Nonhematopoietic NADPH oxidase regulation of lung eosinophilia and airway hyperresponsiveness in experimentally induced asthma

Abstract

Pulmonary eosinophilia is one of the most consistent hallmarks of asthma. Infiltration of eosinophils into the lung in experimental asthma is dependent on the adhesion molecule vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells. Ligation of VCAM-1 activates endothelial cell NADPH oxidase, which is required for VCAM-1-dependent leukocyte migration in vitro. To examine whether endothelial-derived NADPH oxidase modulates eosinophil recruitment in vivo, mice deficient in NADPH oxidase (CYBB mice) were irradiated and received wild-type hematopoietic cells to generate chimeric CYBB mice. In response to ovalbumin (OVA) challenge, the chimeric CYBB mice had increased numbers of eosinophils bound to the endothelium as well as reduced eosinophilia in the lung tissue and bronchoalveolar lavage. This occurred independent of changes in VCAM-1 expression, cytokine/chemokine levels (IL-5, IL-10, IL-13, IFNgamma, or eotaxin), or numbers of T cells, neutrophils, or mononuclear cells in the lavage fluids or lung tissue of OVA-challenged mice. Importantly, the OVA-challenged chimeric CYBB mice had reduced airway hyperresponsiveness (AHR). The AHR in OVA-challenged chimeric CYBB mice was restored by bypassing the endothelium with intratracheal administration of eosinophils. These data suggest that VCAM-1 induction of NADPH oxidase in the endothelium is necessary for the eosinophil recruitment during allergic inflammation. Moreover, these studies provide a basis for targeting VCAM-1-dependent signaling pathways in asthma therapies.

Fig. 1

Timeline for ovalbumin (OVA) sensitization, OVA challenge, and intratracheal transfer of purified eosinophils. Chimeric mice were sensitized and challenged with OVA according to protocol 1 (A) or protocol 2 (B). C: using protocol 2, groups of OVA-challenged chimeric CYBB mice received eosinophils from bronchoalveolar lavage (BAL) of OVA-challenged CD45.1 donor mice or received purified eosinophils from blood of NJ.1638 mice that were not challenged with OVA. AHR, airway hyperresponsiveness.

Fig. 2

Chimeric CYBB mice have reduced OVA-stimulated lung eosinophilia. Chimeric CYBB mice (CYBB) and chimeric C57BL/6 mice (WT), that had been reconstituted with wild-type (WT) bone marrow from GFP mice, were sensitized once by intraperitoneal on day 0 and challenged intranasally on days 14 and 15 with OVA. On day 16, blood was collected and lungs lavaged. A: blood eosinophils. B: total number of BAL leukocytes. C: number of mononuclear cells, neutrophils, eosinophils, and lymphocytes in BAL. Bars are very small and near x-axis for cells in saline-treated groups. D: data are expressed as percent of total leukocytes. C and D: solid bars, WT saline. Wide-hatched bars, WT OVA. Stippled bars, CYBB saline. Narrow-hatched bars, CYBB OVA. Values are means ± SE of 6–8 mice per group. *P < 0.05 compared with OVA-treated WT mice.

Fig. 3

Endothelial cell vascular cell adhesion molecule-1 (VCAM-1) expression was not altered in the OVA-stimulated chimeric CYBB mice. Tissue sections from mice in Fig. 2 were indirectly immunofluorescence labeled for VCAM-1 and examined by confocal microscopy. A and C: chimeric WT C57BL/6 mouse lung. B and D: chimeric CYBB mouse lung. A and B: OVA challenged. C and D: saline challenged. Isotype antibody control labeled sections were negative (data not shown). Shown are representative tissues. E: sum of fluorescence intensity per μm² of endothelium. Solid bars, CYBB chimera. Hatched bars, WT chimera. Values are means ± SE for 6–8 mice per group. *P < 0.05 compared with saline-treated mice.

Fig. 4

Eosinophils accumulated on the luminal surface of the endothelium in the OVA-stimulated CYBB chimeras. Frozen lung tissue sections from mice in Fig. 2 were fixed and stained with hematoxylin and eosin (A and B) or immunolabeled for major basic protein (MBP) (C–F). A and C: WT chimeric mouse lung, OVA challenged. B and D: CYBB chimeric mouse lung, OVA challenged. E: C57BL/6 chimeric mouse lung, saline challenged. F: CYBB chimeric mouse lung, saline challenged. Shown are representative sections. Arrows, eosinophils attached to luminal surface of endothelium. L, vessel lumen. G: number of eosinophils at the luminal surface of the endothelium per mm of the luminal surface of the endothelium in the tissue sections. Values are means ± SE of 6–8 mice per group. *P < 0.05 compared with OVA-treated WT mice.

Fig. 5

OVA-stimulated chimeric CYBB mice had reduced AHR. Chimeric CYBB mice (CYBB) and chimeric C57BL/6 mice (WT) were generated by reconstitution with CD45.1 bone marrow. The mice were sensitized and challenged with OVA using protocol 2 (Fig. 1). Briefly, these mice were sensitized intraperitoneally with OVA-alum or saline-alum on day 0 and day 7 and challenged intranasally on days 14, 16, and 19 with OVA or saline. On day 20, mice were tested for airway responsiveness to 50 μg acetylcholine/kg. Airway responsiveness is depicted as airway pressure time index (APTI; cmH₂O · s). Values are means ± SE of 6–8 mice per group. *P < 0.05 compared with the other groups.

Fig. 6

Expression of several cytokines was not altered in the OVA-stimulated chimeric CYBB mice. Lung digests from mice in Fig. 5 were nontreated or restimulated with OVA for 48 h. The culture supernatants were examined for cytokines by ELISA: IL-4 (A), IL-5 (B), IL-10 (C), and IL-13 (D). Solid bars, nontreated. Hatched bars, OVA challenged. Values are means ± SE of 6–8 mice per group. There was no statistical difference among the saline groups. *P < 0.05 compared with saline-treated mice.

Fig. 7

Bypassing the endothelium with intratracheal (IT) eosinophils (Eos) resulted in recovery of AHR. A: the number of BAL eosinophils or blood eosinophils transferred to each chimeric CYBB mouse on days 14, 16, and 19. B: the BAL cells were collected after testing for AHR. Shown is the number of eosinophils in the BAL, demonstrating successful intratracheal administration of eosinophils to the chimeric CYBB mice. *P < 0.05 compared with the other groups. C: to examine AHR on day 20, 75 μg acetylcholine/kg was used, since we had determined that the 50 μg/kg used in Fig. 5 induced about only 30% of the maximum AHR, whereas 75 μg/kg was at 70% of the maximum AHR (data not shown). In some bars in C, the “E” indicates intratracheal administration of eosinophils. *P < 0.05 compared with saline-treated mice.

Fig. 8

OVA-stimulated chimeric CYBB mice had reduced perivascular eosinophilia in the lung tissue. The lungs were frozen from mice in Fig. 7, and the tissue sections were stained by immunohistochemistry with anti-MBP. A and B: WT C57BL/6 chimeric mouse lung. C and D: CYBB chimeric mouse lung. E: CYBB chimeric mouse from the group in Fig. 6 that received 4.1 × 10⁶ total BAL eosinophils (CYBB, OVA, IT BAL eos). A and C: saline challenged. B, D, and E: OVA challenged. Isotype antibody control labeled sections were negative (data not shown). Shown are representative tissues. F: number of perivascular eosinophils per high-powered (×40) field. *P < 0.05 compared with the other OVA-treated groups. G: number of eosinophils on the endothelial lumen/vessel. Values are means ± SE of 6–8 mice per group. *P < 0.05 compared with saline-treated mice and OVA-treated WT mice. L, vessel lumen. Arrows, eosinophils attached to luminal surface of endothelium.

Fig. 9

OVA-stimulated chimeric CYBB mice had reduced peribronchial eosinophilia in the lung tissue. The lungs were frozen from mice in Fig. 7, and the tissue sections were stained by immunohistochemistry with anti-MBP and lightly countered stained with methyl green. A and B: WT C57BL/6 chimeric mouse lung. C and D: CYBB chimeric mouse lung. E: CYBB chimeric mouse from the group in Fig. 7 that received 4.1 × 10⁶ total BAL eosinophils (CYBB, OVA, IT BAL eos). A and C: saline-challenged mice. B, D, and E: OVA-challenged mice. Isotype antibody control labeled sections were negative (data not shown). Shown are representative tissues. F: number of peribronchial eosinophils per high-powered (×40) field. *P < 0.05 compared with OVA-treated mice. B, bronchial airway.

Fig. 10

OVA-challenged chimeric CYBB mice did not have altered numbers of T cell subsets, localization of T cells, serum IgE, or serum OVA-specific Igs. From mice in Fig. 7, serum was obtained from the blood, half of the lung was digested, and half of the lung was frozen for tissue sections. A–C: the lung digests were immunofluorescence labeled for CD4, CD8, and TCRβ and examined by flow cytometry. B: open bars, TCRβ⁺; solid bars, CD4⁺TCRβ⁺; hatched bars, CD8⁺. D: the tissue sections were labeled by immunohistochemistry for CD4 or CD8; HPF, high-powered fields; solid bars, CD4⁺ perivascular; open bars, CD4⁺ peribronchial; hatched bars, CD8⁺ perivascular; stippled bars, CD8⁺ peribronchial. E: the serum was examined for total IgE by ELISA. F: the serum was examined by ELISA for OVA-specific IgE (solid bars), OVA-specific IgG₁ (open bars), or OVA-specific IgG_2a (hatched bars). The OVA-challenged groups were significantly greater than the saline groups in all panels (P < 0.05).