The long-term goals of this laboratory are to identify the role of heat shock proteins (hsp) in the mechanisms involved in the neutrophil's interaction with periodontal disease microorganisms and in its subsequent defense of the host against these microbes. One role of hsp has been proposed to be the protection of the neutrophil from the autooxidation due to the oxygen free radicals that are generated as part of its oxygen- dependent killing mechanism. Our preliminary studies indicate that when neutrophils phagocytize periodontal pathogens, such as P. gingivalis, specific hsp (e.g., hsp 70) are not expressed. The work proposed in this application will address the following hypothesis: Oxidation-protective hsp are not expressed by neutrophils following the phagocytosis of specific periodontal pathogens. Absence of hsp expression may be a result of either 1) direct inhibition of hsp synthesis at the transcriptional or translational levels; or 2) failure to induce hsp expression during phagocytosis. In other systems, the failure to express specific hsp has been correlated with the absence of a detectable respiratory burst and the over/under production of specific proinflammatory cytokines (IL-1, IL-6, TNFalpha). To test this hypothesis we propose the following specific aims: 1) to characterize the hsp profiles expressed during the phagocytosis of selected periodontal pathogens by neutrophils obtained from healthy donors; 2) to characterize the expression of hsp mRNA following phagocytosis of selected periodontal pathogens; and, 3) to measure levels of proinflammatory cytokine messages and products produced at the time of phagocytosis. Characterization of hsp profiles expressed by normal human neutrophils following the phagocytosis of microbes such as P. gingivalis will be accomplished by performing [35S]-methionine labeling of neutrophil proteins synthesized following phagocytosis and identifying proteins synthesized by autoradiography and Western blot analysis using monoclonal antibodies to human hsp. The effect of selected microorganisms on the expression of specific hsp mRNA will be performed by Northern blot analysis using cDNA probes for specific human hsp. Inhibitors of transcription and translation will be employed to identify the levels at which specific microorganisms induce or inhibit hsp expression. Expression of proinflammatory cytokines will be examined by detecting selected cytokines and cytokine mRNA before and after phagocytosis.
| Lopatin, D E; Jaramillo, E; Edwards, C A et al. (1999) Cellular localization of a Hsp90 homologue in Porphyromonas gingivalis. FEMS Microbiol Lett 181:9-16 |
