Avian cytokines--an overview.

In recent years the knowledge of avian cytokines has advanced and new data are continuously added. Nevertheless, some discontinuities persist and the correlations between molecular and functional levels are not completely clear. Most of the studies are focused on chicken, and comparative aspects with other avian groups are limited. The existence of T1 and T3 avian cytokines was assessed long ago and the recent relevant demonstration of the existence of T2 cytokines in birds is a further step in depicting a more complete view on avian immunology. The progressive knowledge of avian cytokines can hopefully help in developing new strategies in prophylaxis and therapy of avian diseases, not always completely controlled due to the emergence of more pathogenic strains.

NKR-P1A stimulation of arachidonate-generating enzymes in rat NK cells is associated with granule release and cytotoxic activity.

NKR-P1A protein has been implicated in the triggering of NK-mediated natural killing contributing to target cell recognition by NK cells. The aim of the present work was to assess whether NKR-P1A receptor triggering also induced arachidonic acid (AA) generation and to determine the possible role of this event on granule release and cytotoxicity. We demonstrated that activation of fresh peripheral blood rat NK cells by cross-linking with the anti-NKR-P1A 3.2.3 mAb induced calcium-dependent AA release, which is due to the activation of cytosolic phospholipase A2 (cPLA2), secretory phospholipase A2 (sPLA2), and diacylglycerol/monoacylglycerol lipase. We also documented the presence of a type II sPLA2 activity in the supernatant fluids from NKR-P1A-activated rat NK cells, suggesting that AA and lysophospholipids could be mobilized from the outside of the cell. The involvement of AA-generating enzymes in NKR-P1A-induced cytotoxic functions was also investigated. Treatment of effector cells with arachidonyl trifluoromethylketone, a cPLA2 inhibitor; p-bromophenacylbromide, a sPLA2 inhibitor; or RHC80267, a diacylglycerol lipase inhibitor, led to a partial inhibition of the redirected lysis against P815 target cells as well the granule content release induced by NKR-P1A cross-linking. A complete abolishment of these events was observed when the cells were simultaneously incubated with all three inhibitors. Taken together, our results support a crucial role for the arachidonate-generating enzymes in the induction of lytic activity of NK cells directly or by leading to generation of additional mediators that can play a role in the context of NK cell activation and cytotoxic functions.

Phospholipase A2 activity and calpactin I levels in rat lymphokine-activated killer cells: correlation with the cytotoxic activity.

In the present paper we have shown evidence for a significant increase of type II sPLA2 activity in A-LAK cells. The A-LAK-mediated cytotoxicity against YAC-1 target cells was strongly inhibited by two inhibitors of sPLA2, p-BPB and mepacrine, suggesting the involvement of this enzyme in the lytic mechanism of A-LAK. On the other hand, stimuli such as A23187 ionophore and TPA, which were able to induce in control cells an increased AA release, failed to cause this effect in IL-2-treated cells, suggesting that PLA2 was not active in these cells. Thus, we analyzed the levels of calpactin I, which is considered to be involved in the down-regulation of PLA2 activity. HrIL-2 treatment led to an increased expression of calpactin I at both the RNA and the protein level. A substantial portion of calpactin I was associated with the external surface of A-LAK and was able to exert a strong inhibitory effect on a purified porcine pancreatic PLA2 activity in vitro. Our results suggest that the role of calpactin I could be relevant to regulate PLA2 activity, and to protect the effector cells against a possible toxic effect which this enzyme could exert if present at high levels.

Involvement of phospholipase A2 activation and arachidonic acid metabolism in the cytotoxic functions of rat NK cells.

Arachidonic acid (AA) release via phospholipase A2 (PLA2) activation and generation of eicosanoids have been implicated as playing signaling roles in a variety of cell types. Here we show evidence that interaction of fresh NK cells with membranes from sensitive or antibody (Ab)-coated targets generates eicosanoids through both cyclooxygenase (CO) and lipoxygenase (LO) pathways. Eicosanoid generation is attributable to PLA2 activation since pretreatment with PLA2 irreversible inhibitors, such as mepacrine or para-bromophenacylbromide (pBPB), completely blocks AA metabolite generation. The involvement of PLA2 or AA metabolites in the cytotoxic functions of rat NK cells has also been investigated. Treatment of effector cells with mepacrine or pBPB resulted in complete, irreversible, dose-dependent inhibition of both NK and ADCC activities, which were completely reversed by the addition of exogenous PLA2 or its hydrolysis products, AA and lysophosphatidylcholine (lysoPC). Among the metabolites of AA released by NK cells, the 5-LO product leukotriene B4 (LTB4) seems to play an important role in cytolytic activities of NK cells. Indeed, the LO inhibitor, nordihydroguaiaretic acid (NDGA), totally abrogated both NK and ADCC activities, which were restored by the addition of exogenous LTB4. However, the failure of LTB4 to reverse mepacrine or pBPB-induced inhibition of NK and ADCC suggests that its effects could be mediated by PLA2. The results are consistent with a crucial role for the target-stimulated AA release as a fundamental step in the signal transduction pathway in NK cell. Moreover, LTB4 generation seems to be responsible for further PLA2 activation in a second step leading to the amplification of response.

Preferential involvement of a phospholipase A2-dependent pathway in CD69-mediated platelet activation.

CD69 is a signal transducing disulfide-linked homodimer functionally expressed on platelets, CD3bright thymocytes, and activated lymphocytes. In an attempt to investigate early molecular events in CD69-mediated cell activation we studied the relative contribution of phospholipase A2 (PLA2) and phosphatidylinositol-specific phospholipase C-dependent pathways during platelet activation induced by CD69 stimulation. Thromboxane A2 (TXA2) synthetase inhibitor and TXA2R inhibitor R68070 were able to inhibit platelet aggregation induced by CD69 stimulation, indicating that TXA2 was the main mediator of the response. CD69-induced arachidonic acid release and TXA2 production were essentially PLA2 dependent because they could be blocked by the PLA2 inhibitor quinacrine. Inositol 1,3,4-trisphosphate generation was clearly detectable after CD69 cross-linking, but it was completely abrogated by quinacrine and R68070 and therefore secondary to TXA2 release and TXA2R engagement. Finally, direct measurement of enzymatic activity in vitro using radiolabeled phospholipid vesicles showed that CD69 cross-linking resulted in PLA2-dependent arachidonic acid and lysophosphatidylcholine generation from phosphatidylcholine, which was sensitive to quinacrine but not to R68070. By contrast, CD69-induced 1,2-diacylglycerol release from phosphatidylinositol 4,5-bisphosphate was blocked by both inhibitors. These results indicate a preferential involvement of PLA2 in CD69-dependent signal transduction in platelets and provide evidence for the unique role of PLA2-mediated activation pathways in transmembrane receptor signaling.

Serum and synovial fluid osteocalcin in rheumatic diseases.

The mechanisms involved in juxta-articular bone destruction are poorly understood. Osteocalcin or gamma-carboxyglutamic acid (GLA) protein is a small non-collagenous bone protein. It is a sensitive marker of osteoblastic bone formation. Its seric variations in the serum in such rheumatisms as rheumatoid arthritis remain unclear. Further information on local osteoblastic activity may be obtained by assaying the level of osteocalcin in the synovium. Its serum level can be evaluated by radioimmunoassay. The same method can be used in the synovial fluid. Paired serum and synovial fluid samples have been assayed from 63 patients, 33 patients with inflammatory arthritis (rheumatoid arthritis, psoriasis, chondrocalcinosis, pyogenic arthritis) and 30 patients with mechanical joint effusion (osteoarthritis, meniscal lesions). Serum levels of osteocalcin were the same in the inflammatory group (m: 8.69 +/- 0.68 ng/ml) and in the mechanical group (m: 10.2 +/- 0.67 ng/ml). In the synovial fluid, the levels of osteocalcin were significantly lower in the inflammatory group (m: 3.27 +/- 0.40 ng/ml) than in the mechanical group (m: 6.91 +/- 0.47 ng/ml). The same results were obtained with the ratio of synovial fluid osteocalcin on serum osteocalcin. There was a significant correlation between serum and synovial fluid osteocalcin and an inverse correlation between synovial fluid osteocalcin and the number of synovial fluid cells. The present study suggests that periarticular osteoblastic depression, among patients with inflammatory arthritis, is likely.

Changes of lipo-melanosome membrane leakage versus pH, charge and composition.

Liposome models of melanosomes (lipo-melanosomes) were used to investigate how phospholipid composition, charge and medium pH may affect the lipo-melanosome membrane permeability to active oxygen species or melanin synthesis intermediaries. Active oxygen accumulated only at pH 6.4 and was polarographically monitored using superoxide dismutase and/or catalase. Cholesterol appears to increase the O2- accumulation at pH 6.4 while incorporation of positive phospholipids within lipo-melanosomes results in the loss of latency with respect to tyrosinase substrate and intermediates of melanin synthesis.

5,6-Dihydroxyindole oxidation by mammalian, mushroom and amphibian tyrosinase preparations.

The oxidation of 5,6-dihydroxyindole by tyrosinases from mushroom, Harding-Passey melanoma, bovine eye and Bufo bufo embryo has been investigated. The apparent Km values for this substrate were measured and found to be of the same order of magnitude as those for L-tyrosine and L-3,4-dihydroxyphenylalanine, as reported in the literature (5 x 10(-4) M). The 5,6-dihydroxyindole oxidases of mushroom and T4 melanoma isozyme are sensitive to phenylthiourea, while, on the other hand, those from crude preparations of bovine and B. bufo tyrosinases are not sensitive to the inhibitor in an evident manner. The action of some indole derivatives on the 5,6-dihydroxyindole oxidase of mushroom has also been investigated.

Possible genotoxicity of melanin synthesis intermediates: tyrosinase reaction products interact with DNA in vitro.

The actual cellular target of the cytotoxic intermediates of melanin synthesis is not yet known. In the present paper it is shown that eukaryotic DNA binds in vitro to soluble reaction products of tyrosinase (EC 1.14.18.1) and is physically modified, as ascertained by the following criteria: (a) buoyant density in cesium chloride density gradients; (b) polyacrylamide gel electrophoresis; (c) deoxyribonuclease (EC 3.1.4.5) test; (d) electron microscopy. The results reported here support the view that DNA itself may be a target for the cytotoxic intermediates of melanin synthesis.

Tyrosinase-like activity in normal human substantia nigra.

A tyrosinase-like activity was found in human substantia nigra by polyacrylamide gel electrophoresis of fractions prepared from homogenates of the substantia nigra. The enzyme activity was detected by staining the gels with L-3,4-dihydroxyphenylalanine, dopamine and 5,6-dihydroxyindole as substrates for tyrosinase (EC 1.14.18.1). A case of parkinsonism does not show the L-3,4-dihydroxyphenylalanine and dopamine oxidase activities.

Harding-passey mouse-melanoma tyrosinase inactivation by reaction products and activation by L-epinephrine.

1. Harding-Passey mouse-melanoma tyrosinase (EC 1.14.18.1) is inhibited during L-3,4-dihydroxyphenylalanine oxidation by reaction products. L-3,4-dihydroxyphenyl 3-[14C]alanine oxidation products bind to the enzyme, as demonstrated by gel electrophoresis and radioactivity measurements. 2. The enzyme interacts with indoles and oxidizes dopamine and norepinephrine. 3. L-epinephrine activates tyrosinase at non hormonal concentrations and bovine serum albumin protects the enzyme from auto-inhibition. 4. The inhibition of the Harding-Passey mouse-melanoma tyrosinase, during substrate oxidation, is very similar to that of mushroom enzyme.

Developmental aspects of hexose metabolism in Bufo bufo.

Due to the close correlation between glucose mobilization and utilization within animal tissues, in this paper, the stages of appearance of phosphorylase, glucose-6-phosphatase and hexokinase as well as the levels of some intermediates of glucose metabolism have been investigated during Bufo bufo development. Phosphorylase first appears at stage 13 and is dominant in the neural part of the embryo, but, after this stage, increases relatively more in the nonneural one. Hexokinase appears at stage 17 and glucose-6-phosphatase soon after. Phosphorylase appearance at stage 13 is correlated with an increase of lactate content in the embryo; this may indicate a metabolization of hexoses. On this basis, the subsequent appearance of hexokinase and glucose-6-phosphatase activities also seems coherent with hexose mobilization and utilization within embryo. No direct causative factor for the changes observed was evident.

A study on melanogenesis and catecholamine biosynthesis during Bufo bufo development.

Tyrosinase and L-DOPA decarboxylase activities have been investigated during Bufo bufo development since catecholamines and melanin are formed from common substrates in homologous cells. Catecholamines first appear at stage 13 (neural plate), but tyrosinase, at a very low level, and L-DOPA decarboxylase are present throughout all of prior development. Hence, L-DOPA decarboxylase activity is not likely to be correlated with the control of catecholamine synthesis, although at stage 17 it is mainly localized in the nonneural part of the embryo. The distribution of young melanosomes and L-DOPA decarboxylase suggest a separation between melanogenesis and catecholamine synthesis.

Some properties of Bufo bufo tyrosinase during development.

Tyrosinase expression during Bufo bufo development has been investigated. Until stage 19, only 1 electrophoretic band is detectable, but at a later stage (25) 3 bands appear. The Km for L-3,4-dihydroxyphenylalanine (L-dopa) was also determined.

Inhibition of tyrosinase by indole compounds and reaction products. Protection by albumin.

Tyrosinase activity decreases as the reaction proceeds and is inhibited by L-3,4-dihydroxyphenylalanine oxidation products. Indole and tryptophan inhibit tyrosinase reaction and bovine albumin protects against end-product(s) inhibition or inactivation. Since the same tyrosinase reaction products are indole compounds and some authors reported the binding of indole derivatives with albumin, it is here suggested that indole intermediates of melanin synthesis inhibit or inactivate tyrosinase.

EFFECTS OF MELANOPROTEINS AND THEIR PRECURSORS ON CELL PROLIFERATION THE PROBLEM OF SELECTIVITY.

The actions of melanin and its precursors on mitotic frequencies, cell division and 3 H-thymidine incorporation in protokaryotic and eukaryotic cells are studied. It was also suggested that the binding of melanin precursors with proteins in the melanosomes is a way of scavenging a cytotoxia activity.