Modulation of tumour cell growth by tumour-infiltrating leucocytes is of high importance for the biological behaviour of malignant neoplasms. In melanoma, tumour-associated macrophages (TAM) and tumour-infiltrating lymphocytes (TIL) are of particular interest as inhibitors or enhancers of cell growth. Recruitment of leucocytes from the peripheral blood into the tumour site is mediated predominantly by chemotaxins, particularly by the group of chemokines. The aim of this study was to identify peptides released by human melanoma cells with monocyte chemotactic properties. To assure the presence of biologically active mediators, biochemical purification and biological characterization of peptides was based on a detection system dependent on bioactive, monocyte chemotactic activity in vitro. Cell culture supernatants of melanoma cells were fractioned by heparin–sepharose followed by preparative reversed-phase HPLC steps to enrich monocyte chemotactic activity in one single band on a sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gel. These purified fractions were shown to react with RANTES-specific antibodies in an enzyme-linked immunosorbent assay (ELISA) as well as in Western blot analysis. Amino acid sequencing of the N-terminal protein fragment confirmed 100% homology to the RANTES protein. Further analysis showed that four out of eight melanoma cell lines constitutively expressed and secreted the β-chemokine RANTES as detected by ELISA. The amount of RANTES protein secreted (up to 50 ng ml −1 ) was about 5–50 times higher than interleukin 8 (IL-8), determined in the same supernatant samples. Tumour necrosis factor α (TNF-α), not, however, IL-2, interferon-γ (IFN-γ), or α-melanocyte-stimulating hormone (α-MSH) was able to up-regulate RANTES and interleukin 8 secretion. Furthermore, higher levels of RANTES secretion in vitro were associated with increased tumour formation upon S . C . injection of six human melanoma cell lines in nude mice. Our data provide evidence that a subset of melanoma cells express mRNA and secrete RANTES protein which may be partly responsible for the recruitment of monocytes, T-cells and dendritic cells into the tumours. However, transplantation experiments in nude mice suggest that effects of RANTES may also benefit tumour progression. Further studies are needed to dissect the underlying mechanisms. © 1999 Cancer Research Campaign

Human eosinophils produce upon treatment with 5-oxo-eicosatetraenoic acid or (5S,15S)-dihydroxyeicosatetraenoic acid a potent eosinophil-chemotactic eicosanoid, 5-oxo-15-hydroxy-(6E,8Z,11Z,13E)-eicosatetraenoi c acid (5-oxo-15-HETE). 5-Oxo-15-HETE induces human eosinophil (Eo) chemotaxis at nanomolar concentrations with an efficacy in vitro comparable to that seen for platelet activating factor. Comparison of Eo chemotactic activities of several structurally related eicosanoids with different substituents and/or double bound geometry led to the conclusion that maximal potency and efficacy of eosinophil-chemotactic and chemokinetic activity is present in 5-oxo-(6E,8Z,11Z,14Z)-eicosatetraenoic acid (5-oxo-ETE). The presence of a hydroxyl group at position C-15 is not necessary for potent chemotactic activity, whereas a geometric isomer having trans instead of cis double bond at C-atom 8, as well as esterified 5-oxo-ETE usually show a 5-10-fold lower potency. 5-Oxo-eicosanoids elicit a dose-dependent transient rise of intracellular Ca2+ levels in human Eos, however, in contrast to some other Eo chemotaxins do not induce degranulation. Cross-desensitization of Ca2+ mobilization and Eo chemotaxis revealed that the geometric isomers of 5-oxo-eicosanoids, 5(S)-HETE, and (5S,15S)-di-HETE cross-deactivate Eo responses to each other, whereas other, unrelated stimuli did not interfere with these lipids indicating that 5-oxo-eicosanoids activate Eos via a separate receptor.

Neutrophil cell responses and signal pathways elicted by the chemotactic arachidonic acid metabolites (6E, SZ, 1 lZ, 14Z)-5-oxo-icosatetraenoic acid and (6E, SZ, 1 IZ, 13E)-5-oxo-15-hydroxy-icosatetraenoic acid were studied and compared with those of other chemotaxins. Polyphosphoinositol lipid analysis revealed activation of phosphatidylinositol-bisphosphate 3-kinase by both agonists. Experiments with Fura-2 in the presence of EGTA indicated Ca2+ mobilization from intracellular stores by both 5-OXOicosanoids. A transient actin response and production of small amounts of superoxide anions upon stimulation with both agents was detected. The changes induced by 5-0x0-icosanoids were more moderate and transient than those obtained by other chemotaxins. Desensitization studies indicated cross-desensitization between both 5-oxo-icosanoids, but no interference with the response of other chemotaxins. All cell responses elicted by 5-0x0-icosanoids were inhibited by pertussis toxin suggesting involvement of Gproteins, a common activation mechanism for all known potent chemotaxins. In contrast to other chemotaxins, 5-0x0-icosanoids at concentrations 500-fold higher than the ED,, of other functions did not induce up-regulation of CD11 b and N-formyl-peptide receptors at the cell surface, and failed to potentiate Nformyl-peptide-induced superoxide anion production. These results indicate that 5-0x0-icosanoids trigger a unique pattern of neutrophil responses.Keywords: neutrophils ; 5-0x0-icosanoids ; Caz+ transients ; F actin; superoxide anions.Accumulation of neutrophils in tissue is characteristic of inflammation and observed in a large variety of pathological conditions. Leukocyte infiltration is presumably caused by different chemotaxins, which are generated by different mechanisms [I]. Extensively characterized chemotaxins are N-formyl peptides, the complement split product C5a (C5a), the chemokines interleukin-8 (IL-8) and GROa as well as the arachidonic acid metabolite leukotriene B, (LTB,) 11-61. In addition to chemotaxis, these substances stimulate with different capacity proinflammatory activities such as production of reactive oxygen metabolites, up-regulation of membrane proteins such as the complement receptor type 3 (CDllb) or the N-formyl-peptide receptor and priming of neutrophil responses [l -61. Activation of neutrophils by chemotaxins requires binding to ligand-specific cell-surface receptors [7]. The receptors for most chemotaxins interact with pertussis-toxin-sensitive or cholera-toxin-sensitive heterotrimeric guanine-nucleotide-binding proteins (G-proteins) 11, 7-91. Activation of G-proteins leads into different signal pathways initiated by phospholipase C-p2, phosphatidylinositol-bisphosphate (PtdInsP,) 3-kinase and actin polymerization [I, 2, 10, 1 I]. The second messengers generated by these differend signal pathways regulate the effector functions of leukocytes in concert [I, 2, 12-15].Recently, 5-oxo-icosanoids, i.e. (6E, SZ, 1 lZ, ~~Z ) -~-O X Oicosatetraenoic acid (5oETE) and (6E, 82, l l Z , 13E...

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