In vitro inhibition of human neutrophil histotoxicity by ambroxol: evidence for a multistep mechanism

Abstract

Neutrophils are major culprits for the protease/antiprotease imbalance during various lung diseases, that is, chronic obstructive pulmonary disease, cystic fibrosis, idiopathic pulmonary fibrosis and adult respiratory distress syndrome. Thus, these cells are presently considered an ideal target for the pharmacologic control of tissue injury during these diseases. This study was planned in order to investigate if ambroxol and its precursor bromhexine are actually capable of preventing alpha-1-antitrypsin (A1AT) inactivation by stimulated neutrophils and possibly to look into the mechanisms underlying this event. Ambroxol inhibited the production of superoxide anion by activated neutrophils, whereas bromhexine had no inhibitory effect. Ambroxol decreased the production of hypochlorous acid (HOCl) from activated neutrophils with high efficiency, whereas bromhexine had a modest activity. Ambroxol and bromhexine were capable of limiting the chlorination of monochlorodimedon by HOCl, displaying the capacity of directly scavenging the oxidant. Ambroxol decreased the release of elastase and myeloperoxidase from activated neutrophils, whereas bromhexine was ineffective. Ambroxol prevented the A1AT inactivation by neutrophils, whereas bromhexine was completely ineffective. Among drugs currently available for in vivo use in humans, ambroxol is unique by virtue of its ability to prevent neutrophil-mediated A1AT inactivation via inhibition of HOCl production as well as HOCl scavenging. Also taking into account its capacity for curbing elastase release, the drug displays the potential to lessen the burden of oxidants/proteases and to increase the antiprotease shield at the site of inflammation. Thus, ambroxol appears to be a good candidate for raising attempts to develop new therapeutic histoprotective approaches to inflammatory bronchopulmonary diseases.

Figure 1

Chemical structure of ambroxol and bromhexine.

Figure 2

Effect of different doses (abscissa) of ambroxol and bromhexine on the production of superoxide anion (O₂⁻) by neutrophils. Results are expressed as mean±1 s.e.m. (ambroxol: n=4, bromhexine: n=5). ^*P<0.05; ^**P<0.01 vs control without the drug (0); ANOVA followed Dunnett's multiple comparison test.

Figure 3

Effect of different doses (abscissa) of ambroxol and bromhexine on the production of HOCl from neutrophils. Results are expressed as mean±1 s.e.m., n=5. ^*P<0.05; ^**P<0.01 vs control without the drug (0); ANOVA followed Dunnett's multiple comparison test.

Figure 4

Inhibitory effect of various concentrations of ambroxol and bromhexine on the chlorination of MCD by reagent HOCl. The abscissa shows the ratio between the concentration of each drug and that of MCD. Results are expressed as mean±1 s.e.m., n=3. ^*P<0.05; ^**P<0.01 vs control without the drug; ANOVA followed Dunnett's multiple comparison test.

Figure 5

Effect of ambroxol on neutrophil primary granule exocytosis. Results are expressed as percent inhibition of MPO (mean±1 s.e.m., n=3) and elastase (mean±1 s.e.m., n=6) release from fMLP-activated neutrophils. ^*P<0.05; ^**P<0.01 vs control without the drug; ANOVA followed Dunnett's multiple comparison test.

Figure 6

Effect of different doses (abscissa) of ambroxol and bromhexine on the inactivation of A1AT by neutrophils. After incubation with neutrophils, the activity of A1AT was measured as EIC using PPE. The percent EIC of A1AT (ordinate) was calculated by comparison of control samples of A1AT incubated in the absence of cells. Results are expressed as mean±1 s.e.m. (ambroxol: n=5, bromhexine: n=3). ^*P<0.05; ^**P<0.01 vs control without the drug; ANOVA followed Dunnett's multiple comparison test.

Figure 7

Analysis of the interaction of PPE with A1AT, incubated with neutrophils in the absence or presence of ambroxol. Lane 1=native A1AT; lane 2=A1AT plus PPE; lane 3=neutrophils-exposed A1AT plus PPE; lane 4=A1AT exposed neutrophils in the presence of 100 μM ambroxol plus PPE. The lanes shown are from a representative experiment of three experiments^**.

Figure 8

Proposed model for histoprotective properties of ambroxol. (a) Pathways of neutrophil-mediated tissue injury: extracellular release of elastase (1); chlorinated oxidant production by H₂O₂/MPO pathway (2,3); inactivation of A1AT by HOCl (4). These pathways converge to proteolytic (5) and oxidative (6) tissue injury. (b) Inhibitory effect of ambroxol of histotoxic pathways of neutrophils. The drug is capable of inhibiting the release of elastase (1); to diminish the bioavailability of HOCl by impairing the production of the oxidant precursor O₂⁻ (2), by curbing the release of the catalytic enzyme MPO (3) and by directly scavenging HOCl (4); to restore the antielastase activity of A1AT by neutrophil-mediated inactivation (5). Taken together, these activities result in ambroxol-mediated tissue rescue from neutrophil histotoxicity (6).