Role of tumor necrosis factor-α and its receptors in diesel exhaust particle-induced pulmonary inflammation

Abstract

Inhalation of diesel exhaust particles (DEP) induces an inflammatory reaction in the lung. However, the underlying mechanisms remain to be elucidated. Tumor necrosis factor alpha (TNF-α) is a pro-inflammatory cytokine that operates by binding to tumor necrosis factor receptor 1 (TNFR1) and tumor necrosis factor receptor 2 (TNFR2). The role of TNF-α signaling and the importance of either TNFR1 or TNFR2 in the DEP-induced inflammatory response has not yet been elucidated. TNF-α knockout (KO), TNFR1 KO, TNFR2 KO, TNFR1/TNFR2 double KO (TNFR-DKO) and wild type (WT) mice were intratracheally exposed to saline or DEP. Pro-inflammatory cells and cytokines were assessed in the bronchoalveolar lavage fluid (BALF). Exposure to DEP induced a dose-dependent inflammation in the BALF in WT mice. In addition, levels of TNF-α and its soluble receptors were increased upon exposure to DEP. The DEP-induced inflammation in the BALF was decreased in TNF-α KO, TNFR-DKO and TNFR2 KO mice. In contrast, the inflammatory response in the BALF of DEP-exposed TNFR1 KO mice was largely comparable with WT controls. In conclusion, these data provide evidence for a regulatory role of TNF-α in DEP-induced pulmonary inflammation and identify TNFR2 as the most important receptor in mediating these inflammatory effects.

Figure 1

Exposure to DEP induces inflammation in BALF in a dose-dependent manner. C57BL/6 J WT mice were exposed to saline (white bar) or 12.5 µg, 25 µg, 50 µg and 100 µg DEP (black bars). BALF was collected 48 hours after the last exposure. (A) Total cell numbers in BALF. (B) Neutrophils in BALF were determined on cytospin. (C–F) Monocyte (CD11c^low, CD11b⁺, Ly6C⁺ and Ly6G⁻) (C); dendritic cell (CD11c^high, low autofluorescent and MHCII⁺) (D); CD4⁺ T-cell (CD3⁺, CD8⁻, CD4⁺) (E) and CD8⁺ T-cell (CD3⁺, CD4⁻, CD8⁺) (F) numbers in BALF were determined by flow cytometry. (G) Airway resistance (R) of saline- (broken line) and 100 µg DEP- (full line) exposed mice was measured in response to increasing doses of carbachol. Results are expressed as mean ± SEM. n = 8 mice per group. *p < 0.05.

Figure 2

TNF-α and its soluble receptors are increased in WT mice exposed to DEP. C57BL/6 J WT mice were exposed to saline (white bar) or 100 µg DEP (black bar). BALF and lung tissue were collected 48 hours after the last exposure. (A–C) TNF-α (A), sTNFR1 (B) and sTNFR2 (C) levels were determined in BALF using ELISA. Results are expressed as mean ± SEM. n = 7–9 mice per group. *p < 0.05. Data are representative of two independent experiments. (D–F) TNF-α (D), TNFR1 (E) and TNFR2 (F) mRNA expression in hematopoietic (CD45⁺) and non-hematopoietic (CD45⁻) lung cells were determined using qRT-PCR. n = 5–8 mice per group.

Figure 3

DEP-induced inflammation is TNF-α dependent. C57BL/6 J WT and TNF-α KO were exposed to saline (white bar) or 100 µg DEP (black bar). BALF was collected 48 hours after the last exposure. (A) Total cell numbers in BALF. (B–F) Neutrophil (CD11c^low, CD11b⁺, Ly6C⁺ and Ly6G⁺) (B); monocyte (CD11c^low, CD11b⁺, Ly6C⁺ and Ly6G⁻) (C); dendritic cell (CD11c^high, low autofluorescent and MHCII⁺) (D); CD4⁺ T-cell (CD3⁺, CD8⁻, CD4⁺) (E) and CD8⁺ T-cell (CD3⁺, CD4⁻, CD8⁺) (F) numbers in BALF were determined by flow cytometry. (G–I) CXCL1 (G), CCL2 (H) and CCL20 (I) levels were determined in BALF using ELISA. Results are expressed as mean ± SEM. n = 5–8 mice per group. *p < 0.05.

Figure 4

DEP-induced pulmonary inflammation is dependent on TNF receptor signaling. C57BL/6 J WT and TNFR-DKO mice were exposed to saline (white bar) or 100 µg DEP (black bar). BALF was collected 48 hours after the last exposure. (A) Total cell numbers in BALF. (B–F) Neutrophil (CD11c^low, CD11b⁺, Ly6C⁺ and Ly6G⁺) (B); Monocyte (CD11c^low, CD11b⁺, Ly6C⁺ and Ly6G⁻) (C); dendritic cell (CD11c^high, low autofluorescent and MHCII⁺) (D); CD4⁺ T-cell (CD3⁺, CD8⁻, CD4⁺) (E) and CD8⁺ T-cell (CD3⁺, CD4⁻, CD8⁺) (F) numbers in BALF were determined by flow cytometry. (G–I) CXCL1 (G), CCL2 (H) and CCL20 (I) levels were determined in BALF using ELISA. Results are expressed as mean ± SEM. n = 7–8 mice per group. *p < 0.05. BALF inflammatory cell data are representative of two independent experiments (except for BALF neutrophils).

Figure 5

DEP-induced monocytes and lymphocytes are TNFR2 dependent. C57BL/6 J WT, TNFR1 KO and TNFR2 KO mice were exposed to saline (white bar) or 100 µg DEP (black bar). BALF was collected 48 hours after the last exposure. (A) Total cell numbers in BALF. (B–F) Neutrophil (CD11c^low, CD11b⁺, Ly6C⁺ and Ly6G⁺) (B); monocyte (CD11c^low, CD11b⁺, Ly6C⁺ and Ly6G⁻) (C); dendritic cell (CD11c^high, low autofluorescent and MHCII⁺) (D); CD4⁺ T-cell (CD3⁺, CD8⁻, CD4⁺) (E) and CD8⁺ T-cell (CD3⁺, CD4⁻, CD8⁺) (F) numbers in BALF were determined by flow cytometry. (G–I) CXCL1 (G), CCL2 (H) and CCL20 (I) levels were determined in BALF using ELISA. Results are expressed as mean ± SEM. n = 7–9 mice per group, except the TNFR2 KO mice receiving saline: n = 3 mice. *p < 0.05. BALF inflammatory cell data are representative of two independent experiments (except for BALF neutrophils).