Chlorinated lipid species in activated human neutrophils: lipid metabolites of 2-chlorohexadecanal

Abstract

Neutrophils are important in the host response against invading pathogens. One chemical defense mechanism employed by neutrophils involves the production of myeloperoxidase (MPO)-derived HOCl. 2-Chlorohexadecanal (2-ClHDA) is a naturally occurring lipid product of HOCl targeting the vinyl ether bond of plasmalogens. Previous studies have shown that exogenously-added 2-ClHDA is oxidized to 2-chlorohexadecanoic acid (2-ClHA) and reduced to 2-chlorohexadecanol (2-ClHOH) by endothelial cells. These studies show that both 2-ClHA and 2-ClHOH are produced in activated neutrophils in an MPO- and time-dependent manner and are released by neutrophils into media. 2-ClHDA levels peak following 30 min of phorbol 12-myristate-13-acetate stimulation. In contrast, 2-ClHA and 2-ClHOH levels steadily increased over 60 min, suggesting a precursor-product relationship between 2-ClHDA and both 2-ClHA and 2-ClHOH. Additional experiments using wild-type CHO.K1 and CHO.K1 cells deficient in fatty aldehyde dehydrogenase (FALDH), FAA.K1A, demonstrated that 2-ClHDA oxidation to 2-ClHA is dependent on FALDH activity. Furthermore, mice exposed to intranasal Sendai virus displayed lung neutrophil recruitment, as well as elevated 2-ClHA levels in plasma and bronchoalveolar lavage compared with control-treated mice. Taken together, these data demonstrate, for the first time, that metabolites of 2-ClHDA are produced both in vivo as well as in isolated human neutrophils.

Fig. 1.

Temporal course of 2-ClHDA, 2-ClHOH, and 2-ClHA production in PMA-stimulated human neutrophils. Isolated human neutrophils (1 × 10⁶) were incubated for indicated times with or without 200 nM PMA. At the end of incubations, lipids were extracted by a modified Bligh and Dyer technique (21), and 2-ClHDA and 2-ClHOH were converted to their PFB oxime and PFB ester, respectively, followed by GC-MS quantification employing NICI and SIM as described in Experimental Procedures. 2-ClHA was quantified by LC-MS employing SRM in the negative ion mode as described in Experimental Procedures. Error bars represent SEM, n = 3. *P < 0.05 versus 2-ClHA time 0, **P < 0.01 versus 2-ClHOH time 0 and 15 min.

Fig. 2.

MPO-dependent production of 2-ClHA and 2-ClHOH in isolated neutrophils. Isolated human neutrophils (1 × 10⁶) were either pretreated with 10 mM ATZ or vehicle (ethanol, < 0.1%) alone for 5 min preceding a 30 min incubation with or without 200 nM PMA. At the end of incubations, lipids were extracted by a modified Bligh and Dyer technique (21), and 2-ClHA was quantified by LC-MS employing SRM in the negative ion mode (A), and 2-ClHOH was converted to its PFB ester and quantified by GC-MS employing NICI and SIM (B) as described in Experimental Procedures. Error bars represent SEM, n = 3. A: **P < 0.01 versus +PMA +ATZ, ***P < 0.001 versus negative control. B: ***P < 0.001 versus negative control and + PMA + ATZ.

Fig. 3.

2-ClHDA metabolism to 2-ClHA is dependent on long-chain fatty alcohol:NAD⁺ oxidoreductase. CHO.K1 and FAA.K1A (deficient in long-chain fatty alcohol:NAD⁺ oxidoreductase) cell lines were treated with 2-ClHDA (1 μM) for indicated time intervals. At the end of each time point, cellular (A) and media (B) lipids were extracted in the presence of 2-Cl-[d₄]HA as an internal standard and processed for analyses as described in Experimental Procedures. Error bars represent SEM, n = 3.

Fig. 4.

Long-chain fatty alcohol:NAD+ oxidoreductase defective cells produce more 2-ClHOH from 2-ClHDA than control cells. CHO.K1 (CHO) and FAA.K1A (FAA) cell lines were treated with 2-ClHDA (1 μM) between 30 min and 4 h. At the end of each time point, cellular (A) and media (B) lipids were extracted in the presence of 2-Cl-[d₄]HOH as an internal standard and processed for analyses as described in Experimental Procedures. Error bars represent SEM, n = 3. Cells: *P < 0.05 versus CHO at time 30 min, **P < 0.01 (CHO at time 30 and 60 min) versus FAA at time 30 and 60 min, respectively, ***P < 0.001 (FAA at time 120 and 240 min vs. FAA at time 30 and 60 min, CHO at time 30 min vs. FAA at time 60 min, CHO at 30 to 240 min vs. FAA at 120 and 240 min). Media: *P < 0.05 (FAA at 120 min and CHO at 30 min vs. FAA 60 min, CHO at 240 min vs. FAA at 30 min), **P < 0.01 (CHO at 120 min vs. FAA at 120 min and CHO at 240 min vs. CHO at 30 and 60 min), ***P < 0.001 (FAA at 120 and 240 min vs. FAA at 30 min, FAA at 240 min vs. FAA at 60 min, FAA at 240 min and CHO at 30 and 60 min vs. FAA at 120 min, CHO at 30 to 240 min vs. FAA at 240 min).

Fig. 5.

Release of 2-ClHA and 2-ClHOH into media from PMA-stimulated isolated human neutrophils. Isolated human neutrophils (1 × 10⁶) were incubated for indicated times with or without 200 nM PMA. At the end of each time point, cells were pelleted as described in Experimental Procedures. Both cells and media were extracted by a modified method of Bligh and Dyer (25) in the presence of deuterated internal standards before being subjected to quantification of 2-ClHA by LC-MS employing SRM in the negative ion mode (A) or converted to the PFB ester and subjected to quantitation of 2-ClHOH by GC-MS employing NICI and SIM (B) as described in Experimental Procedures. Error bars represent standard error, n = 3. A: *P < 0.05 versus cell associated at 15 min, **P < 0.01 versus cell associated at 0.5 min. B: **P < 0.01 versus cell associated at 30 min, ***P < 0.001 versus cell associated at 0.5 and 15 min.

Fig. 6.

2-ClHDA metabolites, 2-ClHA and 2-ClHOH, do not induce neutrophil chemotaxis. The lower wells of a multiwell Boyden chamber contained either chemotaxis buffer alone, chemotaxis buffer with DMSO, 100 nM fMLP (positive control), 100 nM 2-ClHA or 2-ClHOH, or a combination of 100 nM fMLP with 10 or 100 nM 2-ClHA or 2-ClHOH. 2 × 10⁵ neutrophils were loaded into each upper well. Error bars represent SEM, n = 3. A: *P < 0.05 or (B) **P < 0.01 versus 100 nM fMLP.

Fig. 7.

Plasma and BALF levels of 2-ClHA increase in mice exposed to SeV. Adult C57BL/6 mice were infected with SeV (14,000 pfu) intranasally; controls received sterile PBS. 2-ClHA was converted to its PFB ester and quantified by GC-MS employing NICI and SIM. Error bars represent SEM; control mouse BALF (n = 3), virus infected mouse BALF (n = 4) (A), control mouse plasma (n = 4), and virus infected mouse plasma (n = 7) (B). Total RNA was isolated from lungs of control (n = 3) and SeV-infected (n = 4) mice, and the level of SeV-specific RNA was quantified by densitometry of real-time PCR products run on agarose gel. Data were normalized to 36B4 mRNA levels (C). The presence of inflammatory cells in the lungs of control and SeV-infected mice was examined using an MPO activity assay (D). *P < 0.05 (A,B,D) and ***P < 0.005.