In periodontal disease and other diseases, resistance to infection is enhanced when neutrophils respond to lipopolysaccharide (LPS) from bacteria and become primed. Priming results in increased ability of neutrophils to produce microbicidal oxygen radicals and enzymes when they phagocytize microbes. However, to limit inflammation and to avoid tissue damage by oxygen radicals and other products of primed neutrophils, the bacteria must be efficiently killed, and their LPS must be rapidly inactivated. The objective of this proposal is to investigate the inactivation of LPS by neutrophils. In preliminary experiments, LPS that had been pre- incubated with neutrophils lost its ability to prime fresh neutrophils. The LPS was not absorbed or destroyed, because its ability to gel Limulus lysate (a standard test for LPS) was not impaired. In preliminary work, a protein of approximately 80 kDa that inactivated LPS was isolated from neutrophil culture supernatant.
Specific Aim 1 is to identify the enzyme or binding factor from neutrophils that inactivates LPS. The LPS inactivating protein will be purified. The purified protein will be characterized and partially sequenced, to see if it is a novel protein.
Specific Aim 2 is to identify any chemical change in LPS that results from inactivation by neutrophils. Inactivated LPS will be purified and chemically characterized by chromatography and mass spectroscopy.
Specific Aim 3 is to identify factors in serum that protect LPS against inactivation. The affinity and concentration of LPS protecting factors in serum will be compared with affinity and concentration of the LPS in activating factor of neutrophils. This might help to clarify the role of serum exudate in potentiating neutrophil-mediated inflammatory responses in tissues.
Specific Aim 4 is to determine the biological activity of inactivated LPS on monocytes. LPS inactivated by neutrophils might be active on monocytes. The ability of monocytes to inactivate LPS will also be examined.
Specific Aim 5 is to determine if LPS's from periodontal pathogens are inactivated normally by neutrophils. LPS from a pathogen might resist inactivation, leading to chronic inflammation and loss of connective tissue and bone. Alternatively, LPS from a pathogen might already exist in a form that appears inactivated to neutrophils. Such LPS might allow a bacterium to avoid priming neutrophils and therefore avoid being efficiently killed by neutrophils. This investigation might lead to stratagems for increasing resistance to infection and decreasing tissue damage in periodontal disease and other chronic infections.
