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Rate of Phagocytosis in Macrophages
Stimulated by Astragalus membranaceus
Curtis Dobrowolski
Department of Biology and Marine Science
Jacksonville University
Introduction
 Macrophages are the first line of defense in the human
immune system
 Phagocytose
 Important antigen presenting cells, required for adaptive
immune response
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
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Introduction
 Astragalus membranaceus
 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
 Huáng qí- 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/
50 Fundamental Chinese Medicinal Herbs
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Agastache rugosa
Alangium chininese
Anemone chininese
Anisodus tanguticus
Aridisia j aponica
Aster tataricus
Astragalus membranaceus
Camillia sinesis- Traditional Black Tea
Cannabis sativa- Marijuana
Cathamus tinctorius
Cinnamomum cassia- Cinnamon
Cissampelous pareira
Coptis chinensis
Corydalis ambigua
Croton tiglium
Daphne genkwa
Datura metel
Datura tatula
Dendrobium nobile -Orchid
Dichroa febrifuga
Ephedra sinica
Eucommia ulmoides
Euphorbia pekinensis
Flueggea suffruticosa
Forsythia suspensa
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Gentiana loureiroi
Gleditsia sinensis
Glycyrrhiza uralensis
Hydnocarpus anthelmintica
Ilex purpurea
Leonurus japonicus
Ligusticum wallichii
Lobella chinensis
Phellodendron amurense
Platycladus orientalis
Pseudolarix amabilis
Psilopeganum sinense
Pueraria lobata
Rauwolfia serpentina
Rehmannia glutenosa
Rheum officinale
Rhododendron tsinghaiense
Saussurea costus
Schisandrac hinensis
Scutellaria baicalensis
Stemona tuberosa
Stephania tetrandra
Styphnolobium japonicum
Trichosanthes kirilowii
Wiksotroemia indica
Hypothesis
 In vitro phagocytosis in macrophages
increases when subjected to A. membranaceus
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
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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
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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 microchamber
 Incubated for minimum of 3 hours
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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
Control
EtOH
Control
1%A.M.
1%A.M.
Control
EtOH
Control
1%A.M.
1%A.M.
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
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
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
Results
3.00%
Rate of Phagocytosis of Any Number of Beads
Media Alone
2.50%
N=9
EtOH Control
1% A.M. Media for 60 Min
Rate of Phagocytosis
2.00%
1% A.M. Media for 90 Min
1.50%
1.00%
0.50%
0.00%
P Value
0.396169
P Value
0.000538
P Value
0.000463
Results
0.60%
Rate of Phagocytosis of More than One Bead
Media Alone
Rate of Phagocytosis of More than One Bead
0.50%
N=9
EtOH Control
1% A.M. Media for 60 Min
0.40%
1% A.M. Media for 90 Min
0.30%
0.20%
0.10%
0.00%
-0.10%
P value
0.3867711
P value
0.445056
P value
0.0167912
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
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
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
2. 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
1.