-6 levels increase significantly during progressive disease. Until this problem is resolved and these assays are validated, studies which attempt to quantitate IL-6 and other growth factors in serum should be treated with some caution.     

The use of a commercially available immunoassay to determine the level of interleukin-6 in the serum of patients with myeloma

Interleukin-6 (IL-6) is the B cell growth factor which stimulates the final differentiation of B cells to plasma cells and has been suggested to be an autocrine growth factor in patients with multiple myeloma. We have compared the concentration of IL-6 in the serum of patients with myeloma at diagnosis (n = 11), in plateau phase (n = 10) and with progressive disease (n = 13) using Intertest-6, a commercially available enzyme-linked immunoassay (ELISA). Longitudinal studies were performed with 6 patients. IL-6 levels were normal in all myeloma sera studied and did not change with disease progression. Serum IL-6 levels did not correlate with disease activity as determined by serum thymidine kinase (r = 0.07) nor did serum IL-6 provide any useful prognostic data. These results contradict previous studies using bioassays which reported that IL-6 levels increase significantly during progressive disease. Until this problem is resolved and these assays are validated, studies which attempt to quantitate IL-6 and other growth factors in serum should be treated with some caution.     

A possible growth factor role of IL-6 in neuroectodermal tumours

Preliminary data have shown that IL-6 may act as an autocrine growth factor to control proliferation. We further characterised the role of IL-6 in tumour growth as an autocrine/paracrine growth factor in neuroectodermal tumours. We evaluated the production and secretion of IL-6 by seven human melanoma, five neuroblastoma and one glioblastoma cell lines. Moreover, we determined their IL-6-dependent growth in serum free-medium or under minimal growth-supplement conditions: IL-6 dependent growth was observed in two non-IL-6 producing melanoma and in one neuroblastoma cell lines. In addition, expression of IL-6 mRNA and peptide was increased by retinoic acid. The data support the hypothesis that IL-6 contributes to neuroectodermal tumour growth, even though it shows a less potent effect than other reported growth factor such as IGF-II.