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* Institute for Medical Microbiology and Hygiene and
Institute for Physiology, Medical University of Lübeck, Lübeck, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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24 h. The antiapoptotic
effect of the parasites could not be transferred by the supernatants,
despite secretion of IL-8 by PMN upon coculture with L.
major. In vivo, intact parasites were found intracellularly in
PMN collected from the skin of mice 3 days after s.c. infection. This
finding strongly suggests that infection with Leishmania
prolongs the survival time of neutrophils also in vivo. These data
indicate that Leishmania induce an increased survival of
neutrophil granulocytes both in vitro and in vivo. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Immediately after infection with Leishmania major in the skin, a local inflammatory process is initiated, which involves the accumulation of leukocytes. In the earliest wave of infiltration, polymorphonuclear neutrophil granulocytes (PMN)3 are recruited within the first hours after infection, followed by monocytes/macrophages 2–3 days later (3, 4). Although the uptake and intracellular survival of Leishmania in macrophages are well established, little is known about the role of PMN as host cells for the parasites.
PMN play a vital role by phagocytosing and destroying microorganisms such as bacteria and fungi. Neutrophils were reported to phagocytose also Leishmania promastigotes (5) using both opsonin-dependent and opsonin-independent uptake mechanisms (6). Leishmania were reported to interfere with the production of reactive oxygen intermediates by neutrophils (7, 8). Consequently, not all internalized parasites are killed immediately in PMN, as demonstrated by the finding that morphologically intact parasites were detected in granulocytes of infected animals (9). Moreover, infected neutrophils were observed in chronic lesions of mice after infection with L. major (10). Therefore, after being recruited to the site of infection, granulocytes can internalize Leishmania and possibly can provide an intracellular milieu for the survival of the parasites.
Neutrophil granulocytes are inherently short-lived cells with a
t1/2 of only 6–10 h in the
circulation, after which they undergo apoptosis (11, 12).
It is an active and well-regulated process that is characterized by
specific phenomena such as cell shrinkage, chromatin condensation,
internucleosomal DNA fragmentation, membrane blebbing, and, finally,
the decay into apoptotic bodies (12, 13). Although
apoptosis is an intrinsic cell process, the life span of mature
neutrophils can be extended in vitro by incubation with either
proinflammatory cytokines including GM-CSF, G-CSF (14),
IL-8 (15), IL-1
(16), glucocorticoids
(17), or bacterial products such as LPS and fMLP
(16, 18).
Moreover, several microbial pathogens have been reported to influence this process as common strategy to overcome host defenses (for review, see Ref. 19). Thus, apoptosis induction in cells of the innate or adaptive immunity appears to be a way to escape antimicrobial defense of the host. Microbial pathogens such as Escherichia coli (20) and Candida albicans (21) were found to induce apoptosis of PMN. Induction of apoptotic death of both mononuclear and polymorphonuclear leukocytes by Fusobacterium nucleatum was suggested to mediate immunosuppression in periodontal disease (22). In contrast, inhibition of apoptosis of host cells by intracellular pathogens such as Theileria (23), Toxoplasma (24), or the agents of human granulocytic ehrlichiosis (HGE) (25) can provide an intracellular niche for the pathogens by extending the life span of the host cells.
In the present study, we investigated whether also Leishmania can prolong the lifetime of neutrophils to secure an intracellular environment for its survival. We found that coincubation with L. major promastigotes results in a delay of neutrophil apoptosis, which is associated with a decrease in caspase-3 activity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peripheral blood was collected by venipuncture from healthy adult volunteers using lithium-heparin, which was reported to be the optimal anticoagulant for preserving granulocyte viability (26). Blood was layered on Histopaque 1119 (Sigma-Aldrich, Deisenhofen, Germany) and centrifuged for 20 min at 800 x g. The plasma and the interphase consisting mainly of lymphocytes and monocytes were discarded. The granulocyte-rich layer below the interphase was collected, leaving the erythrocyte pellet on the bottom of the tube. Granulocytes were washed twice in RPMI 1640 medium (Seromed-Biochrom, Berlin, Germany), and further fractionated on a discontinuous Percoll (Pharmacia, Uppsala, Sweden) gradient consisting of layers with densities of 1105 g/ml (85%), 1100 g/ml (80%), 1093 g/ml (75%), 1087 g/ml (70%), and 1081 g/ml (65%). After centrifugation for 20 min at 800 x g, the interface between the 80% and 85% Percoll layers was collected and washed twice in RPMI 1640 medium. All procedures were conducted at room temperature. The preparations contained >99% granulocytes, of which >95% were neutrophils and 1–4% were eosinophils, as determined by Giemsa staining of cytocentrifuged (Shandon, Pittsburgh, PA) samples. Eosinophil granulocytes were identified as cells containing numerous small orange-red-stained intracellular granula. Cell viability was >98%, as determined by trypan blue exclusion.
Leishmania and coincubation experiments
The origin and propagation of the cloned virulent line of L. major (MHOM/IL/81/FEBNI) have been described elsewhere (27). Stationary phase promastigotes were collected from in vitro cultures in biphasic NNN blood agar medium. PMN were coincubated at 37°C in a volume of 1 ml RPMI 1640 medium (Life Technologies Laboratories, Eggenstein, Germany) supplemented with 50 µM 2-ME, 2 mM L-glutamine, 10 mM HEPES, 100 µg/ml penicillin, and 160 µg/ml gentamicin (all Seromed-Biochrom), and with 10% FCS (Life Technologies Laboratories) with L. major promastigotes at a parasite-PMN ratio of 5:1 in a humidified atmosphere containing 5% CO2.
Coincubation experiments were also conducted by using killed promastigotes or parasite lysates. L. major promastigotes were killed by treatment with ethanol, as described (28). Briefly, stationary phase promastigotes at a concentration of 1 x 107/ml were incubated in PBS containing 30% ethanol for 30 min at room temperature, followed by washing twice in PBS. Parasite lysates were obtained by freezing and thawing L. major promastigotes (1 x 107/ml in PBS) five times. In the coincubation experiments, killed Leishmania and parasite lysate were used at a parasite-PMN ratio of 5:1.
To assay the antiapoptotic effect of the supernatants from L. major-PMN cocultures, neutrophils were coincubated with L. major promastigotes for 24 h, as described above. Supernatants were collected and freshly isolated, and pelleted PMN were resuspended in these supernatants (supernatant concentration = 100%) for 24 h.
Assessment of PMN apoptosis
Morphological assessment of apoptosis. In neutrophils, morphological changes of apoptosis are striking and include separation of nuclear lobes and darkly stained pyknotic nuclei (12, 13). Accordingly, the morphological criteria for neutrophil apoptosis were one or more densely stained nuclear fragments and the absence of chromatin within nuclear lobes/fragments. Nuclear morphology was assessed on Giemsa-stained cytocentrifuge slides. Cell morphology was examined under oil immersion light microscopy, and a minimum of 200 cells/slide was examined and graded as apoptotic/nonapoptotic.
Annexin V binding
Annexin V exhibits calcium-dependent binding to phosphatidylserine (PS) expressed in the outer membrane leaflet of apoptotic PMN (29). Labeling of apoptotic cells with annexin V FITC (Roche Molecular Biologicals, Mannheim, Germany) was performed, as recommended by the manufacturer. Labeled cells were analyzed by flow cytometry using a FACSCalibur with CellQuest software (BD Biosciences, San Diego, CA).
TUNEL assay of chromatin fragmentation
The TUNEL assay (In Situ Cell Death Detection kit; Roche Molecular Biologicals) was used to detect apoptotic cell death by enzymatic labeling of DNA strand breaks with fluorescein-dUTP and TdT (30). Briefly, cytocentrifuge slides containing 1 x 105 cells were fixed in 4% formaldehyde/PBS (pH 7.4) for 60 min at room temperature, washed in PBS, and then suspended in permeabilization solution (0.1% Triton X-100/0.1% sodium citrate) for 3 min on ice. Cells were washed again, resuspended in 50 µl TUNEL-reaction mixture or in 50 µl label solution alone (negative control), and incubated in a humidified dark chamber at 37°C, followed by washing in PBS. The green fluorescence of apoptotic nuclei was detected by fluorescence microscopy.
Gel electrophoresis of low m.w. DNA
Internucleosomal DNA fragmentation was assessed using the ApopLadder Ex kit (Takara Biomedicals, Takara Shuzu, Otsu, Shiga, Japan). Briefly, neutrophil granulocytes were incubated with or without L. major promastigotes, and the fragmented DNA was separated from intact chromatin of 1 x 106 granulocytes. Proteins and RNA were eliminated by enzymatic digestion. Low m.w. DNA was precipitated with ethanol and analyzed in 1.8% agarose gels.
Fluorometric analysis of caspase-3 activity
Neutrophil granulocytes were incubated with/without L. major promastigotes, as described above. A total of 1 x 106 cells was counted and collected by centrifugation and resuspended in 100 µl lysis buffer (100 mM HEPES, pH 7.5, 10% sucrose, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 1 mM EDTA, and 1 mM PMSF). Cell lysate was incubated with 12 µM fluorogenic peptide substrate DEVD-AMC (7-amino-4-methylcoumarin; Biomol, Hamburg, Germany) in a 96-well microtiter plate (Greiner, Frickenhausen, Germany) at room temperature. The cleavage of DEVD-AMC was monitored by AMC (Sigma-Aldrich) liberation in CytoFluor 2350 plate reader (Millipore, Bedford, MA) using 360-nm excitation and 460-nm emission wavelength. Fluorescence was measured every 2 min during a 60-min period, and fluorescence units were converted to pmol amounts of AMC using a standard curve generated with free AMC. To assess the specificity of the reaction, the competitive inhibitor of caspase-3 DEVD-CHO (50 nM) was added to the samples. This treatment blocked DEVD-AMC cleavage (not shown).
SYTO-16/annexin V double staining to visualize simultaneously viable parasites and apoptotic PMN
Neutrophil granulocytes were coincubated with L. major promastigotes at a parasite-PMN ratio of 5:1 at 37°C in a humidified atmosphere containing 5% CO2 in a volume of 1 ml RPMI 1640 medium supplemented with 10% FCS (see above). After 18 h, cells were stained with SYTO-16 (Molecular Probes, Leiden, The Netherlands) and annexin V-Alexa 568. Staining with 5 µmol SYTO-16 for 10 min at room temperature was used to visualize viable intracellular parasites in PMN. This dye penetrates cell membranes and stains DNA. SYTO-16 stains, therefore, nuclei and kinetoplast of viable cells/parasites green. SYTO-16 has been reported to be useful to visualize also apoptotic nuclei in live cells (31). Subsequent to the SYTO-16 staining, the cells were stained with annexin V to reveal apoptotic cells, as described above.
Cytokine assays
Neutrophil granulocytes were coincubated with L.
major promastigotes at a parasite-PMN ratio of 5:1 at 37°C in a
humidified atmosphere containing 5% CO2 in a
volume of 1 ml RPMI 1640 medium supplemented with 10% FCS (see above).
Culture supernatants were collected after 18 and 24 h and stored
at -20°C until cytokine determination. IL-8, GM-CSF, and TNF-
were measured using ELISA (R&D Systems, Wiesbaden, Germany), according
to the manufacturer’s instructions.
Subcutaneous air-pouch technique
Female BALB/c mice were obtained from Charles River Breeding Laboratories (Sulzfeld, Germany) and were used at 8–12 wk of age. Air pouches were raised on the dorsum by s.c. injections of 4 ml sterile air on days 0 and 3, as previously described (32). On day 6, 1 x 107 L. major promastigotes in 1 ml PBS were injected into the air pouch. As control, 107 latex beads (3 µm diameter; Sigma-Aldrich) in 1 ml PBS or as a sterile inflammatory agent 0.5% oyster type II glycogen in 1 ml PBS (Sigma-Aldrich) (33) were injected into the pouch. Three days after infection, the mice were killed and 1 x 105 exudate cells were centrifuged on microscope slides at 200 x g for 5 min using a cytocentrifuge. The slides were air dried, fixed with methanol, and then stained with Giemsa.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Morphological assessment of PMN apoptosis.
Neutrophils undergo constitutive apoptosis when aging in vitro. Aging
granulocytes exhibit classical features of apoptosis, such as cell
shrinkage, cytoplasmic condensation, and condensation of nuclear
heterochromatin (11, 12, 13). Accordingly, neutrophil
apoptosis can be assessed by various parameters, including changes in
cellular morphology. Using these criteria, the ratio of apoptotic cells
was determined in highly purified granulocytes (purity >99%) cultured
in vitro in the absence or presence of L. major
promastigotes. The majority of neutrophil granulocytes (63 ±
11%, n = 8, range of 50–80%) were found to have an
apoptotic nuclear morphology after incubation in vitro for 24 h
(Figs. 1
B and
2). The rate of apoptosis was strongly
reduced when neutrophils were coincubated with promastigotes of
L. major; 30 ± 6% of the cells
(n = 8, range of 15–45%) had apoptotic morphology
after 24 h of coincubation (Figs. 1A and 2). The ratio
of infected PMN was 74 ± 9%. Infected granulocytes contained at
average 2.5 ± 0.5 intracellular parasites in a range of one to
seven parasites/cell. After 24 h of coincubation with L.
major, most of the apoptotic PMN were those without intracellular
parasites. However, a small proportion (5.5 ± 3.3%) of infected
granulocytes also had apoptotic nuclear morphology.
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Detection of nuclear DNA nicks using the TUNEL staining
In addition to the analysis of apoptotic nuclear morphology, the
inhibitory effect of L. major on neutrophil apoptosis was
investigated by the TUNEL assay, which reveals the apoptotic
fragmentation of nuclear DNA. After incubation in vitro for 24 h,
most of the neutrophils showed an intensive TUNEL staining (Fig. 3B). The majority of
neutrophils that were coincubated with L. major
promastigotes remained TUNEL negative (Fig. 3A).
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Electrophoresis of extracted low m.w. DNA from PMN populations
aged in vitro for 24 h showed a ladder pattern of internucleosomal
cleavage indicative of endonuclease activation. The analysis of
internucleosomal DNA degradation revealed an inhibition of apoptosis in
PMN by L. major, as shown by the marked reduction of low
m.w. chromatin fragments after coincubation with the parasites as
compared with neutrophils incubated in medium alone (Fig. 4).
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Visible changes of nuclear morphology are associated with
progressed stage of cellular apoptosis. Similarly, TUNEL positivity and
the appearance of typical apoptotic low molecular DNA ladder are
associated with the later stage of the apoptotic process. An earlier
marker of PMN apoptosis is the appearance of PS on the outer membrane,
a process that is called membrane flip. PS can be detected by staining
with annexin V. Annexin V staining revealed high PS expression in the
majority of neutrophils after incubation in vitro for 18 h (Fig. 5). Coincubation with L. major
resulted in a marked decrease in annexin V binding (Fig. 5).
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The spontaneous apoptosis was inhibited only in the presence of
viable L. major promastigotes. Coincubation of neutrophils
with killed promastigotes or a lysate of parasites did not lead to a
decrease in the rate of apoptotic cells after 24 h of in vitro
culture (Fig. 2). The antiapoptotic activity of Leishmania
did not strictly depend on the phagocytosis of the parasites because
some neutrophils without intracellular Leishmania also
showed a nonapoptotic nuclear morphology after 24 h of
coincubation with L. major. The uptake of
Leishmania appeared to be a major factor of delayed
apoptosis because microscopical examination of Giemsa-stained
cytocentrifuge preparates, as shown above, revealed only a minor
population (5.5 ± 3.3%) of infected neutrophils that was
apoptotic. To confirm this observation, a double staining was applied
to visualize both live parasites and apoptotic neutrophils. Neutrophils
were coincubated with L. major for 18 h and then
stained with the live stain SYTO-16 to visualize viable cells. The
cells were then stained with annexin V PE to detect apoptotic cells.
The majority (>95%) of neutrophils that contained green-stained
Leishmania were nonapoptotic viable cells, as detected by
the positive staining with SYTO-16 and lack of reactivity with annexin
V (as shown in Fig. 6). Almost none of
the red-stained apoptotic neutrophils harbored Leishmania
(Fig. 6). However, a minor population (2%) of apoptotic neutrophils
was infected (not shown). These findings suggest that the
internalization of living Leishmania promastigotes is a
strong inducer of delayed spontaneous apoptosis in neutrophils.
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Caspase-3 is one of the key enzymes involved in spontaneous
apoptosis of neutrophils (34). The enzymatic activity of
caspase-3 in lysates of neutrophils was measured by using a synthetic
substrate of caspase-3, giving rise of fluorescent product after
cleavage by the enzyme. A strong caspase-3 enzymatic activity was
observed in PMN after incubation in vitro for 18 h (Fig. 7). Coincubation with L. major
reduced the caspase-3 activity by 50% (Fig. 7).
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). The low caspase-3 activity
at this time point suggests that the cell death program had been
completed in many neutrophils by 42 h of in vitro culture. Indeed,
after 42 h, many cells were dead, as detected by staining with
trypan blue (not shown), as the consequence of cellular disintegration
of neutrophils upon extended incubation in vitro.
A delay of cell death could be observed in the presence of L.
major promastigotes, in which the majority of neutrophils were
still trypan blue negative after 42 h of culture (not shown).
Importantly, neutrophils coincubated with L. major expressed
a higher caspase-3 activity after 42 h as compared with the enzyme
activity in these cells after 18 h of coincubation (Fig. 7). The
level of caspase-3 activity of PMN after 42 h of coincubation with
L. major was comparably high as the enzyme activity measured
in the absence of Leishmania already 18 h after in
vitro culture. This finding clearly shows that the apoptotic process of
neutrophils is not blocked, but only delayed in the presence of
L. major.
L. major induces the release of IL-8, but not of GM-CSF by PMN
Various cytokines are among the proteins that are synthesized by
neutrophils. Among these, IL-8 (15) and GM-CSF
(14) have been reported to be able to inhibit neutrophil
apoptosis. To investigate whether L. major can affect PMN
apoptosis by inducing the secretion of antiapoptotic cytokines, IL-8
and GM-CSF were measured in culture supernatants of neutrophils after
coculture with L. major. Viable promastigotes induced the
production of high levels of IL-8 within the first 24 h of
coincubation (Fig. 8). The finding that
the IL-8 content of the supernatants markedly increased between 18 and
24 h (Fig. 8) indicates clearly that the PMN were not apoptotic,
but functionally still fully active during this period of coincubation.
In contrast to viable promastigotes that induced IL-8 in the range of
2000–3000 pg/ml (Fig. 8), killed parasites induced only marginal
(50–100 pg/ml) amounts of IL-8 in PMN cultures (not shown). The GM-CSF
content of all supernatants tested remained below the detection level
of 10 pg/ml (not shown).
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). This finding suggests that, although PMN produce
IL-8 upon coculture with viable L. major, the induction of
antiapoptotic cytokines appears not to be the mechanism of
antiapoptotic effect of Leishmania. Leishmania survive in PMN in vivo
The finding that Leishmania induces the delay of
neutrophil apoptosis in vitro suggests that this may occur also in
vivo, resulting in the survival of the parasites intracellularly in
neutrophils in the first days of infection. To test this hypothesis,
L. major promastigotes were injected s.c. into air pouches
of BALB/c mice. After inflammatory stimuli, neutrophils migrate into
this type of pouch within hours. This early influx of neutrophils is
then followed by a massive infiltration of mononuclear cells. To
investigate the intracellular survival of Leishmania in this
in vivo model, inflammatory exudate cells were collected
from the air pouch 3 daysafter Leishmania infection.
Giemsa-stained cytospin slides were assessed for the presence of
neutrophils. Both apoptotic and nonapoptotic neutrophil granulocytes
were detected among the infiltrating cells. However, the rate of
apoptotic PMN was as low as 4.6% ± 3.3%. The rate of
L. major infection in the exudate PMN was 14.3 ± 6%.
In control experiments, inert latex beads or the sterile inflammatory
agent glycogen were injected into air pouches of BALB/c mice. The latex
bead-induced exudate contained 10.6 ± 4.1% apoptotic
neutrophils. This figure, however, is valid only for the PMN containing
few beads. Because latex beads are not transparent under light
microscopy, a clear differentiation between apoptotic and nonapoptotic
nuclear morphology was difficult in PMN harboring several beads. In
glycogen-induced exudate, the percentage of apoptotic PMN was 8.0
± 3.4%, thus somewhat higher than in the L. major-induced
exudate. These data suggest that Leishmania induce a delayed
PMN apoptosis also in vivo. Importantly, intracellular parasites were
seen only in nonapoptotic neutrophils, but not in those with apoptotic
nuclear morphology (Fig. 9). This finding
suggests that while noninfected PMN become apoptotic, the spontaneous
apoptosis of infected neutrophils is delayed in vivo. It is likely that
the infected nonapoptotic PMN 3 days after infection represent the PMN
that ingested Leishmania in the first hours after infection
and whose apoptosis was delayed by the parasites. The intracellular
parasites were morphologically intact, demonstrating clearly that
Leishmania can survive in neutrophil granulocytes in vivo
for as long as 3 days.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Coincubation of PMN with L. major promastigotes in vitro led to an inhibition of their spontaneous apoptosis, as assessed by morphological analysis of PMN, annexin V binding, as well as by detecting the apoptosis-associated DNA fragmentation. Several intracellular microorganisms have been shown to delay the apoptosis of their host cells. Theileria parva infects and transforms bovine T cells. The parasite-induced mechanisms play a crucial role in the survival of transformed T cells by conveying protection against an apoptotic signal that accompanies parasite-mediated transformation (23). The molecular mechanisms underlying the inhibition of induced host cell apoptosis are not known yet. Toxoplasma gondii protects murine lymphoma cell lines from apoptosis, which requires the presence of live organisms (24). However, T. gondii inhibits the induced and not the spontaneous apoptosis of host cells. In this study, we described the inhibition of spontaneous rather than induced apoptosis of PMN by Leishmania. The differences between the spontaneous and induced apoptotic processes suggest that the various infectious agents may use more than one mechanism to achieve the extended survival of their host cells. Whether similar mechanisms are involved in the antiapoptotic effect of T. gondii and L. major remains to be clarified.
In our present study, we demonstrated that Leishmania
affects the survival of neutrophils via a mechanism involving the
inhibition of caspase-3 activation. Caspase-3 activation was found to
be associated with the neutrophil apoptosis induced by various agents
such as TNF- treatment (35), glutathione depletion
(36), or infection with C. albicans
(21), therefore being one of the key molecules in the
induced neutrophil apoptosis. In addition, the spontaneous apoptosis of
PMN was also shown to involve caspase-3 (35). Freshly
isolated neutrophils have a high expression of procaspase-3, which is
cleaved during spontaneous apoptosis (37). We showed that
the Leishmania-mediated delay of neutrophil apoptosis is
associated with a marked decrease in caspase-3 activity. This suggests
that L. major affects the transition of procaspase-3 to
enzymatically active caspase-3. Infection with C. albicans
was reported to enhance caspase-3 activity in PMN, resulting in the
accelerated apoptosis of these cells (21). Therefore, the
transition of procaspase-3 to caspase-3 appears to be a target pathway
of pathogens both to inhibit and to accelerate the spontaneous
apoptosis of PMN.
Previously, Leishmania donovani has been reported to inhibit
the apoptosis of macrophages (38). However,
Leishmania appear to use distinct pathways to inhibit
apoptosis in neutrophils and macrophages. First, Leishmania
infection induces TNF- secretion by macrophages, and TNF- in turn
can inhibit macrophage apoptosis. Therefore, TNF- was suggested as
one possible mediator of delayed macrophage apoptosis by L.
donovani (38). In contrast to macrophages, however,
TNF- accelerates PMN apoptosis, leading to rapid apoptotic death of
these cells (35, 39). Second, L. donovani
promastigotes were shown to induce the secretion of GM-CSF in
macrophages (38), a cytokine that can delay induced
apoptosis of macrophages. In our present study, PMN did not produce
GM-CSF upon coculture with L. major. This is in accordance
with previous findings that macrophages, but not neutrophils, are a
major source of GM-CSF (40). Third, in our experiments,
only live L. major promastigotes inhibited the PMN
apoptosis. Killed promastigotes or a lysate of the parasites had no
antiapoptotic effect. The antiapoptotic effect of L.
donovani in macrophages did not depend on live parasites; killed
promastigotes, parasite lysate, supernatants of parasite cultures as
well as lipophosphoglycan had all antiapoptotic activity
(38).
To date, the agent of HGE was the only known microorganism able to inhibit spontaneous neutrophil apoptosis (25). The mechanism of how this intracellular bacterium can prolong the life span of neutrophils remains fully unknown. However, it appears to be different from the mechanism used by Leishmania. Although only viable L. major displayed an antiapoptotic effect, not only live, but also killed HGE agent as well as bacterial supernatant were effective. The antiapoptotic principle of HGE agent was not identified; preformed proteins of the bacteria were suggested to be responsible for the observed effect (25). The reported data did not rule out that LPS or related bacterial products, such as the major surface protein P44 (41) or an atypical bacterial component found in HGE agents (42), mediate the antiapoptotic effect of these intracellular bacteria. This could explain that not only live, but killed bacteria and bacterial supernatant delayed the PMN apoptosis.
IL-8 has also been shown to delay the apoptosis of neutrophils
(15, 43). Although PMN produced high amounts of IL-8 upon
coculture with Leishmania, the IL-8-containing supernatants
had no antiapoptotic effect on freshly isolated neutrophils. Therefore,
the antiapoptotic effect of Leishmania is not mediated by
soluble mediators such as IL-8. However, the issue of IL-8-mediated
inhibition of PMN apoptosis is controversial. Whereas in one report
(43) IL-8 was shown to inhibit PMN apoptosis in all
concentrations in the range of 1–1000 ng/ml, others demonstrated that
only high, but not low concentrations (below 80 ng/ml) of IL-8 have
this activity (15). The supernatants of PMN after
conincubation with viable L. major promastigotes contained
2–3 ng/ml IL-8. In our studies, this concentration of rIL-8 did not
affect PMN apoptosis, although a significant inhibition of PMN
apoptosis was achieved by IL-8 concentrations higher than 100 ng/ml
(not shown). It is also possible that the supernatants contain
additional factors, such as TNF-, which could promote cell death and
counteract the effects of IL-8. However, using ELISA, TNF- was not
detectable in the supernatants (not shown).
After having established the inhibition of PMN apoptosis in vitro, we aimed to evaluate its relevance in the course of in vivo infection. An experimental murine model of L. major infection was applied to investigate whether Leishmania infection can delay the PMN apoptosis in vivo. Within the first 10–24 h after infection with L. major, PMN migrate rapidly into the infected skin. However, during the second and third day of infection, primarily macrophages are recruited and dominate in the cellular infiltrate (3). Using the s.c. air-pouch technique, infiltrate cells were collected from the infected skin 3 days after infection with L. major. Importantly, morphologically intact nonapoptotic PMN were observed, and a population of these cells contained intracellular Leishmania. The facts that, on the one hand, no apoptotic cells were among the infected ones and, in contrast, no Leishmania were seen in apoptotic PMN, suggest that the infection led to the extended survival of these cells in vivo. These are the first experimental data suggesting that the observed inhibition takes place not only in vitro, but also in vivo in the early phase of infection.
L. major did not block, but rather delayed the spontaneous
PMN apoptosis by 24 h. The infected cells became apoptotic during
the second day of culture, as demonstrated by the high caspase-3
activity in PMN after 42 h of coincubation with
Leishmania. Considering the different kinetics of
recruitment of neutrophils and macrophages to the infected skin, the
delay of PMN apoptosis can have significant consequences for disease
development. No significant numbers of macrophages, the ultimate host
cells of the parasites, are present in the infected skin at this early
time point of infection (3, 4). It is conceivable that, in
the absence of macrophages, PMN phagocytose Leishmania and
hence provide them an intracellular niche for survival within the first
hours of infection. Due to the short lifetime of PMN, however, the
parasites can survive the first day intracellularly only if the PMN
apoptosis is delayed. In this study, we have demonstrated that infected
PMN become eventually also apoptotic, with a delay of 24 h. PS is
exposed on the surface of apoptotic PMN (29), leading to
their recognition and phagocytosis by macrophages (11).
Accordingly, we hypothesize that infected PMN, which become apoptotic
with a delay of at least 24 h, are phagocytosed by macrophages
that are recruited into the infected skin 1–2 days after infection. In
this context, it is important to note that the uptake of apoptotic
cells does not activate the antimicrobial effector mechanisms of
macrophages (44). According to this hypothesis, the uptake
of infected apoptotic neutrophils could be a way of silent entry of the
parasites into macrophages. This fits into the "safe target"
theory, which suggested that myeloid cells provide intracellular niche
for the survival of Leishmania (45), and can
explain the reported disease-promoting effect of neutrophil
granulocytes in experimental L. major infection
(46).
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Acknowledgments |
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ELISA. We also thank Nicole Jahnke and Nina Hermann for expert
technical assistance. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Tamás Laskay, Institute for Medical Microbiology and Hygiene, Medical University of Lübeck, Ratzeburger Allee 160, D-23538 Lübeck, Germany. E-mail address: Tamas.Laskay{at}hygiene.ukl.mu-luebeck.de
3 Abbreviations used in this paper: PMN, polymorphonuclear neutrophil granulocyte; AMC, 7-amino-4-methylcoumarin; HGE, human granulocytic ehrlichiosis; PS, phosphatidylserine.
Received for publication August 20, 2001. Accepted for publication April 17, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-mediated apoptosis of human neutrophils. Kidney Int. 53:84.[Medline]
RIII and acquire annexin V binding sites during apoptosis in vitro. Blood 85:532.
. J. Biol. Chem. 274:37329.
B activates dual inhibition sites in the regulation of tumor necrosis factor--induced neutrophil apoptosis. Eur. J. Pharmacol. 407:211.[Medline]
B activation is a critical regulator of human granulocyte apoptosis in vitro. J. Biol. Chem. 274:4309., tumor necrosis factor , and interleukin-6 in human peripheral blood leukocytes exposed to human granulocytic ehrlichiosis agent or recombinant major surface protein P44. Infect. Immun. 68:3394.This article has been cited by other articles:
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  A. B. Guimaraes-Costa, M. T. C. Nascimento, G. S. Froment, R. P. P. Soares, F. N. Morgado, F. Conceicao-Silva, and E. M. Saraiva Leishmania amazonensis promastigotes induce and are killed by neutrophil extracellular traps PNAS, April 21, 2009; 106(16): 6748 - 6753. [Abstract] [Full Text] [PDF]
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 M. Halle, M. A. Gomez, M. Stuible, H. Shimizu, W. R. McMaster, M. Olivier, and M. L. Tremblay The Leishmania Surface Protease GP63 Cleaves Multiple Intracellular Proteins and Actively Participates in p38 Mitogen-activated Protein Kinase Inactivation J. Biol. Chem., March 13, 2009; 284(11): 6893 - 6908. [Abstract] [Full Text] [PDF]
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 S. Chanchamroen, C. Kewcharoenwong, W. Susaengrat, M. Ato, and G. Lertmemongkolchai Human Polymorphonuclear Neutrophil Responses to Burkholderia pseudomallei in Healthy and Diabetic Subjects Infect. Immun., January 1, 2009; 77(1): 456 - 463. [Abstract] [Full Text] [PDF]
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 N. C. Peters, J. G. Egen, N. Secundino, A. Debrabant, N. Kimblin, S. Kamhawi, P. Lawyer, M. P. Fay, R. N. Germain, and D. Sacks In Vivo Imaging Reveals an Essential Role for Neutrophils in Leishmaniasis Transmitted by Sand Flies Science, August 15, 2008; 321(5891): 970 - 974. [Abstract] [Full Text] [PDF]
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 L. Afonso, V. M. Borges, H. Cruz, F. L. Ribeiro-Gomes, G. A. DosReis, A. N. Dutra, J. Clarencio, C. I. de Oliveira, A. Barral, M. Barral-Netto, et al. Interactions with apoptotic but not with necrotic neutrophils increase parasite burden in human macrophages infected with Leishmania amazonensis J. Leukoc. Biol., August 1, 2008; 84(2): 389 - 396. [Abstract] [Full Text] [PDF]
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Y. Osorio, D. L. Bonilla, A. G. Peniche, P. C. Melby, and B. L. Travi Pregnancy enhances the innate immune response in experimental cutaneous leishmaniasis through hormone-modulated nitric oxide production J. Leukoc. Biol., June 1, 2008; 83(6): 1413 - 1422. [Abstract] [Full Text] [PDF]
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 T. A. Fuchs, U. Abed, C. Goosmann, R. Hurwitz, I. Schulze, V. Wahn, Y. Weinrauch, V. Brinkmann, and A. Zychlinsky Novel cell death program leads to neutrophil extracellular traps J. Cell Biol., January 16, 2007; 176(2): 231 - 241. [Abstract] [Full Text] [PDF]
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 M. C. MONTEIRO, H. C. LIMA, A. A. A. SOUZA, R. G. TITUS, P. R. T. ROMAO, and F. DE QUEIROZ CUNHA EFFECT OF LUTZOMYIA LONGIPALPIS SALIVARY GLAND EXTRACTS ON LEUKOCYTE MIGRATION INDUCED BY LEISHMANIA MAJOR Am J Trop Med Hyg, January 1, 2007; 76(1): 88 - 94. [Abstract] [Full Text] [PDF]
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 C. Allenbach, C. Zufferey, C. Perez, P. Launois, C. Mueller, and F. Tacchini-Cottier Macrophages Induce Neutrophil Apoptosis through Membrane TNF, a Process Amplified by Leishmania major. J. Immunol., June 1, 2006; 176(11): 6656 - 6664. [Abstract] [Full Text] [PDF]
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B. Mehrad, S. J. Park, G. Akangire, T. J. Standiford, T. Wu, J. Zhu, and C. Mohan The lupus-susceptibility locus, sle3, mediates enhanced resistance to bacterial infections. J. Immunol., March 1, 2006; 176(5): 3233 - 3239. [Abstract] [Full Text] [PDF]
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 N. Berkova, S. Lair-Fulleringer, F. Femenia, D. Huet, M.-C. Wagner, K. Gorna, F. Tournier, O. Ibrahim-Granet, J. Guillot, R. Chermette, et al. Aspergillus fumigatus conidia inhibit tumour necrosis factor- or staurosporine-induced apoptosis in epithelial cells Int. Immunol., January 1, 2006; 18(1): 139 - 150. [Abstract] [Full Text] [PDF]
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K.-S. Choi, J. T. Park, and J. S. Dumler Anaplasma phagocytophilum Delay of Neutrophil Apoptosis through the p38 Mitogen-Activated Protein Kinase Signal Pathway Infect. Immun., December 1, 2005; 73(12): 8209 - 8218. [Abstract] [Full Text] [PDF]
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 L. Eidsmo, S. Nylen, A. Khamesipour, M.-A. Hedblad, F. Chiodi, and H. Akuffo The Contribution of the Fas/FasL Apoptotic Pathway in Ulcer Formation during Leishmania major-Induced Cutaneous Leishmaniasis Am. J. Pathol., April 1, 2005; 166(4): 1099 - 1108. [Abstract] [Full Text] [PDF]
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G. van Zandbergen, M. Klinger, A. Mueller, S. Dannenberg, A. Gebert, W. Solbach, and T. Laskay Cutting Edge: Neutrophil Granulocyte Serves as a Vector for Leishmania Entry into Macrophages J. Immunol., December 1, 2004; 173(11): 6521 - 6525. [Abstract] [Full Text] [PDF]
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P. Brest, F. Betis, N. Cuburu, E. Selva, M. Herrant, A. Servin, P. Auberger, and P. Hofman Increased Rate of Apoptosis and Diminished Phagocytic Ability of Human Neutrophils Infected with Afa/Dr Diffusely Adhering Escherichia coli Strains Infect. Immun., October 1, 2004; 72(10): 5741 - 5749. [Abstract] [Full Text] [PDF]
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M. Aleman, P. Schierloh, S. S. de la Barrera, R. M. Musella, M. A. Saab, M. Baldini, E. Abbate, and M. C. Sasiain Mycobacterium tuberculosis Triggers Apoptosis in Peripheral Neutrophils Involving Toll-Like Receptor 2 and p38 Mitogen Protein Kinase in Tuberculosis Patients Infect. Immun., September 1, 2004; 72(9): 5150 - 5158. [Abstract] [Full Text] [PDF]
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P. Kropf, N. Freudenberg, C. Kalis, M. Modolell, S. Herath, C. Galanos, M. Freudenberg, and I. Muller Infection of C57BL/10ScCr and C57BL/10ScNCr mice with Leishmania major reveals a role for Toll-like receptor 4 in the control of parasite replication J. Leukoc. Biol., July 1, 2004; 76(1): 48 - 57. [Abstract] [Full Text] [PDF]
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K. Akarid, D. Arnoult, J. Micic-Polianski, J. Sif, J. Estaquier, and J. C. Ameisen Leishmania major-mediated prevention of programmed cell death induction in infected macrophages is associated with the repression of mitochondrial release of cytochrome c J. Leukoc. Biol., July 1, 2004; 76(1): 95 - 103. [Abstract] [Full Text] [PDF]
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D. J. Costa, C. Favali, J. Clarencio, L. Afonso, V. Conceicao, J. C. Miranda, R. G. Titus, J. Valenzuela, M. Barral-Netto, A. Barral, et al. Lutzomyia longipalpis Salivary Gland Homogenate Impairs Cytokine Production and Costimulatory Molecule Expression on Human Monocytes and Dendritic Cells Infect. Immun., March 1, 2004; 72(3): 1298 - 1305. [Abstract] [Full Text] [PDF]
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S. Lotz, E. Aga, I. Wilde, G. van Zandbergen, T. Hartung, W. Solbach, and T. Laskay Highly purified lipoteichoic acid activates neutrophil granulocytes and delays their spontaneous apoptosis via CD14 and TLR2 J. Leukoc. Biol., March 1, 2004; 75(3): 467 - 477. [Abstract] [Full Text] [PDF]
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G. van Zandbergen, J. Gieffers, H. Kothe, J. Rupp, A. Bollinger, E. Aga, M. Klinger, H. Brade, K. Dalhoff, M. Maass, et al. Chlamydia pneumoniae Multiply in Neutrophil Granulocytes and Delay Their Spontaneous Apoptosis J. Immunol., February 1, 2004; 172(3): 1768 - 1776. [Abstract] [Full Text] [PDF]
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H. Suttmann, N. Lehan, A. Bohle, and S. Brandau Stimulation of Neutrophil Granulocytes with Mycobacterium bovis Bacillus Calmette-Guerin Induces Changes in Phenotype and Gene Expression and Inhibits Spontaneous Apoptosis Infect. Immun., August 1, 2003; 71(8): 4647 - 4656. [Abstract] [Full Text] [PDF]
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S. Ibata-Ombetta, T. Idziorek, P.-A. Trinel, D. Poulain, and T. Jouault Candida albicans Phospholipomannan Promotes Survival of Phagocytosed Yeasts through Modulation of Bad Phosphorylation and Macrophage Apoptosis J. Biol. Chem., April 4, 2003; 278(15): 13086 - 13093. [Abstract] [Full Text] [PDF]
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H. Scaife, Z. Woldehiwet, C. A. Hart, and S. W. Edwards Anaplasma phagocytophilum Reduces Neutrophil Apoptosis In Vivo Infect. Immun., April 1, 2003; 71(4): 1995 - 2001. [Abstract] [Full Text]
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