Improved superoxide-generating ability by interferon γ due to splicing pattern change of transcripts in neutrophils from patients with a splice site mutation inCYBB gene

Abstract

Chronic granulomatous disease (CGD) is an inherited disorder of host defense against microbial infections caused by defective activity of the phagocyte NADPH oxidase. Based on an increase of neutrophil superoxide-generating ability in response to interferon ␥ (IFN-␥) in a single patient with CGD, multicentered group studies demonstrated a beneficial effect of prophylactic IFN-␥. However, no apparent increase of the phagocyte superoxide generation was found in patients enrolled in these studies. The present report offers an additional kindred in whom an IFN-␥-dependent increase in neutrophil superoxide production was observed in 3 affected patients. The defect in the CYBB gene for gp91-phox was identified as an otherwise silent mutation adjacent to the third intron of the CYBB gene that alters messenger RNA splicing. By molecular analysis, significant differences were found in the splicing pattern of CYBB gene transcripts in patient neutrophils between 1 and 25 days after administration of IFN-␥. IntroductionChronic granulomatous disease (CGD) is an inherited disorder of host defense against bacterial and fungal infections; affected patients suffer from severe recurrent and intractable infections beginning in early childhood. 1 Phagocytes from patients with CGD show impaired microbicidal activity due to defects in the superoxidegenerating phagocyte oxidase. 1 It is known that the mutations responsible for CGD reside within the genes for 4 essential components of the oxidase designated as gp91-phox (phagocyte oxidase), p22-phox, p47-phox and p67-phox. 2,3 The gp91-phox forms membrane cytochrome b 558 together with p22-phox and plays an essential role in the transfer of electrons following assembly of the active oxidase with the cytoplasmic p47-and p67-phox components. Patients with CGD with gp91-phox defects account for the majority of cases, and in most instances the cytochrome is reduced or absent in phagocytes and B lymphocytes. The CYBB gene that encodes gp91-phox is localized to Xp21.1 and encompasses 13 exons spanning approximately 30 kilobases (kb). Mutations in the CYBB gene are heterogenous, and include missense, nonsense, deletion, insertion, and splicing defects. 1,4,5 Various cytokines have been studied for enhancement of the neutrophil superoxide generation, showing that IFN-␥ may be an effective agent. 6,7 Ezekowitz and coworkers first demonstrated that phagocytes of a patient with X-linked CGD were responsive to interferon-␥ (IFN-␥), which almost completely corrected the oxidase defect in vitro and in vivo. 8,9 These findings motivated multicenter groups to perform double-blinded clinical studies of IFN-␥ as a prophylactic agent in CGD, which demonstrated its clinical benefit in the majority of patients with CGD. 10,11 In these group studies, however, no apparent increases in phagocyte superoxide generation were observed. The patient studied by Ezekowitz and colleagues, therefore, has been considered to be an exceptional case. Recently, the mutation in this patient has been identified as a single b...

NOX-2S is a new member of the NOX family of NADPH oxidases

“…The pattern of NOX-2S expression was markedly different to that of NOX-2 in Ramos cells. A number of reports have indicated that IFN-g treatment alters the splicing pattern of some RNA transcripts including HLA class I (He and Le, 1995), nitric oxide synthase (Eissa et al, 1996), and NOX-2 (Condino-Neto and Newburger, 2000;Ishibashi et al, 2001). Thus, IFN-g could affect the splicing pattern of NOX-2 transcripts.…”

Human neutrophils produce interferon gamma upon stimulation by interleukin-12

“…IFNg prophylaxis improved the PMN oxidative burst in two patients with chronic granulomatous disease, by partially correcting the abnormal splicing of NADPH-oxidase CYBB gene transcripts. 41,42 Also, inhaled IFNg restored normal pulmonary immune status in patients with severe trauma and immune paralysis. 43 Finally, our data may explain the intriguing role of PMN in antitumor reactions, as reviewed by Di Carlo et al…”

Invasive Fungal Infections in Patients with Chronic Granulomatous Disease

“…A subgroup of variant X-linked CGD patients (i.e., with very low, but detectable, baseline superoxide-generating activity), who have splice site mutations, has been shown to be responsive to INF-γ [154,155]. Treatment resulted in improved splicing efficiency and an increase in cytrochrome b expression, allowing near normal levels of superoxide production and bactericidal activity of neutrophils and monocytes [156][157][158].…”