Characterization of neutrophil bactericidal activity in three-dimensional fibrin gels

I developed and validated experimentally a simple, reproducible, three-dimensional in vitro fibrin gel system that is useful for many different types of analyses of interactions of leukocytes with one another, with microbes, and with other types of cells (e.g. tumor cells). These gels can be formed within minutes in sizes ranging from 50 mu--1500 mu in thickness and 6--100 mul in volume by addition of thrombin to solutions containing fibrinogen at concentrations similar to those found in plasma or exudates (i.e. 1--3 mg/ml). These gels can be lysed within minutes with trypsin or plasmin, allowing >99% recovery of mammalian and/or bacterial cells embedded within them.; I have used these fibrin gels to identify the involvement of various components of plasma (i.e. IgG, the third and fifth components of complement and their cleavage products), chemoattractants (i.e. C5a, LTB4 and fMLP), neutrophil membrane receptors (e.g. beta2 integrins), and cytosolic components (e.g. granules) in killing of Staphylococcus epidermidis by neutrophils. These experiments show that, (1) opsonization of S. epidermidis with C3 is required for neutrophil to kill these bacteria efficiently in fibrin gels; (2) C5a formed by C5 convertases on the surfaces of S. epidermidis increases the efficiency of neutrophil bactericidal activity at neutrophil concentrations <4 x 107/ml in fibrin gels, but not in stirred suspension. Optimal neutrophil bactericidal activity requires each bacterium to release C5a from its surface, thereby creating a gradient of C5a that directs neutrophils toward it; (3) fMLP, at concentrations found in human colon, inhibits neutrophil migration, thereby reducing the efficiency of neutrophil bactericidal activity in fibrin gels; (4) neutrophil bacterial killing in fibrin gels requires activated ERK but not beta2 integrins.; I developed a generally applicable mathematical model for assessing neutrophil bactericidal efficiency in stirred suspensions and in tissue-like environments at neutrophil concentrations ranging from 105 to 10 7/ml, and at bacterial concentrations ranging from 103 to 108 colony forming units (CFU)/ml.; Using my mathematical model I identified a new parameter for assessing the minimal neutrophil concentration needed to control bacterial growth. I have termed this new parameter the critical neutrophil concentration (CNC). The CNC is the neutrophil concentration at which the rate of neutrophil bacterial killing is precisely equal to the rate of bacterial growth. Thus at the CNC, the bacterial concentration remains constant. (Abstract shortened by UMI.)...