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*
Department of Microbiology, and
The Arthritis Center, Department of Medicine, School of Medicine, Boston University, Boston, MA 02118; and
The Schepens Eye Research Institute, Boston, MA 02114
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, MIP-2,
MIP-1
, and MIP-1. In vitro culture of purified peritoneal
populations identified Mac1high cells as the major
cytokine/chemokine producers in response to mFasL-VP. Purified
Mac1high cells exposed to FasL could restore the ability of
Fas-deficient mice to mount an inflammatory response. Our data
demonstrate that the FasL-mediated inflammatory response starts with
the production of proinflammatory mediators by preapoptotic resident
tissue macrophages and suggest a general mechanism responsible for
neutrophil inflammation seen in cases of FasL-expressing
allografts. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As with other TNF family members, FasL can be cleaved by a metalloproteinase to release a soluble product, sFasL. In vitro analyses initially suggested that sFasL could mediate inflammation directly by establishing a neutrophil chemotactic gradient (11, 12). However, in vivo studies involving tumor cells transfected with experimentally modified murine FasL constructs clearly demonstrated that membrane-bound FasL (mFasL) could effectively induce an inflammatory response (9, 13). These data indicated that FasL worked indirectly, triggering the release of proinflammatory cytokines or chemokines by engagement of a Fas+ target cell. This premise was supported by studies demonstrating that reconstitution of a Fas-deficient animal with Fas-sufficient resident peritoneal washout cells (PWC) could restore its ability to mount a FasL-triggered inflammatory response. However, PWC are a highly heterogeneous population and the actual target population and relevant cytokines and/or chemokines were not identified (13).
We have addressed these specific questions in the current study by extending the previously described peritonitis model in which syngeneic mFasL-expressing tumor cells elicit a vigorous Fas-dependent neutrophil response. An important advantage of this model is that it allows us to readily sample the effect of FasL on peritoneal cell populations at various time points after challenge. To facilitate a biochemical analysis of peritoneal cells, a cell-free preparation of membrane-bound FasL vesicles (mFasL-VP) was used in place of the tumor cell transfectants (14).
We found that Mac1high resident peritoneal macrophages were the major population targeted by FasL in vivo. These resident peritoneal macrophages were highly susceptible to FasL-induced apoptosis and the apoptotic demise of this population in the peritoneum preceded neutrophil inflammation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Four- to 6-wk-old female A/J, C3H/HeJ, DBA/2J, and MRL+/+ mice were purchased from The Jackson Laboratory (Bar Harbor, ME). MRL-lpr/gld double-mutant mice were bred at the Boston University School of Medicine animal facility (15).
Cell lines and reagents
Neuro2a- (A/J) derived N2-mFasL and N2-neo cells were established as described previously (14) and were maintained in 10% FCS-DMEM supplemented with 1x penicillin/streptomycin/glutamine, 1x nonessential amino acids, and 1 mg/ml geneticin-selective antibiotic (G418; Life Technologies, Rockville, MD). The L5178Y-R- (DBA/2J) derived transfectant clones expressing different forms of mouse FasL (wild-type, L5-wtFasL; membrane only, L5-mFasL; soluble extracellular domain, L5-sFasL.EX; naturally cleaved soluble product, L5-sFasL; and empty vector control, L5-neo) have also been described previously (13). The L5 lines were maintained in 10% FCS-RPMI supplemented with 10 mM of HEPES, 50 µM of 2-ME, 1x penicillin/streptomycin/glutamine, and 1 mg/ml G418 (Life Technologies).
Vesicle preparation
N2a-mFasL and N2a-neo cells were expanded in G418-free medium to 70% confluence. Cell culture supernatants were collected 48 h later and centrifuged at 250 x g for 10 min at 5°C to remove any detached cells. Residual cell debris in the culture supernatant was removed by further centrifugation at 20,000 x g for 30 min at 4°C. The cell-free supernatant was then centrifuged at 90,000 x g for 3 h at 4°C. The resulting vesicle pellet was resuspended in serum-free RPMI medium to 10% of the original volume and passed through a 0.45-µm sterile filter. The in vitro killing activity and physiochemical properties of mFasL-VP have been described (14). mFasL-VP specific cytotoxic activity was determined in a standardized 5-h 51Cr release assay using A20 target cells as previously described (13); 1 U of activity was determined as the amount of mFasL-VP necessary to achieve 50% maximal cell death of 3 x 104 cells cultured at 1.5 x 105 cells/ml. In each experiment, control groups were always treated with comparable volumes of mFasL-VP and neo-VP isolated from parallel cultures.
Induction, isolation, and characterization of peritoneal exudate cells (PEC) and PWC
At various times after i.p. injection of 200 µl (7–14 U) of mFasL-VP or neo-VP, mice were euthanized by carbon dioxide asphyxiation. PEC were harvested with 2 ml of serum-free RPMI containing 0.1% endotoxin-free BSA (Sigma-Aldrich, St. Louis, MO) and 1% ITS+1 (Sigma-Aldrich) culture supplement (a combination of insulin, transferrin, selenium, linoleic acid, and BSA; BD Biosciences, Bedford, MA). The collected sample was centrifuged and the supernatant, referred to as the peritoneal exudate fluid (PEF), was stored at -20°C for the subsequent determination of cytokine and chemokine content. The PEC were washed and either stained immediately for flow-cytometric analysis or placed in culture as described below. To identify macrophage and neutrophil populations, FcRs were blocked by pretreatment with mAb 2.4G2 (anti-CD16/CD32) and then stained with FITC-conjugated anti-Gr1 (Ly-6G), PE-conjugated anti-Mac1(M1/70 mAb), and biotinylated anti-F4/80 followed by streptavidin-PerCP (BD PharMingen, San Diego, CA and Caltag Laboratories, Burlingame, CA). In the peritoneal cavity, anti-Gr1 specifically stains neutrophils whereas anti-F4/80 specifically stains macrophages. Mac1 is expressed on both granulocytes and macrophages. In some experiments, F4/80-stained PEC were further incubated on ice for 45 min with FITC-conjugated annexin V in 1x binding buffer (Trevigen, Gaithersburg, MD). Alternatively, unstained PEC were incubated on ice for 45 min with FITC-conjugated annexin V in 1x binding buffer plus 5 µg/ml propidium iodide (PI) to distinguish early and late apoptotic cells. Cells were then analyzed on a FACScan flow cytometer (BD Biosciences). Acquired data was plotted using CellQuest software (BD Biosciences) with contour plot settings of 10% probability and smoothing factor-5.
RNase protection assay
Total RNA from freshly isolated PEC was prepared using a TRIzol
reagent according to the manufacturer’s instructions (Life
Technologies) and analyzed using the RiboQuant Multi Probe RNase
Protection Assay System (BD PharMingen). The MultiProbe template sets
mCK2b and mCK5b were used to assay for IL-1, IL-1, IL-1RA,
MIP-1, MIP-1, MIP-2, monocyte chemotactic protein (MCP)-1,
TCA-3, L32, and GAPDH. Radio-labeled protected probes were resolved
using the Sequagel sequencing gel system (National Diagnostics,
Atlanta, GA). Dried gels were exposed to Kodak Biomax MR films (Kodak,
Rochester, NY) at -80°C.
In vitro culture of PEC, PWC, or purified cell populations
Resident PWC were collected as described above for PEC and
stained with Mac1 and Gr1. A MoFlo cell sorter (Cytomation, Fort
Collins, CO) was used to purify Gr1-,
Mac1high, and Gr1- Mac1
low/intermediate cells (Mac1low) (Tufts
University, School of Medicine, Boston, MA). Total PWC or sorted PWC
subpopulations were incubated in serum-free RPMI ITS+1-BSA medium with
mFasL-VP at a final concentration of 14 U/ml, or with a similar
dilution of neo-VP, for 1–4 h. In some studies, PWC were incubated for
18 h with tumor cells expressing different forms of FasL. Some
samples were incubated with 100 ng/ml LPS (protein-free,
phenol/water-extracted LPS was purified from Escherichia
coli K235 as described (16) and kindly provided by
Dr. M. Fenton, Boston University, School of Medicine) or 200 pg/ml
staurosporin (Sigma-Aldrich). To prevent adhesion-induced activation,
tissue-culture plates for some of these studies were precoated with 1%
agarose. Molecular biology-certified agarose (Kodak) dissolved in PBS
was autoclaved and 200 µl or 400 µl was added to the wells of 48-
or 24-well tissue-culture plates, respectively. To equilibrate the
agarose, four changes of medium were added to the solidified agarose
over the course of a 48 h incubation at 37°C. Supernatants were
collected and IL-1 and MIP-2 protein contents were quantified with
cytokine-specific ELISAs (R&D Systems, Minneapolis, MN) and cells were
analyzed by flow cytometry.
IL-1, MIP-2 ELISA
PEF or in vitro cell culture supernatants were collected and
stored at -20°C for the subsequent determination of cytokine and
chemokine content. Experimental samples were diluted in 0.1% BSA/RPMI
and assayed by IL-1- and MIP-2 capture ELISA (R&D Systems) according
to the manufacturer’s instructions.
Adoptive transfer of Mac1 subpopulations
Fas/FasL-deficient MRL-lpr/gld mice were coinjected with 1 x 106 L5178Y-R transfectants and 1 x 106 purified cells sorted from the total PWC population. PWC from MRL+/+ mice or MRL-lpr/gld mice were stained with Mac1, Gr1, and CD11c. Mac1high, Gr1-, CD11c- cells and Mac1int, Gr1-, CD11c- cells were purified using a Coulter Epic Elite cell sorter (Schepens Eye Research Institute, Harvard University, Boston, MA). Eighteen hours after tumor inoculation, mice were euthanized by carbon dioxide asphyxiation. PEC were harvested and washed with Hank’s phosphate buffered saline containing 2% FCS. Aliquots of fresh PEC were pretreated with 2.4G2, stained with FITC-conjugated anti-Gr1 and PE-conjugated anti-Mac1 (BD PharMingen), and then analyzed on the FACScan flow cytometer as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Significant levels of IL-1 protein had previously been detected
in the PEF of mice inoculated with mFasL-, but not neo-transfected,
cells (9, 13). However, IL-1 per se has little or no
direct chemotactic or activating activity for neutrophils
(17, 18, 19) and therefore we reasoned that other factors were
involved. Chemokine/cytokine production can be elicited by subtle
perturbations in tissue homeostasis and it was important to distinguish
the direct effects of FasL target cell engagement from the factors
produced and elicited by viable tumor cells. Membrane vesicles isolated
from mFasL transfectants (mFasL-VP) have been shown to be a highly
effective source of cell-free FasL when tested in vitro for their
ability to kill standard Fas+ target populations
(14). We found that mFasL-VP could also elicit an in vivo
inflammatory response. Injection of mFasL-VP, i.p., into A/J mice
induced peritoneal extravasation of neutrophils comparable to that
induced by 6 x 105 L5-mFasL tumor cells
(Fig. 1
A). Neo-VP elicited
only a negligible response, comparable to that of buffer alone.
Therefore, mFasL-VP and neo-VP were used for all subsequent studies on
cytokine/chemokine induction in vivo.
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, MIP-2, MIP-1, and MIP-1 RNA was detected in PEC
collected from mice injected with mFasL-VP, but not in PEC from
noninjected or neo-VP injected mice. MCP-1 was also induced in the
mFasL-VP mice and appeared to persist at elevated levels (Fig. 1
B). These data demonstrate that cells recoverable by
peritoneal lavage are responsible for the production of neutrophil
chemotactic factors. Rapid effects of mFasL-VP on Mac1high peritoneal macrophages
MIP-1 and MIP-2 are recognized murine neutrophil chemoattractants
(20, 21, 22, 23, 24, 25) while MCP-1 is predominantly a
monocyte/macrophage-specific chemokine (26, 27). Given the
persistent expression of MCP-1, one might expect increased numbers of
both neutrophils and monocytes/macrophages at 18 h. However, as
indicated in Fig. 1A, the neutrophil accumulation in the
peritoneum, apparent 4–18 h after mFasL-VP injection, was associated
with a notable loss of Mac1high macrophages. This
trend suggested that the Mac1high population was
either particularly sensitive to the proapoptotic effects of mFasL-VP
or that it was induced to down-regulate the level of Mac1 expression.
To more directly analyze the effects of mFasL-VP on peritoneal
macrophages, PEC were triple-stained for Mac1, Gr1, and F4/80 at
various times after VP injection. The cytometer FL1 gain was slightly
adjusted relative to earlier experiments such that the
Mac1+, Gr1- cells could be
viewed as a distinct cell population. Neutrophil (Mac1/Gr1
double-positive, F4/80 negative) inflammation was apparent as early as
2 h after injection of mFasL-VP. As the neutrophils began to
accumulate, there was a decrease in the number of
Mac1high, Gr1- cells by 2
and 4 h (Fig. 2A).
Gr1-positive cells were excluded from the analysis in Fig. 2B by gating on Gr1- cells. The
Mac1-F4/80 analysis revealed that the disappearance of
Mac1high/F4/80+ cells from
the peritoneum at 4 h was preceded by a simultaneous decrease in
Mac1 and F4/80 surface expression (Fig. 2B). The reduction
in Mac1 and F4/80 expression correlated with a decreased
forward-scatter profile, indicating a loss in cell size.
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A).
Most of F4/80-positive peritoneal
macrophages from the mFasL-VP mice became annexin V-positive within 15
min of mFasL-VP injection (Fig. 3B). This shift preceded the
loss of F4/80 expression starting at 1 h after injection as well
as the neutrophil accumulation starting at 2 h. At 1 h, 100%
of peritoneal macrophages were annexin V-positive and the majority of
these cells seemed to disappear from the peritoneum by 4 h. No
significant changes in the degree of annexin V staining were observed
at any of the time points in cells collected from mice injected with
neo-VP vesicles. None of the other cell populations in the peritoneum
became annexin V-positive as a result of the mFasL-VP injection when
compared with the neo-VP control. In a separate experiment, PEC were
stained with annexin V and PI to distinguish early from late apoptotic
cells. A significant increase in annexin V+ and
PI- cells was observed at 0.5 h and
1.5 h (Fig. 3C). Annexin V+,
PI+ double-positive cells could be observed in
the PEC 1.5 h after VP-FasL injection.
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, mFasL-VP induced the apoptotic cell death of peritoneal F4/80-positive
macrophages in vitro. In contrast to the in vivo experiment, the
apoptotic cells persisted and could be readily detected at 18 h.
LPS did not cause any significant cell death, whereas staurosporin did.
Overall, the data demonstrate that resident peritoneal macrophages are
rapidly induced to undergo apoptosis by FasL-VP.
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As shown above, i.p. challenge with FasL-VP was associated with
increased PEC IL-1 and MIP-2 mRNA expression (Fig. 1B).
To test whether MIP-2 protein levels were elevated in vivo in response
to mFasL-VP injection, we collected PEF samples at different times
after injection of mFasL-VP or neo-VP. The MIP-2 protein concentration
was determined by capture ELISA. Perturbation of the peritoneal cavity
by injection of 0.2 ml of neo-VP caused a slight increase in the PEF
MIP-2 protein concentration at 1.5 and 3 h (31 pg/ml and 90 pg/ml,
respectively). In contrast, injection of 14 U of mFasL-VP resulted in
an 18-fold higher FasL-specific release of MIP-2 (574 pg/ml) into the
PEF at 1.5 h (Fig. 5). Increased
MIP-2 levels in PEF correlated with advanced apoptosis of
Mac1high peritoneal macrophages (see Fig. 2C). At 3 h, when most of the macrophages were deleted
from the peritoneum, MIP-2 levels were below the neo-VP control
injection (39 pg/ml vs 90 pg/ml).
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A), as well as examination of
H&E-stained cytospins of each fraction (Fig. 6B), was
used to assess the purity of each fraction. The
Mac1high fraction contained a homogeneous
population of peritoneal macrophages, whereas the
Mac1low-sorted population represented a
heterogeneous population containing all other
Gr1- PWC. The Mac1high
cells and Mac1low cells were incubated in vitro
with mFasL-VP or neo-VP for 4 h (Fig. 6C) or they were
incubated with LPS or L5 transfectants, expressing different forms of
FasL, for 18 h (Fig. 6D). IL-1 protein
concentrations were determined in the cell culture supernatant by
capture ELISA. Only peritoneal macrophages
(Mac1high), and none of the other peritoneal cell
populations (Mac1low), were able to respond to
FasL in the form of mFasL-VP, L5-wild-type FasL, or L5-mFasL by
secreting significant levels of IL-1, comparable to those seen with
LPS stimulation (Fig. 6, C and D). These results
are consistent with the linked induction of macrophage
cytokine/chemokine production and apoptosis.
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The data above clearly demonstrate that FasL can induce the
production of inflammatory mediators in Mac1high
resident peritoneal cells. However, it was important to verify that
this macrophage response was sufficient to trigger neutrophil
extravasation in vivo. Resident peritoneal macrophages were collected
from either MRL+/+ (Fas+)
or MRL-lpr/gld (Fas-) mice and
stained with a combination of PE-Mac1, FITC-Gr1, and FITC-CD11c.
Mac1high and Mac1int
subpopulations that were Gr1 and CD11c negative were isolated with a
cell sorter and reassessed by flow cytometry (Fig. 7, top panel). This enrichment
protocol excluded any low-level contamination with neutrophils or
myeloid dendritic cells which might contribute to the overall level of
cytokine/chemokine production. Fas+ or
Fas- Mac1high cells
(1 x 106) or Fas+
Mac1int cells were mixed with either L5-neo or
L5-mFasL transfectants and coinjected i.p. into MRL-lpr/gld
recipient mice as indicated in Fig. 7. The next day, neutrophil
extravasation was assessed by flow cytometry. Only the coinjection of
Fas+ Mac1high and L5-mFasL
cells resulted in neutrophil extravasation (Fig. 7, bottom
panel). Coinjection of L5-mFasL cells and either
Fas+ Mac1int cells or
Fas- Mac1high cells failed
to elicit a neutrophil response (Fig. 7, bottom panel).
Overall, these data support the premise that FasL specifically triggers
resident macrophages to release neutrophil chemotactic factors.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and chemokines
MIP-2, MIP-1, MIP-1, and MCP1 early in the response. Lymphocytes
could be excluded as a potential target, because we found that
neutrophil extravasation in response to mFasL tumor cells was
comparable in lymphocyte-deficient BALB/c-SCID and BALB/c wild-type
mice (data not shown). A significant decrease in the number of Mac1high cells in the PEC population was noted at 4 h after mFasL-VP injection. More detailed studies using a combination of Mac1, F4/80, and annexin V as well as annexin V/PI flow-cytometric staining demonstrated the rapid and complete apoptotic demise of peritoneal macrophages in vivo and in vitro in response to mFasL-VP. Mac1high peritoneal macrophages acquired annexin V positivity in vivo within 30 min of mFasL-VP inoculation, decreased their volume and surface expression of Mac1 and F4/80 shortly thereafter, and were absent from the peritoneum by 4 h. When peritoneal macrophages were incubated in vitro with mFasL-VP, a comparable extent of apoptotic cell death was observed, but the dying cells persisted. This is probably due to the fact that apoptotic cells are efficiently eliminated in vivo by neighboring phagocytic cells (28).
The dramatic proapoptotic effect of mFasL-VP on resident peritoneal
macrophages was not anticipated, as previous in vitro data suggested
that thioglycolate-elicited peritoneal macrophages were resistant to
FasL-mediated apoptosis and could only be sensitized through IFN-
or
TNF- pretreatment (29, 30). We have also found that
freshly prepared thioglycolate-elicited peritoneal macrophages are
resistant to mFasL-VP (data not shown). The distinction between
resident and elicited macrophage populations probably reflects
differences in their activation and/or differentiation status. Human
peripheral blood monocytes undergo spontaneous apoptosis in vitro in
the presence and absence of serum that is partially dependent on FasL
(31, 32, 33) whereas in vitro monocyte-to-macrophage
differentiation results in FasL resistance and correlates with
increased Fas-associated death domain-like IL-1-converting enzyme
inhibitory protein (FLIP) expression (32). To our
knowledge, the current study is the first report on the proapoptotic
effect of FasL on macrophages in a noninflamed tissue in vivo.
Once it had been clearly shown that FasL could induce resident
peritoneal macrophages to apoptose, it was also important to determine
whether the same cells were responsible for the production of
neutrophil chemotactic factors. Earlier in vivo studies had shown that
significant amounts of IL-1 could be detected in the PEF of mice
18 h after injection with FasL-expressing tumor cells and in vitro
experiments demonstrated FasL-dependent IL-1 secretion by
thioglycolate-elicited neutrophils (9). Fas engagement of
dendritic cells has also been shown to elicit IL-1 production
(34). IL-1 has no chemotactic or activating activity on
neutrophils per se, (17, 18, 19) but neutrophil recruitment
has been shown to be mediated by IL-1-induced secretion of MIP-2
(25, 35). Macrophage-, and possibly neutrophil-, derived
IL-1 might serve to amplify neutrophil inflammation because the
response in IL-1 knockout mice is reduced but not eliminated
(9). The major neutrophil chemoattractants identified in
the mouse are MIP-1 and , MIP-2, and murine CXC chemokine
KC (20, 24, 35, 36, 37, 38, 39, 40). MIP-1 and and MIP-2 RNA
were rapidly induced in mice injected with mFasL-VP. When we sampled
the peritoneal fluid of mice injected with mFasL-VP, a sharp and
transient increase in MIP-2 protein was detected. The peak MIP-2
activity was found 1.5 h after injection, at a time when most
Mac1high peritoneal macrophages showed signs of
apoptosis, as assessed by annexin V positivity, loss of Mac1 and F4/80
expression and the inability to exclude PI. At 3 h, most apoptotic
macrophages appeared to be cleared from the peritoneal space and the
level of detectable MIP-2 returned to baseline, either because
production decreased or because the protein was bound up by the
incoming neutrophils. This correlation between macrophage apoptosis and
cytokine production suggested that dying macrophages were responsible
for producing the factor(s) responsible for the initiation of the
neutrophil response. Subsequent in vitro studies demonstrated that FasL
engagement of purified Mac1high peritoneal
macrophages led to the secretion of IL-1 protein, demonstrating
directly that proinflammatory cytokines could be secreted by the
Mac1high population. Purified
Mac-1low cells failed to secrete IL-1 in
response to FasL. Furthermore, the adoptive transfer of
Mac1high, Gr1-,
CD11c- MRL+/+ resident
peritoneal cells into Fas-deficient recipients restored the capacity of
these mice to mount a neutrophil response to L5-mFasL cells. Because
cells expressing either Gr1 (granulocytes) or CD11c (dendritic cells)
were excluded from this sorted population, it is highly likely that the
Mac1high macrophages initiated the neutrophil
response.
There are two possible mechanisms that could link macrophage death to
cytokine/chemokine release. One possibility is that the Fas-signaling
pathway could activate cytoplasmic mediators that regulate chemokine
production and release as well as caspases. The fact that, at least in
vitro, FasL-induced secretion of proinflammatory cytokines can be
uncoupled from cell death in a variety of different homogeneous cell
populations directly implicates the Fas-signaling pathway in the
induction of cytokine/chemokine production (34, 41, 42, 43, 44).
Several of these studies involved cells expressing elevated levels of
FLIP and it has been suggested that FLIP may serve as a link to NF-
B
activation (45). However, resident peritoneal macrophages
do not express FLIP (A. M. Hohlbaum, unpublished observation) and
rapidly undergo apoptosis after Fas cross-linking, so it is unlikely
that FLIP is involved in the cytokine/chemokine response described
above. An alternative explanation is suggested by the failure of crmA
transgenic mice or mice deficient in the expression of caspase 8 or the
Fas adapter protein Fas-associated death domain to develop peripheral
lymphadenopathy, splenomegaly, and autoimmune disease characteristic of
Fas-deficient lpr mice even though T cells from these mice
are resistant to FasL-mediated apoptosis (46, 47, 48, 49). One
interpretation of these observations is that the autoimmune parameters
are dependent on agonistic or pro-proliferative activities emanating
from the Fas pathway and suggest that other adaptor proteins may be
involved. It is also possible that the initial apoptotic cells might be
recognized by other macrophages that are then induced to secrete
chemotactic factors as a means for recruiting additional phagocytic
cells to cope with the cell debris. However, the uptake of apoptotic
cells by phagocytic macrophages has been shown to inhibit the
production of proinflammatory cytokines and actually triggers the
release of the anti-inflammatory cytokine TGF (50).
Thus, our results most likely reflect a direct effect of FasL on
resident macrophages leading to the release of proinflammatory
cytokines/chemokines and suggest a general mechanism responsible for
neutrophil inflammation seen in cases of FasL-expressing
allografts.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ann Marshak-Rothstein, Department of Microbiology, School of Medicine, Boston University, 80 East Concord Street, Boston, MA 02118. E-mail address: amrothst{at}bu.edu
3 Abbreviations used in this paper: FasL, Fas ligand; mFasL, membrane-only FasL; sFasL, soluble FasL natural cleavage product; PWC, peritoneal washout cells; VP, vesicle preparation; G418, geneticin selective antibiotic; PEC, peritoneal exudate cells; PEF, peritoneal eluate fluid; PI, propidium iodide; MCP, monocyte chemotactic protein; FLIP, Fas-associated death domain-like IL-1-converting enzyme inhibitory protein.
4 M. S. Gregory, A. C. Repp, A. M. Hohlbaum., R. R. Saff, A. Marshak-Rothstein, and B. R. Ksander. Membrane Fas ligand activates innate immunity and terminates ocular immune privilege. Submitted for publication.
Received for publication July 18, 2001. Accepted for publication September 26, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in macrophages that have ingested apoptotic cells. J. Immunol. 163:6164.This article has been cited by other articles:
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S. Cuzzocrea, E. Mazzon, R. Di Paola, A. Peli, A. Bonato, D. Britti, T. Genovese, C. Muia, C. Crisafulli, and A. P. Caputi The role of the peroxisome proliferator-activated receptor-{alpha} (PPAR-{alpha}) in the regulation of acute inflammation J. Leukoc. Biol., May 1, 2006; 79(5): 999 - 1010. [Abstract] [Full Text] [PDF]
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C. Shi, J. Wolfe, J. Q. Russell, K. Fortner, N. Hardin, J. Anguita, and R. C. Budd Fas Ligand Deficiency Impairs Host Inflammatory Response against Infection with the Spirochete Borrelia burgdorferi Infect. Immun., February 1, 2006; 74(2): 1156 - 1160. [Abstract] [Full Text] [PDF]
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H. Kidoya, M. Umemura, T. Kawabe, G. Matsuzaki, A. Yahagi, R. Imamura, and T. Suda Fas Ligand Induces Cell-Autonomous IL-23 Production in Dendritic Cells, a Mechanism for Fas Ligand-Induced IL-17 Production J. Immunol., December 15, 2005; 175(12): 8024 - 8031. [Abstract] [Full Text] [PDF]
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 M. Perl, C.-S. Chung, J. Lomas-Neira, T.-M. Rachel, W. L. Biffl, W. G. Cioffi, and A. Ayala Silencing of Fas, but Not Caspase-8, in Lung Epithelial Cells Ameliorates Pulmonary Apoptosis, Inflammation, and Neutrophil Influx after Hemorrhagic Shock and Sepsis Am. J. Pathol., December 1, 2005; 167(6): 1545 - 1559. [Abstract] [Full Text] [PDF]
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A. E. Ryan, F. Shanahan, J. O'Connell, and A. M. Houston Addressing the "Fas Counterattack" Controversy: Blocking Fas Ligand Expression Suppresses Tumor Immune Evasion of Colon Cancer In vivo Cancer Res., November 1, 2005; 65(21): 9817 - 9823. [Abstract] [Full Text] [PDF]
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C. Collins, J. Wolfe, K. Roessner, C. Shi, L. H. Sigal, and R. C. Budd Lyme Arthritis Synovial {gamma}{delta} T Cells Instruct Dendritic Cells via Fas Ligand J. Immunol., November 1, 2005; 175(9): 5656 - 5665. [Abstract] [Full Text] [PDF]
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 Z. Guo, M. Zhang, H. Tang, and X. Cao Fas signal links innate and adaptive immunity by promoting dendritic-cell secretion of CC and CXC chemokines Blood, September 15, 2005; 106(6): 2033 - 2041. [Abstract] [Full Text] [PDF]
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S. Baumann, A. Dostert, N. Novac, A. Bauer, W. Schmid, S. C. Fas, A. Krueger, T. Heinzel, S. Kirchhoff, G. Schutz, et al. Glucocorticoids inhibit activation-induced cell death (AICD) via direct DNA-dependent repression of the CD95 ligand gene by a glucocorticoid receptor dimer Blood, July 15, 2005; 106(2): 617 - 625. [Abstract] [Full Text] [PDF]
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 M. Engelbert and M. S. Gilmore Fas Ligand but Not Complement Is Critical for Control of Experimental Staphylococcus aureus Endophthalmitis Invest. Ophthalmol. Vis. Sci., July 1, 2005; 46(7): 2479 - 2486. [Abstract] [Full Text] [PDF]
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M. S. Gregory, S. Koh, E. Huang, R. R. Saff, A. Marshak-Rothstein, S. Mukai, and B. R. Ksander A Novel Treatment for Ocular Tumors Using Membrane FasL Vesicles to Activate Innate Immunity and Terminate Immune Privilege Invest. Ophthalmol. Vis. Sci., July 1, 2005; 46(7): 2495 - 2502. [Abstract] [Full Text] [PDF]
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T. A. Neff, R.-F. Guo, S. B. Neff, J. V. Sarma, C. L. Speyer, H. Gao, K. D. Bernacki, M. Huber-Lang, S. McGuire, L. M. Hoesel, et al. Relationship of Acute Lung Inflammatory Injury to Fas/FasL System Am. J. Pathol., March 1, 2005; 166(3): 685 - 694. [Abstract] [Full Text] [PDF]
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M. Giroux and F. Denis CD1d-unrestricted human NKT cells release chemokines upon Fas engagement Blood, January 15, 2005; 105(2): 703 - 710. [Abstract] [Full Text] [PDF]
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N. Vij, L. Roberts, S. Joyce, and S. Chakravarti Lumican Regulates Corneal Inflammatory Responses by Modulating Fas-Fas Ligand Signaling Invest. Ophthalmol. Vis. Sci., January 1, 2005; 46(1): 88 - 95. [Abstract] [Full Text] [PDF]
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 S. D. Reynolds, A. Giangreco, K. U. Hong, K. E. McGrath, L. A. Ortiz, and B. R. Stripp Airway injury in lung disease pathophysiology: selective depletion of airway stem and progenitor cell pools potentiates lung inflammation and alveolar dysfunction Am J Physiol Lung Cell Mol Physiol, December 1, 2004; 287(6): L1256 - L1265. [Abstract] [Full Text] [PDF]
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S. Xiao, U. S. Deshmukh, S. Jodo, T. Koike, R. Sharma, A. Furusaki, S.-s. J. Sung, and S.-T. Ju Novel Negative Regulator of Expression in Fas Ligand (CD178) Cytoplasmic Tail: Evidence for Translational Regulation and against Fas Ligand Retention in Secretory Lysosomes J. Immunol., October 15, 2004; 173(8): 5095 - 5102. [Abstract] [Full Text] [PDF]
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M. Umemura, T. Kawabe, K. Shudo, H. Kidoya, M. Fukui, M. Asano, Y. Iwakura, G. Matsuzaki, R. Imamura, and T. Suda Involvement of IL-17 in Fas ligand-induced inflammation Int. Immunol., August 1, 2004; 16(8): 1099 - 1108. [Abstract] [Full Text] [PDF]
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R. R. Saff, E. S. Spanjaard, A. M. Hohlbaum, and A. Marshak-Rothstein Activation-Induced Cell Death Limits Effector Function of CD4 Tumor-Specific T Cells J. Immunol., June 1, 2004; 172(11): 6598 - 6606. [Abstract] [Full Text] [PDF]
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 Z. Hao, B. Hampel, H. Yagita, and K. Rajewsky T Cell-specific Ablation of Fas Leads to Fas Ligand-mediated Lymphocyte Depletion and Inflammatory Pulmonary Fibrosis J. Exp. Med., May 17, 2004; 199(10): 1355 - 1365. [Abstract] [Full Text] [PDF]
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S. H. Burnett, E. J. Kershen, J. Zhang, L. Zeng, S. C. Straley, A. M. Kaplan, and D. A. Cohen Conditional macrophage ablation in transgenic mice expressing a Fas-based suicide gene J. Leukoc. Biol., April 1, 2004; 75(4): 612 - 623. [Abstract] [Full Text] [PDF]
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 S. Hayley, S. J. Crocker, P. D. Smith, T. Shree, V. Jackson-Lewis, S. Przedborski, M. Mount, R. Slack, H. Anisman, and D. S. Park Regulation of Dopaminergic Loss by Fas in a 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine Model of Parkinson's Disease J. Neurosci., February 25, 2004; 24(8): 2045 - 2053. [Abstract] [Full Text] [PDF]
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A. Zanin-Zhorov, R. Hershkoviz, I. Hecht, L. Cahalon, and O. Lider Fibronectin-Associated Fas Ligand Rapidly Induces Opposing and Time-Dependent Effects on the Activation and Apoptosis of T Cells J. Immunol., December 1, 2003; 171(11): 5882 - 5889. [Abstract] [Full Text] [PDF]
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 M. Dias-Baruffi, H. Zhu, M. Cho, S. Karmakar, R. P. McEver, and R. D. Cummings Dimeric Galectin-1 Induces Surface Exposure of Phosphatidylserine and Phagocytic Recognition of Leukocytes without Inducing Apoptosis J. Biol. Chem., October 17, 2003; 278(42): 41282 - 41293. [Abstract] [Full Text] [PDF]
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M. Fukui, R. Imamura, M. Umemura, T. Kawabe, and T. Suda Pathogen-Associated Molecular Patterns Sensitize Macrophages to Fas Ligand-Induced Apoptosis and IL-1{beta} Release J. Immunol., August 15, 2003; 171(4): 1868 - 1874. [Abstract] [Full Text] [PDF]
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 D. R Balkundi, J. A Ziegler, J. F Watchko, C. Craven, and M. Trucco Regulation of FasL/Fas in Human Trophoblasts: Possible Implications for Chorioamnionitis Biol Reprod, August 1, 2003; 69(2): 718 - 724. [Abstract] [Full Text] [PDF]
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D. R. Park, A. R. Thomsen, C. W. Frevert, U. Pham, S. J. Skerrett, P. A. Kiener, and W. C. Liles Fas (CD95) Induces Proinflammatory Cytokine Responses by Human Monocytes and Monocyte-Derived Macrophages J. Immunol., June 15, 2003; 170(12): 6209 - 6216. [Abstract] [Full Text] [PDF]
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J. Yang, S. P. Jones, T. Suhara, J. J. M. Greer, P. D. Ware, N. P. Nguyen, H. Perlman, D. P. Nelson, D. J. Lefer, and K. Walsh Endothelial Cell Overexpression of Fas Ligand Attenuates Ischemia-Reperfusion Injury in the Heart J. Biol. Chem., April 18, 2003; 278(17): 15185 - 15191. [Abstract] [Full Text] [PDF]
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W.-S. Hor, W.-L. Huang, Y.-S. Lin, and B.-C. Yang Cross-talk between tumor cells and neutrophils through the Fas (APO-1, CD95)/FasL system: human glioma cells enhance cell viability and stimulate cytokine production in neutrophils J. Leukoc. Biol., March 1, 2003; 73(3): 363 - 368. [Abstract] [Full Text] [PDF]
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K. Roessner, J. Wolfe, C. Shi, L. H. Sigal, S. Huber, and R. C. Budd High Expression of Fas Ligand by Synovial Fluid-Derived {gamma}{delta} T Cells in Lyme Arthritis J. Immunol., March 1, 2003; 170(5): 2702 - 2710. [Abstract] [Full Text] [PDF]
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S. Xiao, S. Jodo, S.-s. J. Sung, A. Marshak-Rothstein, and S.-T. Ju A Novel Signaling Mechanism for Soluble CD95 Ligand. SYNERGY WITH ANTI-CD95 MONOCLONAL ANTIBODIES FOR APOPTOSIS AND NF-kappa B NUCLEAR TRANSLOCATION J. Biol. Chem., December 20, 2002; 277(52): 50907 - 50913. [Abstract] [Full Text] [PDF]
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M. S. Gregory, A. C. Repp, A. M. Holhbaum, R. R. Saff, A. Marshak-Rothstein, and B. R. Ksander Membrane Fas Ligand Activates Innate Immunity and Terminates Ocular Immune Privilege J. Immunol., September 1, 2002; 169(5): 2727 - 2735. [Abstract] [Full Text] [PDF]
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 V. M. Borges, M. F. Lopes, H. Falcao, J. H. Leite-Junior, P. R. M. Rocco, W. F. Davidson, R. Linden, W. A. Zin, and G. A. DosReis Apoptosis Underlies Immunopathogenic Mechanisms in Acute Silicosis Am. J. Respir. Cell Mol. Biol., July 1, 2002; 27(1): 78 - 84. [Abstract] [Full Text] [PDF]
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F. H. Igney and P. H. Krammer Immune escape of tumors: apoptosis resistance and tumor counterattack J. Leukoc. Biol., June 1, 2002; 71(6): 907 - 920. [Abstract] [Full Text] [PDF]
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