Cellular association of antiproteases in lavages from ventilated preterm human neonates.

Abstract

Lung antiprotease activity is routinely assayed in the supernatant of bronchoalveolar lavage fluid (BALF). In this study the cellular fraction of lavages was also analyzed. Functionally active acid-resistant inhibitors with molecular masses characteristic of the mucus proteinase inhibitor (MPI, 14 kDa) and elastase-specific inhibitor (ESI, 7 kDa) were demonstrated by gel chromatography. Immunocytochemical studies of cells obtained at various postnatal time points from lavages of 10 premature infants with chronic lung disease showed that the inhibitors were confined to neutrophils and macrophages. At each time point, about 70% and 21% of these cells, respectively, stained positively. The polyclonal antibodies usually used to detect MPI did not distinguish between MPI and ESI. Because of this cross reactivity, it was not possible to differentiate between MPI and ESI. Analysis with reverse transcriptase-polymerase chain reaction (RT-PCR) of cells from lavages and of nucleated cells isolated from the peripheral blood showed the production of ESI only, but not of MPI. Nevertheless, MPI can associate with neutrophils and macrophages, as was shown in binding studies with the recombinant protein. These data suggest that when assaying bronchoalveolar lavages (BALs) for these antiproteases in the supernatant only, the total pool of inhibitors may be underestimated.

Inhibitors of Elastase in Airway Lavage Samples from Ventilated Preterm Human Neonates

“…A substantial fraction of the acid-resistant inhibitors is associated with the cellular fraction of lavage samples, as previously shown (6). Elastase-specific inhibitor was expressed by these cells, whereas MPI was merely bound to neutrophils or macrophages, without expression of a significant amount of mRNA.…”

“…Besides synthesis by mucosal cells in the airways (5), our previous study in a comparable group of preterm neonates suggested the synthesis of ESI, but not of MPI, and the association of MPI with a substantial fraction of the neutrophils and macrophages of the cellular lavage fraction (6). We did not find expression of MPI in pilot experiments on unstimulated human neutrophils from peripheral blood or macrophages from healthy adult volunteers (6). Recently, it was demonstrated that neutrophils and peritoneal macrophages from mice synthesize MPI (or SLPI) after lipopolysaccharide (LPS) stimulation (15).…”

“…Porcine pancreatic elastase (EC 3.4.21.36) was obtained from Serva (Heidelberg, Germany). Neutrophil elastase (EC 3.4.21.37) was isolated from leukocytes as described earlier (6). Cathepsin G from leukocytes (EC 3.4.21.20), N -succinyl-di-alanylprolyl-phenylalanyl-4-nitroanilide came from Sigma (Deisenhofen, Germany).…”

Reduced proteolysis of surfactant protein A and changes of the bronchoalveolar lavage fluid proteome by inhaled α1-protease inhibitor in cystic fibrosis

“…The enzymatic activity of the human leukocyte elastase (HLE) was determined spectrophotometrically with the specific substrate N-Suc-Ala-Ala-Ala-p-nitroanilide (Elastin Products, Owensville, MO, USA), similarly as described previously [21]. All samples were assessed in triplicate.…”