Th17 development and autoimmune arthritis in the absence of reactive oxygen species.

Dendritic cells (DC) express a functional NADPH oxidase and produce reactive oxygen species (ROS) upon interaction with microbes and T cells. Exposure to ROS leads to DC activation and maturation, as evidenced by phenotypic and functional changes. We have evaluated how endogenous ROS production affects the cytokine secretion pattern and T cell-activating capacity of bone marrow-derived murine DC. DC treated with ROS scavengers, as well as DC from mice that lack a functional NADPH oxidase (and thereby inherently deficient in ROS production) produced significantly increased levels of IL-1beta, IL-6, TNF-alpha and TGF-beta in response to microbial activation. DC deficient in ROS production induced high levels of IFN-gamma and IL-17 in responding T cells after Ag-specific or superantigen-induced activation. Finally, we show that ROS deficiency affected the induction of a T cell-dependent inflammatory condition, collagen-induced arthritis (CIA). C57BL/6 mice that lack a functional NADPH oxidase developed a severe and erosive CD4-dependent CIA, whereas the majority of the congenic wild-type animals remained healthy. These data suggest that ROS act as immunomodulators in DC-driven T cell activation and perhaps also in T cell-dependent immunopathology.

Diagnostic paradigm for evaluation of male patients with chronic granulomatous disease, based on the dihydrorhodamine 123 assay.

BACKGROUND: Chronic granulomatous disease (CGD) is a phagocyte disorder caused by mutations in nicotinamide dinucleotide phosphate (NADPH) oxidase subunits. The dihydrorhodamine 123 (DHR) assay is an effective test for CGD that for most patients also might help to differentiate between the 2 most common forms, X-linked (X) gp91(phox) defect CGD and autosomal recessive (AR) p47(phox) defect CGD. However, some male patients with X-CGD have DHR patterns that overlap the AR-CGD pattern. OBJECTIVE: The purpose of this investigation was to develop a diagnostic paradigm to delineate male patients with X-CGD expressing a DHR pattern suggestive of p47(phox) deficiency. METHODS: The DHR assay measured change in fluorescence of DHR-loaded granulocytes after phorbol myristate acetate (PMA) stimulation. Western blot analysis measured the presence of NADPH oxidase subunits gp91(phox), p47(phox), p67(phox), and p22(phox). CYBB exonic sequencing was performed on PCR-amplified genomic DNA through use of intronic flanking primers. Ferricytochrome-c assay evaluated specific superoxide production by PMA-stimulated granulocytes. RESULTS: Although 83% of patients with X-CGD have virtually no neutrophil DHR activity, we found that 17% of patients, proven to have X-CGD by other criteria, have modest DHR activity that is most consistent with p47(phox) deficiency. We describe a diagnostic paradigm to deal with such patients, and we present 2 cases, along with results of additional studies, including carrier evaluation, protein assessment, and mutation analysis, that are useful in establishing the genotype under these circumstances. DHR assays from the 2 patients described showed a fluorescence shift most characteristic of p47(phox)-AR-CGD; however, each of the patients' mothers showed mosaicism with a bimodal DHR pattern. Patient 1 had some gp91(phox) protein with a Y41D mutation and modest superoxide activity. Patient 2 had a normal level of gp91(phox) protein with a C537R mutation without detectable superoxide activity, as determined by ferricytochrome-c assay, despite the modest DHR activity. CONCLUSIONS: Evaluation of male patients with CGD with modest DHR activity should initially include evaluation of potential female carriers for mosaicism with the use of the DHR assay. In circumstances in which this is uninformative, patients should be referred to centers capable of additional testing, including Western blot analysis and CYBB mutation analysis, to clarify the disease genotype.

Specific inhibition of plasma kallikrein modulates chronic granulomatous intestinal and systemic inflammation in genetically susceptible rats.

The kallikrein-kinin (K-K) (contact) system is activated during acute and chronic relapsing phases of enterocolitis induced in genetically susceptible Lewis rats by intramural injection of peptidoglycan-polysaccharide (PG-APS). Using the selective plasma kallikrein inhibitor P8720, we investigate whether activation of the K-K system plays a primary role in chronic granulomatous intestinal and systemic inflammation in this model. Group I (negative control) received human serum albumin intramurally. Group II (treatment) received PG-APS intramurally and P8720 orally. Group III (positive control) received PG-APS intramurally and albumin orally. P8720 attenuated the consumption of the contact proteins, high molecular weight kininogen (P<0.03), and factor XI (P<0.04) in group II vs. group III. P8720 decreased chronic intestinal inflammation measured by blinded gross (P<0.01) and histologic (P<0.0005) scores as well as systemic complications (arthritis, splenomegaly, hepatomegaly, leukocytosis, and acute-phase reaction) (P<0.01) in group II as compared with group III. We conclude that relapsing chronic enterocolitis and systemic complications are in part due to plasma K-K system activation, and that inhibition of this pathway is a potential therapeutic approach to human inflammatory bowel disease and associated extraintestinal manifestations.

Activation of O2(-)-generating oxidase in an heterologous cell-free system derived from Epstein-Barr-virus-transformed human B lymphocytes and bovine neutrophils. Application to the study of defects in cytosolic factors in chronic granulomatous disease.

Epstein-Barr-virus-transformed human B lymphocytes (EBV B lymphocytes) stimulated by 4 beta-phorbol 12-myristate 13-acetate exhibit an NADPH-dependent oxidase activity capable of generating the superoxide anion O2-, similar to, but less efficient than that of activated neutrophils. A cell-free system of oxidase activation consisting of a membrane fraction and cytosol from EBV B lymphocyte homogenate supplemented with guanosine 5'-[gamma-thio]triphosphate (GTP[S]), arachidonic acid and Mg2+ was found to be competent in the production of O2-, assessed by the superoxide-dismutase-sensitive reduction of cytochrome c in the presence of NADPH. However, cytochrome c reduction was slow and largely insensitive both to superoxide dismutase, and to iodonium biphenyl, a powerful inhibitor of the oxidase activity in neutrophils. A markedly faster reduction of cytochrome c in the presence of NADPH was obtained with a heterologous system consisting of cytosol from EBV B lymphocytes and bovine neutrophil membranes, GTP[S], arachidonic acid and Mg2+; in this system, reduction of cytochrome c was totally inhibited by superoxide dismutase and iodonium biphenyl. These results show that EBV B lymphocytes contain a substantial amount of cytosolic factors of oxidase activation, and that the limiting factors for O2- production in B lymphocytes are the membrane components of the oxidase complex. The heterologous system of EBV B lymphocyte cytosol and bovine neutrophil membranes provided a rapid and convenient method to diagnose cytosolic defects in autosomal forms of chronic granulomatous disease. In addition, it might be a useful tool to explore the mechanism of action of the cytosolic factors in oxidase activation.

The phagocyte 47-kilodalton cytosolic oxidase protein is an early reactant in activation of the respiratory burst.

Activation of the phagocyte NADPH oxidase requires participation of membrane-bound cytochrome b558 and cytosol proteins of 47 kDa (p47) and 67 kDa (p67). We examined the sequence of participation of p47 and p67 in activation of the oxidase using an arachidonate-activated cell-free superoxidase (O2-) generating assay requiring phagocyte membrane and cytosol. Neutrophil cytosol from patients with certain forms of autosomal recessive chronic granulomatous disease (CGD) lack either p47 or p67. Initial incubation of membrane and arachidonate with CGD cytosol deficient in either p47 or p67 fails to generate superoxide in the cell-free assay until addition of complementary cytosol. CGD cytosol was incubated with arachidonate and membrane for 5-15 min and the lag time of O2- generation was measured after addition of complementary CGD cytosol. The lag time is shortened when p47, but not p67, is present in the initial incubation. We have previously shown that the peptide, RGVHFIF, corresponding to a cytoplasmic carboxyl-terminal domain of the large subunit of cytochrome b558, inhibits activation of NADPH oxidase in the cell-free assay, but does not affect the enzyme activity of fully assembled oxidase. Experiments with sequential addition of complementary CGD cytosols were performed as above, except that RGVHFIF was added after the initial incubation. The peptide failed to inhibit when added after initial incubation if p47 was present during that incubation. In contrast, the peptide markedly inhibited oxidase activity if p47 was absent during the initial incubation. These results suggest that p47, but not p67, is a participant with membrane and/or other cytosol components in early arachidonate-dependent reactions. In the absence of p67, these reactions culminate in the irreversible formation of a metastable activation intermediate that is insensitive to inhibition by RGVHFIF. After addition of p67, this activation intermediate subsequently reacts to form the active NADPH oxidase.