Expression and function of adenosine receptors in human dendritic cells.

Dendritic cells (DCs) are specialized antigen-presenting cells characterized by their ability to migrate into target sites, process antigens, and activate naive T cells. In this study, we analyzed the biological activity and intracellular signaling of adenosine by using reverse transcriptase-polymerase chain reaction assays to investigate mRNA expression of A(1), A(2a) and A(3) adenosine receptors in immature and mature human DCs. Functional experiments on adenosine stimulation showed chemotaxis, intracellular calcium transients, and actin polymerization, but no activation of adenylate cyclase in immature DCs. Experiments with receptor isotype-selective agonists and antagonists as well as pertussis toxin revealed that chemotaxis, calcium transients, and actin polymerization were mediated via G(i-) or G(0-)protein-coupled A(1) and A(3) receptors. Maturation of DCs induced by lipopolysaccharide (LPS) resulted in down-regulation of A(1) and A(3) receptor mRNAs, although A(2a) receptor mRNA was still expressed. However, in LPS-differentiated DCs, adenosine and an A(2a) receptor agonist stimulated adenylate cyclase activity, enhanced intracellular cAMP levels, and inhibited interleukin 12 (IL-12) production. These effects could be completely prevented by pretreatment with A(2) receptor antagonist. These findings strongly suggest that adenosine has important but distinct biological effects in DCs activity as a chemotaxin for immature DCs and as a modulator of IL-12 production in mature DCs. These effects can be explained by differential expression of adenosine receptor subtypes.

Functional characterization of P2Y and P2X receptors in human eosinophils.

Activation of purinoceptor by ATP induces in eosinophils various cell responses including calcium transients, actin polymerization, production of reactive oxygen metabolites, CD11b-expression, and chemotaxis. Here, the effect of ion channel-gated P2X and/or G protein-coupled P2Y receptor agonists ATP, ATPgammaS, alpha,beta-meATP, 2-MeSATP, BzATP, ADP, CTP, and UTP on the intracellular Ca(2+)-mobilization, actin polymerization, production of reactive oxygen metabolites, CD11b expression and chemotaxis of human eosinophils were measured and the biological activity was analyzed. Although all tested nucleotides were able to induce all these cell responses, the biological activity of the analyzed nucleotides were distinct. Agonists of the G protein-coupled P2Y receptors such as 2-MeSATP, UTP, and ADP have a higher biological activity for production of reactive oxygen metabolites, actin polymerization and chemotaxis in comparison to the ion channel-gated P2X agonists alphabeta-meATP, BzATP, and CTP. In contrast, P2Y and P2X agonist showed similar potencies in respect to intracellular calcium transient and CD11b up-regulation. This conclusion was further supported by experiments with receptor iso-type antagonist KN62, EGTA or with the G(i) protein-inactivating pertussis toxin. These findings indicate participation of different purinorecptors in the regulation of cell responses in eosinophils.

The role of the urokinase-type plasminogen activator (uPA) and its receptor (CD87) in lipodermatosclerosis.

BACKGROUND: Lipodermatosclerosis refers to a sclerosing panniculitis and dermopathy of the lower extremities sometimes seen in association with venous ulceration. Matrix metalloproteinases are implicated in the pathogenesis of venous leg ulcers and the in vitro activation of recombinant MMP-2 is controlled by the plasminogen activation system. To better understand the role of plasminogen activation in the pathogenesis of venous leg ulcers we investigated fibrinolytic factors and their inhibitors in tissue samples of lipodermatolsclerosis. METHODS: The expression and the functional state of the urokinase-type plasminogen activator (uPA), the tissue-type plasminogen activator (tPA), the urokinase receptor (CD87), the plasminogen activator inhibitors-1 and -2 (PAI-1 and PAI-2) were assayed using reverse transcription polymerase chain reaction, Western blot, fibrin zymography and immunohistochemistry analyses in tissue samples of lipodermatosclerosis. RESULTS: Our results provide direct evidence of elevated expression of uPA (p<0.01) and CD87 (p<0.01) mRNA and protein level in lipodermatosclerosis in comparison with healthy skin. By immunohistochemistry, elevated expression of uPA and CD87 could be detected. Fibrin zymography showed significantly elevated endogenous uPA activity (p<0.01) in liposclerotic lesions compared to healthy controls. CONCLUSION: Our findings indicate that elevated plasminogen activation in lipodermatosclerotic tissue may play a crucial role in the pathogenesis of venous leg ulceration.

Distinct amplification of the C5a-receptor pathways in normodense and hypodense eosinophils of patients with atopic dermatitis.

In patients with atopic dermatitis two different types of blood eosinophils with distinct density can be isolated. The normodense cells represent the huge majority in count, whereas the hypodense eosinophils are characterized by higher effector activity. To understand the altered functional responsiveness of these two cell subtypes, the expression of C5a receptors as well as C5a-induced signal pathways and the production of reactive oxygen metabolites have been analyzed. Chemiluminescence measurements revealed significant higher production of reactive oxygen metabolites in hypodense eosinophils in comparison to normodense cells. However, no difference in the expression level of C5a receptors as well as in the C5a-induced Ca2+-transients between normodense and hypodense eosinophils were found. In contrast, hypodense eosinophils showed a significantly higher actin polymerization response and phosphatidylinositol 4,5 bisphosphate 3-kinase activation after stimulation with C5a than normodense eosinophils. Therefore, normodense and hypodense eosinophils from the blood of patients with atopic dermatitis are characterized by differential amplification of C5a-receptor signal pathways, which might explain the differences in their proinflammatory activity.

Inflammation in stasis dermatitis upregulates MMP-1, MMP-2 and MMP-13 expression.

Stasis dermatitis is a common disorder, which is a consequence of impaired venous drainage of the legs. It is characterized histologically by proliferation of small blood vessels in the papillary dermis. This neovascularization may lead occasionally to the formation of discrete papules due to inflammatory processes. In order to evaluate the role of matrix metalloproteinases (MMPs) in the acute phase of chronic venous insufficiency, we examined the production of MMP-1, -2, -13 and tissue inhibitors of metalloproteinase (TIMP)-1 and -2 in lesional skin of stasis dermatitis. A total of 19 patients affected by stasis dermatitis were included in this experimental study. Polymerase chain reaction, western blot and immunohistochemical studies on tissue specimen were performed. In lesional skin of stasis dermatitis, there was elevated gene expression and immunoreactivity for MMP-1, -2 and -13 in comparison to healthy controls. In contrast, genexpression and immunoreactivity for TIMP-1 and -2 were diminished in stasis dermatitis in comparison with healthy controls. Overexpression and production of MMP-1, -2 and -13 without inhibitory effects could be the result of cytokine mediated induction. Matrix metalloproteinases (MMPs) may play an important role in the remodeling of lesional skin in stasis dermatitis.

Fractalkine induces chemotaxis and actin polymerization in human dendritic cells.

OBJECTIVE AND DESIGN: Dendritic cells (DCs) are considered as the principle initiators of immune responses by virtue of their ability to migrate into target sites, process antigens and activate naive T cells. Here, the chemotactic activity and intracellular signaling of fractalkine was analyzed and compared to well known chemotaxins. METHODS: The mRNA-expression of G protein-coupled CX3CR1 was analyzed by RT-PCR. Chemotaxis was measured in 48-well Boyden chambers and actin polymerization by flow cytometry. RESULTS: The mRNA-expression of CX3CR1 in immature and mature DCs was revealed. Fractalkine elicited actin polymerization and chemotaxis in a dose-dependent manner in DCs independent of their state of maturation. CONCLUSIONS: These results show that immature and mature DCs express mRNA for the CX3CRI and that fractalkine induces chemotaxis and migration associated actin polymerization in immature as well as in mature DCs, contrasting with the action of other chemokines such as RANTES or MIP-3beta which act only on distinct maturation states of DCs.

P2 purinergic receptors of human eosinophils: characterization and coupling to oxygen radical production.

Extracellular nucleotides elicit multiple responses in eosinophils but no information on expression of purinergic receptors in these cells is available so far. In the present study we show that human eosinophils express the following P2Y and P2X subtypes: P2Y(1), P2Y(2), P2Y(4), P2Y(6), P2Y(11), and P2X(1), P2X(4), P2X(7), whose stimulation results in intracellular Ca(2+) increase and production of large amounts of reactive oxygen intermediates. These events are stimulated or inhibited, respectively, by P2 receptor agonists or antagonists.

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.

The P2 purinergic receptors of human dendritic cells: identification and coupling to cytokine release.

We investigated the expression of purinoceptors in human dendritic cells, providing functional, pharmacological, and biochemical evidence that immature and mature cells express P2Y and P2X subtypes, coupled to increase in the intracellular Ca(2+), membrane depolarization, and secretion of inflammatory cytokines. The ATP-activated Ca(2+) change was biphasic, with a fast release from intracellular stores and a delayed influx across the plasma membrane. A prolonged exposure to ATP was toxic to dendritic cells that swelled, lost typical dendrites, became phase lucent, detached from the substrate, and eventually died. These changes were highly suggestive of expression of the cytotoxic receptor P2X(7), as confirmed by ability of dendritic cells to become permeant to membrane impermeant dyes such as Lucifer yellow or ethidium bromide. The P2X(7) receptor ligand 2',3'-(4-benzoylbenzoyl)-ATP was a better agonist then ATP for Ca(2+) increase and plasma membrane depolarization. Oxidized ATP, a covalent blocker of P2X receptors, and the selective P2X(7) antagonist KN-62 inhibited both permeabilization and Ca(2+) changes induced by ATP. The following purinoceptors were expressed by immature and mature dendritic cells: P2Y(1), P2Y(2), P2Y(5), P2Y(11) and P2X(1), P2X(4), P2X(7). Finally, stimulation of LPS-matured cells with ATP triggered release of IL-1 beta and TNF-alpha. Purinoceptors may provide a new avenue to modulation of dendritic cells function.

Autologous platelet-derived wound healing factor promotes angiogenesis via alphavbeta3-integrin expression in chronic wounds.

Healing of venous leg ulcers depends on the adhesive interaction and formation of new vascular cells. Angiogenesis on the surface of angiogenic blood vessels requires the vascular integrin alphavbeta3 also known as the vitronectin receptor. Autologous platelet-derived wound healing factor (autologous PDWHF) has been described to regulate the wound healing process by forming granulation tissue in the early healing phase. Here we analysed the influence of autologous PDWHF on the expression of the alphavbeta3 integrin in tissue specimen of venous leg ulcers in comparison with placebo treated controls by using reverse transcriptase-polymerase chain reaction and immunohistochemistry. Our investigations provide evidence that mRNA and protein expression of alphavbeta3 were significantly increased in healing venous leg ulcers after 96 h treatment (p<0.05), whereas the total amount of alphavbeta3 mRNA and protein was not altered in placebo treated patients. In healing leg ulcers the alphavbeta3 integrin was predominantly localized around capillary vessels preferentially at sites of newly formed granulation tissue. Placebo controlled patients displayed no altered expression of the alphavbeta3 integrin in biopsy specimen. These findings suggest that topical autologous platelet-derived wound healing factor influences the process of angiogenesis/revascularization via alphavbeta3 integrin-expression hereby promoting granulation tissue formation in healing leg ulcers.

Downregulation of platelet-activating factor responsiveness during maturation of human dendritic cells.

Dendritic cells (DCs) are specialized antigen-presenting cells characterized by their ability to migrate into target sites, process antigens, and activate naive T-cells. Biological activities of platelet-activating factor (PAF) and the cytokine macrophage inflammatory protein-3beta (MIP-3beta) as well as the mRNA expression of their receptors were characterized in human DCs during lipopolysaccharide (LPS)-promoted maturation. Platelet-activating factor induced calcium transients, migration-associated actin polymerization response, and chemotaxis in immature human dendritic cells differentiated in vitro from monocytes with interleukin-4 and granulocyte macrophage colony stimulating factor. In addition, RT-PCR experiments indicated mRNA expression of the PAF receptor in these immature DCs. Cell studies and mRNA analyses further revealed that immature DCs neither respond to MIP-3beta nor express its specific receptor, CCR7. Induction of cell differentiation by LPS led to the loss of the mRNA expression of the PAF receptor, accompanied by decreasing intracellular calcium release, actin polymerization, and migration after stimulation with PAF. In contrast, LPS treatment induced increasing responsiveness toward MIP-3beta and mRNA expression of CCR7. Comparable data regarding mRNA expression of PAF receptor and PAF responsiveness were also obtained with another maturation protocol using TNFalpha instead of LPS. The direct comparison between the two different protocols showed a slower decrease of PAF responsiveness induced by TNFalpha than by LPS. These results show the loss of PAF responsiveness associated with downregulation of PAF receptor mRNA expression during LPS- and TNFalpha-induced maturation in human DCs. Therefore, these findings point to a functional relevance of PAF in recruiting immature DCs, whereas MIP-3beta might regulate the migration of DCs at a later stage of maturation.

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.

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.

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.

Actin polymerization in human eosinophils, unlike human neutrophils, depends on intracellular calcium mobilization.

Eosinophils represent major effector cells in the allergic inflammation. In contrast to neutrophils, the mechanism of eosinophil activation during the inflammatory response is poorly understood. In this study, the relation between calcium fluxes, chemotaxis, and actin polymerization in eosinophils from healthy non-atopic donors was investigated. Pre-incubation of eosinophils with the intracellular calcium chelator BAPTA dose-dependently prevented an increase in the intracellular calcium concentration ([Ca2+]i), whereas the depletion of extracellular calcium in the test medium had no effect. The chemotactic response of eosinophils, which was measured by the modified boyden chamber technique upon stimulation with RANTES, C5a and PAF, was dose-dependently inhibited by the chelation of intracellular calcium as well as inactivation of the cells in Ca2+ -depleted medium. To evaluate whether other cell functions which are involved in the migratory response of eosinophils might be dependent on intracellular and extracellular calcium, actin polymerization was investigated. Flow-cytometric measurement of F-actin with NBD-phallacidin revealed that actin polymerization in human eosinophils in response to RANTES, C5a, and PAF was dose-dependently inhibited by the intracellular calcium chelator BAPTA. Since it is well known that actin polymerization in neutrophils is not affected by chelation of intracellular calcium, actin polymerization in these cells was investigated under the same conditions as for eosinophils. In contrast to eosinophils, BAPTA did not inhibit actin polymerization in neutrophils. In summary, these data demonstrate that intracellular calcium fluxes represent a prerequisite for eosinophil chemotaxis and actin polymerization in human eosinophils. Furthermore, regulation of actin polymerization in eosinophils differed from that of neutrophils on the level of intracellular calcium fluxes.

Activation of the respiratory burst in human eosinophils by chemotaxins requires intracellular calcium fluxes.

Eosinophils represent major effector cells in the allergic inflammatory response. Following activation, these cells are capable of mediating tissue damage, particularly by the release of reactive oxygen species. In this study, the role of extracellular and intracellular calcium in the induction of the respiratory burst of human eosinophils was investigated in healthy non-atopic individuals. Pre-incubation of Fura-2-loaded eosinophils with the intracellular calcium chelator 2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid prevented the increase of the [Ca++]i following stimulation by RANTES, C5a and PAF, in concentration-dependent fashion, whereas depletion of extracellular calcium in the test medium by ethyl=eneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid was ineffective. To investigate the potential role of extracellular and intracellular calcium on the production of reactive oxygen species, flow-cytometric measurement of H2O2 production by dihydrorhodamine 123 and lucigenin-dependent chemiluminescence were carried out. Chelation of both intracellular and extracellular calcium prevented production of reactive oxygen species after stimulation with C5a, PAF, or RANTES. However, production of reactive oxygen species after stimulation by phorbol myristate acetate, which bypasses post-receptor events by direct activation of protein kinase C, was prevented only after chelation of intracellular but not extracellular calcium. This suggested a Ca(++)-sensitive form of protein kinase C in the activation process of the respiratory burst. These data demonstrate that intracellular and extracellular calcium represent a prerequisite of chemotaxin-induced activation of the respiratory burst in human eosinophils. Thus, intracellular calcium seems to play a central role in the modulation of the respiratory burst in eosinophils and might therefore be an interesting target for drugs that interfere with calcium homeostasis and reduce the tissue destructive power of eosinophils.