Metalloelastase in lungs and alveolar macrophages is modulated by extracellular substance P in mice

Abstract

Metalloelastase (MMP-12), mainly produced by macrophages, has been shown to play a key role in the pathogenesis of emphysema in animal models. Chronic cigarette smoke increases pulmonary MMP-12, which is closely correlated with an elevation of pulmonary substance P (SP). Because alveolar macrophages (AMs) contain the neurokinin-1 receptor (NK1R), we tested whether SP was able to trigger the upregulation of MMP-12 synthesis in AMs by acting on the NK1R. AMs isolated from bronchoalveolar lavage cells in C3H/HeN mice were cultured with control medium or SP that was coupled without or with NK1R antagonists (CP-99,994 or aprepitant) for 24 h. We found that SP significantly increased the mRNA of MMP-12 and NK1R by 11-fold and 82%, respectively, in AMs (P<0.05), and these responses were abolished by NK1R antagonists with little change in the cells' viability. Because pulmonary SP is primarily released by bronchopulmonary C-fibers (PCFs), we further asked whether destruction of PCFs would reduce SP and MMP-12. Two groups of mice were pretreated with vehicle and neonatal capsaicin (NCAP) to degenerate PCFs, respectively. Our results show that NCAP treatment significantly decreased mRNA and protein levels of SP associated with a reduction NK1R and MMP-12 in the lungs and AMs. These findings suggest that SP has a modulatory effect on pulmonary MMP-12 by acting on NK1R to trigger MMP-12 syntheses in the AMs.

Fig. 1.

Effects of substance P (SP) and CP-99,994 (CP) or aprepitant (AP) on metalloelastase (MMP-12), matrix metalloproteinase-9 (MMP-9), and tissue inhibitor of matrix metalloproteinase (TIMP)-1 mRNA expression and protein levels in alveolar macrophages (AMs) in vitro. The representative mRNA bands of MMP-12, MMP-9, and TIMP-1 (172, 390, and 333 bp, respectively) and their protein bands (54, 98, and 27 kDa, respectively) are displayed in A and B, respectively. The mRNA group data for MMP-12 and MMP-9 (the ratio of MMP-12/MMP-9 to TIMP-1) are shown in C and D, respectively. Values are means ± SE; n = 5 trials. *P < 0.01 compared with control culture medium (CM). †P < 0.01, SP vs. CP, CP+SP, AP, or AP+SP.

Fig. 2.

Effects of SP and CP on neurokinin-1 receptor (NK1R) mRNA expression and protein level in AMs in vitro. The representative NK1R mRNA (365 bp) and protein (53 kDa) from AMs are displayed in A and B, respectively. The group data of NK1R mRNA are shown in C. Values are means ± SE; n = 5 trials. *P < 0.01 compared with CM. †P < 0.01, SP vs. CP or CP+SP.

Fig. 3.

Effects of SP and CP on protein levels of interleukin (IL)-1β (A) and tumor necrosis factor (TNF)-α (B) secreted from AMs in vitro. Values are means ± SE; n = 5 trials. *P < 0.01 compared with CM. †P < 0.01, SP vs. CP or CP+SP.

Fig. 4.

Effects of SP, CP, and AP on the viability of AMs in vitro. Cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and expressed as the percentage of control (dashed line). Values are means ± SE; n = 5 trials for CP and AP, respectively.

Fig. 5.

Dose and time dependence of SP-induced MMP-12 mRNA expression in the RAW264.7 cell line. The group data for dose and time dependence of SP-induced modulation of MMP-12 mRNA measured using real-time PCR are exhibited in A and B, respectively, whereas the representative bands of MMP-12 mRNA (172 bp) detected using RT-PCR are shown in C and D. Values are means ± SE; n = 6 trials. *P < 0.01 compared with control. †P < 0.01 compared with 10⁻¹⁰ M SP in A and 1.5 h in B. ‡P < 0.01 compared with 10⁻⁹ M SP in A and 3.0 h in B. Note that the data for MMP-12 mRNA obtained after 3 h of SP culture are significantly higher than those for the control (P < 0.05) in B.

Fig. 6.

Effect of neonatal capsaicin (NCAP) treatment on preprotachykinin-A (PPT-A) mRNA expression and SP protein levels in lung tissues and AMs. A and B, top: 4 representative mRNA expressions of PPT-A (364 bp) in lungs (A) and AMs (B) of control (CON) and NCAP-treated mice. Bottom: relevant group data for PPT-A mRNA and SP protein. Values are means ± SE; n = 12 and 6 per group for lungs and AMs, respectively. **P < 0.01, CON vs. NCAP-treated mice.

Fig. 7.

Effect of NCAP treatment on MMP-12 and TIMP-1 mRNA expression and protein levels in lung tissues and AMs. Four representative mRNA expressions of MMP-12 and TIMP-1 (172 and 333 bp, respectively) and their protein bands (54 and 27 kDa, respectively) are displayed in A and B, respectively. The group data for MMP-12 and the ratio of MMP-12 to TIMP-1 are exhibited in C and D, respectively. Values are means ± SE; n = 12 and 6 per group for lungs and AMs, respectively. *P < 0.05; **P < 0.01, CON vs. NCAP-treated mice.

Fig. 8.

Comparison of NK1R mRNA and protein in lung tissues and AMs between CON and NCAP-treated mice. Four representative NK1R protein (53 kDa) and gene (365 bp) expressions in lungs and AMs are displayed in A and B, respectively. The group data for the NK1R mRNA expression in the lung (left) and AMs (right) are shown in C. Values are means ± SE; n = 12 and 6 per group for lungs and AMs, respectively. **P < 0.01, CON vs. NCAP-treated mice.

Fig. 9.

Comparison of bronchoalveolar lavage (BAL) total (A) and differential cells [AMs (B), neutrophils (C), and lymphocytes (D)] between CON and NCAP-treated mice. Values are means ± SE; n = 6 per group. *P < 0.05, CON vs. NCAP-treated mice.