Flow Cytometry in the Study of the Interaction between Murine Macrophages and the

Protozoan Parasite Leishmania amazonensis

Author(s): A. L. Bertho, L. Cysne and S. G. Coutinho
Source: The Journal of Parasitology , Aug., 1992, Vol. 78, No. 4 (Aug., 1992), pp. 666-671
Published by: Allen Press on behalf of The American Society of Parasitologists

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 J. Parasitol., 78(4), 1992, p. 666-671
9 American Society of Parasitologists 1992

FLOW CYTOMETRY IN THE STUDY OF THE INTERACTION

BETWEEN MURINE MACROPHAGES AND THE PROTOZOAN
PARASITE LEISHMANIA AMAZONENSIS

A. L. Bertho, L. Cysne, and S. G. Coutinho

Department of Protozoology, Oswaldo Cruz Institute, Fiocruz, Av. Brasil, 4365, Pav. Carlos Chagas 50 an
Manguinhos, Rio de Janeiro RJ, Brazil 21045

ABSTRACT: A flow cytometry method was adapted to study interaction between murine mac
Leishmania amazonensis. Using this method it was possible to detect internalization of parasite
increase in macrophage granularity (side scatter), with the latter indicating the presence of p
parasitophorus vacuoles. A quenching technique was used to confirm the feasibility of the m
distinguish between internalized and externally attached parasites. Experiments using fixed-label
unlabeled parasites gave similar results, demonstrating that granularity was an adequate parameter
of parasite-macrophage interaction, when compared with labeling methods. Experiments that me
nalization using only the increase in macrophage granularity as an indicator showed that living L
was internalized to a greater extent than killed-unlabeled parasites. This finding suggests that th
an active role in the process of internalization. The 2 methods in combination, flow cytometry a
can be used to study murine peritoneal cell-L. amazonensis interactions and to sort phagocyto
phagocytosing subpopulations of macrophages for further studies.

Leishmaniasis is caused by obligate intracel- Bassoe and Solberg, 1984), and an FCM fluo-
lular protozoans of the genus Leishmania
replicate inside the parasitophorus vacuoles
infected cutaneous and visceral phagocytic
There is evidence that cell-mediated immunity
is the principal mechanism involved in the
ing or progression of murine disease (Mauel
al., 1981; Titus et al., 1984; Liew and Dhaliwal, metric analysis and fluorescence quenching tech-
1987). The various ways in which macrophages
interact with Leishmania amazonensis, phage
the parasite, and present antigens to T lympho-
cytes are considered fundamental in determining
the outcome of disease (Mauel et al., 1981; Cou-
tinho et al., 1984). Several in vitro methods are
available for the study of macrophage-L.
zonensis interactions. However, most of them
suffer from disadvantages, such as the labori-
ousness of cell-staining techniques and the
jective and time-consuming nature of micros-
copy, when establishing parasitization ratios.
Flow cytometry (FCM) provides an automated
means of single-cell multiparametric analysis.
addition, flow systems easily can analyze cells in
numbers several times larger than is possible
microscopy and offer more accurate and reliable
data. Furthermore, different cell populations
be sorted for further use in other assays.
Several phagocyte-microorganism interac-
tions already have been investigated using flow2% formaldehyde for 15 min, washed, and incubated
systems (Bassoe et al., 1980; Derer et al., 1983; with the primary antibody for 1 hr at 37 C. Afterward
Received 28 October 1991; revised 2 March 1992;
accepted 2 March 1992.
rescence
that quenching technique has been used in
the study
of of parasite attachment and internali-
zation (Bjerknes and Bassoe, 1984).
cells.
In the present study we made use of advantages
offered by flow cytometric assays (Bjerknes and
heal-
Bassoe,et1984), which combine cell multipara-
niques, to a study of L. amazonensis-macro-
destroy interactions in vitro.
MATERIALS AND METHODS
Ten-to-twelve-week-old BALB/c mice, bred in the
central animal house of the Microbiology Institute of
the Federal University of Rio de Janeiro, were used
ama-
throughout.
After isolation from skin lesions of experimentally
infected mice, Leishmania amazonensis (MHOM/Br/
77/LTB0016)
sub- was cultured to stationary phase in
McNeal, Novy, and Nicolle (N.N.N.) medium. The
N.N.N. liquid layer consisted of RPMI-1640 medium
(Sigma, St. Louis, Missouri) supplemented with 5%
fetal calf serum (Microbiol6gica, Rio de Janeiro, Rio
de Janeiro,
In Brazil), overlaid on the N.N.N. solid layer.
Leishmania amazonensis promastigotes were harvest-
ed from the liquid layer and washed in RPMI medium
by
by centrifugation at 1,400 g for 5 min.
Leishmania amazonensis promastigotes were la-
beled with
canfluorescein-isothiocyanate (FITC), using in-
direct immunofluorescence. Briefly, sera from L. ama-
zonensis promastigote-immunized rabbits were used
as the primary antibody. The parasites were fixed with
they were washed once in phosphate-buffered saline
(PBS) and incubated with a goat anti-rabbit-FITC con-
jugate antibody (Sigma) for 1 hr at 37 C. They then
were washed 3 times in PBS and resuspended in RPMI
666

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 BERTHO ET AL.-MACROPHAGE-L. AMAZONENSIS INTERACTION 667

cn
- WINDOW 1
u)

n-
I0 a.
LL
0

w
m
0
IL

z
-a WINDOW 2

0 101 10 10
SIDE SCATTER

FIGURE 1. Two-dimensional computer contour plot GREEN FLUORESCENCE

showing murine peritoneal macrophages (window 1)
FIGURE 2. Green fluorescence histogram showing
and free Leishmania amazonensis promastigotes (win- efficiency ofLeishmania amazonensis pro-
the labeling
dow 2), distinguished by size (forward angle mastigotes.
light scat- The frequency of stained parasites was 90%.
ter) and granularity (side scatter).

Formaldehyde-fixed FL or unlabeled living L. ama-

medium. Labeling efficiency was checked byzonensis FCM. Ali-
in RPMI culture medium, in a cell-parasite
quots of formaldehyde-fixed FITC-labeled (FL) L. ratio of 1:10, was used. Cultures were kept at 37 C in
amazonensis were stored at - 20 C for subsequent use. a 5% CO2 incubator for 28 hr, with occasional shakings.
Peritoneal cells (PC) from normal BALB/c mice were At several times (0, 4, 8, 12, 24, 28 hr), aliquots were
collected after intraperitoneal (i.p.) injection of 5 ml withdrawn from each culture, placed in silicone tubes,
RPMI culture medium. Peritoneal fluid was drawn
and kept in an ice-water bath until FCM analysis.
through the abdominal wall with a 20-gauge needle. Statistical analysis was made using Mann-Whitney's
Fluids from several animals were pooled and centri- U-test. Any P value ?0.05 was considered significant.
fuged at 400 g for 10 min at 4 C. Washed PC suspen-
sions were adjusted to 1-6 x 106 cells/ml in culture RESULTS
medium and placed in polypropylene tubes to avoid
macrophage adhesion (Nacy and Diggs, 1981). Preliminary experiments were carried out to
FCM measurements were made using an EPICS see 751
if free Leishmania parasites and murine peri-
(Coulter Electronics, Hialeah, Florida), with a 488-nm
laser excitation at 250 mW. For the phagocytosis assay,
FITC fluorescence was measured at 515-575 nm, and 60

both forward angle light scatter and side scatter were

measured at 488 nm. The samples were run using list so-

mode, which makes further analysis possible. To con- uso

u,
firm the efficiency of parasitization, L. amazonensis-
infected or uninfected macrophages were sorted into a0S40-
96-well microtitration plate using an Auto-Clone de-
vice (Coulter Electronics). After sorting, aliquots were
taken from wells, placed on slides, and stained with 20
Giemsa stain. Cell sorting was standardized for scatter o3
and fluorescence by using a suspension of fluorescent
beads (Immunochecks, Coulter Electronics).
Attachments and internalization of FL L. amazo-
nensis into macrophages were determined by an FCM o0 4 8 12 24 28
fluorescence quenching technique, as described else-
HOURS
where (Bjerknes and Bassoe, 1984). Briefly, 200 1l of
dye solution (trypan blue, 1 mg/ml in PBS at pH FIGURE
7.4) 3. The kinetics of formaldehyde-fixed la-
was added to a 200-1l sample taken from the beled Leishmania amazonensis internalization into
cultured
murine macrophages, before (squares) and after (tri
macrophage-parasite suspension. With this method,
angles)
the FITC fluorescence of extracellular (i.e., free andquenching by trypan blue solution. Difference
attached) L. amazonensis was quenched by trypanwereblue
statistically significant (P - 0.05) at 4, 8, 12, and
24 hr.
solution, whereas the fluorescence of internalized The results are given as the mean ? the standar
par-
asites remained unaltered. deviation of 4 experiments.

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668 THE JOURNAL OF PARASITOLOGY, VOL. 78, NO. 4, AUGUST 1992

SI I '- I IA I i IA

LL)

LLI

,. .....S DSIDE
EC..... .
SCATTER
FIGURE 4. The kinetics of living Leishmania amazonensis internalization into mu
scatter histograms gated out from window I show increasing macrophage granularity
after culture incubation periods of A, 0 hr; B, 4 hr; C, 8 hr; D, 12 hr; E, 24 hr; and F, 2
experiment.

toneal macrophages could be distinguished by of fluorescent L. amazonensis-associated mac-

flow cytometry measurements using size and rophages before quenching (i.e., macrophages
granularity as parameters. It was observed that with attached plus internalized parasites) and af-
window 1 contained about 90% of the macro- ter quenching (macrophages with internalized
phages (Fig. 1). This was confirmed by labeling parasites only). The results suggest that after 4
macrophages with the specific monoclonal hran-only macrophages with attached parasites on
tibody anti-MAC-1. Window 2 contained 95% their surface could be detected, as the macro-
of the free promastigotes. phage-associated fluorescence was quenched by
After the FITC-labeling procedures, aliquots trypan blue solution. Internalization of parasites
of promastigotes and amastigotes were run on a increased progressively over time, until after 24-
flow cytometer to check the efficacy of parasite 28 hr almost all the macrophage-associated par-
labeling. Figure 2 shows a typical experiment in asites had been internalized, with little difference
which 90% of the promastigotes were found to in fluorescence before and after quenching. Sim-
be labeled, although some experiments reached ilar results were obtained when the same material
95% of labeling. was examined under a fluorescent optical micro-
Murine peritoneal cells were incubated with scope.
FL promastigotes of L. amazonensis in supple- By measuring the increasing granularity of
mented RPMI-1640 culture medium. The ki- macrophage window 1, it was possible to estab-
netics of parasite uptake were determined lish the bykinetics of unlabeled-living promasti-
measuring the FITC fluorescence gatedgote out internalization.
from Macrophage parasitization
window 1. To distinguish between different was measured
de- by comparing the profile in the
grees of parasite attachment and/or internaliza- side scatter histograms at each sequential time
tion, the FITC fluorescence of extracellular (i.e., the histogram obtained at time 0 (no
point with
free and attached) parasites was quenched parasite
by try-inside the macrophages) (Fig. 4). Using
pan blue solution. Figure 3 shows the percentage this procedure, we observed a progressive in-

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 BERTHO ET AL.-MACROPHAGE-L. AMAZONENSIS INTERACTION 669

crease in macrophage granularity over the course

100

of the experiment, indicating progressive para-

sitization of macrophages. 80

There was a greater internalization of living

so
than of killed-fixed parasites, with the difference
becoming clearer after 24-28 hr of culture (Fig.
5). Despite this difference, however, both types40
showed a progressive increase in internalization
over the entire experimental period.
H 20O
The percentage of parasitization of macro-
phages in experiments evaluated by granularity
using flow cytometry was similar to that obtained 0 4 8 12 24 28

by microscopic examination of Giemsa-stained HOURS

preparations. FIGURE 5. Kinetics of Leishmania amazonensis
promastigote internalization into murine macrophages
DISCUSSION
measured by increases in macrophage granularity (side
scatter) gated out from window 1. Circles indicate cul-
Several studies on cell phagocytosis have been
tures of macrophages plus living promastigotes; squares
carried out using dead microorganismsindicate
(Bassoecultures of macrophages plus killed-fixed pro-
et al., 1983; Sveum et al., 1986) or fluorescent
mastigotes. Differences were statistically significant (P
particles (Scribner and Fahrney, 1976; Dunn
< 0.05)and
at 24 and 28 hr. Triangles indicate cultures of
macrophages without parasites as control. The results
Tyrer, 1981; Steinkamp et al., 1982; Bjerknes
are given as the mean ? standard deviation of 3 ex-
and Bassoe, 1983, 1984). They have shown that
periments.
phagocytes participate in the process of inter-
nalization. It is also possible, however, to use
unlabeled-living microorganisms in investiga- of L. amazonensis organisms. Another interest-
tions into the role of parasites in this phenom- ing point is that attachment, as opposed to in-
enon and in intracellular killing of microorgan- ternalization, was especially apparent after 4 hr
isms (Bjerknes, 1984). of incubation, as revealed by the difference be-
In our study labeled-living L. amazonensis tween the quenching and nonquenching proce-
could not be used because labeling antibodies dures. This notwithstanding, attached parasites
were shed too quickly. As far as we know, there still could be detected after 28 hr in culture, al-
is no available labeling procedure for living par- though in lower proportions.
asites in which staining can be maintained for Whether macrophages engulf leishmanial pro-
long periods in culture. We therefore chose to mastigotes by phagocytosis, or whether the par-
determine the internalization of unlabeled-living asites actively penetrate cells, is a controversial
parasites by measuring increases in macrophagequestion. Some authors have demonstrated that
granularity, and we successfully demonstrated promastigotes of Leishmania donovani enter
that it is feasible. Although the method does not hamster macrophages by first inserting the fla-
discriminate between attached and internalized gellar tip, indicating that parasites actively enter
parasites, we were able to overcome this limi- the macrophages (Pulvertaft and Hogle, 1960;
tation by using the quenching technique (Bjerknes Miller and Twohy, 1967). Lewis (1974) found
and Bassoe, 1984). that Leishmania mexicana promastigotes could
Comparing internalization of killed or living infect sarcoma cells despite the presence of cy-
parasites, as measured by increases in macro-tochalasin B, which suggests that promastigotes
phage granularity, we observed that there was a play an active role in host cell infection. Con-
greater internalization when living parasites wereversely, other model systems have demonstrated
used. This finding shows that living parasites play an abundance of macrophage pseudopods around
an important role in the interaction between the promastigotes, no preferable orientation of
phagocytes and L. amazonensis. Figure 3 shows the parasite during entry, and inhibition of par-
fixed-labeled parasites presenting a similar de- asite entry by cytochalasin B, all of which point
gree ofparasitization to the one established using to an important role for macrophage phagocy-
unlabeled killed parasites (Fig. 5), a finding that tosis (Akiyama and Haight, 1971; Chang, 1979).
demonstrates granularity can be used with con- Aikawa et al. (1982) demonstrated that the mode
fidence as a criterion for detecting internalization of entry of Leishmania panamensis promasti-

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670 THE JOURNAL OF PARASITOLOGY, VOL. 78, NO. 4, AUGUST 1992

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the process of attachment and ingestion, includ- bined measurement of phagocytosis and intracel-
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et Immunologica Scandinavica 91: 341-348.
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mannose, fucose, or N-acetylglycosamine ter- attachment and internalization flow cytometric
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a complement breakdown product, C3bi. CR3
Experimental Parasitology 48: 175-189.
has been shown to be important in both the at-
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tachment and the ingestion of promastigotes ENGERS. 1984. Induction by specific T lympho-
(Wilson and Pearson, 1988). Although several cytes of intracellular destruction of Leishmania
receptor-ligand systems may be responsible for major in infected murine macrophages. Parasite
Immunology 6: 157-170.
L. amazonensis-macrophage interaction, rela-
DfRER, M., C. WALKER, F. KRISTENSEN, AND M. C.
tively little is known about the physical surface REINHARDT. 1983. A simple and rapid flow cy-
aspects of this recognition phenomenon (Saraiva tometric method for routine assessment of baker's
et al., 1989). yeast uptake by human polymorphonuclear leu-
Flow cytometry allows sorting of phagocytic kocytes. Journal of Immunological Methods 61:
359-365.
and nonphagocytic subpopulations for further
DUNN, P. A., AND H. W. TYRER. 1981. Quantitation
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ACKNOWLEDGMENTS
LEWIS, D. H. 1974. Infection of tissue culture cells
The authors are grateful to M. A. Santiago and of low phagocytic ability by Leishmania mexicana
mexicana. Annals of Tropical Medicine and Par-
R. M. P. Pereira for valuable technical assistance,
asitology 68: 327-336.
and to R. Pelegrino for excellent secretarial help.
LIEW, F. Y., AND J. S. DHALIWAL. 1987. Induction of
The research was partially supported by UNDP/ delayed-type hypersensitivity to Leishmania ma-
World Bank/WHO Special Programme for Re- jor and the concomitant acceleration of disease
search and Training in Tropical Diseases. development in progressive murine cutaneous
leishmaniasis. Infection and Immunity 55: 645-
651.
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