Neutrophil Monitoring Laboratory (NML)

Research

Clinical Research Interests

Chronic granulomatous disease (CGD) is a rare genetic disease in which peripheral blood neutrophils fail to generate H2O2, resulting in a lifetime of life-threatening infections and abnormal inflammation (granuloma formation). The NML has analyzed blood samples from >100 patients with CGD. Neutrophils from these patients have been evaluated for production of reactive O2 species by several different assays - cytochrome c reduction, FACS analysis of dihydrorhodamine oxidation, etc, and by Western blot analysis to characterize the specific genetic defect. This extensive database of CGD data has yielded an algorithm to predict the probable phenotype of patients with CGD.

In addition, the NML has shown that, compared to control neutrophils, neutrophils isolated from patients with CGD exhibit increased IL 8 production in response to the formyl peptide, fMLF. The increase in neutrophil IL-8 in CGD patients is not due to increased IL-8 mRNA but rather a prolonged IL-8 mRNA response. The IL-8 mRNA response to fMLF can be modulated in vitro by H2O2. The failure to generate H2O2 in CGD may result in a failure to modulate IL-8 generation, leading to abnormal inflammation, and suggests H2O2 production may be involved in more than bactericidal activity.

The NML has been studying a patient with recurrent bacterial infections who exhibited abnormal responses to lipopolysaccharide, IL-1, and IL-18. In collaboration with Dr. Stephanie Vogel (University of Maryland, Baltimore), these abnormal responses were attributed to a genetic defect in IRAK-4. The NML provided both functional data and biological material in this collaborative effort. No subtle defect that could be discerned in the heterozygous carrier parents and grandparents. This human "knockout" will continue to provide a wealth of information on the role of innate immune system in host defense, septic shock, etc.

Basic Research Interests

Human exudative neutrophils obtained from dermal lesions contain almost 200-fold more cell-associated IL-8 than neutrophils isolated from peripheral blood. This increase in neutrophil IL-8 can be mimicked in vitro by treatment of isolated peripheral blood neutrophils with a chemotactic dose (5x10-9 M) of the formyl peptide, fMLF, in the presence of physiologic concentrations of fibrinogen. Treatment of human neutrophils with fibrinogen plus fMLF results in a dramatic increase in IL-8 mRNA. Similar synergy was observed with the neutrophil chemoattractant, LTB4, while C5a, PAF, and GRO-α fail to demonstrate synergy.

Responding to chemotactic signals, neutrophils leave the blood vessel and migrate to the site of infection. This response is dependent on the ability of the neutrophil to undergo dramatic morphological changes. The NML has characterized a dramatic, but transient, change in neutrophil morphology after treatment with the chemotactic peptide, fMLF, using both light microscopy and transmission and scanning electron microscopy. The morphological change is dose-dependent and is prolonged by co-incubation with fibrinogen. The change in neutrophil morphology can be assessed on a flow cytometer using changes in forward and 90o light scatter. This change in morphology is temporally associated with the capability of the neutrophil to produce another neutrophil chemoattractant, interleukin-8. The production of IL-8 production by migrating neutrophils provides a plausible rationale for the recruitment of additional neutrophils and amplification of the inflammatory response.