Lysophosphatidylcholine (LPC) is a lipid mediator with pro-inflammatory and pro-atherogenic activities. It is believed to be a critical factor underlying cardiovascular diseases,1,2 and several pathological conditions are associated with elevated LPC levels in the circulation.3-5 LPC has a broad-spectrum of pro-inflammatory activities, including promotion of cell growth,6 migration,7,8 secretion of chemokines and cytokines,9,10 generation of reactive oxygen species,11 and upregulation of adhesion molecules such as ICAM-1, VCAM-1, and selectins.12

Evidence implicates involvement of LPC in inflammatory injury of lungs, such as acute lung injury and chronic diseases such as asthma. In lungs from late stage adult respiratory distress syndrome,13 lungs challenged with antigen14 or instilled with lipopolysaccharide,15 elevated amounts of LPC are reported in the broncho-alveolar lavage fluids (BALF). Furthermore, the administration of exogenous LPC into lungs of animal models promotes eosinophil infiltration,16 increases airway resistance,16,17 and increases airway epithelial permeability.18,19 These findings suggest that elevated levels of LPC in the extracellular lung fluids can induce inflammatory injury on the airway and alveolar cell populations. To date, the role of LPC in lung diseases is incomplete.

Asthma is a chronic inflammatory disease of the airways characterized by epithelial injury, leukocytic infiltration, airway hyper-reactivity, and airway wall remodeling. We postulate that the chronic inflammation is attributed, at least in part, to elevated amounts of LPC in the lungs. In lungs of asthmatic subjects14,20-22 and in animal models of asthma,23,24 the LPC-generating enzyme, phospholipase A₂ (PLA₂) is significantly increased. PLA₂ hydrolyzes membrane phosphatidylcholines and oxidized phosphatidylcholines at the sn-2 position, generating LPC following release of the free fatty acid.25 However, it is not known whether the inherent LPC content in lungs is increased in asthmatics. To begin to investigate the involvement of LPC in asthma, this study determined LPC levels in the BALF from asthmatic subjects.

Table 1 summarizes the subjects' clinical characteristics, of which the lung function data (FEV1) were used to categorize subjects into non-asthmatic control, mild- or moderate-asthmatics according to guidelines from the Global Initiative for Asthma.26 As expected, the BALF in the mild- and moderate-asthmatics presented with several indices of inflammation (Table 1). A cell differential analysis of BALF from mild-asthmatics indicated higher percentages of eosinophils and lymphocytes relative to non-asthmatic controls. These proportions of leukocytes further increased in the moderate-asthmatics, which now included neutrophils. An upward trend was observed in the total cell count and protein content in the BALF of mild- and moderate-asthmatics. We also found a progressive increase of IL-8 content in the non-asthmatics to the mild- and moderate-asthmatics. In summary, the BALF of asthmatics contain several markers of inflammation that progress with degree of asthma severity.

We found that non-asthmatics have basal levels of all 3 LPC species at a range of 180-250 ng/mL of BALF (Fig. 1). The saturated species LPC16:0 and LPC18:0 were significantly increased in asthmatic subjects with FEV1 characteristic of moderate asthmatics (Table 1) when compared with the non-asthmatic controls (Fig. 1). Asthmatic subjects with less severe FEV1 representing the mild-asthmatics showed no increases of the LPC species relative to controls. The findings indicate that the lung mucosal surface of moderate-asthmatics is likely to be chronically exposed to ~1.3 to 1.9-fold higher amounts of LPC16:0 and LPC18:0, respectively, than in non-asthmatics.

We next determined whether the increased LPC content in lungs of moderate-asthmatics could be attributed to secreted PLA₂, hydrolyzing phosphatidylcholines of cell membranes or surfactant. Measures of the PLA₂ activity of BALF indicated that moderate-asthmatics, but not mild-asthmatics, show a 3-fold increase over non-asthmatics (Fig. 2A). The infiltrated lung leukocytes as well as the airway epithelium are the likely cell populations that secrete PLA₂ into the lung luminal fluids. Quantitative RT-PCR evaluation of the cell fraction of the BALF indicated that subtype secretory sPLA₂-X mRNA was upregulated in moderate-asthmatics, but there were no changes in other subtypes (sPLA₂-XII, cPLA₂-IVa and iPLA₂-VI) (Fig. 2B). These findings suggest that sPLA₂-X is likely a key subtype in the generation of LPC in asthmatic subjects.

The central new finding is that inherent levels of LPC16:0 and LPC18:0, but not LPC18:1, were significantly elevated in lung lining fluids of subjects with lung function impairment characteristic of moderate asthma. These corresponded to 1.3- and 2-fold respective increases in LPC16:0 and LPC18:0 over non-asthmatic controls. Interestingly, in atopic asthmatic subjects challenged with antigen, LPC16:0 is increased in BALF when compared to saline challenge;14 however, the LPC level in non-asthmatic control was not reported to ascertain the effects in the absence of antigen challenge in asthmatics. Reports indicate that among acyl-lysophospholipid species, LPC16:0 and LPC18:0 exert the greatest pro-inflammatory activities, such as eosinophil adhesion,27 neutrophil priming,28 and cytokine secretion.9,10 Altogether, the results implicate a pathogenic role of LPC in the chronic inflammatory injury of the asthmatic airway.

We can only speculate on the inflammatory potential of an elevated LPC content in lung lining fluids of moderate-asthmatics, since precise in vivo LPC concentrations are unknown and its bioactivity is dependent on the free form (not bound to albumin or other proteins).1 In in vitro culture studies, LPC concentrations of 1-100 µM are effective in inducing a wide range of inflammatory activities, such as cell proliferation,6 migration,7,8 increases in endothelial permeability,29 leukocyte adhesion,27,30 neutrophil priming,28 and cytokine secretion.9,10 The calculation of molar units of LPC from our data indicated 70 µM of LPC16:0 and 140 µM of LPC18:0 from moderate asthmatics, which are values comparable to those used in in vitro studies. This finding suggests that the increased LPC content in lungs of moderate-asthmatics could be sufficient in promotion of airway inflammatory injury.

The results indicate that the increased LPC content in lungs of moderate-asthmatics was accompanied by significant increases in the PLA₂ activity in the lung lining fluids, providing evidence that increased generation of LPC was likely attributed to secreted PLA₂. Moreover, the PLA₂-induced increase in LPC is expected to be accompanied by concomitant equimolar amounts of free fatty acids, which can be arachidonic acid depending on the substrate phosphatidylcholine species. The well-recognized pathway of arachidonic acid metabolism by cyclooxygenases and lipoxygenases will, in turn, produce respective prostaglandins and leukotrienes, which are believed to be important lipid mediators in asthma. Thus, increases in LPC together with increases in arachidonic acid metabolites suggests that the inflammatory potential of PLA₂ is likely significantly greater than recognized previously.

Furthermore, the increased PLA₂ activity was attributed to, at least in part, to upregulated sPLA₂-X mRNA in the leukocytic infiltrate, a finding consistent with the report of overexpression of sPLA₂-X in airway epithelial cells and bronchial macrophages of human asthmatic subjects.20 Although our protocol did not allow us to identify the cell type(s) responsible for the upregulated sPLA₂-X, analysis of the cell differential indicated that in moderate-asthmatics, the infiltrate population shifted towards increasing eosinophils, neutrophils, and lymphocytes (Table 1).

In summary, the lung lining fluids in moderate-asthmatics showed an inherently elevated content of LPC16:0 and LPC18:0. Such increased LPC is a potential injurious mediator in the initiation and/or progression of airway inflammatory injury in asthma.