In the present report, we have conducted comparative proteomic analysis to identify proteins relevant to the functionality of AMs at the exudative phase and recovery phase during the course of ALI/ARDS. Our data support that AMs undergo a functional property switch during ALI/ARDS initiation and recovery, in which AMs initiate, amply and perpetuate inflammatory responses in the early stage (exudative phase) of ALI/ARDS, while AMs manifest antiinflammatory properties to prevent further tissue injury and perpetuated inflammation in the recovery phase of ALI/ARDS. Therefore, those proteins characterized through current report could be valuable biomarkers for assessing ALI/ARDS progression and prognosis.
Of note, cathepsin B, a lysosomal cysteine proteinase, was found to be significantly upregulated in the exudative phase of AMs, which could be caused by the reduced lysosomal membrane stability, increased permeability and even membrane rupture. Given the role of cathepsin B played in enhancing inflammatory response, copious amount of cathepsin B released from AM lysosome into the cytoplasm or tissue space would promote ALI/ARDS progression by exacerbating inflammatory response in the lung, and therefore, cathepsin B could serve as a biomarker for early diagnosis of ALI/ARDS. In line with this assumption, cathepsin B has been suggested to a potential prognostic marker for inflammatory breast cancer .
Similarly, heat shock protein 27 (HSP27, spot 882) was characterized to be upregulated in the exudative phase of AMs, but significantly downregulated in the recovery phase of AMs. HSP27 belongs to the heat shock protein (HSP) family, which has been noted to be widely involved in many biological processes such as cell proliferation, differentiation and apoptosis [17–20]. During the course of an inflammatory response, increased synthesis of cytokines induces the expression of heat shock proteins to prevent endoreticular (ER) stress. More recently, heat shock proteins have also been recognized to be potent mediators of inflammation and immunity . Particularly, similar as HMGB1, passively released heat shock proteins are considered to be innate alarmins for the initiation of tissue repair or clearance of invaded pathogens [22, 23]. In the exudative phase of ALI/ARDS, activated AMs are presumed to secrets copious amount to pro-inflammatory cytokines to perpetuate inflammatory response in the lung, which requires the expression of heat shock proteins such as HSP27 to prevent ER stress. On the other hand, those induced heath shock proteins can be passively released from the damaged AMs, which then amplify or exacerbate inflammatory responses along with ALI/ARDS progression. Therefore, similar as cathepsin B, HSP27 possesses the properties to be a biomarker for ALI/ARDS early diagnosis.
Of note, unlike cathepsin B and HSP27, neutrophil elastase (NE) inhibitor or serine protease inhibitor (spot 505) was characterized to be significantly increased in the recovery phase of AMs. NE is the major protease released by PMNs during the course of ALI/ARDS. It impacts epithelial integrity to induce lung injury by digestion and degradation of the extracellular matrix. As a result, NE has been considered to be a main effector in ALI/ARDS inflammatory cascade . Indeed, studies in ALI/ARDS patients have consistently revealed that NE expression is associated with disease progression. In sharp contrast, NE inhibitor attenuates the release of inflammatory mediators to effectively suppress inflammatory cytokine cascades. Particularly, it antagonizes microbial activities through inhibition of TNF-a/IL-1β expression and attenuation of NF-kB activation. Indeed, recombinant human NE inhibitor has been found to provide protection for rats against cystic fibrosis induced lung injury . In line with this notion, a variety of NE inhibitors have been employed in the settings against severe infections . Thus far, three categories of protease inhibitors are found to be naturally distributed in the bronchus and lung tissues, the a1-proteinase inhibitor (a1-Pi), the secretory leukocyte protease inhibitor (SLPI) and the specific protease inhibitors (Elafin). All together, it is plausible to assume that NE inhibitor could be an ideal biomarker for ALI/ARDS prognosis.
Interestingly, S100-A8 (spot 896) and S100-A9 (spot 805) were found to be highly expressed in the recovery phase of AMs as well. S100 is a group of low-molecular-weight calcium-binding proteins. Although a great deal of effort has been devoted to the studies of S100 proteins, their functional relevance is still remained obscure . Torre and colleagues found that S100 proteins interact with several key factors implicated in ALI/ARDS pathogenesis including TNF-а, IL-6 and p38MAPK, but has no obvious connections with other non-inflammatory proteins , suggesting that S100 proteins (mainly S100A8 and S100A9) could be a potential biomarker for ALI/ARDS prognosis and a therapeutic target in the settings of patients with ALI/ARDS.
Of note, although the above identified proteins have great potential to serve as biomarkers for ALI/ARDS early diagnosis and prognosis, a simulation dataset with new ALI/ARDS patients would be essential for validation of their feasibility. Furthermore, this report only characterized 27 informative proteins, and it is likely that more proteins should be implicated in this functional switch for AMs during the course ALI/ARDS. Therefore, additional studies with more patients and advanced technologies would be necessary to further address this issue. Also, we only selectively conducted Western blot analysis for S100A9 and HSP27, and follow up studies aimed at confirming the expression changes for the rest proteins are needed.