Respiratory Bursts in Garlic Treated Macrophages

Shannon Proctor

Departments of Chemistry and Biology

Jacksonville University

MacrophagesGreek for “big eaters”Originate from white blood cells called monocytesThey play many roles in the bodyMajor role in immunity and the immune response

Electron micrograph of a macrophage attacking bacteria.

Representation of macrophages that I worked with using an inverted microscope.

www.hartnell.edu/biology

BackgroundGarlic has been used since ancient times as an

herbal remedy.Heart diseaseTumorsThe PlagueAntibacterial properties

BackgroundPrevious research has shown that garlic enhances

macrophage activity.Leishmania major, Ghazanfari 2005Augmentation of function, Lau 1991Enhanced immunocompetence, Lamm 2001

Our lab has shown that garlic does increase phagocytosis in macrophages

Internalization vs. DigestionMacrophages will phagocytose many things

Engulf the particleMay or may not destroy

There is a chemical change when they digestO2 uptake increasesRapid release of reactive oxygen speciesThis is what we want to measure

AbstractAn increase in the respiratory burst would be

significant

Nitro-blue tetrazolium (NBT)Yellow solution that turns blue

Cell CultureIC-21 Cells

Cultured in RPMI media

Incubated at 37°C, 5% CO2

Phagocytosis AssaySubcultured at

100% confluency

DetachedResuspended in control and media with garlic (0.5% or

1%)Incubation for 3hrsAddition of latex beads and fresh media

Phagocytosis AssayRepeated every 30, 60, and 90 minutesIncubation throughoutAssay stopped by washingStaining using Hema 3 kitInverted microscope for viewing175-250 cells were counted and analyzed

8-Well Slide

Results

Results

Percent phagocytosis measured using the following equation:(# of macrophages with engulfed bead ÷ total # macrophages) X 100

D. Lindsey and K. Jackson, 2006

IC-21 Phagocytosis5 ul/ml Garlic

0.00

5.00

10.00

15.00

20.00

25.00

30 60

Time (minutes)

% o

f to

tal c

ells

p

hag

ocy

tose

*= Control (60 % ethanol)

= Garlic

* P=.055, Student’s T-test

ResultsNBT Test

Dissolve 1 tablet in waterFix cells (no staining)Cover cells with NBT solution and incubate at 37°C for

30 minutesRinse with filtered PBSObserve color under microscope

ConclusionFuture research could use latex beads to ensure

phagocytosis and use a bacterium, for example, to measure the respiratory burst

AcknowledgementsDr. Karen Jackson, advisor

Patricia Roman and Curtis Dobrowolski

Jacksonville University Public Safety

ReferencesBuescher, 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 (Allium Sativum) treatment. Journal of Ethnopharmacology 103 (3), 333-337.

Lindsey, D. and Jackson, K. (2006). Allium sativum Effects on Phagocytic Activity of IC-21 Peritoneal Macrophages in vitro