Role of serine proteases in the regulation of interleukin-877 during the development of bronchopulmonary dysplasia in preterm ventilated infants.

RATIONALE: The chemokine interleukin-8 is implicated in the development of bronchopulmonary dysplasia in preterm infants. The 77-amino acid isoform of interleukin-8 (interleukin-877) is a less potent chemoattractant than other shorter isoforms. Although interleukin-877 is abundant in the preterm circulation, its regulation in the preterm lung is unknown. OBJECTIVES: To study expression and processing of pulmonary interleukin-877 in preterm infants who did and did not develop bronchopulmonary dysplasia. METHODS: Total interleukin-8 and interleukin-877 were measured in bronchoalveolar lavage fluid from preterm infants by immunoassay. Neutrophil serine proteases were used to assess processing. Neutrophil chemotaxis assays and degranulation of neutrophil matrix metalloproteinase-9 were used to assess interleukin-8 function. MAIN RESULTS: Peak total interleukin-8 and interleukin-877 concentrations were increased in infants who developed bronchopulmonary dysplasia compared to those who did not. Shorter forms of interleukin-8 predominated in the preterm lung (96.3% No-bronchopulmonary dysplasia vs 97.1% bronchopulmonary dysplasia, p>0.05). Preterm bronchoalveolar lavage fluid significantly converted exogenously added interleukin-877 to shorter isoforms (p<0.001). Conversion was greater in bronchopulmonary dysplasia infants (p<0.05). This conversion was inhibited by α-1 antitrypsin and antithrombin III (p<0.01). Purified neutrophil serine proteases efficiently converted interleukin-877 to shorter isoforms in a time- and dose-dependent fashion; shorter interleukin-8 isoforms were primarily responsible for neutrophil chemotaxis (p<0.001). Conversion by proteinase-3 resulted in significantly increased interleukin-8 activity in vitro (p<0.01). CONCLUSIONS: Shorter, potent, isoforms interleukin-8 predominate in the preterm lung, and are increased in infants developing bronchopulmonary dysplasia, due to conversion of interleukin-877 by neutrophil serine proteases and thrombin. Processing of interleukin-8 provides an attractive therapeutic target to prevent development of bronchopulmonary dysplasia.

Examination of in vitro epithelial cell lines as models for Francisella tularensis non-phagocytic infections.

Francisella tularensis (F. tularensis), the causative agent of tularemia, has long been known to invade and occupy non-phagocytic epithelial cells. Many epithelial cell infection models have been developed to study this process; however, due to the lack of consensus on infection methods and precise experimental procedures to evaluate invasion and replication, selection of appropriate models to use based on the literature is challenging. To evaluate in vitro non-phagocytic cell infection models, we chose 8 epithelial cultured cell lines from published models to infect with F. tularensis subspecies novicida (F. novicida) and compared the results to a recently developed model that used the mouse hepatocyte BNL CL.2 cell line. We utilized classical gentamicin-based invasion assays to determine total intracellular bacterial loads and employed microscopic examination with staining techniques that distinguished between intracellular and extracellular bacteria to provide an accurate assessment of the proportion of invaded host cells and the degree of bacterial replication. We found that COS-7 cells exhibited the greatest invasion rates; CMT-93 cells contained the largest intracellular bacterial loads; ad HEK-293s were capable of invasion and replication rates at high levels, but required shorter infection incubation times. Although COS-7, CMT-93 and HEK-293 cell lines may be suited to study certain aspects of invasion or replication, we found that BNL CL.2 cells appeared the most appropriate to study the overall pathogenesis of F. novicida when examined in toto.