Pharmacology & Pharmacy, 2011, 2, 248-255
doi:10.4236/pp.2011.24032 Published Online October 2011 (
Copyright © 2011 SciRes. PP
Effect of the Monocyte Locomotion Inhibitory
Factor (MLIF) a Natural Anti-Inflammatory
Produced by E. histolytica on Apoptosis in
Human CD4+ T Lymphocytes
Sara Rojas-Dotor1, Liliana Vargas-Neri2, Francisco Blanco-Favela1
1Unidad de Investigación Médica en Inmunología, Hospital de Pediatría, Centro Médico Nacional Siglo XXI Instituto Mexicano del
Seguro Social, México City, México; 2Facultad de Química, Universidad Nacional Autónoma de México, México City, México.
Received June 4th, 2011; revised July 28th, 2011; accepted August 16th, 2011.
Apoptosis prevents the extravasation of intracellular material and the subsequent inflammatory response. Currently, it
is not known whether Monocyte Locomotion Inhibitor Factor (MLIF), an anti-inflammatory pentapeptide, induces pro-
grammed cell death. We evaluated the effect of MLIF on extrinsic and intrinsic apoptosis pathways human CD4+ T
lymphocytes. Cells were cultured for 24 h in RPMI-1640 medium alone (control) or in RPMI medium containing MLIF
alone, PMA alone, PMA + MLIF or actinomycin D. Annexin V/propidium iodide-stained cells in early apoptosis
showed that cells treated with MLIF or PMA + MLIF were not significantly different from control cells in medium; in
contrast, cells treated with PMA or PMA + MLIF demonstrated significant differences from the control in delayed
apoptosis. Cytochrome c and caspase 3 levels in cells treated with MLIF showed no significant differences from control
cells, however, compared to the control, cells treated with PMA and PMA + MLIF exhibited a significant increase in
cytochrome c and caspase 3 levels, which demonstrates that this weak induction of cell death is regulated by the intrin-
sic pathway of apoptosis. The Fas receptor was not detected in cell culture with any of the treatments employed, sug-
gesting that the extrinsic pathway of apoptosis is not involved. The MLIF per se does not induce apoptosis in human
CD4+ T lymphocytes; there may be an additional effect of PMA + MLIF producing the low levels of cell death re-
corded in the late apoptosis phase. MLIF acts as a natural, biological anti-inflammatory compound produced in axenic
cultures of E. histolytica that does not cause apoptosis or elicit an immune response due to its small size, which could
make it a strong candidate for future clinical applications.
Keywords: MLIF, Apoptosis, Anti-Inflammatory, Intrinsic Pathway, Extrinsic Pathway
1. Introduction
T lymphocytes constitute an essential part of the immune
system. Their generation, activation, proliferation and
survival are subject to tight regulation by several ex-
tracellular factors including cytokines, MHC-antigen
complexes and co-stimulatory ligands; however, internal
or external stimuli, such as over-stimulation, cytotoxicity
or time, can lead to death. The balanced interplay between
these factors determines the fate of a T cell [1-3]. Once the
cytotoxicity or over-stimulation occurs, T cells become
apoptosis-sensitive, leading to the death of most cells and
the survival of only a few memory T cells. Like other cells,
T cells are dependent on signals from their environment
for their survival; apoptosis thus represents a tightly
regulated process by which lymphocyte homeostasis is
controlled, and the generation of autoreactive cells is
prevented [4-6].
Apoptosis or programmed cell death, which plays a
central role in the normal development of multicellular
organisms, is essential for T cell function, the regulation
of B cells, the suppression of autoimmunity, the control
infections, as well as immune surveillance and homeo-
stasis [7,8]. One of the most important physiological
characteristics of cell death by apoptosis is no inflamma-
tory response. Key proteins are released into the cytosol
by mitochondria (intrinsic pathway of apoptosis) or as a
Effect of the Monocyte Locomotion Inhibitory Factor (MLIF) a Natural Anti-Inflammatory Produced by 249
E. histolytica on Apoptosis in Human CD4+ T Lymphocytes
consequence of activating a cell death receptor on the
cell surface (extrinsic pathway of apoptosis) [9]. Both
signalling pathways can converge upon the activation
cascade of effector caspases. Activation of the pro-
apoptotic proteins of the Bcl-2 family leads to the forma-
tion of a pore in the outer membrane of mitochondria,
which allows the release of proteins of including cyto-
chrome c from mitochondrial intramembranous space
into the cytosol; activates the apoptosome complex and
caspase 9; and finally activates effector caspases, such as
caspase-3-6 and 7 which triggers the final stages of apo-
ptosis. In contrast, when a cell dies via necrosis, the cy-
toplasmic contents are released into the interstitial space,
exposing a large number of antigens and triggering a
strong inflammatory response [10,11].
The protozoan parasite Entamoeba histolytica (E. his-
tolytica) produces an anti-inflammatory pentapeptide
(Met-Gln-Cys-Asn-Ser; MLIF) that inhibits human mo-
nocyte locomotion in vivo and in vitro [12]. MLIF de-
presses production of nitric oxide reactive intermediates
by human monocytes and neutrophils, decreases the ex-
pression of adhesion molecules (VL-4 in monocytes and
VCAM-1 in the vascular endothelium), and inhibits de-
layed hypersensitivity reactions in gerbils and guinea
pigs [13-15]. The scrambled pentapeptide (MLIF, Gln-
Cys-Met-Ser-Asn); showed no anti-inflammatory proper-
ties [16]. This anti-inflammatory effect could be attrib-
uted to the chemical activity of the peptide. Ongoing
studies in quantum chemistry have revealed that a phar-
macophore group in the MLIF sequence (… Cys-Asn-
Ser) could be responsible for most of the anti-inflam-
matory properties of this molecule [17]. Currently it is
unknown whether MLIF may have some involvement
with the mechanisms of programmed cell death. The aim
of this study was to evaluate the effect of anti-inflam-
matory MLIF on apoptosis in vitro by studying well-
characterized markers of apoptosis, such as annexin-V/
propidium iodide (IP) binding, activation of caspase-3,
cytochrome c and Fas receptor expression in human
CD4+ T lymphocytes.
2. Materials and Methods
2.1. Monocyte Locomotion Inhibitory Factor
MLIF (Met-Gln-Cys-Asn-Ser), 96% pure was obtained
commercially (American Peptide Co., Sunnyvale, CA,
USA). All working solutions were tested for endotoxin
(LPS < 0.3 pg) using the limulus assay (Amoebocyte
Lysate Endosafe KTA Charles River Endosafe INC,
Charleston, Inc, USA) and were kept at –70˚C until use.
2.2. Cell Purification
Thirty milliliters of heparinized venous blood was ob-
tained from healthy, non-smoking adult volunteers of
both sexes. Blood was diluted 1:2 with phosphate buff-
ered saline (PBS; 0.15 M phosphate buffer), then 10 ml
of the sample was layered over 4 ml of a Ficoll-Hypaque
(Sigma Chemical Co., St. Louis, MO) gradient (
1.077) and centrifuged at 700 × g for 30 min at 21˚C [18].
Peripheral blood mononuclear cells (PBMC) at the inter-
face were removed and washed twice with PBS. CD4+ T
cells were purified using the CD4+ T Cell Isolation Kit II
(Miltenyi Biotec, Germany) an indirect magnetic label-
ling system for the isolation of untouched CD4+ T cells
from human PBMCs, with LS columns and a MidiMACS
separator. Briefly, 1 × 107 PBMC were placed in poly-
propylene tubes with 80 l of PBS-albumin-EDTA and
20 l a cocktail of biotin-conjugated antibodies against
CD8, CD11b, CD16, CD19, CD36, CD56, CD123,
TCRγ/δ and CD235a (glycophorin A), and were incu-
bated for 10 min at 4˚C. These cells were subsequently
magnetically labelled with Anti-Biotin MicroBeads for
depletion. The population of CD4+ T lymphocytes that
was isolated was 95% pure.
2.3. Cell Culture
Five × 105 CD4+ T cells were placed in 24-well plates in:
1) RPMI-1640 medium alone (supplemented with 10%
fetal calf serum (FCS), 2 mM L-glutamine, 100 U/ml
streptomycin, 5 g/ml gentamicin, and 1 mM sodium
pyruvate) (Gibco Laboratories, Grand Island, NY, USA);
2) in medium supplemented with 50 ng/ml phorbol
12-myristate 13-acetate (PMA) (Sigma, Chemical Co, St.
Louis MO); 3) in medium with 50 g/ml MLIF; 4) PMA
+ MLIF; or 5) 1g/ml actinomycin D for 24 h at 37˚C,
5% CO2 [19]. Cell viability was 90% by trypan blue
dye (Sigma) exclusion. The optimal concentrations of
MLIF and PMA were determined from dose-response
2.4. Annexin V/Propidium Iodide Double
Staining by Flow Cytometry
For annexin V/propidium iodide (PI) double staining,
cells were resuspended in 100 ml of binding buffer (10
mM N-(2-hydroxyethyl) piperazine-N-2-ethanesulfonic
acid (HEPES)/NaOH, 140 mM NaCl, 2.5 mM CaCl2, pH
7.4) and stained with 5 ml of both FITC-conjugated an-
nexin V and PI (BD Biosciences). The mixture was in-
cubated for 15 min at room temperature in the dark, fol-
lowed by flow cytometry analysis. Acquisition of 10,000
events was conducted in flow cytometry FACScalibur
(BD Biosciences, Palo Alto, CA). For analysis, Win MDI
Copyright © 2011 SciRes. PP
Effect of the Monocyte Locomotion Inhibitory Factor (MLIF) a Natural Anti-Inflammatory Produced by
E. histolytica on Apoptosis in Human CD4+ T Lymphocytes
2.8 software was used.
2.5. Quantitation of Total Cytochrome c in
Cultured Cells
After a 24 h incubation in the presence of medium alone,
PMA, PMA + MLIF or actinomycin D the CD4+ T cells
were harvested in their culture medium, pelleted by cen-
trifugation at 300 × g for 15 min, rinsed with PBS, and
resuspended in Cell Lysis Buffer 2 (Cell Lysis Buffer 2
solubilises the cytochrome c in both the cytosol and mi-
tochondria) to a final concentration of 1.5 × 106 cells/ml.
The cell lysate was incubated for 1 h and centrifuged at
1000 × g for 15 min. The resulting supernatant cyto-
chrome c was measured with a quantitative sandwich
enzyme immunoassay technique (cytochrome c ELISA
Kit) (Calbiochem® Merck KGaA, Darmstadt, Germany).
Plates were pre-coated with a monoclonal antibody spe-
cific for cytochrome c. Standards and samples were pi-
petted into coated wells, and cytochrome c was bound to
the immobilised antibody. After washing, an enzyme-
linked monoclonal antibody specific for cytochrome c
was added to each well, followed by the substrate. In this
assay, the colour develops in proportion to the amount of
cytochrome c bound in the initial step. Colour develop-
ment was stopped, and the intensity of the colour was
measured. The quantity of human cytochrome c was de-
termined as a change in absorbance at 450 nm using a
microplate reader (Dynatech MR 5000, Maryland, USA).
2.6. Quantitation of Activated Human
Caspase-3 activity was measured by using a human ac-
tive caspase 3 ELISA kit protocol (OptEIA™, BD Bio-
sciences). Briefly, cells were lysed in a hypotonic buffer
(10 mM HEPES [pH 7.4], 50 mM NaCl, 2 mM MgCl2, 5
mM EGTA, 1 mM phenylmethylsulfonyl fluoride, and
protease inhibitor cocktail). After incubating for 45 min
on ice, lysates were centrifuged at 40,000 × g for 45 min
at 4˚C. Cell lysates were diluted with 200 l assay dilu-
ent buffer (50 mM HEPES [pH 7.4], 0.2% CHAPS
sulfonate}, 20% glycerol, 2 mM EDTA, 10 mM dithio-
threitol), was added to each well and incubated for 2 h at
room temperature. Next, 100 l of cytochrome c standard
or sample was added to each well and incubated for 2 h.
Subsequently, 100 l of horseradish peroxidase-conju-
gated streptavidin (1X) and 100 l of substrate solution
were added to each well and incubated for 30 min. Fi-
nally, 50 l of stop solution was added to each well. The
absorbance of the cleaved substrate at 450 nm was
measured with a microplate reader (Dynatech MR 5000,
Maryland, USA).
2.7. Quantification of Fas in the Supernatant of
Cultured Cells
The presence of Fas in the supernatant fluids of cultured
CD4+ T lymphocytes (1 × 106 cells/well) was deter-
mined and quantified using an ELISA kit with an anti-
body that recognises human Fas (Ray Biotech, Inc., At-
lanta, Georgia), according to the manufacturer’s instruct-
tions. For calibration, we used a commercially available,
natural human Fas protein to construct a standard curve
and to obtain absolute values. The concentration of Fas
was measure in six independent assays per treatment,
each in duplicate, and the average of the six measure-
ments was considered to be the final concentration.
2.8. Statistical Analysis
All values were expressed as mean standard deviation
(SD). Commercial SPSS v. 11.0 for Windows (SPSS Inc.,
Chicago, IL) was used for statistical analysis. Statistical
comparisons between groups were performed using the
Mann-Whitney U tests; p values 0.05 were deemed
statistically significant.
3. Results
3.1. Detection of Apoptosis by Annexin
V/Propidium Iodide Staining
To determine whether apoptosis was induced by MLIF,
and to discriminate between early apoptosis and necrosis,
cells were simultaneously stained with annexin V and PI.
Human CD4+ T lymphocytes were cultured in the pres-
ence of RPMI medium (negative control), MLIF, MLIF,
PMA or PMA + MLIF, and actinomycin D served as the
positive control. Results showed that 7% of CD4+ T
lymphocytes underwent spontaneous apoptosis in the
medium control. When the cells were stimulated with
MLIF, 9% of CD4+ T lymphocytes stained positive for
annexin V. When stimulated with PMA, a significant
number of cells (13%) were apoptotic (p < 0.003 com-
pared to medium control), and among cells treated with
PMA+MLIF, the frequency of apoptosis was 10% (not
significant, NS). Actinomycin D treatment increased
early apoptosis to 19% of cells (p < 0.0001 compared to
medium control) (Figure 1). However, in the analysis of
late apoptosis, PMA + MLIF significantly increased the
binding of annexin V (21% of cells) compared to the
control (11%, p < 0.002) (Figure 1), suggesting that the
pro-apoptotic activity of MLIF + PMA is related to the
extrinsic signalling pathway and/or the intrinsic pathway
of apoptosis.
3.2. Production of Cytochrome c and Fas (APO-1)
PMA + MLIF may favour ativation-induced cell death. c
Copyright © 2011 SciRes. PP
Effect of the Monocyte Locomotion Inhibitory Factor (MLIF) a Natural Anti-Inflammatory Produced by
E. histolytica on Apoptosis in Human CD4+ T Lymphocytes
Copyright © 2011 SciRes. PP
Figure 1. Apoptosis in CD4+ T lymphocytes. Lymphocytes were cultured for 24 h in RPMI-1640 medium alone (control) or in
RPMI medium containing MLIF alone, PMA alone, PMA + MLIF or actinomycin D. Cells were stained with FITC-conju-
gated annexin V and propidium iodide (annexin/IP) and processed for cell death determination by flow cytometry. (a) Cali-
bration parameters and positive control (actinomycin D); (b) The numbers in each quadrant of the flow cytometry histo-
grams represent the mean percentage of six independent experiments. The percentage of necrotic cells is indicated in the up-
per left quadrant; apoptotic-necrotic ce lls, in the upper right quadrant; and early apoptotic cells in the lower right quadrant.
Non-stimulated CD4+ T cells (RPMI medium control)
produced 9 g/ml of cytochrome c; cells stimulated with
MLIF produced 12 g/ml; with PMA, 15 g/ml; with
PMA + MLIF, 19 g/ml; and with actinomycin D, 33
g/ml (Figure 2). MLIF per se did not stimulate the re-
lease cytochrome c (NS), but treatment with PMA +
MLIF resulted in a there was a statistically significant
difference compared to the negative control (p < 0.001)
and positive control (p < 0.0001), suggesting that the
intrinsic pathway of apoptosis is activated. Actinomycin
D produced a greater increase in the release of cyto-
chome c (Figure 2). In contrast, Fas receptor expression
was not observed under any of the treatment conditions
(data not shown).
3.3. Determination of Activated Human
Execution of apoptosis requires the activation of the cas-
pase cascade, where activated initiator caspases cleave
and thereby trigger downstream effector caspases that, in
turn, dismantle the cell. Whereas proteolytic cleavage of
an effector caspase is indicative of its activation, the
same is not necessarily true with initiator caspases.
We wondered whether the effect of MLIF on the ex-
pression of cytochrome c converged upon the caspase-3
pathway. In cells treated with medium alone, basal ex-
pression of caspase-3 was 90 U/ml (Figure 3). Exposure
to MLIF increased expression of caspase-3 to 110 U/ml,
and upon PMA treatment, was 196 U/ml. In cells treated
with PMA + MLIF caspase-3 production was 235 U/ml,
and with actinomycin D, it was 329 U/ml. As expected,
actinomycin D treatment was significantly different (p <
0.002) compared to basal levels (Figure 3) and cells
treated with MLIF, PMA, PMA + MLIF (p < 0.001, p <
0.0001 and p < 0.01 respectively). The signalling cascade
of caspase 3 was different between groups, and this
stronger between the PMA and actinomycin D.
4. Discussion
Apoptosis can be induced eier as a consequence of key th
Effect of the Monocyte Locomotion Inhibitory Factor (MLIF) a Natural Anti-Inflammatory Produced by
E. histolytica on Apoptosis in Human CD4+ T Lymphocytes
Figure 2. Quantification of cytochrome c. One × 105 CD4+ T lymphocytes, cultured in the presence of PMA, MLIF or PMA +
MLIF for 24 h, were lysed and centrifuged at 4000 × g, and cytochrome c in the supernatant was measured. Histograms show
mean values ± SD of six independent experiments. Statistically significant differences between all groups and actinomycin D
are indicated with an asterisk , p < 0.05 (Mann-Whitney test).
Figure 3. Quantification of caspase-3. After culturing under the indicated conditions, 105 lymphocytes were ly sed and centri-
fuged at 4000 × g. Released caspase-3 in the supernatant was measured using a commercially available enzyme-linked im-
munosorbent assay (ELISA) kit. The levels of caspase-3 are represented as means of caspase-3 concentrations vs the negative
control group, or the positive control group (ac tinomycin D) in cells treated with MLIF, PMA, PMA + MLIF (p < 0.001, p <
0.0001 and p < 0.01 respectively). NS = no significant difference, n = 6, p < 0.05 (Mann-Whitney Test).
proteins being released into the cytosol by the mitochon-
dria or by the activation of a death receptor on the cell
surface. Both signalling pathways converge at the level
of effector caspases, without causing the inflammation
produced by necrotic cell death [20].
Infection with E. histolytica is associated with an acute
inflammatory response [21]. However, it is not com-
pletely clear how E. histolytica modulates the pro-in-
flammatory response of the host. Previous work studying
the effect of MLIF on inflammation, specifically the in-
hibition of interleukins, such as IL-2, IFN-
and IL-1
indicates that MLIF acts selectively in different cell
lineages. At least two possible explanations could ac-
count for this effect: MLIF may either inhibit soluble
pro-inflammatory or regulate apoptosis mediators [22,
In this study, we determined that MLIF per se does not
induce programmed cell death in human CD4+ T lym-
phocytes, as demonstrated in early apoptosis. Cell death
was slightly increased in the presence of PMA (MLIF +
PMA) but was detected only in late apoptosis (apoptosis/
necrosis). MLIF has powerful but selective anti-inflam-
matory properties; this peptide inhibits locomotion (ran-
dom, chemotactic and chemokinetic) of normal human
peripheral blood mononuclear phagocytes in response to
various chemoattractants, such as C5a-desArg, lym-
Copyright © 2011 SciRes. PP
Effect of the Monocyte Locomotion Inhibitory Factor (MLIF) a Natural Anti-Inflammatory Produced by 253
E. histolytica on Apoptosis in Human CD4+ T Lymphocytes
phokine chemotactic or lymphocyte-derived chemotactic
factor (LDCF), but has no effect on polymorphonuclear
neutrophils [12]. MLIF is capable of downregulating
some pro-inflammatory cytokines, including IFN-
, IL-2,
and IL-6, and of inducing the anti-inflammatory cyto-
kines IL-4 and IL-10 [22]. MLIF delays the arrival of
mononuclear leukocytes into Rebuck chambers applied
to the skin of healthy human volunteers [12]. In this
study, the combination of PMA + MLIF caused an in-
creased susceptibility to late apoptosis in lymphocytes,
implicating the intrinsic apoptosis signalling pathway.
This increase may illustrate an additive effect because
PMA-dependent apoptosis was also slightly increased in
the late apoptosis. The intracellular apoptosis machinery
depends on caspases, which are proteases responsible for
executing programmed cell death. Once activated, a cas-
pase will in turn activate the next pro-caspase down-
stream, creating a proteolytic amplification cascade.
Some of the activated caspases cleave other key proteins
in the cell, such as those that form the nuclear lamina and
the cytoskeleton, leading to the destruction of the nuclear
framework, and activate DNases that degrade nuclear
DNA. These changes underlie the nuclear and cytoplas-
mic structural modifications that occur in apoptotic cells
[24,25]. Release of cytochrome c from mitochondria is
an important step in mitochondria-mediated apoptosis; it
leads to the activation of caspase 9 and then caspase 3
[26]. The fact that the Fas receptor was absent from the
surface of MLIF-treated T cells suggests that there was
no death receptor activation. Although Fas is mostly
thought of as a death receptor that is capable of inducing
the apoptosis of activated T cells via the caspase cascade
[27], this receptor has also been implicated in effective T
cell activation. Fas could act as a receptor with functional
pleiotropy in models of T cell [28]. MLIF interacts with
human leukocytes through a mannose-containing recep-
tor [29], and it causes an increase in the number of
pericentriolar microtubules and in the cAMP concentra-
tion, without a concomitant reduction in cGMP [30]. In
U-937 cells, MLIF inhibits the expression of inflamma-
tory cytokines, such as MIP-1α and MIP-1
, which are
B pathway-regulated proteins, as was described by
Utrera-Barillas [31]. The p65-p50 heterodimer com-
prises the most abundant form of NF-
B in a PMA-in-
duced system. Temporary studies showed that MLIF
induces p50 translocation, which could be due to the fact
that MLIF stimulates cAMP synthesis and phosphoryla-
tion of NF-
B by protein kinase A, phosphorylation of
B and subsequent NF-
B translocation [32]. MLIF
strongly inhibits inflammation but does not induce early
apoptosis, downregulates the Fas receptor, activate cas-
pase 3 or trigger the release of cytochrome c, indicating
that it is largely non-cytotoxic, however; we must be
caution in extrapolating these results to other population
of lymphocytes such as CD8+ T cells and regulatory T
We propose that this is a novel natural anti-inflam-
matory molecule produced in axenic cultures of E. histo-
lytica that, owing to its small size (five peptides), is non-
antigenic, thus making it a strong candidate for future
clinical applications.
5. Acknowledgements
This research was supported, in part, by a grant from the
Consejo Nacional de Ciencia y Tecnología, (CONA-
CYT): No. 089657. The authors wish to thank Verónica
Vrátný Zuleta for the critical review of the English lan-
guage in the manuscript. The authors are grateful to
laboratory technician José de la Luz Romero Preciado
from the Blood Bank in the Centro Médico Nacional
Siglo-XXI-IMSS, México City, México.
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