Enzymatic basis of macrophage activation. Kinetic analysis of superoxide production in lysates of resident and activated mouse peritoneal macrophages and granulocytes

To compare the kinetics of the O-2-generating enzyme in nonactivated and activated macrophages and granulocytes from the mouse peritoneal cavity, we sought conditions in which the activity of this enzyme in cell lysates was comparable to that in intact cells. Pretreatment of macrophages with 10 mM diethyldithiocarbamate inhibited endogenous superoxide dismutase by 70% and enhanced O-2 secretion up to 15-fold, so that it was comparable to H2O2 secretion. O-2 secretion was terminated by detergent lysis and reconstituted by addition of NAD(P)H to the lysates. Optimal detection of O-2 production in lysates depended on prior stimulation of the respiratory burst, lysis with 0.05% deoxycholate rather than any of 4 other detergents or sonication, acetylation of the cytochrome c used as an indicator, and addition of NADPH rather than NADH. Kinetic analysis using NADPH-reconstituted deoxycholate lysates, together with spectra of oxidized and reduced cells, failed to reveal either marked differences in the Vmax of the O-2-generating enzyme or correlations between O-2 secretion and cytochrome b559 content among 5 macrophage populations whose H2O2 secretion ranged from 0 to 365 nmol/90 min/mg of protein. In contrast, the Km of the oxidase for NADPH varied markedly and inversely with the capacity of the intact cells to secrete O-2 or H2O2: J774G8 histiocytoma cells, 1.43 mM; resident macrophages, 0.41 mM; proteose peptone-elicited macrophages, 0.20 mM; casein-activated macrophages, 0.05 mM; NaIO4-activated macrophages, 0.05 mM; and granulocytes, 0.04 mM. These results suggest that macrophage activation, a process that enhances oxygen-dependent antitumor and antimicrobial functions, may equip the cell to secrete increased amounts of reactive oxygen intermediates largely by increasing the affinity of the oxidase for NADPH.     

Fungal gliotoxin targets the onset of superoxide-generating NADPH oxidase of human neutrophils

Gliotoxin from Aspergillus, bearing a S&bond;S bond in its structure, prevented the onset of O(-)(2) generation by the human neutrophil NADPH oxidase in response to phorbol myristate acetate (PMA). Gliotoxin affected the activation process harder than the activated oxidase, as shown by its stronger inhibition when added to neutrophils prior to, than post-PMA at maximum enzyme turnover. Decreased O(-)(2) generation persisted even if cells treated with gliotoxin were subsequently washed, with half-inhibition concentrations (IC(50)) of 5.3, and 3.5 microM for treatments of 15 and 30 min, respectively. In addition, gliotoxin made neutrophils reduce cytochrome c regardless of absence of PMA, through its reaction with intracellular reductants in an oxygen-dependent process, named redox cycling. Thus, we next tested whether preincubation of neutrophils with gliotoxin under hypoxic conditions would relieve the inhibition of NADPH oxidase. Instead, this prevention of redox cycling significantly favored damage to the NADPH oxidase with an IC(50) of 0.009 microM. Moreover, conversion of gliotoxin to its dithiol derivative by addition of reduced dithiothreitol during incubation protected cells from losing oxidase activity. These findings support that the disulfide form of gliotoxin targets NADPH oxidase activation.     

Monocarboxylate transporter-1 is required for cell death in mouse chondrocytic ATDC5 cells exposed to interleukin-1beta via late phase activation of nuclear factor kappaB and expression of phagocyte-type NADPH oxidase

Interleukin-1β (IL-1β) induces cell death in chondrocytes in a nitric oxide (NO)- and reactive oxygen species (ROS)-dependent manner. In this study, increased production of lactate was observed in IL-1β-treated mouse chondrocytic ATDC5 cells prior to the onset of their death. IL-1β-induced cell death in ATDC5 cells was suppressed by introducing an siRNA for monocarboxylate transporter-1 (MCT-1), a lactate transporter distributed in plasma and mitochondrial inner membranes. Mct-1 knockdown also prevented IL-1β-induced expression of phagocyte-type NADPH oxidase (NOX-2), an enzyme specialized for production of ROS, whereas it did not have an effect on inducible NO synthase. Suppression of IL-1β-induced cell death by Nox-2 siRNA indicated that NOX-2 is involved in cell death. Phosphorylation and degradation of inhibitor of κBα (IκBα) from 5 to 20 min after the addition of IL-1β was not affected by Mct-1 siRNA. In addition, IκBα was slightly decreased after 12 h of incubation with IL-1β, and the decrease was prominent after 36 h, whereas activation of p65/RelA was observed from 12 to 48 h after exposure to IL-1β. These changes were not seen in Mct-1-silenced cells. Forced expression of IκBα super repressor as well as treatment with the IκB kinase inhibitor BAY 11-7082 suppressed NOX-2 expression. Furthermore, Mct-1 siRNA lowered the level of ROS generated after 15-h exposure to IL-1β, whereas a ROS scavenger, N-acetylcysteine, suppressed both late phase degradation of IκBα and Nox-2 expression. These results suggest that MCT-1 contributes to NOX-2 expression via late phase activation of NF-κB in a ROS-dependent manner in ATDC5 cells exposed to IL-1β.     

An Fc-receptor activity of plasma membranes from guinea-pig peritoneal polymorphonuclear leucocytes.

Plasma membranes prepared from guinea-pig peritoneal polymorphonuclear leucocytes showed an immune-complex-binding activity that corresponded well with the activity in intact cells. The characteristics of this activity were reversible binding, dependence on the Fc portion of antigen-complexed IgG (immunoglobulin G), competition with aggregated IgG and independence from energy metabolism. These results support the conclusion that the binding activity found in the isolated plasma membranes is an Fc-receptor activity of guinea-pig peritoneal polymorphonuclear leucocytes. The activity showed Kd = 6.7 x 10(-8) M-IgG and maximum binding of 17 pmol of IgG/mg of membrane protein when measured with an immune complex of alpha-amylase and homologous guinea-pig anti-(alpha-amylase) IgG. Inhibition of the Fc-receptor activity by a series of various salts indicated the contribution of the hydrophobic interaction to the binding. Inhibitory effects of salts or metal-chelating reagents on the Fc-receptor activity were also observed on superoxide generation by these cells induced by the immune complex, suggesting a role of the Fc receptor as the immune-complex-binding site responsible for the initiation of superoxide generation.     

Expression of a p67(phox) homolog in Caco-2 cells giving O(2)(-)-reconstituting ability to cytochrome b(558) together with recombinant p47(phox)

Human normal and transformed (Caco-2) colon tissues as well as guinea pig gastric mucosal cells express Nox1, which is a homolog of the phagocyte NADPH oxidase subunit, gp91(phox) of membrane-bound cytochrome b(558). It was reported that Nox1-transfection to NIH 3T3 cells could provide O(2)(-)-generating ability, independently of regulatory cytosolic factors (Rac2, p67(phox), and p47(phox)) that are obligatory in the phagocyte oxidase system. Here, we detected and sequenced a p67(phox) homolog in Caco-2 almost identical to the neutrophil sequence, except for three nucleotide substitutions, two of which changed lysines 181 and 328 to arginines. Investigation of its ability to support O(2)(-)-generation in cell-free reconstitution experiments combining with neutrophil cytochrome b(558) showed O(2)(-)-generation, provided that recombinant p47(phox) was added. This result demonstrates that the intrinsic p67(phox) homolog of Caco-2 was able to function as a phagocyte p67(phox) for cytochrome b(558). The requirement of p47(phox) addition suggested that this component was absent in Caco-2 cells. Caco-2 membranes, used as a source of Nox1 in place of cytochrome b(558), did not show significant O(2)(-)-generation, which was mainly explained by their very little Nox1 expression.     

Factor Structure of the Japanese Version of the Edinburgh Postnatal Depression Scale in the Postpartum Period

Background

The Edinburgh Postnatal Depression Scale (EPDS) is a widely used screening tool for postpartum depression (PPD). Although the reliability and validity of EPDS in Japanese has been confirmed and the prevalence of PPD is found to be about the same as Western countries, the factor structure of the Japanese version of EPDS has not been elucidated yet.

Methods

690 Japanese mothers completed all items of the EPDS at 1 month postpartum. We divided them randomly into two sample sets. The first sample set (n = 345) was used for exploratory factor analysis, and the second sample set was used (n = 345) for confirmatory factor analysis.

Results

The result of exploratory factor analysis indicated a three-factor model consisting of anxiety, depression and anhedonia. The results of confirmatory factor analysis suggested that the anxiety and anhedonia factors existed for EPDS in a sample of Japanese women at 1 month postpartum. The depression factor varies by the models of acceptable fit.

Conclusions

We examined EPDS scores. As a result, “anxiety” and “anhedonia” exist for EPDS among postpartum women in Japan as already reported in Western countries. Cross-cultural research is needed for future research.     

Role of nicotinamide adenine dinucleotide phosphate oxidase 1 in oxidative burst response to Toll-like receptor 5 signaling in large intestinal epithelial cells

The NADPH oxidase 1 (Nox1) is a gp91(phox) homologue preferentially expressed in the colon. We have established primary cultures of guinea pig large intestinal epithelial cells giving 90% purity of surface mucous cells. These cells spontaneously released superoxide anion (O(2)(-)) of 160 nmol/mg protein/h and expressed the Nox1, p22(phox), p67(phox), and Rac1 mRNAs, but not the gp91(phox), Nox4, p47(phox), p40(phox), and Rac2 mRNAs. They also expressed novel homologues of p47(phox) and p67(phox) (p41(nox) and p51(nox), respectively). Human colon cancer cell lines (T84 and Caco2 cells) expressed the Nox1, p22(phox), p51(nox), and Rac1 mRNAs, but not the other NADPH component mRNAs, and secreted only small amounts of O(2)(-) (<2 nmol/mg protein/h). Cotransfection of p41(nox) and p51(nox) cDNAs in T84 cells enhanced PMA-stimulated O(2)(-) release 5-fold. Treatment of the transfected T84 cells with recombinant flagellin (rFliC) from Salmonella enteritidis further augmented the O(2)(-) release in association with the induction of Nox1 protein. The enhanced O(2)(-) production by cotransfection of p41(nox) and p51(nox) vectors further augmented the rFliC-stimulated IL-8 release from T84 cells. T84 cells expressed the Toll-like receptor 5, and rFliC rapidly phosphorylated TGF-beta-activated kinase 1 and TGF-beta-activated kinase 1-binding protein 1. A potent inhibitor for NF-kappaB (pyrrolidine dithiocarbamate) significantly blocked the rFliC-primed increase in O(2)(-) production and induction of Nox1 protein. These results suggest that p41(nox) and p51(nox) are involved in the Nox1 activation in surface mucous cells of the colon, and besides that, epithelial cells discern pathogenicities among bacteria to appropriately operate Nox1 for the host defense.