We have used three-dimensional fibrin and collagen I gels to compare neutrophil (PMN) bactericidal activity in tissue-like environments vs. stirred suspensions and discovered that a critical PMN concentration (CNC) is required to block the growth of bacteria in fibrin and collagen gels and in stirred suspensions. The concept that a critical leukocyte concentration is required to carry out specific immune effector functions is both novel and important. It unifies a large body of literature on host defense against bacterial infections, and provides a conceptual framework for assessing quantitatively the concentration of any class or type of immune cell that must be delivered to a tissue to execute a specific function. We have derived an equation that enables us to calculate the CNC for all bacterial concentrations under all experimental conditions so far tested. Using it we found the CNC in suspension (approximately 4 x 105 PMN/ml), is almost identical to that known to predispose neutropenic humans to sepsis, while that in fibrin gels is 2.5 - 10-fold higher (e.g., 1-4 x 106PMN/ml). Using these gels we have extended our previous observation that specific matrix proteins (e.g., fibrin) and chemoattractants (e.g., fMLP) block PMN migration by showing that in the presence of fibrin, fMLP blocks PMN bactericidal activity. Moreover, we discovered that PMN genetically deficient in a2-integrins phagocytose and kill S.epidermidis as efficiently as wild-type PMN in fibrin gels. We also have discovered that contrary to conventional wisdom, PMN have the capacity to invade and kill >98% of S. epidermidis in mature (5-day old) biofilms. We seek continued support to extend these studies, and to identify mechanisms that regulate emigration of PMN and monocytes from the blood into tissues, processes central to limiting tissue damage, and to delivering sufficient PMN and monocytes to eradicate planktonic and biofilm bacteria, and cytotoxic lymphocytes to kill cancer cells. This application has three Specific Aims.
Aim #1. To measure the critical concentration of monocytes for killing S. epidermidis and E. coli in fibrin and collagen I gels in vitro, and the critical concentrations of PMN and of monocytes for killing of S. epidermidis and E.coli in vivo, and to identify the mechanisms that signal cessation of entry of PMN and of monocytes into dermal sites of bacterial infection.
Aim #2. To identify the immunological mechanisms that impede PMN and monocyte killing of biomaterials-associated S. epidermidis biofilms.
Aim #3. To determine whether the concept of a critical leukocyte concentration also applies to the tumoricidal activities of cytotoxic lymphocytes and monocytes. In other words, must cytotoxic lymphocytes reach a critical concentration within a tumor bed to effect a reduction in tumor mass?
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