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Jacinta E. Cooper, Ph.D.

Janoff Lab


Complete Title of Thesis:
"Complement-independent killing of antibody-opsonized Streptococcus pneumoniae by activated human neutrophils"

Prepared under the direction of: Edward N. Janoff


Capsule-specific IgG and IgA support killing of S. pneumoniae in the presence of complement and phagocytes (neutrophils and alveolar macrophages). However, levels of complement in the lung are low, and pneumococci employ several mechanisms to evade complement. We hypothesize that activation of phagocytes elicited by pathogen-induced local inflammation may abrogate the need for high levels of complement for antibody-dependent killing in the lung. We stimulated neutrophils with TNFα or C5a to characterize activation by changes in cell surface markers (esp. CD11b and CD62L) and oxidation of dihydrorhodamine by flow cytometry. We quantified uptake of FITC-labeled pneumococcus by human neutrophils by flow cytometry. We utilized opsonophagocytosis assays to measure the ability of activated neutrophils to reduce colony counts in the absence of complement and to enhance binding and uptake of pneumococci.

Pretreatment of neutrophils with TNFα or C5a supported dose-dependent killing by capsule-specific human monoclonal IgG (100 ng/ml) without complement, comparable to results with complement. These reductions required the presence of antibody, as treated neutrophils alone or in the presence of complement, were unable to facilitate killing. We extended these findings by determining that pretreatment of neutrophils with TNFα increased the binding and uptake of FITC-labeled bacteria by flow cytometry, and enhance killing of the organism. Killing likely occurred intracellularly because treating the neutrophils with cytochalasin D, a drug that prevents remodeling of the actin cytoskeleton abolished killing in both the presence and absence of complement. Complement-independent killing by TNFα- and C5a-stimulated neutrophils was dependent on serine proteases as pretreating the neutrophils with AEBSF, a serine protease inhibitor, abrogated complement-independent killing. In contrast, DPI, a NAPDH oxidase inhibitor had no effect. Further, chloroquine enhanced complement-independent killing and was partially blocked by AEBSF, indicating serine proteases play a role in pneumococcal killing in the absence of complement. In conclusion, despite low levels of complement in the lung, mucosal antibodies may support killing of S. pneumoniae by phagocytes in the presence of local inflammatory mediators.