Expression of functional NK1 receptors in human alveolar macrophages: superoxide anion production, cytokine release and involvement of NF-kappaB pathway

Abstract

1 Substance P (SP) is deeply involved in lung pathophysiology and plays a key role in the modulation of inflammatory-immune processes. We previously demonstrated that SP activates guinea-pig alveolar macrophages (AMs) and human monocytes, but a careful examination of its effects on human AMs is still scarce. 2 This study was undertaken to establish the role of SP in human AM isolated from healthy smokers and non-smokers, by evaluating the presence of tachykinin NK(1) receptors (NK-1R) and SP's ability to induce superoxide anion (O(2)(-)) production and cytokine release, as well as activation of the nuclear factor-kappaB (NF-kappaB) pathway. 3 By Western blot analysis and immunofluorescence, we demonstrate that authentic NK-1R are present on human AMs, a three-fold enhanced expression being observed in healthy smokers. These NK-1R are functional, as SP and NK(1) agonists dose-dependently induce O(2)(-) production and cytokine release. In AMs from healthy smokers, SP evokes an enhanced respiratory burst and a significantly increased release of tumor necrosis factor-alpha as compared to healthy non-smokers, but has inconsistent effects on IL-10 release. The NK(1) selective antagonist CP 96,345 ((2S,3S)-cis-2-diphenylmethyl-N[(2-methoxyphenyl)-methyl]-1-azabicyclo-octan-3-amine)) competitively antagonized SP-induced effects. 4 SP activates the transcription factor NF-kappaB, a three-fold increased nuclear translocation being observed in AMs from healthy smokers. This effect is receptor-mediated, as it is reproduced by the NK(1) selective agonist [Sar(9)Met(O(2))(11)]SP and reverted by CP 96,345. 5 These results clearly indicate that human AMs possess functional NK-1R on their surface, which are upregulated in healthy smokers, providing new insights on the mechanisms involved in tobacco smoke toxicity.

Figure 1

Immunocytochemical analysis of the expression and location of NK-1R in human AMs. (a) Phase contrast. (b) Immunofluorescence of anti-NK-1R polyclonal Ab followed by FITC-conjugated anti-rabbit immunoglubulins (green). (c) Nuclear staining with Hoechst 33258 (blue). (d) Merge of B and C. Fluorescence was visualized by a vertical fluorescence microscopy (100-fold magnification; Eclipse E600, Nikon, Tokyo, Japan).

Figure 2

NK-1R expression in human AMs from healthy smokers (S) and non-smokers (NS). In (a), Western blot analysis of NK-1R in AM extracts of two smokers and two non-smokers. The blots are assembled from different single experiments in which AMs from S or NS have been evaluated. As a positive control for the presence of NK-1R, the macrophage cell line J774.A1 was used (for clarity, only one blot of J774.A1 cells, representative of seven others, is shown). The same protein amount (30 μg) was used in each experiment with AMs from smokers and non-smokers. An arrowhead indicates the 68-kDa band corresponding to the receptor. The migration of protein standards of known sizes is shown on the left. In (b), quantitative evaluation of NK-1R expression by densitometry. Intensity of the specific band of NK-1R in the macrophage J774.A1 cells amounted to 8710±250 intensity units (means±s.e.m. of eight experiments) and was taken as 100%. Results are expressed as % expression of the positive control; mean+s.e.m. of four experiments for S and NS.

Figure 3

NK-1R agonists evoke O₂⁻ production in human AMs isolated from healthy smokers (a) and non-smokers (b). Cells were challenged with increasing concentrations of SP, [Sar⁹Met(O₂)¹¹]SP and Pro⁹SP for 30 min. Results are means±s.e.m. of five to six experiments in duplicate. ^*P<0.05 vs non-smokers.

Figure 4

The NK₁ selective antagonist CP96,345 competitively antagonizes SP-induced O₂⁻ production (a) and [Sar⁹Met(O₂)¹¹]SP-induced O₂⁻ production (b) in AMs from healthy smokers. Cells were preincubated with CP 96,345 at 1 nM for 15 min and then challenged with the NK₁ agonists for further 30 min. Results are means±s.e.m. of four experiments in duplicate.

Figure 5

NK-1R stimulation induces TNF-α release in human AMs isolated from healthy smokers and healthy non-smokers. SP-induced TNF-α release in (a); [Sar⁹Met (O₂)¹¹]SP-induced release in (b); reversal by CP 96,345 1 nM of SP-induced release in smokers in (c). Data are means±s.e.m. of five to six experiments in duplicate. ^*P<0.01 vs non-smokers. See text for further details.

Figure 6

Effects of SP on IL-1β release (a) and IL-10 release (b) in human AMs isolated from healthy smokers and healthy non-smokers. PMA-induced release is shown for comparison. Data are means±s.e.m. of five to six experiments in duplicate.

Figure 7

SP induces NF-κB activation in human AMs from healthy non-smokers (a) and healthy smokers (b) in a time- and dose-dependent manner. AMs were stimulated with SP (10⁻⁶ and 10⁻⁸ M) or PMA 10⁻⁶ M for 1 or 2 h, in the presence or absence of CP 96,345. The NK-1R antagonist was evaluated at 1 μM and preincubated for 15 min. Nuclear extracts (5 μg) were prepared and assayed for NF-κB activity by EMSA (see text for further details). In (a) (non-smoker): lane 1=PMA 2 h; lane 2=PMA 1 h; lane 3=SP 10⁻⁶ M 1 h; lane 4=SP 10⁻⁶ M 2 h; lane 5=SP 10⁻⁸ M 1 h; lane 6=SP 10⁻⁸ M 2 h; lane 7=control, unstimulated AM; lane 8=CP 96,345 + SP 10⁻⁸ M 2 h; lane 9=CP 96.345 + SP 10⁻⁶ M 2 h; lane 10=CP 96,345 alone; lane 11=CP 96,345 + PMA 2 h. In (b) (smoker): lane 1=PMA 2 h; lane 2=PMA 1 h; lane 3=SP 10⁻⁶ M 2 h; lane 4=SP 10⁻⁶ M 1 h; lane 5=SP 10⁻⁸ M 2 h; lane 6=SP 10⁻⁸ M 1 h; lane 7=control, unstimulated AM; lane 8=CP 96,345 + SP 10⁻⁸ M 2 h; lane 9=CP 96.345 + SP 10⁻⁶ M 2 h; lane 10=CP 96,345 alone; lane 11=CP 96,345 + PMA 2 h. This experiment was performed three times with similar results.

Figure 8

The NK₁ selective agonist [Sar⁹Met(O₂)¹¹]SP induces NF-κB activation in human AMs from healthy smokers and its effects are reduced by the NK-1R antagonist CP 96,345. AMs were stimulated with [Sar⁹Met(O₂)¹¹]SP 10⁻⁸ or 10⁻⁶ M for 2 h, in the presence or absence of CP 96,345 at 1 μM. The effects of SP 10⁻⁶ M are shown for comparison. Nuclear extracts (5 μg) were prepared and assayed for NF-κB activity by EMSA (see text for further details). Lane 1=control, unstimulated AM; lane 2=Sar⁹ 10⁻⁸ M; lane 3=Sar⁹ 10⁻⁶ M; lane 4=SP 10⁻⁶ M; lane 5=CP 96,345 + Sar⁹ 10⁻⁶ M. This experiment was performed three times with similar results.

Figure 9

Western blots of p50 and p65 subunits in AMs from both non-smokers (NS) and smokers (S). The nuclear translocation of p50 is reported in (a); the translocation of p65 is reported in (b). Beta-actin is shown for comparison. Lane 1=control; lane 2=PMA 10⁻⁶ M; lane 3=SP 10⁻⁶ M; lane 4=SP 10⁻⁸ M; lane 5=Sar⁹ 10⁻⁶ M.

Figure 10

NK-1R stimulation induces the translocation of p50 (a) and p65 (b) subunits in human AMs from healthy smokers and healthy non-smokers. PMA-induced translocation is shown for comparison. AMs were challenged for 2 h with the stimuli; nuclear and cytosolic extracts were prepared and evaluated for their content in p50 and p65 subunits. Results are expressed as the nuclear/cytosolic ratio (ratio N/C) for both p50 and p65 subunits. Data are means±s.e.m. of five experiments in duplicate. ^*P<0.05 vs non-smokers.