Respiratory Bursts in Garlic Treated Macrophages

1. Respiratory Bursts in Garlic Treated Macrophages Shannon Proctor Departments of Chemistry and Biology Jacksonville University

2. Macrophages • Greek for “big eaters” • Originate from white blood cells called monocytes • They play many roles in the body • Major role in immunity and the immune response

3. Electron micrograph of a macrophage attacking bacteria. www.hartnell.edu/biology Representation of macrophages that I worked with using an inverted microscope.

4. Background • Garlic has been used since ancient times as an herbal remedy. • Heart disease • Tumors • The Plague • Antibacterial properties

5. Background • Previous research has shown that garlic enhances macrophage activity. • Leishmania major, Ghazanfari 2005 • Augmentation of function, Lau 1991 • Enhanced immunocompetence, Lamm 2001 • Our lab has shown that garlic does increase phagocytosis in macrophages

6. Internalization vs. Digestion • Macrophages will phagocytose many things • Engulf the particle • May or may not destroy • There is a chemical change when they digest • O2 uptake increases • Rapid release of reactive oxygen species • This is what we want to measure

7. Abstract • An increase in the respiratory burst would be significant • Nitro-blue tetrazolium (NBT) • Yellow solution that turns blue

8. Cell Culture • IC-21 Cells • Cultured in RPMI media • Incubated at 37°C, 5% CO2

9. Phagocytosis Assay • Subcultured at 100% confluency • Detached • Resuspended in control and media with garlic (0.5% or 1%) • Incubation for 3hrs • Addition of latex beads and fresh media

10. Phagocytosis Assay • Repeated every 30, 60, and 90 minutes • Incubation throughout • Assay stopped by washing • Staining using Hema 3 kit • Inverted microscope for viewing • 175-250 cells were counted and analyzed

11. 8-Well Slide

13. Results

14. Results * = Control (60 % ethanol) = Garlic * P=.055, Student’s T-test Percent phagocytosis measured using the following equation: (# of macrophages with engulfed bead ÷ total # macrophages) X 100 D. Lindsey and K. Jackson, 2006

15. Results • NBT Test • Dissolve 1 tablet in water • Fix cells (no staining) • Cover cells with NBT solution and incubate at 37°C for 30 minutes • Rinse with filtered PBS • Observe color under microscope

16. Conclusion • Future research could use latex beads to ensure phagocytosis and use a bacterium, for example, to measure the respiratory burst

17. Acknowledgements • Dr. Karen Jackson, advisor • Patricia Roman and Curtis Dobrowolski • Jacksonville University Public Safety

18. References • Buescher, E., Alling, D., and Gallin, J. (1985) Use of an x-linked human neutrophil marker to estimate timing of lyonization and size of the dividing stem cell pool. Journal of Clinical Investigation 76 (4), 1581-1584. • Ghazanfari, T., Hassan, Z., and Khamesipour, A. (2005). Enhancement of peritoneal macrophage phagocytic activity against Leishmania major by garlic (AlliumSativum) treatment. Journal of Ethnopharmacology 103 (3), 333-337. • Lindsey, D. and Jackson, K. (2006). AlliumsativumEffects on Phagocytic Activity of IC-21 Peritoneal Macrophages in vitro