Neutrophils from Both Susceptible and Resistant Mice Efficiently Kill Opsonized Listeria monocytogenes

Abstract

Inbred mouse strains differ in their susceptibility to infection with the facultative intracellular bacterium , largely due to delayed or deficient innate immune responses. Previous antibody depletion studies suggested that neutrophils (polymorphonuclear leukocytes [PMN]) were particularly important for clearance in the liver, but the ability of PMN from susceptible and resistant mice to directly kill has not been examined. In this study, we showed that PMN infiltrated the livers of BALB/c/By/J (BALB/c) and C57BL/6 (B6) mice in similar numbers and that both cell types readily migrated toward leukotriene B4 in an chemotaxis assay. However, CFU burdens in the liver were significantly higher in BALB/c mice than in other strains, suggesting that PMN in the BALB/c liver might not be able to clear as efficiently as B6 PMN. Unprimed PMN harvested from either BALB/c or B6 bone marrow killed directly, and pretreatment with autologous serum significantly enhanced killing efficiency for both. were internalized within 10 min and rapidly triggered intracellular production of reactive oxygen species in a dose-dependent manner. However, PMN from gp91-deficient mice also readily killed , which suggested that nonoxidative killing mechanisms may be sufficient for bacterial clearance. Together, these results indicate that there is not an intrinsic defect in the ability of PMN from susceptible BALB/c mice to kill and further suggest that if PMN function is impaired in BALB/c mice, it is likely due to locally produced modulating factors present in the liver during infection.

Neutrophils Are More Effective than Monocytes at Phagosomal Containment and Killing ofListeria monocytogenes

“…Neutrophils are more effective at L. monocytogenes killing than inflammatory monocytes Phagosomal containment of L. monocytogenes serves little to no purpose if the bacteria are not ultimately killed. Neutrophils and macrophages have been identified as having the primary function of bacteria killing (10,15,36), although there have been some studies that concluded that monocytes also kill during infection (17,33). LAMP-1 is a common lysosomal marker and also a necessary component of the mature phagosome (i.e., the phagolysosome) (37).…”

“…Neutrophils also generated more ROS than monocytes when stimulated. However, this may not necessarily be the manner by which they kill, as a recent finding by Pitts et al (15) concluded that ROS is not required for killing of L. monocytogenes by neutrophils. Mice lacking the gp91 subunit of the NADPH oxidase had previously been demonstrated to be more susceptible to L. monocytogenes (18,19).…”

“…Mice lacking the gp91 subunit of the NADPH oxidase had previously been demonstrated to be more susceptible to L. monocytogenes (18,19). Interestingly, the recent study by Pitts et al (15) showed that neutrophils from the gp91 mice were able to kill as effectively as neutrophils from wild-type mice. Therefore, although ROS may be contributing to the efficiency of neutrophil killing, it might not be the primary manner by which L. monocytogenes is killed.…”

“…For in vitro studies, the bacteria were diluted to the desired multiplicity of infection (MOI) in phenol red-free DMEM lacking antibiotics and supplemented with L-glutamine, 10% FBS, and vitamins (Invitrogen). Bacteria were opsonized by adding mouse serum to the solution at a final concentration of 10% and placed on ice for 30 min before being added to the cells (15). Mice were inoculated i.v, for in vivo infections, with bacteria diluted in PBS to the desired concentration.…”

“…Depletion of both cells concurrently or separately during L. monocytogenes infection leads to an increase in host susceptibility (12)(13)(14). Phagocytosis and bacterial killing of L. monocytogenes are functions that have been attributed primarily to neutrophils during the early stages of infection (10,15). Specific depletion of neutrophils with use of the Ly6G (1A8) Ab led to increased bacterial burden during infection which is indicative of a lack of bacterial uptake and killing (12,16).…”