206 research outputs found
Relationships between translocation and phosphorylation of p47phox and p67phox NADPH oxidase components
No full textThe p47 phox is required for the translocation of p67phox from the cytosol to the membranes of leukocytes taking place during NADPH oxidase activatio
Activation of NADPH oxidase of human neutrophils involves the phosphorylation and the translocation of cytosolic p67phox
No full textActivation of human neutrophil NADPH oxidase requires the interaction of cytosolic and membrane-associated components. Evidence has been accumulated that in phorbol 12-myristate 13-acetate (PMA)-stimulated neutrophils, the translocation to the plasma membrane of the cytosolic components p47phox and p67phox and the phosphorylation of p47phox are essential steps in activation of NADPH oxidase. No direct evidence has been presented to date as to whether p67phox is also phosphorylated. To address this problem we have immunoprecipitated p67phox from neutrophil cytosol and membrane fractions. The results indicate that, very soon after activation with PMA (20 s), p67phox was present in a phosphorylated form in the cytosol and in the membranes. At later times (1-3 min) the extent of p67phox phosphorylation continuously increased both in the cytosol and in the membrane fraction, while oxygen consumption reached the maximal rate within 40 s, and then remained linear. p67phox was also phosphorylated in formyl-methionyl-leucyl-phenylalanine-activated neutrophils. That the phosphorylated p67 protein we identified in immunoprecipitation experiments was p67phox was confirmed by the observation that no phosphorylated band of 67 kDa was immunoprecipitated from the cytosol and membranes of PMA-stimulated neutrophils from a p67phox-deficient chronic granulomatous disease patient. In this case, p47phox was normally phosphorylated. These data demonstrate that: (1) the phosphorylation of p67phox is correlated with activation of NADPH oxidase, and (2) continuous phosphorylation of p67phox is required in order to maintain the linearity of the respiratory burst
Induction of Th1/Th17 immune response by Mycobacterium tuberculosis: role of cytokines produced by human dendritic cells and pathogen recognition receptors involved
No full textMtb influences DC activity and T cell-mediated immune
responses. We show that the treatment of immature
monocyte-derived DC with Mtb elicited the formation of
mature DC, producing TNF-, IL-1, IL-6, and IL-23 and
instructing CD4 cells to secrete IFN- and IL-17. Mtb-induced
cytokine release by DC depended on dectin-1 receptor
engagement, whereas MR or DC-SIGN stimulation
inhibited this process. A selective dectin-1 binding by the
receptor agonist glucan was sufficient to enable DC to
generate Th1/Th17 lymphocytes, showing features comparable
with those induced by Mtb-treated DC. Interestingly,
DC-SIGN or MR engagement inhibited Th17 and increased
Th1 generation by glucan- or Mtb-treated DC.
Our results indicate that Mtb modulates the lymphocyte
response by affecting DC maturation and cytokine release.
Dectin-1 engagement by Mtb enables DC to promote
a Th1/Th17 response, whereas DC-SIGN and MR
costimulation limits dectin-1-dependent Th17 generation
and favors a Th1 response, probably by interfering with
release of cytokines
Mechanisms of NADPH oxidase activation: translocation of p40phox, rac 1 and rac 2 from the cytosol to the membranes in human neutrophils lacking p47phox or p67phox.
No full textOn neutrophil stimulation, the cytosolic components of NADPH oxidase, p67-phox, p47-phox, p40-phox, as well as the Ras-related G-proteins Rac1 and Rac2, are translocated from the cytosol to cell membranes where they associate with a flavocytochrome b, forming a functional complex responsible for the production of oxygen radicals in phagocytes. In this paper we show that (a) in neutrophils from a patient with a form of chronic granulomatous disease (CGD) in which p67-phox is absent, p47-phox and Rac2, but not p40-phox and Rac1 were translocated from the cytosol to the membrane on stimulation with formylmethionylleucylphenylalanine (fMLP) or phorbol 12-myristate 13-acetate (PMA); (b) in neutrophils from a patient with a form of CGD in which p47-phox is absent, p67-phox, p40-phox and Rac1 failed to associate with the membrane on stimulation with fMLP or PMA, whereas Rac2 was translocated as in normal neutrophils. We also show that in neutrophils from a patient lacking p67-phox, the amount of cytosolic p40-phox was decreased by about 40%. These findings indicate that, on neutrophil stimulation, p67-phox mediates the translocation of p40-phox and Rac1 from the cytosol to cell membranes and that Rac2 associates with the membranes independently of p47-phox and p67-phox
A possible role for MAP kinases and for a 75 kDa protein in the activation of NADPH oxidase
No full textPhosphorylation by MAP kinases could have a role in the mechanisms of NADPH oxidase assembl
Effects of Mycobacterium tuberculosis on DC apoptosis, maturation and oxygen free radicals production
No full textM. tuberculosis affects on oxygen free radicals production by dendritic cell
Prostate carcinoma cells LNCaP differentially regulate cytokine release in dectin-1 and Toll-like receptor-stimulated human monocyte-derived dendritic cells
No full textGlucan and LNCaP cooperate in induction of cytokine synthesis by DC. LNCaP enhance IL-1β, IL-23, IL-6 and TNF-α secretion by decreasing glucan-dependent NADPH oxidase activity, whereas glucan increases IL-12 production through NADPH oxidase-unrelated mechanisms. This cooperation is essential to elicit a substantial NK cells and CD4+ lymphocytes activity, pointing out a potential relevance of glucan in prostate cancer therapy
Mechanisms of NADPH oxidase assembly and regulation in human neutrophils
No full textThe p47 phox is requested for the assembly of NADPH oxidase, whereas p67phox is not necessary. Both proteins are phosphorylate
Tyrosine phosphorylation and activation of NADPH oxidase in human neutrophils: a possible role for MAP kinases and for a 75 kDa protein
No full textChallenge of neutrophils with concanavalin A (ConA), formyl-methionyl-leucyl-phenylalanine (FMLP), and phorbol 12-myristate 13-acetate (PMA) induced the tyrosine phosphorylation of several proteins. Among these proteins we have identified two mitogen-activated protein kinase (MAPK) isoforms of 43 kDa (p43 MAPK) and 45 kDa (p45 MAPK) molecular mass. Moreover here we show that: (1) FMLP induced the tyrosine phosphorylation of the p43 MAPK, and ConA that of p45 MAPK, while PMA induced the tyrosine phosphorylation of both p43 and p45 MAPK; all these agonists induced the tyrosine phosphorylation of a 75 kDa protein (p75). (2) With FMLP or ConA as agonists, tyrosine phosphorylations of MAPK and p75 can be involved in the process of NADPH oxidase activation. On the contrary, PMA can activate the respiratory burst independently of these phosphorylations. (3) In Ca(2+)-depleted neutrophils, where phospholipid hydrolysis did not take place, ConA or FMLP did not activate the respiratory burst, but while ConA induced the tyrosine phosphorylation of p45 MAPK and p75, FMLP was not able to phosphorylate p43 MAPK and p75. (4) As previously observed in our laboratory, a double stimulation of Ca(2+)-depleted neutrophils with ConA plus FMLP induced a respiratory burst in the absence of activation of second messengers derived from phospholipase C, D and A2 activity. This respiratory burst was accompanied by tyrosine phosphorylation of both p43 and p45 MAPKs. These results indicate that when FMLP is the agonist, both the tyrosine phosphorylation of p43 MAPK and p75, and the activation of NADPH oxidase, are coupled to Ca(2+)-dependent mechanisms. On the contrary, ConA can induce the tyrosine phosphorylation of p45 MAPK and p75 independently of calcium, but an unknown Ca(2+)-dependent mechanism is necessary for the activation of NADPH oxidase by this agonist. This mechanism could be substituted by the induction of tyrosine phosphorylation of both p43 MAPK and p45 MAPK when Ca(2+)-depleted neutrophils are stimulated with ConA plus FMLP
Studies on the nature and activation of O2- -forming NADPH oxidase of leukocytes. II. Relationships between phosphorylation of a component of the enzyme and oxidase activity
No full textThe activation of O2- -formation by neutrophil NADPH oxidase is associated with phosphorylation of several membrane and cytosolic proteins. In the membranes a phosphoprotein of 32 kDa belonging to the NADPH oxidase-cytochrome b-245 system (P. Bellavite et al., Free Rad. Res. Commun., 1, 11 (1985] showed the highest relative increase of 32Pi incorporation. Concomitant with the phosphorylation, a shift of the apparent molecular mass of the protein from 31 to 32 kDa occurred. The time-course, the sensitivity to trifluoperazine and the dose-dependence of phosphorylation were similar to those of O2- forming activity, except that the latter showed a longer lag-time than the former. The increase of the 32 kDa phosphoprotein was also comparable to the kinetics of cytochrome b-245 reduction by anaerobically activated neutrophils. The phosphorylation and the NADPH oxidase were triggered by various stimulants including phorbol myristate acetate, opsonized zymosan, arachidonic acid and sodium fluoride. With arachido
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