p47phox deficiency induces macrophage dysfunction resulting in progressive crystalline macrophage pneumonia

Abstract

Nicotinamide dinucleotide phosphate oxidase-deficient (p47(phox-/-)) mice are a model of human chronic granulomatous disease; these mice are prone to develop systemic infections and inflammatory diseases. The use of antibiotic (Bactrim) prophylaxis in a specific pathogen-free environment, however, impedes infection in the majority of p47(phox-/-) mice. We examined infection-free p47(phox-/-) mice between 1 and 14 months of age and found that they developed proliferative macrophage lesions containing Ym1/Ym2 protein and crystals in lung, bone marrow, lymph nodes, and spleen. Here, we show that the lung lesions progressed from single macrophages with intracellular Ym1/Ym2 protein crystals to severe diffuse crystalline macrophage pneumonia without histological evidence of either granulation tissue or pulmonary fibrosis. Ym1/Ym2 is a chitinase-like secretory protein that is transiently induced in alternatively activated macrophages during T-helper (Th)2-biased pathogenesis and during chemical and traumatic inflammation. Bronchoalveolar lavage from p47(phox-/-) mice contained significantly higher levels of Th-1 (interferon-gamma), Th-2 (interleukin-4), and Th-17 (interleukin-17)-associated cytokines than wild-type mice, as well as copious amounts of interleukin-12, indicating that Ym1-secreting p47(phox-/-) macrophages are also integrated into classically activated macrophage responses. These results suggest that p47(phox-/-) macrophages are extremely pliable, due in part to an intrinsic dysfunction of macrophage activation pathways that allows for distinct classical or alternative activation phenotypes.

Figure 1

A–F: Histology of macrophage crystalline pneumonia. A: Lung, early crystalline pneumonia, 4-month-old p47^phox−/− mouse, H&E, 1000x magnification. B: Lung, severe lesions with abundant crystalline material within alveoli and bronchioles, 14-month-old p47^phox−/− mouse, H&E, original magnification ×200. C: Lung, higher magnification demonstrating intra and extracellular giant crystals admixed with macrophages, 14-month-old p47^phox−/− mouse, H&E, original magnification ×400. D: Spleen, electron microphotograph of crystalline material showing electron-dense spicules, >12-month-old p47^phox−/− mouse (original magnification ×11,880). E: Lung, immunostaining against Ym1/Ym2 protein visualized in pulmonary macrophages with few crystals, 9-week-old p47^phox−/− mouse, IHC, original magnification ×1000. F: Lung, immunostaining against macrophage mannose receptor, 9-week-old p47^phox−/− mouse, IHC, original magnification ×400.

Figure 2

A–F: H&E histology and immunohistochemical labeling for Ym1/Ym2 in extrapulmonary lesions with histiocytic proliferation in p47^phox−/− mice. A, B: Lymph node, original magnification ×200. C, D: Spleen, original magnification ×200, ×400, respectively. E, F: Bone marrow, original magnification ×200.

Figure 3

p47^phox−/− bronchoalveolar lavage (BAL) cytokine milieu. BAL was obtained from wild-type (WT) and p47^phox−/− mice. The amount of IL-2, IL-4, IL-6, IL-10, IL-12p40, IL-17, IFN-γ, and TNFα in the BAL was quantified using specific ELISA. Scatter plots show the responses of six individual WT (open circle) or p47^phox−/− (filled squares) mice. Horizontal bars represent mean cytokine levels. *P < 0.005.

Figure 4

Cytokine production by lymph node T cells stimulated with anti-CD3/anti-CD28. Purified lymph node CD4⁺ and CD8⁺ lymphocytes from WT (open histograms) or p47^phox−/− (filled histograms) were stimulated with plate-bound anti-CD3/anti-CD28 in the presence of IL-2 for 18 hours. Concentrations of IL-4, IL-17, and IFN-γ were analyzed using specific ELISA. Each histogram shows the mean (± SEM) results obtained for four independent experiments with pooled cells from three mice per experiment. *P < 0.05, **P = 0.007.

Figure 5

Enhanced IL-1α production by classically and alternatively activated p47^phox−/− macrophages in vitro. Thioglycollate activated peritoneal macrophages were stimulated with media alone, LPS, or IL-4 for 18 hours. The concentration of IL-1 was analyzed using specific ELISA. Each histogram shows the mean (± SEM) results obtained for four independent experiments with pooled cells from three to four mice per experiment. *P < 0.0005.

Figure 6

Enhanced Ym1 expression in p47^phox−/− macrophages. Thioglycollate induced peritoneal and bone marrow derived macrophages were incubated with 1 μg/ml LPS or 1300u/ml IL-4 for 18 hours. Equal amount of proteins extracted from the cells were subjected to Western blot analysis using rat antibody against eosinophilic chemotactic factor-L and mouse antibody against actin. A: Thioglycollate induced peritoneal macrophage Ym1 expression at baseline (B), and following stimulation with media alone (–), LPS (L), or IL-4 (4). One representative experiment of four is shown. B: Bone marrow derived macrophage Ym1 expression following stimulation with media alone (–) or IL-4. For the chase Ym1 response, IL-4 stimulated macrophages were washed out of the IL-4 containing media and restimulated with media alone (N) or LPS (L). One representative experiment of two is shown.