Specific monoclonal antibody to detect the C-terminal peptide of alpha1-antitrypsin: clinical importance

Chronic obstructive pulmonary disease (COPD) is associated with chronic neutrophilic inflammation and pathological changes in circulating levels of alpha1-antitrypsin (AAT), an inhibitor of neutrophil proteases. Variants of C-terminal fragments of AAT have also been identified in human body fluids and tissues, including lungs. However, their biological role and clinical relevance remain largely unknown. Originally it has been thought that these fragments are generated during unspecific cleavage of AAT by metalloproteases. The results of our studies led to unexpected finding that human peripheral blood leukocytes express and release short transcripts, corresponding to 36 amino acid C-terminal peptide of AAT (C-36). Here we show that the hydrophobic C-36 peptide of AAT added to human neutrophil cultures stimulates cell migration and adhesion and induces free radical and cytokine release. Moreover, C-36 forms amyloid-like fibrils, in vitro, which can activate neutrophils. We hypothesize that the C-fragment of AAT may serve as a new marker in COPD patients with and without inherited AAT deficiency. In this study we present data on monoclonal antibody that specifically recognizes C-36 peptide but does not react with full-length native or modified forms of AAT such as oxidized, polymeric or complexed with elastase. The applications we used for antibody validation include dot blots, western blots, flow cytometry, and immunohistochemistry. We also provide evidence that cellular expression of C-36 is associated with more severe stages of COPD. This finding encourages to re-evaluate the significance of AAT fragments.