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Max von Pettenkofer Institute for Hygiene and Medical Microbiology, Munich, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B in macrophages, which
suppresses NF-B-dependent antiapoptotic activities. The simultaneous
initiation of proapoptotic signaling by yersiniae infection or LPS
treatment results in macrophage apoptosis. In this study, we used YopP
as a tool to dissect survival- and death-inducing pathways in
bacteria-faced macrophages. We cotransfected J774A.1 macrophages with
expression plasmids for YopP and dominant-negative mutants of signal
transmitters of the NF-B cascade downstream from the LPS receptor
complex. Dominant-negative myeloid differentiation factor 88 (MyD88) or
IL-1R-associated kinase (IRAK) 2 diminished LPS-induced apoptosis in
YopP-transfected macrophages, suggesting implication of MyD88 and IRAK2
in signaling cell death. In contrast, dominant-negative IRAK1 and
TNFR-associated factor 6 (TRAF6) did not provide protection, but
augmented LPS-mediated apoptosis in the absence of YopP, which
indicates roles of IRAK1 and TRAF6 in the antiapoptotic signal relay of
the NF-B cascade. The distinct functions of IRAK members in
macrophage survival were reflected by opposing effects of
dominant-negative IRAK1 and IRAK2 on Y.
enterocolitica-mediated apoptosis. Yersiniae- and LPS-dependent
cell death were substantially attenuated by a specific caspase-8
inhibitory peptide or by dominant negative Fas-associated death domain
protein (FADD). This suggests, that Yersinia-induced
apoptosis involves a proapoptotic signal relay through MyD88 and IRAK2,
which potentially targets the Fas-associated death domain
protein/caspase-8 apoptotic pathway, whereas IRAK1 and TRAF6 counteract
the bacteria-induced cytotoxic response by signaling macrophage
survival. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-8, and mediates macrophage apoptosis (5, 6, 7, 8, 9).
YopP/YopJ inhibits the activities of several parallel host cell
signaling cascades. It targets and blocks the NF-B-activating IB
kinase IKK
, resulting in impairment of activation of transcription
factor NF-B (10). In addition, YopP/YopJ binds and
inhibits members of the mitogen-activated protein kinase (MAPK) kinase
family, which leads to disruption of MAPK pathways (10).
It was recently proposed that YopP/YopJ possesses a ubiquitin-like
protein protease activity that may contribute to these suppressive
effects by disturbing the processing of intracellular signaling
complexes (11). As the MAPK and NF-B signaling cascades
synergistically control the synthesis of host inflammatory cytokines,
the inversion of these pathways by Yersinia spp. accounts
for the cytokine suppressive effect (12, 13).
Yersinia-induced apoptosis also crucially relies on
inhibition of the NF-B signaling pathway by YopP/YopJ (10, 13, 14). NF-B mediates survival of eukaryotic cells by the
prevention of apoptosis (15, 16, 17, 18, 19, 20, 21, 22). It transcriptionally
up-regulates the synthesis of antiapoptotic proteins, such as
inhibitors of apoptosis and Bcl-2 family members (19, 20, 21, 22).
These proteins counteract proapoptotic signals elicited by diverse
extracellular stimuli, which consequently preserves cellular viability
(16, 17, 18, 19, 20, 21, 22). Several studies demonstrated that the
antiapoptotic function of NF-B is essential for the mediation of
self-defense and survival of macrophages when encountered with bacteria
or LPS (13, 14, 23, 24). LPS simultaneously activates pro-
and antiapoptotic signaling pathways in macrophages, and the rapid
activation of NF-B provides protection against the cytotoxic
response. Consequently, the inhibition of NF-B activation sensitizes
macrophages to undergo apoptosis upon LPS treatment (13, 14, 23). Our previous study showed, that Y.
enterocolitica exploits this mechanism to compel the macrophage to
undergo apoptosis (14). We demonstrated that Y.
enterocolitica YopP disrupts the NF-B survival pathway in
J774A.1 macrophages at the level of IKK (14). IKK-
phosphorylates the NF-B inhibitory IB proteins, which sequester
nonactivated NF-B in the cytoplasm (15, 16).
Phosphorylated IBs are degraded through the proteasome pathway,
which mediates release and nuclear translocation of NF-B upon
cellular activation. The transfection of J774A.1 macrophages with a
YopP expression vector mediates moderate apoptosis, but this effect is
substantially enhanced by the additional initiation of LPS signaling
(14). This reveals a synergism between LPS-induced signal
transduction and inhibition of NF-B by YopP, leading to severe
apoptosis in macrophages. Overexpression of the transcriptionally
active p65 NF-B subunit reverses Y. enterocolitica- and
YopP- plus LPS-mediated cell death. Thus, the disruption of the
antiapoptotic functions of NF-B by YopP along with the activation of
LPS signaling critically determines the fate of the Y.
enterocolitica-infected macrophage (14). As LPS by
itself mediates macrophage NF-B activation, which is blocked by YopP
at the IKK level, the proapoptotic LPS signal must occur upstream
from IKK.
In this study, we used YopP as a tool to seek for signaling molecules
that are potentially involved in the yersiniae- and LPS-induced
proapoptotic response. In macrophages and monocytes, LPS initiates a
signaling cascade that involves the transmembrane Toll-like receptors
(TLR) 2 and 4, the surface receptor glycoprotein CD14, and the helper
molecule MD-2 (25, 26, 27, 28, 29, 30). The receptor-dependent signals
converge on the adapter protein myeloid differentiation factor
88 (MyD88), which binds to the cytoplasmic receptor domain
(25, 26, 27, 28, 29, 30, 31, 32). MyD88 then recruits members of the
IL-1R-associated kinase (IRAK) family to the receptor complex. Three
IRAK molecules, IRAK1, IRAK2, and IRAK-M (33, 34, 35, 36), can
relay the NF-B-activating signal via TNFR-associated factor 6
(TRAF6) and TGF--activated kinase 1 to the IKK complex, which
ultimately mediates NF-B activation (25, 26, 27, 28, 29). Recently,
another adapter, designated MyD88-adapter-like (Mal) or Toll-IL
I receptor domain-containing adapter protein (TIRAP), was
identified which signals TLR4-responsive NF-B activation
independently from MyD88 (37, 38).
We transfected J774A.1 macrophages with dominant negative constructs of
components of the NF-B signaling cascade for LPS. Our data point out
differential roles of these signaling intermediates in determining the
fate of the macrophage that faces a bacterial pathogen. The
proapoptotic signal relay exploited by Y. enterocolitica
YopP potentially involves MyD88 and IRAK2, which appear to target the
Fas-associated death domain protein (FADD)/caspase-8 apoptotic pathway.
Inversely, IRAK1 and TRAF6 are mediators of macrophage survival upon
LPS treatment. This indicates that bifurcation of death-inducing and
-preventing signals in bacteria-challenged macrophages depends on MyD88
and IRAK family members.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Y. enterocolitica wild-type (WT) strain WA-314,
serotype O8, and the respective yopP-knockout mutant
WA-314
yopP were grown as described previously
(14). The murine macrophage cell line J774A.1 was cultured
as described elsewhere (14), treated with 10 µg/ml LPS
from Escherichia coli O55:B5 (Sigma-Aldrich, Munich,
Germany), or infected as indicated. Infections were performed at a
ratio of 50 bacteria per cell. For incubation times longer than 90 min,
bacteria were killed by addition of gentamicin (100 µg/ml) after 90
min of infection. In some experiments, the cells were pretreated with
the proteasome inhibitory peptide Z-Leu-Leu-Leu-CHO (Mg-132, 2.5 µM;
Biomol, Plymouth Meeting, PA), or with inhibitors for caspase-2
(z-VDVAD-fmk), caspase-8 (z-IETD-fmk), caspase-9 (z-LEHD-fmk; all from
Calbiochem, La Jolla, CA), caspase-10 (z-AEVD-fmk; Biovision, Palo
Alto, CA), or with broad-spectrum caspase inhibitor (z-VAD-fmk; Bachem,
Heidelberg, Germany) at 40 µM for 1 h, then stimulated as
indicated. Elicited peritoneal macrophages were obtained from male
C3H/HeN mice 3 days after i.p. inoculation of 1 ml of 10% proteose
peptone broth (13). Peritoneal exudate cells were washed
and cultured at 37°C in cell growth medium. After 3 h,
nonadherent cells were removed by repeated washing, and remanent
macrophages were treated as indicated.
Expression vectors, J774A.1 cell transfection, and analysis of quantity and morphology of transfected cells
A total of 5 x 105 J774A.1
cells/well was transfected with 4 µl ExGen 500 according to the
manufacturer’s instructions (Fermentas, Hanover, MD). ExGen 500 is a
nonliposomal gene delivery reagent consisting of linear
polyethylenimine molecules that share high cationic-charge density
potential (39). Its gene transfer activity is related to
its capacity for condensing DNA, interacting with anionic proteoglycans
of the cell membrane, entering cells by endocytosis, and protecting DNA
from lysosomal degradation. ExGen 500 appeared appropriate for our
experimental approach because of low toxicity on J774A.1 cells. The
interexperimental transfection efficiencies usually varied between 1
and 10%, providing around 500-5000 transfected cells/sample. Unless
indicated otherwise, transfections were conducted as cotransfection
experiments using 1 µg of plasmid DNA. In dependence on the
experiment, 0.33 µg of pSV--galactosidase expression vector
(Promega, Madison, WI) was combined with 0.66 µg of the plasmid of
interest, or 0.2 µg of pSV--galactosidase expression vector were
combined with 0.4 µg of two different plasmids of interest
(14). -Galactosidase expression correlates with
expression of proteins encoded by cotransfected plasmids in the
transfected cells (14). Most of the expression plasmids
used in this study have been characterized and described before. They
included expression vectors for YopP (14);
dominant-negative TRAF6 and WT-TRAF6 (Ref. 40 ;
kindly provided by J. Inoue, Institute of Medical Science,
University of Tokyo, Tokyo, Japan); dominant-negative
MyD88152–296(152–296), IRAK11–215(1–215), IRAK21–96(1–96) (Refs. 35, 41 ; kindly provided by M. Muzio, Department of Immunology
and Cell Biology, Mario Negri Institute, Milan, Italy);
dominant-negative IRAK-M1–161(1–161) Ref. 36 ; kindly
provided by Z. Cao and H. Wesche, Tularik, South San Francisco, CA);
dominant-negative FADD (42; kindly provided by C. Vincenz,
Department for Pathology, University of Michigan Medical School);
WT-MAPK kinase (MKK) 1 (Ref. 43 ; kindly provided by N.
Ahn, Howard Hughes Medical Institute, University of Colorado, Boulder,
CO); pNF-B-d2EGFP (NF-B reporter plasmid encoding a destabilized
variant of GFP with reduced half-life under control of the enhancer
element; Clontech Laboratories, Palo Alto, CA). Empty expression
vectors containing no inserts were used as negative controls. For
expression of green fluorescent protein (GFP)-labeled YopP, the
yopP gene was amplified from Y. enterocolitica
WA-314 and cloned into the multicloning site of plasmid pEGFP-C1
(Clontech Laboratories). The resulting plasmid produce YopP in fusion
to the C terminus of enhanced GFP under control of the CMV promoter.
Eighteen hours after transfection, cells were treated with LPS or
infected with yersiniae as indicated. To identify transfected
macrophages, cells were immunofluorescent labeled or stained with
5-bromo-4-chloro-3-indolyl -D-galactoside (X-gal)
after certain points of time. For assessment of cell death in
cotransfected cells, blue transfected cells were counted and the
morphology of the transfected cells was determined using light
microscopy (14, 17, 18, 19, 20). Every single transfected cell was
analyzed for an apoptotic appearance. A minimum of eight microscopic
fields was investigated for each sample. For quantification, the number
of apoptotic blue cells was assayed in relation to the total number of
transfected cells. Results are expressed as mean percentages ± SD
from three independent experiments. For accurate comparison of the
different dominant-negative IRAK members, the cDNA for IRAK-M was
amplified and inserted into the multicloning side of plasmid pcDNA3.1
(Invitrogen, San Diego, CA). pcDNA3.1 was used as expression plasmid
for IRAK1 and IRAK2 (41). All PCR products were
checked for sequence accuracy by sequencing.
Fluorescent labeling of transfected and apoptotic cells
To quantify cell death in J774A.1 and mouse peritoneal macrophages in response to Yersinia infection, apoptotic cells were labeled with fluorescein-conjugated annexin V (Boehringer Mannheim, Mannheim, Germany) as described previously (2). The cells transfected with the GFP plasmids were stained with Cy3-labeled annexin V (Sigma-Aldrich). Annexin V binds with high affinity to phosphatidylserine exposed on the outer leaflet of apoptotic cells and confers fluorescein- or Cy3-dependent fluorescence to the cells undergoing apoptosis. Viability of cells after treatment with specific caspase inhibitory peptides and Yersinia was assessed by exclusion of the DNA stain propidium iodide (2). The rate of cell death was determined by visual scoring of a minimum of 200 cells per sample in a fluorescence microscope. Results are expressed as mean percentages ± SD of apoptotic/dead fluorescent cells vs the total number of cells from three independent experiments.
For immunofluorescent labeling of transfected cells, the cells were
stimulated as indicated, washed twice with PBS, fixed with 3.7%
paraformaldehyde, permeabilized with 0.02% Nonidet P-40, and blocked
with goat serum (14). To specifically label single
proteins, the following primary IgG Abs were used: goat polyclonal
anti-p65 (Santa Cruz Biotechnology, Santa Cruz, CA), mouse
monoclonal anti-AU-1 (Covance Research Products, Richmond, CA;
toward IRAK2 and MyD88), mouse monoclonal anti-HA (Cell
Signaling Technology, Beverly, MA; toward WT-MKK1), rabbit polyclonal
and mouse monoclonal anti-FLAG (Sigma-Aldrich; toward TRAF6,
WT-TRAF6, and IRAK-M), mouse monoclonal anti-c-myc
(Clontech Laboratories; toward IRAK1), and rabbit polyclonal
anti--galactosidase (Clontech Laboratories). Primary Abs were
stained with appropriate fluorescein- or rhodamine-conjugated secondary
anti-IgG Abs (Sigma-Aldrich).
Statistical analysis
Data were expressed as mean percentages ± SD. Statistical
analysis was performed using Epi Info (version 6, Center for Disease
Control, Atlanta, GA). Statistical significances of differences were
determined by the 
2 test with a p
value <0.01 considered to be significant (44).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Y. enterocolitica efficiently suppresses NF-B
activation and triggers apoptosis in the murine J774A.1 macrophage cell
line (13). Identical effects have been demonstrated in
primary mouse peritoneal macrophages, in which the NF-B inhibitory
action of Y. enterocolitica similarly correlates with the
induction of apoptosis (13). Survival of LPS-challenged
macrophages depends on de novo protein synthesis (45, 46),
and these observations suggest, that onset of apoptosis is related to
inhibition of the antiapoptotic NF-B pathway by Yersinia
infection. A link between artificial suppression of NF-B activation
and the induction of cell death by bacterial infection has been
demonstrated in several macrophage cell lines (13, 23). In
our hands, pretreatment of J774A.1 macrophages with the proteasome
inhibitory peptide Z-Leu-Leu-Leu-CHO (MG-132) sensitizes the cells to
undergo apoptosis upon subsequent stimulation with LPS or nonvirulent,
yopP-negative yersiniae (13). MG-132 suppresses
degradation of the NF-B inhibitory IB proteins through the
proteasome pathway (47), which substantially inhibits
NF-B activation in J774A.1 macrophages (13). To
investigate whether a similar effects occurs in primary macrophages, we
treated elicited mouse peritoneal macrophages with MG-132 and
subsequently stimulated with Yersinia strains or LPS. The WT
Y. enterocolitica strain WA-314 efficiently conferred cell
death in both J774A.1 and peritoneal macrophages (Fig. 1
). Similarly, 60–75% of the peritoneal
macrophages significantly underwent apoptosis upon challenge with LPS
or the yopP-negative mutant WA-yopP when the
cells were pretreated with MG-132. These effects were similar to those
observed in J774A.1 cells. These results confirm a potential
antiapoptotic role of NF-B in bacterial infection of macrophages and
suggest that the observed effects in J774A.1 cells closely reflect the
situation in primary macrophages.
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Stimulation with LPS efficiently triggers apoptosis in J774A.1
macrophages that were transiently transfected with a YopP expression
plasmid (14). To investigate the roles of well-known LPS
signal transducers in this process, we cotransfected dominant-negative
constructs of components of the NF-B signaling cascade for LPS along
with the YopP expression plasmid. For identification of the transfected
cells, the cells were cotransfected with a -galactosidase-encoding
reporter vector (14, 17, 18, 19, 20). Staining with X-gal allows
the detection of -galactosidase expression by conferring a blue
color to transfected cells. The number of transfected cells and their
morphology were scored by microscopy. Apoptotic cell death is
characterized by typical cellular shrinkage and condensation (14, 17, 18, 19, 20). Cells that were transfected with the empty expression
vector control and treated with LPS exhibited blue color and a normal
cellular shape (Fig. 2A,
top). These cells apparently remained viable. In contrast,
LPS challenge of YopP-transfected macrophages conferred a
characteristic apoptotic phenotype, the majority of the cells being
reduced in size and condensed as a result of apoptotic destruction
(Fig. 2A, bottom). The dying cells have been
shown to display apoptotic DNA fragmentation (14). We
additionally analyzed phosphatidylserine exposure in these cells using
Cy3-conjugated annexin V. Cy3-annexin V binds with high affinity to
phosphatidylserine in the outer leaflet of apoptotic cells and confers
red fluorescence. For identification of the transfected cells, YopP was
expressed as fusion protein with GFP, which mediates green
fluorescence. In correlation with the results obtained by
-galactosidase coexpression, cells transfected with the empty GFP
vector control remained viable upon LPS treatment and did not stain
with Cy3-annexin V (Fig. 2B, top). A single
apoptotic cell characterized by red fluorescence is included in Fig. 2B (top). In contrast, YopP-transfected
and LPS-treated cells became Cy3-annexin V positive, which indicates
occurrence of apoptosis (Fig. 2B, bottom).
Interestingly, YopP-GFP expression and Cy3-annexin V labeling behaved
antagonistically. Strong expression of GFP-YopP correlated with weak
Cy3-annexin V staining and vice versa (Fig. 2B,
bottom). Thus, the expression of YopP-GFP was reduced in the
later stages of apoptosis, which are characterized by enhanced
phosphatidylserine exposure. Accordingly, Fig. 2B
(bottom) displays the apoptotic body of a fragmented,
YopP-transfected cell exhibiting weak green GFP fluorescence, but
substantial red annexin V staining. This suggests that YopP-GFP is
degraded during the apoptotic process. Since -galactosidase
expression appeared more stable than expression of GFP in
YopP-transfected cells, we considered -galactosidase expression as
the more suitable reporter for the cotransfection experiments with
YopP.
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MyD88, IRAK1, and
TRAF6. To quantify the degree of apoptosis in the cotransfected
cells, the number of -galactosidase-expressing apoptotic cells was
determined in relation to the total number of transfected cells (Fig. 2
C). Cells cotransfected with YopP and the empty vector
control efficiently underwent apoptosis upon exposure to LPS (Fig. 2C, YopP plus vector: 85 ± 6% apoptosis). Treatment
of the cells with the broad-spectrum caspase inhibitor z-VAD-fmk (40
µM), which prevents activation of caspase cysteine proteases during
the apoptotic response, substantially suppressed cell death and only
20–30% of the cells underwent apoptosis. A dominant-negative mutant
version of the adapter protein MyD88 (MyD88), which abrogates
LPS-induced NF-B activation (31, 32), provided partial
protection against LPS-promoted apoptosis: Hence, MyD88
significantly reduced LPS-enhanced apoptosis in YopP-transfected cells
(Fig. 2C, YopP plus MyD88: 54 ± 6% apoptosis).
This indicates that MyD88 is able to block a proapoptotic signal
that is elicited by LPS stimulation. However, both dominant-negative
IRAK1 and TRAF6, mutant constructs of signaling molecules
downstream from MyD88, did not suppress YopP plus LPS-mediated cell
death (Fig. 2C, YopP plus IRAK1: 96 ± 3%
apoptosis; YopP plus TRAF6: 92 ± 6% apoptosis). IRAK1 and
TRAF6 slightly augmented apoptosis in YopP-transfected cells, which
may point out a synergistic action of IRAK1 and TRAF6 with YopP
in impairing cellular viability. In expectation that a
dominant-negative version of an apoptosis-inducing signaling molecule
reduces cell death, these results rule out implication of IRAK1 and
TRAF6 in signaling a death-triggering response. Inversely, these data
indicate, that relay of the LPS-dependent proapoptotic signal depends
on MyD88, but not on IRAK1 and TRAF6.
IRAK2 potentially confers NF-B activation and cell death in
infected macrophages
The IRAK family includes two other members beside IRAK1, which are
IRAK2 and IRAK-M (34, 35, 36). Both IRAK2 and IRAK-M share
structural as well as functional similarities with IRAK1 and can relay
a MyD88-dependent NF-B-activating signal upon LPS treatment
(36). We investigated involvement of these molecules in
the macrophage apoptotic response evoked by Y.
enterocolitica and YopP/LPS treatment. Surprisingly,
dominant-negative IRAK2 produced different results in cotransfection
experiments with YopP as compared with IRAK1 (Fig. 3A). In relation to YopP and
control vector-transfected cells (Fig. 3A, YopP plus vector:
82 ± 7% apoptosis), cells transfected with YopP and IRAK2
significantly survived subsequent treatment with LPS (Fig. 3A, YopP plus IRAK2: 44 ± 6% apoptosis). Thus,
unlike IRAK1, IRAK2 apparently exerts a protective effect against
enhancement of apoptosis through LPS stimulation. The dominant-negative
IRAK-M construct provided slight protection. Another set of
experiments revealed that IRAK2 not only attenuated
YopP/LPS-mediated apoptosis, but also cell death elicited by yersiniae
infection (Fig. 3B). Whereas WT Y. enterocolitica
efficiently triggered apoptosis in cells transfected with the empty
control vector or with IRAK1, apoptosis was significantly blocked in
IRAK2 transfected cells. Thus, IRAK2 obviously disrupts the
transmission of an apoptotic signal that is activated by LPS treatment
or yersiniae infection. This suggests that IRAK2 may participate
besides MyD88 in relaying the apoptotic signal.
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IRAK1 and IRAK2 on the NF-B survival pathway, we
analyzed the impacts of overexpression of dominant-negative IRAK1
and IRAK2 on LPS-induced NF-B activation. NF-B activation was
investigated at the single cell level by fluorescence microscopy. To
preclude nonspecific fluorescent labeling, we cotransfected IRAK1,
IRAK2, and the empty vector control with the -galactosidase
reporter plasmid. The samples were simultaneously probed with
anti--galactosidase (red fluorescence) and
anti-c-myc or anti-AU-1 (green fluorescence) Abs to
detect -galactosidase and IRAK1 (anti-c-myc) or
IRAK2 (anti-AU-1) in the transfected cells. As displayed in Fig. 3C, cells transfected with the -galactosidase and the
empty control vector selectively exhibited red fluorescence (staining
of -galactosidase; Fig. 3C, top), whereas
IRAK2-transfected cells were additionally characterized by yellow
fluorescence, resulting from superimposition of red and green
fluorescent signals (staining of IRAK2; Fig. 3C,
bottom). Similar results were obtained for IRAK1 (data
not shown). Cross-processing of IRAK1-transfected cells with
anti-AU-1 and IRAK2-transfected cells with
anti-c-myc Abs did not produce any conspicuous
fluorescence signals (data not shown). These results indicate that the
mouse monoclonal anti-AU-1 and anti-c-myc Abs used
in these studies specifically label overexpressed constructs bearing
the respective Ab epitope tags. Furthermore, cotransfection with the
-galactosidase plasmid seems to be an appropriate approach to
identify transfected cells.
We first analyzed nuclear translocation of NF-B using goat
polyclonal anti-NF-B p65 Abs (Fig. 3D, green
fluorescence). The samples were additionally probed with the
anti-c-myc or anti-AU-1 Abs to detect IRAK1 or
IRAK2 expression in the transfected cells (Fig. 3D, red
fluorescence). Overexpressed IRAKs apparently accumulated in the
perinuclear and nuclear region, which agrees with previous observations
(48). In nonstimulated cells, the major amount of NF-B
was localized in the cytoplasm (Fig. 3D, top). No
NF-B-dependent fluorescent signal was detectable after processing
the samples with secondary fluorescein-conjugated anti-rabbit
instead of anti-goat Abs. The secondary Ab alone, without
appropriate primary Ab, did not produce any conspicuous fluorescent
signals (data not shown). Upon treatment with LPS, NF-B moved to the
nucleus and exhibited a nuclear staining pattern in the nontransfected
cells, which indicates NF-B activation (Fig. 3D,
bottom). In contrast, overexpression of dominant-negative
IRAK2 obviously impeded NF-B in its ability to move to the
nucleus and NF-B was predominantly detected in the cytoplasm of
transfected cells (Fig. 3D, bottom). The same
held true for overexpression of dominant-negative IRAK1 (data not
shown). To substantiate a potential inhibitory action of IRAKs on
induction of the NF-B pathway, we additionally monitored NF-B
activation using a GFP-NF-B reporter vector (pNF-B-d2EGFP). Use
of this plasmid allows real time analysis of binding of endogenous
NF- to the enhancer in the promoter region of the vector,
resulting in transcription of the gfp gene. The NF-B
reporter vector was cotransfected with IRAK1, IRAK2, WT-TRAF6, or
WT-MKK1. The cells were processed with Abs against the epitope tags of
the respective overexpressed proteins for identification of the
transfected constructs by fluorescence microscopy (red fluorescence).
Expression of gfp in dependence on NF-B activation was
simultaneously assayed (green fluorescence). The percentage of green
and red fluorescent cells, indicating transfected cells with activated
NF-B, was determined in relation to the total number of transfected
cells (cells with red or red and green fluorescence; Fig. 3E). Although a multitude of the transfected cells exhibited
nonspecific NF-B activation in the absence of stimulation
(30–50%), cells transfected with WT-TRAF6 and WT-MKK1 significantly
responded to treatment with LPS by additional NF-B induction,
resulting in increased numbers of green and red fluorescent cells (Fig. 3E). In contrast, NF-B activation in cells overexpressing
IRAK1 or IRAK2 was not significantly enhanced upon LPS treatment.
These results confirm an inhibitory effect of the dominant-negative
IRAK constructs on the NF-B pathway, which was demonstrated in
previous publications (31, 32, 35, 36, 41). Thus, although
both dominant-negative IRAK1 and IRAK2 share the capacity to
suppress NF-B activation, the outcomes on cellular survival and
apoptosis are obviously different. IRAK2 rather exerts a protective
effect on yersiniae- and YopP/LPS-mediated cell death, in contrast to
IRAK1. This implies that a proapoptotic signal elicited by bacterial
infection may bifurcate at the IRAK level by involving IRAK2.
IRAK1 and TRAF6 potentially mediate survival of macrophages upon LPS treatment
The data provided here suggest a role of MyD88 and IRAK2 in
signaling a LPS-dependent apoptotic response under conditions when
NF-B activation is inhibited by YopP. On the other hand, MyD88 and
IRAK2, as well as IRAK1, IRAK-M, and TRAF6, are known to participate in
transmitting the NF-B-dependent survival signal (31, 36), which suggests that the transfection of dominant-negative
versions of these molecules alone should influence survival of
LPS-treated macrophages. To check this, we transfected J774A.1
macrophages with the respective dominant-negative constructs and
assessed cellular viability upon LPS treatment in the absence of YopP
or yersiniae (Fig. 4A).
MyD88 did not considerably affect viability of LPS-treated
macrophages, indicating that the cytotoxic pathways remain silent and
that potential inhibition of NF-B activation does not lead to
apoptosis. In the same manner, overexpression of dominant-negative
IRAK2 only marginally provoked cell death, although it impairs the
NF-B pathway (Fig. 4A). In contrast, subversion of
NF-B activation by overexpression of IRAK1 significantly
triggered apoptosis in LPS-challenged macrophages (Fig. 4A,
76 ± 6% apoptosis). Similar results were obtained for TRAF6,
acting downstream from the IRAKs in the NF-B cascade (Fig. 4A, 71 ± 4% apoptosis). Apoptosis in IRAK1- and
TRAF6-transfected and LPS-stimulated cells was considerably
suppressed by pretreatment with the broad-spectrum caspase inhibitor
z-VAD-fmk (28 ± 3% and 29 ± 7% apoptotic cells,
respectively). IRAK-M exerted an intermediate effect on macrophage
viability as compared with IRAK1 and IRAK2. Immunofluorescence
labeling with Abs toward the epitope tags of the overexpressed proteins
revealed that MyD88, TRAF6, and IRAK-M were expressed in the
transfected cells (data not shown). Furthermore, these
dominant-negative LPS signaling constructs were similarly to IRAK1
and IRAK2 able to impair induction of the cotransfected GFP-NF-B
reporter (pNF-B-d2EGFP) upon LPS stimulation. Accordingly, the
NF-B-dependent increase in fluorescent cell numbers was <30% in
stimulated vs nonstimulated cells, in contrast to 80–90% for
WT-MKK1. This confirms an inhibitory effect of MyD88, TRAF6, and
IRAK-M on NF-B activation (31, 32, 35, 36, 40, 41).
These data indicate that disruption of the NF-B pathway by IRAK1
and TRAF6, but not by MyD88 or IRAK2, actively supports cell
death in LPS-treated macrophages. These effects resemble the action of
YopP, enabling LPS to mediate apoptosis in macrophages when NF-B is
inhibited. Apparently during suppression of the NF-B survival signal
by IRAK1, TRAF6, or YopP, upstream death-triggering signals still
proceed and subsequently mediate macrophage apoptosis. On the contrary,
MyD88 and IRAK2 apparently also block the death-promoting
pathway, which consequently does not reduce viability of LPS-stimulated
macrophages. These data indicate differential roles of LPS signal
transducers in determining the fate of the LPS-challenged macrophage.
IRAK1 and TRAF6 are obviously essential in transmitting the survival
signal, whereas MyD88 and IRAK2 appear to be implicated in both the
antiapoptotic NF-B pathway and the cell death-inducing pathway.
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IRAK2 and MyD88 should be able
to moderate the cytotoxic effect induced by LPS in IRAK1-transfected
macrophages. As indicated in Fig. 4B, transfection of
IRAK2 as well as of MyD88, but not of the empty vector control,
significantly decreased apoptosis in IRAK1-transfected and
LPS-treated macrophages. Thus, IRAK2 and MyD88 can counteract
IRAK1/LPS-mediated cell death, which confirms differential roles of
these signal transmitters in conferring apoptosis. Y. enterocolitica-induced apoptosis involves the FADD/caspase-8 signaling complex
We attempted to characterize a potential downstream apoptotic
signal relay transmitted through MyD88 and IRAK2. The execution of
apoptosis in eukaryotic cells depends on the activation of caspases, a
family of cysteine proteases that accomplish the apoptotic response in
a proteolytic cascade (49). To characterize the roles of
single caspases in Y. enterocolitica- and YopP-mediated
apoptosis, we used specific caspase inhibitory peptides. J774A.1
macrophages were pretreated with the caspase inhibitors and
subsequently infected with yersiniae (Fig. 5A). In accordance with
previous studies, the broad-spectrum caspase inhibitor z-VAD-fmk (lane
designated 
caspases, Fig. 5A) efficiently prevented
loss of viability in yersiniae-infected macrophages (2).
Furthermore, the inhibitors of caspase-8 and 9 provided at least
partial protection against yersiniae-induced cell death (Fig. 5A, 27 ± 3% and 39 ± 5% apoptosis vs 86
± 9% apoptosis in untreated cells), in contrast to inhibitors of
caspase-2 and 10. This points out particular involvement of caspase-8
as well as caspase-9 in apoptosis triggered by Y.
enterocolitica. The FADD is a crucial mediator of caspase-8
activation (50, 51). To substantiate a connection to the
FADD/caspase-8 apoptotic pathway, we cotransfected J774A.1 cells with
dominant-negative FADD and YopP expression plasmids or treated the
cells with z-VAD-fmk or the caspase-8 inhibitory peptide. Fig. 5B shows that FADD (37 ± 6% apoptosis) and the
caspase-8 inhibitor (44 ± 3% apoptosis) significantly reduced
cell death mediated by YopP and LPS stimulation, although to a lesser
extent as compared with z-VAD-fmk (26 ± 4% apoptosis). This
suggests that FADD and caspase-8 may indeed be involved in the
yersiniae- and LPS-activated proapoptotic response.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B rescues macrophages from apoptosis
mediated by bacterial infection or LPS treatment (13, 14, 23, 24). Y. enterocolitica takes advantage of this
mechanism to trigger apoptosis in macrophages (14).
Translocated YopP down-regulates the activities of the NF-B
signaling cascade by blockage of IKK, which is the major
LPS-responsive NF-B-activating kinase in macrophages
(52). The subsequent initiation of proapoptotic LPS
signaling upstream from IKK compels the macrophage to undergo
apoptosis. In this study, we used YopP as a tool to investigate the
signal relay that mediates apoptosis of infected macrophages under
conditions when NF-B activation is suppressed. The innate immune
system engages a number of TLRs that recognize diverse conserved
microbial products, such as LPS, peptidoglycan, bacterial lipoproteins,
or flagellin (25, 26, 27, 28, 29, 53). All of these TLRs signal
through the adaptor protein MyD88, which activates an signaling cascade
that ultimately leads to nuclear translocation of NF-B.
To identify potential proapoptotic LPS-responsive signal transducers in
Yersinia- and YopP-mediated apoptosis, we cotransfected
J774A.1 macrophages with dominant-negative constructs of proximal LPS
signaling molecules along with a YopP expression plasmid. We analyzed
occurrence of apoptosis in transfected cells upon LPS stimulation. In
our hands, a dominant-negative version of MyD88 provided partial
protection against LPS-induced apoptosis in YopP-transfected cells.
This suggests a role of MyD88 in transmitting a LPS-dependent
proapoptotic signal. Dominant-negative versions of IRAK1 and TRAF6,
signaling molecules downstream from MyD88, failed to suppress
LPS-enhanced apoptosis. On the contrary, IRAK1 and TRAF6
conferred macrophage cell death after LPS stimulation in the absence of
YopP. This indicates that inhibition of the NF-B cascade by IRAK1
or TRAF6 can replace the NF-B-suppressive effect of YopP, which
sensitizes J774A.1 macrophages to LPS-induced apoptosis. Thus, IRAK1
and TRAF6 appear to play important roles in the survival-mediating
branch of the LPS-signaling networks, but do not signal LPS-responsive
apoptosis. In contrast, a dominant-negative version of IRAK2 diminished
YopP- plus LPS- and also Yersinia-mediated cell death.
Furthermore, IRAK2 as well as MyD88, only marginally evoked cell
death in LPS-treated macrophages in the absence of YopP. IRAK2 and
MyD88 on the contrary attenuated IRAK1-mediated apoptosis in
cotransfected cells. These results suggest implication of IRAK2 besides
MyD88 as a proapoptotic signal transducer in LPS signaling. This seems
to be a specific role of IRAK2, since the NF-B suppressive effect of
IRAK2 in LPS-stimulated J774A.1 macrophages was comparable to that
of IRAK1, confirming common functional properties of IRAK1 and IRAK2
in NF-B activation. The third IRAK member, IRAK-M, displayed
intermediate effects on cellular viability, as compared with those of
IRAK1 and IRAK2.
Alignment of the amino acid sequences of IRAK1, IRAK2, and IRAK-M
revealed 31–38% similarity in between the single IRAK members
(36). Selectively, IRAK1 possesses a kinase activity that
induces potent autophosphorylation (34, 36). In contrast,
IRAK2 lacks key residues in the conserved protein kinase subdomains,
making it catalytically inactive (35, 36, 54, 55). A weak
kinase activity was reported for IRAK-M (36). These
distinct features of the IRAK members imply that they may
differentially influence cellular signaling processes. A unique role of
IRAK1 was demonstrated in the control of p38 MAPK activation and
spreading of macrophages upon LPS treatment (56). This
process requires the IRAK1 kinase activity, whereas the kinase activity
is dispensable for NF-B activation (36, 54, 55, 57).
IRAK1 has been shown to mediate phosphorylation of the Toll-interacting
protein (Tollip) (58). Tollip forms a complex with IRAK
members in resting cells, thereby preventing NF-B activation.
Cellular stimulation mediates recruitment of Tollip-IRAK complexes to
the cytoplasmic receptor, which leads to phosphorylation of IRAK1 and
Tollip and dissociation of the receptor-associated complex
(58, 59, 60). This event may be required for transmission or
termination of activating signals. The data provided in our study
indicate opposing effects of IRAK1 and IRAK2 on macrophage cytotoxic
pathways in response to bacterial infection. This suggests that the
bifurcation of apoptosis-promoting and -preventing signals depends on
IRAK family members. A unique role in cellular signaling was also
reported for IRAK2. The MyD88-related adapter protein Mal/TIRAP
activates NF-B selectively through IRAK2, but not through IRAK1
(37, 38). This confirms distinct functions of IRAK
molecules in the cellular signaling networks in response to bacterial
infection and tempts to ask for the roles of Mal/TIRAP and Tollip in
the preservation of cellular viability, which were not yet
investigated.
The apoptosis-preventing effect of the most upstream dominant-negative
signaling intermediate investigated, MyD88, was less pronounced than
that of IRAK2. This may support a model in that MyD88, as
common upstream activator of both IRAK1 and IRAK2 (35, 61), participates in the regulation of both divergent pathways.
A report from Aliprantis et al. (62) indicates implication
of MyD88 in proapoptotic signaling in monocytic cells that were treated
with bacterial lipoproteins. They found that MyD88 is coupled to the
apoptotic machinery through binding and engagement of the death
receptor adaptor protein FADD. A corresponding pathway was recently
identified in Drosophila (63). FADD mediates
the onset of apoptosis by activating the initiator caspase-8 (50, 51). A study on endothelial cells suggests that also bacterial
LPS is able to engage a FADD-dependent apoptotic pathway
(64). In an attempt to characterize downstream apoptotic
processes in our infection model, we investigated the impact of
specific caspase inhibitory peptides on Y.
enterocolitica-induced cell death. Inhibitors of caspase-8 and
caspase-9 provided substantial protection against apoptosis due to
yersiniae infection. In addition, a dominant-negative version of the
caspase-8 activator FADD considerably suppressed YopP/LPS-conferred
apoptosis. These results suggest implication of the FADD/caspase-8
module as well as of caspase-9 in the Y. enterocolitica- and
YopP/LPS-induced apoptotic response. Aliprantis et al.
(62) demonstrated binding of MyD88 to FADD through death
domain interactions. Since IRAK members also harbor a death domain, we
speculated that IRAK2 may similarly interact with FADD. However,
preliminary immunoprecipitation experiments with overexpressed FADD and
IRAK2 rule out direct in vivo interaction in between the two molecules
(data not shown). A study by Denecker et al. (65) revealed
that Y. enterocolitica induces cleavage of the proapoptotic
Bcl-2 family member Bid. Truncated Bid bridges death receptor signaling
to the mitochondrial apoptotic pathway, resulting in the release of
cytochrome c from the mitochondrion and in the activation of
caspase-9. Since upstream processing of Bid is efficiently mediated by
caspase-8 (50, 51), these findings may coincide with our
results showing involvement of the FADD-caspase-8 complex and of
caspase-9 in the bacteria-induced proapoptotic response. Together, our
study indicates that bacterial infection of macrophages results in
activation of a MyD88- and IRAK2-dependent apoptotic signal relay,
which potentially targets the FADD/caspase-8 death signaling pathway.
IRAK1 and TRAF6 on the contrary signal macrophage survival to
counteract the cytotoxic response. Interestingly, the proposed
functions of MyD88 and IRAK1 seem to differ in endothelial cells.
Bannerman et al. (66) recently found involvement of both
MyD88 and IRAK1 in signaling endothelial apoptosis upon LPS treatment.
This cytotoxic pathway does apparently not engage FADD, although FADD
was also reported to confer LPS-induced endothelial apoptosis
(64). This suggests that there are differences in the
regulation of macrophage and endothelial cell survival in response to
bacterial infection, which may be related to the distinct functions of
the two cell types within the host immune response. Alternatively, the
signals that regulate entry to apoptosis downstream from MyD88 and
IRAK1 are less prominent in LPS-stimulated macrophages than in
endothelial cells. Fig. 6 gives an
overview on the postulated model, how Y. enterocolitica
exploits the signaling networks of innate immunity in macrophages to
mediate macrophage apoptosis.
|
|
Acknowledgments |
|---|
MyD88, IRAK1, and
IRAK2 cDNAs; Z. Cao and H. Wesche for dominant-negative
IRAK-M cDNA; J. Inoue for dominant-negative TRAF6 and
WT-TRAF6 cDNA; C. Vincenz for dominant-negative FADD cDNA; and N.
Ahn for WT-MKK1 cDNA. We thank G. Pfaffinger for expert technical
assistance, J. Heesemann, M. Aepfelbacher, B. Rouot, and C. Barz for
constructive discussions, and L. Leitritz for help in statistical
analysis. |
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Klaus Ruckdeschel, Max von Pettenkofer Institute for Hygiene and Medical Microbiology, Pettenkoferstrasse 9a, 80336 Munich, Germany. E-mail address: ruckdeschel{at}m3401.mpk.med.uni-muenchen.de
3 Abbreviations used in this paper: Yop, Yersinia outer protein; FADD, Fas-associated death domain protein; GFP, green fluorescent protein; IKK, IB kinase; IRAK, IL-1R-associated kinase; MyD88, myeloid differentiation factor 88; Mal, MyD88 adapter-like protein; MAPK, mitogen-activated protein kinase; MKK, MAPK kinase; Tollip, Toll-interacting protein; TIRAP, Toll-IL I receptor domain-containing adapter protein; TLR, Toll-like receptor; TRAF, TNFR-associated factor; WT, wild type; X-gal, 5-bromo-4-chloro-3-indolyl -D-galactoside.
Received for publication July 9, 2001. Accepted for publication February 22, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) or pretreated with broad-spectrum caspase
inhibitor z-VAD-fmk (