Chapter II. Trichomonas vaginalis-induced apoptosis in RAW264.7 cells is regulated
T. vaginalis treatment affected expression of Bcl-x L in RAW264.7 cells
Anti-apoptotic protein Bcl-xL also has been reported to play critical roles in inhibiting the
release of cytochrome c and subsequent apoptosis (Pan et al., 1998). To test this, I examined the correlation between Bcl-xL expression and T. vaginalis-induced apoptosis. Whereas
untreated RAW264.7 cells showed high expression of Bcl-xL, Bcl-xL expression in T.
vaginalis-treated cells was decreased after 8 h of culture time (Figure II-5A). These data
suggest that alteration of the Bax/Bcl-xL ratio by downregulating Bcl-xL and stabilizing Bax expression may be critical for induction of apoptosis by T. vaginalis.
Previous work has shown that caspase-3 is involved in the regulation of Bcl-2 expression (Clem et al., 1998). To test this correlation between caspase-3 and Bcl-2 family, I examined T.
vaginalis-treated RAW264.7 cells in the presence or absence of caspase inhibitors
(Boc-D-FMK and Z-DEVD-(Boc-D-FMK). Subsequent western blot analysis of Bcl-xL expression showed that
T. vaginalis-induced downregulation of Bcl-xL was significantly abolished in the presence of caspase inhibitors. These data, the cleavage of Bcl-xL can be blocked by the addition of the
caspase-3 inhibitors, suggest that the cleavage of Bcl-xL in response to T. vaginalis may be a caspase-3-mediated event (Figure II-5B).
Figure II-5. T. vaginalis treatment affected Bcl-xL expression in RAW264.7 cells. (A) T.
vaginalis was added to RAW264.7 cells for the indicated times and lysed, and whole cell
lysate was electrophoresed and probed by western blot with anti-Bcl-xL antibody. (B) 50 µM Boc-D-FMK, and 50 µM Z-DEVD-FMK were pretreated for 30 min prior to T. vaginalis
adhesion. After T. vagianlis treatment for 8 h, RAW 264.7 cells were subjected to immunoblotting with anti-Bcl-2, anti-Bcl-xL, and anti-Bax antibodies. Results are
representative of three separate experiments with comparable outcomes. Actin was used as loading control. Immunoblotting with anti-β-actin antibody was used as a loading control.
Transient overexpression of Bcl-xL inhibited T. vaginalis-induced apoptosis in a dose-dependent manner.
My observation that T. vaginalis down regulates Bcl-xL protein suggests that loss of Bcl-xL
is essential for T. vaginalis-induced apoptosis in RAW 264.7 cells. If so, prevention of this loss
would be expected to protect cells from T. vaginalis-induced apoptosis. To confirm the result above, RAW 264.7 cells were transiently transfected with Bcl-xL-Flag and then treated with T.
vaginalis for 8 h. Following treatment, apoptotic cells were quantified. T. vaginalis-induced
apoptosis was dramatically prevented in RAW 264.7 cells by transfection with Bcl-xL-Flag,
whereas a control vector (pcDNA3) had no effect on the ability of T. vaginalis to induce apoptosis (Figure II-6B). These data suggest that Bcl-xL may regulate apoptosis induced by T.
vaginalis in RAW 264.7 cells.
Because I have observed that T. vaginalis induces cytochrome c release and activates
caspase-9, We examined whether Bcl-xL overexpression could prevent these T. vaginalis-regulated effects. Western blot analysis revealed that Bcl-xL overexpression inhibited
cytochrome c release, activation of caspase-9, and cleavage of PARP (Figure II-6C, D).
Figure II-6. Overexpression of Bcl-xL blocked T. vaginalis-induced apoptosis. RAW 264.7 cells were transiently transfected with an empty vector (pcDNA3) or Bcl-xL-Flag. Then, T.
vaginalis was added to RAW264.7 cells for 8 h. (A) Overexpressed Bcl-xL expression was determined by immunoblotting using anti-Flag antibody. (B) Cells were stained with Annexin
V-FITC and analyzed by flow cytometry. (C) Western blot was conducted with the anti-cytochrome c antibody as indicated. Overexpressed Bcl-xL expression was determined by
immunoblotting using anti-Flag antibody. (D) Cleavage of caspase-9 and PARP was detected by immunoblotting with corresponding antibody. The results are representative of three
separate experiments with comparable outcomes. Immunoblotting with anti-β-actin antibody was used as a loading control.
4. Discussion
It is well known that intracellular protozoan parasites such as T. cruzi and T. gondii inhibit
host cell apoptosis in infected cells. Those studies reported that no significant change of the amount of intracellular Bcl-2 was observed in infected cells (Clark et al., 1999; Goebel et al.,
2001). In contrast, E. histolytica induce apoptosis in target cells, which is not blocked by Bcl-2 (Ragland et al., 1994).
In this study, to evaluate the relationship between T. vaginalis infection and cell death, we investigated the roles of Bcl-2 family on induction of apoptosis in T. vaginalis-treated RAW
264.7 cells. The expression of Bcl-2 protein remained almost constant upon treatment with T.
vaginalis (Figure II-2), and overexpression of Bcl-2 did not block T. vaginalis-induced
apoptosis in RAW264.7 cells (Figure II-3). Other effects of mitochondrial involvement in induction of apoptosis are the release of cytochrome c and the mitochondrial membrane
potential (Ceende et al., 1993). My previous studies demonstrated the release of cytochrome c in T. vaginalis-treated RAW264.7 cells. Here, I have shown that significant change of the
membrane potential was observed in T. vaginalis-treated RAW264.7 cells (Figure II-1B).
Moreover, in Bcl-2-transfected RAW264.7 cells, overexpression of Bcl-2 not only did not
inhibit T. vaginalis-induced cytochrome c release and Bax activity (Figure II-4A), but also did not alter elevation of the membrane potential in T. vaginalis-treated RAW264.7 cells (Figure
of its localization at mitochondrial membrane (Cuende et al., 1993- Zhu et al., 1996).
Therefore, my findings indicate that T. vaginalis-induced apoptosis in RAW264.7 cells may be
independent of Bcl-2 expression. In addition, it is possible that the inability of Bcl-2 overexpression to inhibit T. vaginalis-induced apoptosis may be induced by other Bcl-2 family
rather than Bcl-2 dependent apoptotic pathways.
In addition to mitochondrial involvement, I also examined the relationship between Bcl-2
family activity and the cleavage of caspases, a key event of apoptotic pathway in T. vaginalis-treated RAW 264.7 cells. A family of caspases related to the C. elegans CED-3 plays a central
role in driving the apoptotic pathways triggered by a variety of stimuli. (Ellis et al., 1986).
Activation of caspases through transmission of diverse apoptotic signals leads to cleavage of
target proteins and execution of the apoptotic program (Los et al., 1999). Recent study has shown that overexpressed Bcl-2 can dramatically inhibit the change of mitochondria
membrane potential and activation of both caspase-9 and –3, and thus Bcl-2 overexpression inhibits TCHQ-induced apoptosis in NIH3T3 cells (Lin et al., 2004). Not expected, in this
study, the cleavages of pro-caspase-9, -3 and PARP was not inhibited by overproduction of Bcl-2 (Figure II-4C). This finding supports a hypothesis that T. vaginalis-induced apoptosis
seems to be controlled by very distinct mechanisms from the ones that operate in other studies
showed that although a decrease in the ratio of Bcl-2 to Bax was not apparent, the expression level of Bcl-xL dramatically was decreased with the culture time (Figure II-2, 5A). These
results are consistent with previous observation, which that only Bcl-xL, and not Bcl-2, was observed to inhibit apoptosis in WEHI-231.7 cells in response to cross-linking of IgM and
other stimuli (Gottschalk et al., 1994). Interestingly, the downregulation of Bcl-xL was almost abolished by the treatment with both caspase inhibitors (Figure II-5B). Cheng et al. similarly
reported that the anti-apoptotic protein Bcl-2, a homologue of Bcl-xL, serves as a substrate for caspase-3 in cells undergoing apoptosis and that the cleavage product of Bcl-2 exerts a
death-promoting function (30). In general, caspase-3 activation is though to require Apaf-1 and cytochrome c release from mitochondria. This mechanism is regulated by the Bcl-2 family
(Zou et al., 1997). Thus, my result suggests that caspase-3 may play pivotal role in the process of apoptosis as well as the downregulation of Bcl-xL by T. vaginalis.
Several studies have demonstrated that Bcl-xL can associate with caspase-1, caspase-8, and caspase-9 in mammalian cells (Chinnaiyan et al., 1997; Hu et al., 1998). Here when Bcl-xL
-overexpressing RAW 264.7 cells are treated with T. vaginalis, I have shown that Bcl-xL
associated with cytochrome c release, caspase-9 and caspase-3 (Figure II-6C, D). Therefore,
even though the involvement of Apaf-1 is unclear, it is possible that Bcl-xL could inhibit T.
vaginalis-induced apoptotic cell death. It might be through alteration of the interaction of Apaf-1 with cytochrome c and /or inhibition of conformational changes in the
Apaf-1/caspase-pathway (Giri et al., 2003). The protozoan parasite E. histolytica-induced DNA fragmentation is unaffected in caspase 8-deficient Jurkat cells (Huston et al., 2000). In previous my study, I
could not find cleavage of a caspase-8 and Bid activity in T. vaginalis-treated RAW264.7 cells, indicating that the death receptor-mediated apoptotic pathway was not involved in T.
vaginalis-induced apoptosis.
In conclusion, my study suggests that induction of apoptosis by T. vaginalis was found to be
associated with a reduction of expression of anti-apoptotitc Bcl-xL protein, not Bcl-2. In addition, the activation of caspase-9 suggests that mitochondria play a role in T.
vaginalis-induced RAW264.7 cell death. The downregulation of Bcl-xL expression critically involves activation of caspase-3, which downregulated Bcl-xL, and thereby provides a positive
feedback mechanisms that may be responsible for the T. vaginalis-induced apoptotic cell death.
Therefore, it is plausible that the lack of Bcl-2 protection implies that the functions of Bcl-xL
with caspase-3 may be important to regulate the T. vaginalis-induced apoptotic pathway with biological relevance.
CHAPTER III
Trichomonas vaginalis inhibits proinflammatory cytokine production in macrophages by suppressing
NF- κB activation
1. Introduction
Trichomonas vaginalis is a flagellate protozoan parasite, which infects the genito-urinary
tract of humans. It is responsible for trichomoniasis, one of the most common nonviral sexually transmitted disease (STD) in the world (Petrin et al., 1998). The vaginal infection
caused by T. vaginalis is severe due to the various factors involved in the development of infection. It has been associated with adverse outcomes of pregnancy (Cotch et al, 1997),
cervical cancer (Kharsany et al., 1993), and the increase in the transmission of human immunodeficiency virus (HIV) (Draper et al., 1998). It is well known that T. vaginalis adheres
to epithelial cells and survives in reproductive tracts by scavenging nutrients from the host (Alderete et al., 1995). Their detachment from epithelial cell results in the cytological change
observed in trichomoniasis (Gupta et al., 1990). It causes a variety of symptoms from a state of severe inflammation and irritation with a frothy malodorous discharge to a relatively
asymptomatic carrier state (Petrin et al., 1998).
In the female reproductive tract, the mucosal immune system is the first stage of the defense
against pathogenic organisms (Underdown and Schiff, 1986). It is mediated by both innate and adaptive immune responses including the humoral and cell-mediated immunities, leading to
of protozoan infection (Scharton-Kersten et al., 1997). Some clinical trials have been reported by treating econazole in combination with ibuprofen isobuthanolammonium to improve
submucosal macrophage cytotoxicity against T. vaginalis (Martinotti et al., 1983; Drage et al., 2000) indicate that the important role of macrophage as a compartment of host immune
system. However, the role of macrophage in T. vaginalis infection is not clear.
Macrophages play a key role against infection as an innate immune system, and are capable of producing TNF-α and IL-12 (Butcher et al., 2001). TNF-α is a multifunctional cytokine
that trnasduces signals of survival, differentiation, and cellular death in diverse cell types and
that elicits diverse biological events by inducing the expression of various genes (Roulston et al., 1998). It also has been reported that TNF-α plays a major role in defense against
mycobacterial infection (Sugawara et al., 1999). IL-12 production by macrophages is significant in acquiring the potential of directing acquired immunity toward a Th-1 biased
response that is crucial to controlling microbial infections (Hsieh et al., 1993). Nuclear factor-κB (NF-factor-κB) is implicated in the regulation of cell growth, differentiation, inflammatory responses, and apoptosis (Medzhitov et al., 1997; Baeuerle and Henkel, 1994). NF-κB
consists of a family of dimeric factors and is reported to get involved in the transcriptional regulation of many cytokine genes for TNF-α, IL-1β, and IL-12 in response to extracellular
signals (Murphy et al., 1995; Cogswell et al., 1994). In unstimulated cells, NF-κB remains as an inactive form in the cytoplasm in association with the inhibitory family of IκB molecules.
(Michael and Yinon, 2000; Read et al., 1994). Thus, the activation of NF-κB is strongly
related to the production of cytokines and the activation of effector molecules in association
with innate immunity.
In this study, I investigated whether the inflammatory response of macrophages elicited by T.
vaginalis infection can be characterized by NF-κB activation. I show here that T. vaginalis induces rapid NF-κB activation in RAW264.7 macrophage during the early adhesion.
However, the activation was not maintained but resulted in blocking the production of proinflammatory cytokines such as TNF-α and IL-12. Furthermore, T. vaginalis infection
induced a state of non-responsiveness to subsequent stimulation with bacterial LPS. Together, MG-132 blocking IκB-α degradation curtailed apoptotic cell death in cells adhered with T.
vaginalis demonstrates that the NF-κB activation appears to be related to anti-apoptosis. These results suggest T. vaginalis is able to induce inhibitory mechanism to avoid or delay the
immune responses by host cells.
2. Materials and Methods
Materials
DMEM (Dulbecco’s modified Eagle’s medium) and FBS (fetal bovine serum) were purchased from sigma (St. Louis, USA). Antibodies for IκB-α and β-actin were purchased
from Santa Cruz Biotechnology (Santa Cruz, USA) and for p50 and p65 from Calbiochem (San Diego, USA). Annexin V/PI kit was purchased from Pharmingen (San Diego, USA).
Parasites
Trichomonas vaginalis strain KT-4 (kindly provided by J. S. Ryu, Department of Parasitology, University of Han-yang, Korea) was used in this study. Trichomonads were
cultured in Diamond's Trypticasc-yeast extract-maltose (TYM) medium (Flynn et al., 1995) and supplemented with 10% heat-inactivated horse serum in 5% CO2 environment for 24 h at
37℃. Cultured parasites were always monitored for motility and only those viable parasites (>99%) were for experiments.
Cell lines culture and in vitro infection
The murine monocyte/macrophage cell line, RAW 264.7, was cultured in DMEM supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 U/ml) in 5% CO2
medium at desired time interval.
Transient transfection and luciferase reporter gene assay
The pGL3-NF-κB promoter constructs were transfected into RAW264.7 cells by
LipofectAmine 2000 (Invitrogen, Hercules, USA). Luciferase activity was assayed by using a luciferase assay kit according to the manufacturer's instructions (Promega, Madison, USA).
Cell extracts were prepared in 500 µl 1× reporter lysis buffer. The lysates were centrifuged at
13,000g for 5 min and supernatant was used for detection of luciferase activity in a
Microlumat LB96P Luminometer (Perkin Elmer, Wallac, Inc., USA). The luciferase reporter assay was repeated at least three times. The results are reported as mean ±standard error (SE).
Electrophoretic mobility shift assays (EMSAs)
Assays were performed with the gel shift assay system (Promega) according to the manufacturer’s protocol, with 5 to 10 µg of nuclear protein. Cells (2 × 107) were lysed in 200 µl lysis buffer (20 mM HEPES (pH 7.9), 10 mM NaCl, 3 mM MgCl2, 0.1% Nonidet P-40,
10% glycerol, 0.2 mM EDTA, 1 mM DTT, 0.4 mM PMSF, and 1 µg/ml leupeptin). The lysates
were incubated on ice for 15 min and then centrifuged at 2000 rpm for 5 min. The pellets were
supernatants were stored at -70℃. Sequences of double stranded consensus oligonuclotides used in gel shift reactions were as follows: NF-κB (Promega), 5’-AGT TGA GGG GAC TTT CCC AGG C-3’. Probe labeling was carried out as specified by the manufacturer with [γ-32P]
ATP (3,000 Ci/mmol; 10 mCi/ml) (Amersham). Specificity studies were performed with a
50-fold molar excess of unlabeled oligonucleotide added to the reaction mixtures prior to the addition of radiolabeled oligonucleotides. Reaction mixtures were analyzed on 5%
nondenatured polyacrylamide gels with 0.5x TBE (89 mM Tris-HCl [pH 8.0], 89 mM boric acid, 2 mM EDTA) as the running buffer. The gels were electrophoresed at 100 V for 3 h,
dried in gel dryer, and subjected to autoradiographic exposure for 12 to 48 h.
RT-PCR
RAW264.7 cells were used for reverse transcriptase polymer chain reaction (RT-PCR) analysis to examine TNF-α and IL-12 mRNA expression levels RAW264.7 cells samples
during T. vaginalis adhesion. These samples were snapfrozen in liquid nitrogen and stored at
-80℃ until use. For RNA extraction, the RAW264.7 cells were treated with total RNA isolation reagent, TRIzol reagent (Gibco BRL, Hercules, USA) as specified by the
manufacturer. After isolation, total RNA was reverse transcriptase (Gibco BRL, Hercules, USA) following measurement of the total RNA concentration, and agarose gel electrophoresis was performed. PCR was performed with gene-specific primer sets for TNF-α, IL-12p40, and
23 cycles of denaturation (94℃ for 1 min), annealing (65℃ for 1 min), and extension (72℃
for 2 min). The primer sequences and PCR product sizes were as follows: for TNF-α, 5'-ATG
AGC ACA GAA AGC ATG ATC-3' (sense) and 5'-TAC AGG CTT GTC ACT CGA ATT-3' (anti-sense), 276 bp; for IL12p40, 5'-ATC TCC TGG TTT GCC ATC GTT TTG-3' (sense) and 5'-TCC CTT TGG TCC AGT GTG ACC TTC-3' (anti-sense), 527 bp; for β-actin, 5'-TGT
GAT GGT GGG AAT GGG TCA-3' (sense) and 5'-TTT GAT GTC ACG CAC GAT TTC C-3'
(anti-sense), 514 bp. Amplification was carried out with a thermal cycler (model 480, Perkin-Elmer Cetus). 10 µl of each PCR product was used for electrophoresis in a 4%
agarose-NuSieve GTG (1:3) gel and visualized using ethidium bromide staining.
Quantitative real-time PCR
To quantify mRNA levels of cytokines, quantitative real-time PCR was performed using the SYBRGreen PCR core reagents mix (Applied Biosystems, Foster City, USA) containing 1 ×
SYBRGreen PCR buffer; 3 mM MgCl2; 100 µM dATP, dCTP, and dGTP; 200 µM dUTP;
0.025 U/µl AmpliTaq Gold DNA polymerase; 0.01 U/µl AmpErase UNG ; and 2 pmol/µl
gene-specific sense and anti-sense primers. The primers and probes were used as described in
previously (Overbergh et al., 2003). The reaction conditions were as follows: 2 min at 50°C
ELISA
In order to quantify specific cytokine concentrations in the culture supernatants, commercial available ELISA kits were used. Culture supernatants was harvested after 8 h of T. vaginalis infection and frozen at -70℃ until use. The amounts of released murine TNF-α and IL-12
proteins were analyzed using ELISA-kits purchased from R&D Systems. All assays were
performed in accordance to manufacturer's specifications.
Quantitative analysis of apoptosis by flow cytometry
Cells were trypsinized, washed with PBS, and resuspended in a binding buffer (10 mM
Hepes, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). After 15 min of incubation with annexin V-fluorescein isothiocyanate (Sigma, St. Louis, USA) and propidium iodide (Pharmingen, San
Diego, USA) at room temperature, the fluorescence emitted by cells (10,000 cells/sample) was analyzed on a FACScan flow cytometer (Becton-Dickinson, USA). Ten thousand events were
evaluated using the Cell Quest Program.
Western blot analysis
RAW 264.7 cells were seeded in 35-mm plastic dishes (3 × 105 cells per dish) and incubated
with T. vaginalis for different time periods. Cells were lysed in the lysis buffer (50 mM
Tris-centrifugation at 13,000 × g at 4℃ for 30 min, 20 µg of supernatant lysates from each sample
was run on 10% SDS-polyacrylamide gel and then electrophoretically transferred to PVDF
(polyvinylidene difluoride) membranes. PVDF membranes were rinsed in TBST (10 mM Tris-HCl (pH 7.4), 0.9% NaCl, 0.05% Tween 20, and 1 mM EDTA) and blocked in blocking buffer (TBST containing 5% bovine serum albumin) overnight at 4℃. After washing (3 times for 15
min) with TBST, PVDF membranes were incubated with primary antibodies overnight at 4℃.
Membranes were washed three times with TBST, incubated with goat rabbit HRP or anti-mouse HRP for 1 h at room temperature, washed and developed with ECL substrate
(Amersham Pharmacia Biotech, Arlington Heights, USA), and exposed to Biomax MS autoradiography x-ray film (Kodak, Rochester, USA).
Statistical evaluation
All experiments were performed at least three times. Results are presented as means ±
standard deviation (SD) if not otherwise indicated. Significance of the results was analyzed by
the Student’s t-test.
3. Results
Transient translocation of transcription factor NF-κB was induced in murine macrophage cells during early adhesion by T. vaginalis.
I examined NF-κB promoter activity whether T. vaginalis adhesion influences NF-κB
activation in RAW264.7 macrophage cells. T. vaginalis adhesion initially induced the binding activity of NF-κB promoter and which was declined after 8 h in comparison of LPS-induced activation of NF-κB (Figure III-1A). Translocation of NF-κB to the nucleus was also
confirmed by EMSA using 32P-end labeled DNA oligonucleotides derived from the Igκ chain
gene, which binds to both p65:p50 heterodimer and non-transactivating p50:p50 homodimer.
As shown in Figure III-1B, gel shift analysis showed that NF-κB translocation was also
rapidly proceeded by T. vaginalis adhesion from 1 h, which was similar to LPS treatment as a positive control. However, this translocation was dramatically inhibited after 8 h post-adhesion, suggesting that NF-κB activation in the nuclei is modulated by T. vaginalis adhesion. Since
the transcription factor NF-κB consists of a variety of homo- and heterodimers, gel supershift
analysis was performed to determine the subunit compositions of T. vaginalis-induced DNA complexes (Figure III- 1C). Antibodies against p50 and p65 subunits were added to the
nuclear extracts of adhesive RAW264.7 cells after 3 h post-adhesion. The fact that anti-p50 and -p65 retarded the NF-κB-specific DNA complex, may indicate the presence of p50 and
Figure III-1. Effect of T. vaginalis on the activation of NF-κB in RAW264.7 cells. (A) After
transient transfection with NF-κB-dependent luciferase reporter gene for 24 h, cells were incubated with or without LPS (10 ng/ml), or T. vaginalis for the indicated time and then
luciferase activity was determined in lysates. Results are expressed as means ±SD and are representative of three independent experiments (P<0.05). (B) Nuclear extracts were prepared
and subjected to EMSA analysis using a radiolabeled probe the κB consensus site for the Ig κ light chain gene promoter in comparision with LPS triggering for 30 min. The specificity of
complex formation was confirmed by addition of a 10-fold molar excess of unlabeled oligonucleotide (+cold). (C) To identify p65:p50 and p50:p50 dimers, nuclear extracts were