Rate of Phagocytosis in Macrophages Stimulated by Astragalus membranaceus

1. Rate of Phagocytosis in Macrophages Stimulated by Astragalus membranaceus Curtis Dobrowolski Department of Biology and Marine Science Jacksonville University

2. Introduction • Macrophages are the first line of defense in the human immune system • Phagocytose • Important antigen presenting cells, required for adaptive immune response

3. Introduction • Advantages of increased phagocytosis • Removal of pathogen • More T cell interaction, faster adaptive immune response • More memory • More Antibody production • More cytokines released • increased inflammation and response from other immune cells www.phototakeusa.com

4. Introduction • Astragalusmembranaceus • Shrub native to east Asia, primarily China • A. membranaceus is a common medicinal herb used in eastern medicine • One of the 50 fundamental Chinese medicinal herbs • Huángqí- root tea • Used primarily when symptoms of an infection first arise • Shown to increase antibody production in mice (1) • Increased cytokine production (2) http://nccam.nih.gov/health/astragalus/

6. Hypothesis • In vitro phagocytosis in macrophages increases when subjected to A. membranaceus

7. Methods • IC-21 macrophages were cultured in tissue culturing flasks • Macrophages grown RPMI 1640 media supplemented with 10% fetal bovine serum and 1% pen/strep antibiotic • Incubated at 37°C with 5% CO2 until cells were fully confluent www.millan.com

8. Methods • Macrophages were lifted with Ca2+ depleted PBS Solution • PBS= phosphate buffered saline • PBS/macrophage solution was stained with trypan blue vital dye and counted using a hemocytometer • Using (Q1+Q2+Q3+Q4)*104=cells/ml equation concentrations were determined www.ruf.rice.edu

9. Methods • Using the count from the hemocytometer we placed 300,000 cells in each micro-chamber • Equal amount of RPMI 1640 media were added to each micro-chamber • Incubated for minimum of 3 hours www.usascientific.com

10. Methods • Media/PBS solution was removed • Macrophages were incubated for a minimum of 24 hours to allow the medias to have its effects on the macrophages • Media alone • 0.3% EtOH Control • 1% astragalus EtOH Control Control 1%A.M. 1%A.M. EtOH Control Control 1%A.M. 1%A.M.

11. Methods • Media was removed • 1μ diameter fluorescently tagged latex beads were layered on top of macrophages, at a concentration of 20 beads per cell • Media alone, EtOH and astragalus media were then added to the respective wells

12. Methods • Media and beads were removed and wells were washed 3 times • To remove beads that have not been phagocytosed • Wells were removed and slides were stained with protocol hema 3 stain • Increasing phagocytosis • More cells phagocytosing • Subset of cells phagocytosing more

13. Methods • Data collected • Total number of cells (75-175 cells counted per experimental well) • Number of cells that phagocytosed any beads • Number of cells that phagocytosed >1 bead • Rate of phagocytosis calculated

14. Results

15. Results

16. Conclusions • The rate of in vitro phagocytosis in macrophages increases when subjected to A. membranaceus • Could be beneficial in helping immuno-compromised patients • HIV/AIDS patients • Genetic disorders • More research is need to fully understand A. membranaceus’ immuno-stimulating properties and benefits

17. Acknowledgements • I would like to thank Dr. Karen Jackson PhD, for all of her help in culturing macrophages in a less than ideal environment • Also I would like to thank Sally and Judy in the Science and Math division office, for signing me out a lab key nearly everyday of the year

18. References • Zhao, K S, Mancini, C, et al., “Enhancement of the immune response in mice by Astragalus membranaceus extracts.”, Immunopharmacology, 1990 Nov-Dec; 20(2): 225-233 • Bedir E, Pugh N, et al., “Immunostimulatory effects of cycloartane-type triterpene glycosides from astragalus species.”, Biol Pharm Bull, 2000 Jul; 23(7): 834-7