Using p47 PHOX -deficient mice, a recent study proposed that hyperinflammation of CGD was due to defective production of kynurenine, 8 an anti-inflammatory tryptophan metabolite produced during inflammation by indolamine 2,3-dioxygenase (IDO) using O 2 . as a required cofactor. 9 In these CGD mouse studies, the defective conversion of tryptophan into N-formylkynurenine by superoxide-dependent mouse IDO could be circumvented by providing exogenous kynurenine and interferon ␥ (IFN␥) thereby dramatically reversing inflammation. 8 Consequently, the IDOkynurenine pathway was rapidly proposed as a therapeutic target to control inflammation in human CGD. 10 Tryptophan and kynurenine are important immunoregulatory modulators of tolerance during pregnancy, infection, and autoimmunity. 11 While the bulk of systemic tryptophan conversion is performed by the constitutively expressed hepatic tryptophan 2,3-dioxygenase enzyme, inflammatory stimuli such as IFN␥ strongly up-regulate IDO activity in cells such as macrophages. 12 Furthermore, several tryptophan metabolites are antibacterial 13 and may contribute to controlling some protozoan parasites, 14 raising the possibility that absence of such metabolites in CGD could contribute to infections. Given the pressing clinical need to control hyperinflammation in CGD and better understand regulatory mechanisms of human inflammation in general, we examined whether O 2. played a similar regulatory role in the tryptophan/kynurenine pathway in leukocytes from human CGD subjects.
Methods
PatientsHuman subjects included X-linked (gp91 PHOX -deficient, X-CGD) and autosomal (p47 PHOX -deficient, A-CGD) patients and normal volunteers (Institutional Review Board-approved protocol NIH#05-I-0213).
Sample preparation and stimulationPolymorphonuclear leukocytes (PMNs) were isolated as previously described 15 and purity exceeded 90%. For the Western blot experiments, PMN were further purified with magnetic affinity cell sorting (MACS) to a purity of 99%. Monocytes were prepared using the Dynal Monocyte Negative Isolation bead kit (Invitrogen) mean 87% purity by FACS using CD45: FITC, CD14:PE; Becton Dickinson). PMN and monocytes were suspended in 0.5 mL (5 ϫ 10 6 /mL and 1 ϫ 10 6 /mL, respectively) RPMI 1640/10% fetal calf serum/50M Trp and incubated with buffer or indicated doses of IFN␥ (Actimmune) at 37°C and 5% CO 2 for the indicated periods of time. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734.
1755BLOOD, 9 SEPTEMBER 2010 ⅐ VOLUME 116, NUMBER 10For personal use only. on May 7, 2018. by guest www.bloodjournal.org From Dendritic cells generated from monocytes in RPMI 1640, fetal calf serum, penicillin/streptomycin L-glutamine, 25mM HEPES (N-2-hydroxyethylpiperazine-NЈ-2-ethanesulfonic acid), 1% nonessential amino acids, sodium pyruvate, and 50nM 2-mercaptoethanol, with 50 ng/mL interleukin-4 (IL-4) and 100 U/mL granulocyte/...
