Platelet-activating factor (PAF), a phospholipid mediator of inflammation, is present in breast cancer tissue and correlates with microvessel density. In the present study, we investigated the biological significance of PAF synthesized within breast cancer. In vitro, we observed the production of PAF by two estrogen-dependent (MCF7 and T-47D) and an estrogen-independent (MDA-MB231) breast cancer cell lines after stimulation with vascular endothelial growth factor, basic fibroblast growth factor, hepatocyte growth factor, tumor necrosis factor, thrombin but not with estrogen, progesterone, and oxytocin. The sensitivity to agonist stimulation and the amount of PAF synthesized as cell-associated or released varied in different cell lines, being higher in MDA-MB231 cells, which are known to be highly invasive. We further demonstrate, by reverse transcriptase-polymerase chain reaction and cytofluorimetry, that all of the breast cancer cells express the PAF receptor and respond to PAF stimulation in terms of proliferation. Moreover, in MDA-MB231 cells PAF elicited cell motility. In vivo, two structurally different PAF receptor antagonists WEB 2170 and CV 3988 significantly reduced the formation of new vessels in a tumor induced by subcutaneous implantation of MDA-MB231 cells into SCID mice. In conclusion, these results suggest that PAF, produced and released by breast cancer cells, can contribute to tumor development by enhancing cell motility and proliferation and by stimulating the angiogenic response.

The cysteinyl (Cys) leukotrienes (LT)C4, LTD4, and LTE4, are lipid mediators that have been implicated in the pathogenesis of asthma. The human LTD4 receptor (CysLT1R) was recently cloned and characterized. The present work was undertaken to study the potential modulation of CysLT1R expression by the Th2 cytokines IL-13 and IL-4. In this study, we report that IL-13 up-regulates CysLT1R mRNA levels, with consequently enhanced CysLT1R protein expression and function in human monocytes and monocyte-derived macrophages. CysLT1R mRNA expression was augmented 2- to 5-fold following treatment with IL-13 and was due to enhanced transcriptional activity. The effect was observed after 4 h, was maximal by 8 h, and maintained at 24 h. IL-4, but not IFN-γ, induced a similar pattern of CysLT1R up-regulation. Monocytes pretreated with IL-13 or IL-4 for 24 h showed enhanced CysLT1R protein expression, as assessed by flow cytometry using a polyclonal anti-CysLT1R Ab. They also showed enhanced responsiveness to LTD4, but not to LTB4, in terms of Ca2+ mobilization, as well as augmented chemotactic activity. Our findings suggest a possible mechanism by which IL-13 and IL-4 can modulate CysLT1R expression on monocytes and macrophages, and consequently their responsiveness to LTD4, and thus contribute to the pathogenesis of asthma and allergic diseases.

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Platelet-activating factor (PAF) is a potent inflammatory mediator that has been shown to be produced by human keratinocytes and is thought to play a role in cutaneous inflammation. Immunofluorescence and radioligand binding studies were used to characterize PAF receptors (PAF-R) on human keratinocytes and the human epidermoid cell lines A-431 and HaCaT. Indirect immunofluorescence studies demonstrated anti-PAF-R staining of primary cultures of human keratinocytes, A-431 cells, and HaCaT cells. Primary cultures of human fibroblasts and the melanoma cell line SK-30 failed to show immunostaining above that seen with control antiserum. With indirect immunofluorescence studies of sections of normal human skin, a granular anti-PAF-R staining pattern was noted on the keratinocyte cell membranes. A-431 cells readily metabolized PAF by deacetylation-reacylation at 37 degrees C, but not at 4 degrees C. Binding studies on crude membrane preparations of A-431 cells conducted at 4 degrees C demonstrated specific binding that reached saturation by 120 min. Scatchard analysis of PAF binding data revealed a single class of high-affinity (KD = 6.3 +/- 0.3 nM) PAF binding sites. The immunofluorescence and radioligand binding sites were shown to be functional PAF-Rs, as 10 pM to 1 microM PAF increased intracellular calcium in primary cultures of human keratinocytes, A-431 cells, and HaCaT cells, whereas PAF treatment of primary cultures of human fibroblasts or the melanoma cell line SK-30 did not result in changes in the intracellular calcium concentration. The structurally dissimilar PAF-R antagonists CV-6209, Ro19-3704, and alprazolam all inhibited the PAF-induced calcium changes in A-431 cells. The CV-6209 inhibition was seen at doses that competed with the PAF binding to these cells. These studies provide the first evidence for the presence of a functional PAF-R expressed on human keratinocytes, suggesting that this lipid mediator may play an important role in normal keratinocytes or in inflammatory dermatology.

CCL11, CCL24, and CCL26 are chemokines involved in the recruitment of eosinophils into tissues and mainly activate CCR3. Whereas the genomic or pharmacological inhibition of CCR3 prevents the development of experimental asthma in rodents, it only impairs the recruitment of eosinophils by ∼40% in humans. As humans, but not rodents, express CCL26, we investigated the impact of CCL11, CCL24, and CCL26 on human eosinophils recruitment and evaluated the involvement of CCR3. The migration of eosinophils of healthy volunteers was similar for the three eotaxins. Eosinophils of mild asthmatics had a greater response to CCL11 and a much greater response to CCL26. Whereas all eotaxins induced the migration of eosinophil of asthmatics from 0 to 6 h, CCL26 triggered a second phase of migration between 12 and 18 h. Given that the CCR3 antagonists SB 328437 and SB 297006 inhibited the 5-oxo-eicosatetraenoate-induced migration of eosinophils and that the CCR3 antagonist UCB 35625 was not specific for CCR3, CCR3 blockade was performed with the CCR3 mAb. This antibody completely blocked the effect of all eotaxins on eosinophils of healthy subjects and the effect of CCL24 on the eosinophils of asthmatics. Interestingly, CCR3 blockade did not affect the second migration phase induced by CCL26 on eosinophils of asthmatics. In conclusion, CCL26 is a more effective chemoattractant than CCL11 and CCL24 for eosinophils of asthmatics. The mechanism of this greater efficiency is not yet defined. However, these results suggest that CCL26 may play a unique and important role in the recruitment of eosinophils in persistent asthma.