Human dendritic cells are a physiological source of the chemotactic arachidonic acid metabolite 5-oxo-eicosatetraenoic acid.

OBJECTIVE: The arachidonic acid metabolite, 5-oxo-eicosatetraenoic acid (5-oxo-ETE), is a potent chemotaxin for neutrophils and eosinophils. The aim of this study was to identify physiological conditions and stimulators of 5-oxo-ETE synthesis, because no such conditions have yet been identified. METHODS: Human neutrophils and monocyte-derived dendritic cells were prepared and 5-oxo-ETE synthesis analyzed using precolumn/reversed-phase HPLC under different conditions and with several physiological and unphysiological stimuli. RESULTS: Incubation of neutrophils with 5-hydroxyeicosatetraenoic acid (5-HETE) resulted in the synthesis of about 3.4 nM 5-oxo-ETE per 10(6) cells in 1 ml under optimal conditions. The synthesis was enhanced about 8-fold with the unphysiological stimuli calcium ionophore A23187 and phorbol 12-myristate 13-acetate (PMA). No significant effect was observed with different physiological activators. Under optimal conditions, human dendritic cells produced about 50 nM 5-oxo-ETE per 10(6) cells in 1 ml. The synthesis could be increased with PMA and A23187 by about 50%. Again, no effect could be observed with physiological agents for dendritic cells such as complement fragment C5a, platelet activating factor, N-formyl peptides and interleukin-5. CONCLUSIONS: These data identified dendritic cells as the only yet known physiological source of relevant amounts of 5-oxo-ETE. This suggests a regulatory function of dendritic cells in the induction of inflammatory neutrophil and eosinophil infiltration caused by 5-oxo-eicosatetraenoic acid.

Metabolism and function of 3-D-phosphorylated phosphoinositides in C5a-stimulated eosinophils.

Eosinophils play a central role in the pathogenesis of parasitic infections, atopic diseases, and bullous dermatoses. To understand the regulative function of phosphatidylinositol 3-kinases in cell responses of eosinophils, phospholipid metabolism and production of reactive oxygen metabolites were followed after stimulation with C5a. Measurements of phosphatidylinositol lipids and analysis of deacylated products of separated lipid extracts showed fast and transient formation of phosphatidylinositol 3,4,5-trisphosphate (PIP(3)). Cell studies in the presence of the tyrosine kinase blocker genistein indicated that C5a-stimulated PIP(3) formation occurred independently of tyrosine kinase activity. To analyze the function of PI4,5P(2)-3-kinase in eosinophils, the influence of wortmannin and LY294002 on production of reactive oxygen metabolites was studied. Both compounds inhibited with similar concentration dependency C5a-induced formation of PIP(3) and production of reactive oxygen metabolites. In summary, these data showed for the first time the involvement of PI4,5P(2)-3-kinase in the production of reactive oxygen metabolites in eosinophils.

Matrix metalloproteinases and venous leg ulceration.

Metalloproteinase-mediated proteolysis plays an important role during the phase of venous ulcer formation and wound repair. Venous ulcers manifest as a breakdown of the collagenous stromal tissue and are highly associated to chronic venous insufficiency. A major change in our understanding of the pathogenesis of venous ulcers occurred with the demonstration of extracellular matrix-degrading activity of matrix metalloproteinases to generate a dermal-epidermal skin defect. These proteases were intensely investigated in preceding stages and during wound repair of venous ulcerations. Different studies have revealed their significance in the process of proteolytic remodeling and recognized their potential importance in finding therapeutic rationales to manage late complications of chronic venous ulcers.

Adenosine triphosphate-induced oxygen radical production and CD11b up-regulation: Ca(++) mobilization and actin reorganization in human eosinophils.

Eosinophils are major effector cells in cellular inflammatory conditions such as parasitic infections, atopic diseases, bullous dermatoses, and vasculitis. Biological activities of adenosine triphosphate (ATP) were characterized in human eosinophils and compared with those of other eosinophil activators such as complement fragment product C5a, platelet-activating factor (PAF), and eotaxin. ATP initiated production of reactive oxygen metabolites, as demonstrated by lucigenin-dependent chemiluminescence. Furthermore, ATP caused up-regulation of the integrin CD11b. In addition, fluorescence microscope measurements labeled with fura-2 (1-[2-(5-carboxy-oxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2' -amino-5' -methyl-phenoxy)-ethane-N, N, N, N'-tetraacetic acid, pentaacetoxymethyl ester) eosinophils in the presence or absence of ethyleneglycotetraacetic acid (EGTA) indicated that there was Ca(++) mobilization from intracellular stores by ATP. Flow cytometric studies showed transient actin polymerization upon stimulation with ATP and its stable analogues adenosine 5'-0-(3-thiotriphosphate) and 2-methylthioadenosine triphosphate tetrasodium (met-ATP). The reactions induced by ATP were comparable to those obtained by C5a, PAF, and eotaxin. Production of reactive oxygen metabolites and actin polymerization after stimulation with ATP was inhibited by pertussis toxin, which indicated involvement of receptor-coupled guanine nucleotide-binding proteins (G(i) proteins). In addition, experiments with oxidized ATP also suggest involvement of P2X receptors in this activation process. The results show that ATP is a strong activator of eosinophils and has biological activity comparable to those of the eosinophil chemotaxins C5a, PAF, and eotaxin. The findings strongly suggest a role of ATP in the pathogenesis of eosinophilic inflammation as an activator of proinflammatory effector functions.

Overexpression of CXC-chemokines and CXC-chemokine receptor type II constitute an autocrine growth mechanism in the epidermoid carcinoma cells KB and A431.

The CXC-chemokines Groalpha and interleukin-8 (IL-8) are well characterized growth factors for melanoma cells. Here the constitutive expression of Groalpha, IL-8 and their receptors (CXCR1 and CXCR2) as well as their functional involvement in the proliferation response were analyzed in normal keratinocytes and epidermoid carcinoma cell lines A431 and KB. Flow cytometric measurements, ELISA and semi-quantitative RT-PCR revealed low constitutive protein secretion and mRNA expression of both CXC-chemokines as well as CXCR1 and 2 in normal keratinocytes, whereas significant higher levels of CXC-chemokines and CXCR2 were deteced in epidermoid carcinoma cells. Proliferation of epidermoid carcinoma cells could be induced by CXC-chemokines and constitutive proliferation could be inhibited by neutralizing antibodies against CXC-chemokines and CXCR2. These studies indicate that constitutive Groalpha, IL-8 and CXCR2 protein expression enable an autocrine growth mechanism in epidermoid carcinoma cells.

Synthesis, biological effects and pathophysiological implications of the novel arachidonic acid metabolite 5-oxo-eicosatetraenoic acid (Review).

Arachidonic acid is rapidly metabolized by several distinct enzymes including the 5-lipoxygenase generating leukotrienes and 5-hydroxyeicosatetraenoic acid (5-HETE). These well studied metabolites cause a variety of physiological and pathophysiological effects in different tissues. Recently, oxidation of 5-HETE to 5-oxo-eicosatetraenoic acid (5-oxo-ETE) by an NADP+-dependent dehydrogenase has been demonstrated. Calcium ionophors and protein kinase C activators stimulate the synthesis of 5-oxo-ETE in neutrophils, eosinophils and monocytes. This novel arachidonic acid metabolite has a potent chemotactic activity for neutrophils and eosinophils. It stimulates adhesion of neutrophils and induces reactive oxygen metabolites in eosinophils. There is evidence that 5-oxo-ETE and 5-HETE interact with a specific G-protein coupled receptor. Since in contrast to 5-oxo-ETE much higher concentrations of 5-HETE are needed to provoke cell responses, 5-oxo-ETE might be the physiological relevant ligand for this putative receptor. Further downstream signalling pathways of this ligand include calcium transients, actin polymerization, activation of phosphatidylinositol-3-kinase and MAP-kinase. 5-oxo-ETE has been extracted from scales of psoriatic patients and injection of 5-oxo-ETE into rabbit subcutis causes a severe edema with an inflammatory cell infiltrate resembling an urticarial lesion. These findings indicate, that 5-oxo-ETE might play a role in different cutaneous inflammatory diseases.