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* Respiratory Medicine Unit, Division of Genomic Medicine and
Cardiovascular Research Group, Division of Clinical Sciences (North), University of Sheffield, Sheffield, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-18 to mature forms but it is unclear whether,
like other caspases, it can induce apoptosis by activation of
downstream protease cascades. Neutrophils are known to express
caspase-1, to release IL-1 and to undergo rapid, caspase-dependent
apoptosis. We examined apoptosis and IL-1 production in peripheral
blood neutrophils of caspase-1-deficient and wild-type mice.
Constitutive apoptosis of caspase-1-deficient neutrophils was delayed
compared with wild-type neutrophils and LPS-mediated inhibition of
apoptosis was absent, but caspase-1-deficient neutrophils were
susceptible to Fas-mediated apoptosis. LPS-stimulated IL-1
production was absent from caspase-1-deficient neutrophils. To
ascertain whether these differences in apoptosis and IL-1 production
would alter the response to acute lung injury, we studied pulmonary
neutrophil accumulation following intratracheal administration of LPS.
Caspase-1-deficient mice showed increased, predominantly neutrophilic
pulmonary inflammation, but inflammation had resolved in both wild-type
and deficient animals by 72 h after LPS instillation. IL-1
production was increased in wild-type lungs but was also detected in
caspase-1-deficient mice. We conclude that caspase-1 modulates
apoptosis of both peripheral blood and inflammatory neutrophils, but is
not essential for IL-1 production in the
lung. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-converting enzyme
(1), was originally identified as the enzyme responsible
for processing IL-1 to its 17-kDa mature form (2).
Caspase-1 is able to induce apoptosis when overexpressed in
vitro (1). However, it is generally thought not to
be a component of cell death machinery (3), but instead to
indirectly influence rates of apoptosis through cleavage of IL-1
(4).
The constitutively short lifespan of the neutrophil, less than 24
h in the circulation, is regulated by the onset of neutrophil apoptosis
(programmed cell death) (5). This process is important for
the normal resolution of inflammation in tissues, because it leads to
recognition and clearance of the apoptotic neutrophils by macrophages
(6). The molecular controls of neutrophil lifespan in
peripheral blood and at inflamed sites are unknown, but modulation of
Bcl-2 proteins and proapoptotic signaling via death receptors may be
important (7). Neutrophil apoptosis is highly susceptible
to modulation by host cytokines, including IL-1 (8),
and is also known to be dependent upon caspase activation and
downstream processing of death substrates (9, 10, 11). IL-1
exerts potent antiapoptotic effects in a number of cell types,
including neutrophils (8, 12).
Recent data has suggested caspase-1 may have a role in pathological
induction of apoptosis. Both Salmonella (13)
and Shigella (14) are able to kill myeloid
cells via generation of specific activators of caspase-1 and the
involvement of caspase-1 in acinar cell death in experimental
pancreatitis has been reported (15). These data are
of particular relevance to neutrophils, because they express caspase-1
(16) and exhibit delayed apoptosis following exposure to
exogenous IL-1 (8) and also following LPS treatment,
the latter effect in part mediated via autocrine production of IL-1
(16).
Therefore, we hypothesized that caspase-1 might regulate apoptosis of
both peripheral blood and inflammatory neutrophils. We studied these
processes in caspase-1-deficient mice compared with wild-type controls.
The results provide evidence for a proapoptotic role for caspase-1 in
apoptosis of unstimulated neutrophils that is reversed in LPS-treated
cells by the antiapoptotic effects of processing of IL-1. In
addition, we studied a model of LPS-mediated lung injury and found that
caspase-1-deficient mice show both a prolonged inflammatory response
and evidence of caspase-1-independent IL-1 production in the
lung.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Breeding pairs of caspase-1-/- homozygous mice and wild-type controls were a kind gift from Dr W. Wong (BASF Research Corporation, Worcester, MA) and have been described elsewhere (17). Briefly, caspase-1-/- mice were created in 129 cells by insertion of a neomycin resistance gene into exon 6, the active site region, rendering all downstream sequence out of frame, and were crossed onto a C57BL/6 background. Mice were kept in a temperature-controlled environment and allowed access to food and water ad libitum. Mice (8–12 wk) were anesthetized with an i.p. injection of ketamine (Ketaset, 100 mg/kg; Willows Francis Veterinary, Crawley, U.K.) and acepromazine (2.5 mg/kg; C-Vet Veterinary Products, Lancashire, U.K.). Heparin (50 U) was administered simultaneously with anesthetic to animals that were used for isolation experiments. All experiments were conducted in accordance with the Home Office Animal (Scientific Procedures) Act 1986.
Materials
Hamster anti-mouse Fas (Jo-2) and hamster IgG group 2, 
isotype control Abs, rat anti-mouse Abs to CD2 (RM2-5), CD5
(53-7.3), and CD45R (RA3-6B2) were all obtained from BD PharMingen
(Oxford, U.K.). Rat anti-mouse F4/80 Ag (Cl:A3-1) was purchased
from Serotec (Kidlington, U.K.). Rat anti-mouse ICAM-1 (YN1/1) was
a gift from Dr. C. Wegner (Abbott Laboratories, Abbott Park, IL). Goat
anti-rat IgG microbeads were obtained from Miltenyi Biotec (Bisley,
U.K.). LPS from Escherichia coli serotype O55:B5 was used in
peripheral blood neutrophil culture experiments and from
Pseudomonas aeruginosa serotype 10 for intratracheal
instillations, both from Sigma-Aldrich (Poole, U.K.). We have confirmed
that these two types of LPS do not differ in their effects on
neutrophil apoptosis (data not shown). The caspase inhibitor,
zYVAD.cmk, was purchased from Bachem (Saffron Walden, U.K.).
Sterile-bottled PBS was purchased from BioWhittaker
(Wokingham, U.K.) and sterile saline from Fresenius Kabi (Warrington,
U.K.). RPMI 1640 was purchased from Sigma-Aldrich and culture media
supplements (FCS, penicillin, streptomycin, and glutamine) from Life
Technologies (Paisley, U.K.). Dextran (T500) was purchased from
Amersham Pharmacia Biotech (Buckinghamshire, U.K.). Diff-Quick rapid
staining set was obtained from Merck (Dorset, U.K.). A Quantikine
murine IL-1 ELISA kit was purchased from R&D Systems (Oxon,
U.K.).
Preparation of peripheral blood neutrophils
Blood (1 ml) was collected from anesthetized mice by cardiac
puncture using a heparinized syringe and transferred into dextran (3
ml, 1.25% w/v in saline). The tubes were filled to 10 ml with dextran
solution, inverted, and the erythrocytes were sedimented for 30 min at
room temperature. The leukocyte-rich supernatants from three mice were
then pooled and washed in buffer (sterile-filtered PBS without cations,
containing 0.5% w/v low-endotoxin BSA, pH 7.4). Neutrophils were then
negatively selected by incubating with the following Abs: anti-CD2,
-CD5, -CD45R, -F4/80 and -ICAM-1, using a previously described method
(18). The final yield was 
1 x
106 cells for each group of three mice.
Neutrophil purity was assessed by differential counts of cytocentrifuge
preparations and samples of 90–95% purity were obtained for
subsequent experiments. Viability of freshly isolated neutrophils was
assessed by trypan blue staining, the number of trypan blue-positive
cells was <1.5%.
Neutrophil culture
Neutrophils were cultured at 1.0 x 106/ml in RPMI 1640 with 10% FCS and penicillin and streptomycin (100 U/L). Aliquots (100 µl) of cells were cultured in the presence of various treatments in nontissue culture-treated Falcon "Flexiwell" plates (BD PharMingen) at 37°C in a 5% CO2 atmosphere. Cells were harvested from culture at 6, 12, and 18 h.
Assessment of neutrophil viability and apoptosis
At the time points indicated, cytocentrifuge preparations were
made and the proportion of neutrophils that had undergone apoptosis was
determined by counting duplicate cytospins stained by Diff-Quick (>300
cells per slide). In agreement with other studies (19), we
found the morphological features of apoptotic and nonapoptotic
neutrophils could be clearly distinguished by light microscopy (see
Fig. 1
A). In addition, necrosis was assessed at all time
points by exclusion of the vital dye trypan blue and was <5% unless
otherwise indicated.
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This model of intratracheal instillation of LPS has been described in detail elsewhere (20). Briefly, a catheter (24-gauge, Jelco; Johnson & Johnson Medical, Ascot, U.K.) was inserted into the trachea of anesthetized mice and LPS (0.3 µg), or PBS as a control, was instilled into the lungs using a pipette gel-loading tip, then flushed through the catheter with air. Animals remained on their backs in a warmed cage until conscious and were then given food and water. At the time points indicated, the experiment was terminated by giving animals an overdose of sodium pentobarbitone.
Bronchoalveolar lavage (BAL)4
The chest cavity was carefully opened to allow the lungs to fully expand. The trachea was exposed and catheterized at the same point of entry as was previously used to instill LPS or PBS. The catheter was tied in place, and heparinized saline (10U/ml) was instilled in 4 x 1-ml aliquots. Lavage fluid was recovered and placed on ice. Ten microliters of each BAL sample was diluted in 90 µl of 3% acetic acid and a total cell count was conducted. Cytocentrifuge preparations were made from each BAL sample (100 µl) and stained with Diff-Quick to provide differential cell counts and to assess the proportion of neutrophils that were apoptotic. The BAL fluid was then centrifuged (300 x g for 6 min) and the supernatant was removed. Both the cell pellet and the supernatant were stored at -70°C for subsequent experiments. Lungs from these mice were snap-frozen in liquid nitrogen for later RNA extractions.
RNase protection assay (RPA)
Murine lung RNA was isolated using guanidine thiocyanate and calcium chloride as previously described (21). Total RNA (5 µg) was used in each RPA sample. RPAs for detection of mRNAs for murine cytokines and caspases were conducted using the RiboQuant protocol following the manufacturer’s instructions (BD PharMingen).
ELISA
Levels of processed IL-1 present in cell culture supernatant
or BAL fluid were determined using a murine IL-1 ELISA kit. This
ELISA recognizes predominantly the mature, processed 17-kDa form of
IL-1, with a limit of detection of 3 pg/ml (22).
Statistical analysis
Results were analyzed for statistical variance using a one- or two-way ANOVA as appropriate followed by Bonferroni’s test for multiple comparisons. Results were considered significant if p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We confirmed that total leukocyte numbers and numbers of
circulating neutrophils did not differ between deficient and wild-type
strains as previously described (17). Moreover, in freshly
isolated neutrophil populations, there was no difference in the
percentage of neutrophils showing light microscopic features of
apoptosis (wild-type, 1.33 ± 0.29 (mean ± SEM);
caspase-1-deficient, 1.51 ± 0.27, p = 0.21 for
six randomly chosen mice for each strain). When cultured in vitro,
neutrophils underwent constitutive apoptosis (Fig. 1
A). However,
caspase-1-deficient mice showed delayed spontaneous apoptosis, with
apoptosis significantly reduced at 6 and 12 h compared with
wild-type controls (p < 0.05) (Fig. 1B). However, at 18 h, there was no significant
difference between wild-type and caspase-1-deficient mice but there
were higher rates of necrosis in wild-type cells (wild-type, 21.7
± 4.6%; caspase-1-deficient, 9.9 ± 2.0%). Therefore,
constitutive apoptosis was delayed, but not prevented, in
caspase-1-deficient neutrophils.
LPS does not inhibit apoptosis of neutrophils from caspase-1-deficient mice
Spontaneous apoptosis of both human (12) and murine
(19) neutrophils is delayed by culture with LPS. Treatment
of wild-type neutrophils with LPS (1 µg/ml), a concentration
previously shown to inhibit apoptosis (8, 12),
significantly delayed their constitutive apoptosis
(p < 0.001 at 6 h and p
< 0.01 at 12 h). In contrast, the constitutive apoptosis of
neutrophils from caspase-1-deficient mice was not reduced at 6 and
12 h in LPS-treated cells compared with their internal controls
(Fig. 2). To confirm that LPS-mediated
inhibition of apoptosis of wild-type neutrophils was caspase-dependent,
wild-type neutrophils were incubated with zYVAD.cmk, a relatively
caspase-1-specific inhibitor (23). A concentration of 100
µM has previously been shown to inhibit LPS-induced IL-1
production in human neutrophils (16, 24). At 6 h,
constitutive apoptosis was 28 ± 2.0%, and this was significantly
reduced to 12.2 ±1.2% by LPS treatment
(n = 3, p < 0.01). Coincubation with
zYVAD abrogated the LPS effect, so that apoptosis was not significantly
different from control (23.4 ± 1.9%, n = 3,
p > 0.05).
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Ligation of the Fas death receptor by its cognate ligand, FasL, is
known to accelerate apoptosis of murine neutrophils (25).
We studied the effect of a receptor cross-linking anti-Fas IgM Ab,
Jo-2. Caspase-1-deficient neutrophils show significant acceleration of
apoptosis following Fas ligation (Fig. 3), although there were higher
percentages of apoptotic cells in the wild-type compared with
caspase-1-deficient neutrophils. At 12 h, there was an increase in
the percentage of trypan blue-positive cells in Jo-2-treated cultures
(up to 15%) but with no difference between wild-type and
caspase-1-deficient mice. The fold increases in apoptosis following Fas
treatment in wild-type compared with caspase-1-deficient neutrophils
suggest the caspase-1-deficient cells were at least as susceptible to
Fas-mediated apoptosis (6 h, wild-type, 2.5 ± 0.3-fold;
caspase-1-/-, 4.9 ± 0.6-fold; 12 h,
wild-type, 2.3 ± 0.4-fold; caspase-1-/-,
2.3 ± 0.75-fold).
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is impaired in neutrophils from
caspase-1-deficient mice
Circulating plasma levels of IL-1 were <5 pg/ml in both
wild-type and caspase-1-deficient mice as measured by ELISA (data not
shown). These concentrations are too low to have any likely
autocrine-modulating effect upon neutrophil apoptosis (17, 25). Release of IL-1 into supernatants was also measured in
cultures of peripheral blood neutrophils (Fig. 4). IL-1 production by unstimulated
cells was higher in wild-type, than caspase-1-deficient, neutrophils,
but this failed to reach statistical significance. In both cases,
IL-1 production was below the level at which any modulating effect
upon apoptosis would be anticipated (8, 17). IL-1
production following LPS treatment was significantly higher in
wild-type compared with caspase-1-deficient neutrophils
(p < 0.001). There was no significant
induction of IL-1 following Fas ligation in either population.
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As previously described (20), intratracheal LPS
induced rapid neutrophil migration into the lungs of challenged mice,
that was assessed by BAL at time points up to 72 h following
instillation. Total cell counts and percent neutrophil counts were
increased in both wild-type and caspase-1-deficient mice 6 h after
LPS instillation, when compared with mice instilled with PBS alone. The
numbers of neutrophils in BAL fluid from control mice was <0.3 x
105 neutrophils at 6 h and at all subsequent
time points in both wild-type and caspase-1-deficient strains. There
was a greater inflammatory response in caspase-1 compared with
wild-type mice, with significantly increased total and percent
neutrophil counts at 24 and 48 h following LPS instillation.
However, by 72 h the counts did not differ significantly between
the two populations (Fig. 5, A
and B). The proportion of the neutrophil BAL population that
had the microscopic appearances of apoptosis was <5% in BAL from both
wild-type and caspase-1-deficient mice at time points up to 48 h.
However, at 72 h there was a significantly higher proportion of
apoptotic neutrophils in the caspase-1-deficient mice (Fig. 5C, caspase-1-deficient, 28.7 ± 3.7% compared with
wild-type 17.9 ± 32.4%, p < 0.001).
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in the inflamed lung in
caspase-1-deficient mice
IL-1 levels in BAL from mice that underwent PBS instillation
were below the level of detection of the ELISA (data not shown).
IL-1 was detected in the BAL of LPS-treated wild-type mice at 6
h, with levels increasing significantly at 12 h
(p < 0.01), then declining thereafter. IL-1
production was also detected in BAL from caspase-1-deficient mice at
6 h following LPS instillation, at similar levels to wild-type
BAL, but there was no significant change in levels at subsequent time
points (Fig. 5D).
Caspase and IL-1 mRNA expression in lungs of wild-type and caspase-1-deficient mice
Caspase mRNA expression was determined in lung homogenates
following intratracheal challenge with LPS or PBS control (Fig. 6A). A caspase-1 transcript
was detected in caspase-1-deficient as well as wild-type mice, although
at lower levels in the samples from caspase-1-deficient animals.
Therefore, we confirmed the previous finding of Li et al.
(17), using genomic PCR, that no coding transcript of
caspase-1 is produced. The band seen likely represents a neo-caspase-1
transcript that would not contain any of the residues required for
caspase-1 enzymatic activity (26). Caspase-11, a close
homolog and possible upstream regulator of caspase-1, was induced
following LPS stimulation in wild-type mice, in keeping with previous
observations (27, 28). However, caspase-11 expression
could not be detected in caspase-1-deficient mice as previously
described (29). The third member of the caspase-1
subfamily in mice, caspase-12, showed no differences in expression
either between wild-type and caspase-1-deficient mice or following LPS
stimulation. Similarly, no alterations were seen in expression of the
effector caspases 3, 6, and 7.
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and IL-1
was also examined by RPA. The
expected induction of both mRNAs following LPS stimulation was seen and
was more marked in caspase-1-deficient than in wild-type mice (Fig. 6B). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-converting enzyme) is confined to cytokine processing
(3) or whether it can, in certain circumstances, also
regulate apoptosis (31). Caspase-1 was the first caspase
to be identified (1), but had previously been described as
the enzyme responsible for processing of the immature form of IL-1
to the active 17-kDa cytokine (2). More recently,
caspase-1 was also shown to process IL-18 (32). Original
descriptions of caspase-1-deficient mice observed abnormalities of
cytokine processing (17, 33) and resistance to endotoxic
shock (17) but only minor, stimulus-specific defects in
apoptosis, e.g., reduced thymocyte apoptosis following Fas ligation but
not following dexamethasone or radiation treatment (33).
Neutrophil apoptosis has not previously been studied in these animals.
However, there is evidence that human neutrophils express caspase-1
which is up-regulated following LPS treatment (16) and
that neutrophils synthesize and release IL-1 following stimulation
(34). Inhibitor studies have shown a role for caspases in
the rapid constitutive death of neutrophils (9, 11) but
have not specifically implicated the caspase-1 family.
In agreement with the original descriptions of these mice
(17), we found no alteration in circulating neutrophil
numbers nor did we find any differences in the small numbers of
apoptotic neutrophils circulating between wild-type and
caspase-1-deficient mice. However, when cultured in vitro,
caspase-1-deficient neutrophils showed delayed apoptosis, under
conditions where there is minimal release of IL-1 from either
wild-type or caspase-1-deficient cells. Only one previous study has
examined apoptosis of murine peripheral blood neutrophils, comparing
wild-type neutrophils to those deficient in A1-a, an antiapoptotic
Bcl-2 protein (19). As with the studies reported here,
abnormalities of neutrophil apoptosis in A1-a-deficient mice were
revealed in vitro despite normal circulating neutrophil numbers. Their
results and ours suggest that an intrinsic defect of neutrophil
apoptosis may not be apparent on peripheral blood counts, perhaps due
to cytokine-mediated positive feedback regulation of neutrophil numbers
or because other regulators of apoptosis may compensate in vivo.
Constitutive neutrophil apoptosis is delayed rather than abolished in
caspase-1-deficient mice, suggesting the apoptotic program can be
engaged via upstream signals other than caspase-1. This could be via
another member of the caspase-1 subfamily, such as caspase-11 or 12.
However, we and others (29), have shown caspase-11 is not
expressed in caspase-1-deficient mice and, although caspase-12 is
expressed, we found no evidence that it is up-regulated, at least at
the mRNA level. Alternatively, neutrophil apoptosis may be initiated
independently of upstream caspases, perhaps via mitochondrial factors
(35) or involvement of serine proteases (9, 36).
LPS-mediated inhibition of peripheral blood neutrophil apoptosis is
completely absent from caspase-1-deficient mice. Although the mRNA for
IL-1 is markedly up-regulated following LPS stimulation of
caspase-1-deficient mice, there is minimal release of IL-1 from
their neutrophils, in keeping with previous studies of peripheral blood
cytokine levels in these mice (17, 33). Previous reports
(16, 24) have also shown that LPS-mediated IL-1
production from wild-type neutrophils is caspase-1-dependent using the
inhibitor zYVAD. Our data support previous observations by Watson et
al. (16) in human peripheral blood neutrophils that LPS
inhibition of apoptosis is dependent upon autocrine production of
IL-1, because it is abrogated by blocking Abs to IL-1 or by IL-1R
antagonist. Other enzymes, including neutrophil products such as
proteinase-3 (37), have been shown to process IL-1 to
its active form (37, 38, 39) and they may account for the low
levels of background IL-1 production by caspase-1-deficient
peripheral blood neutrophils. The levels observed (<20 pg/ml) are
100-fold lower than the lowest concentration at which an
antiapoptotic effect of IL-1 has been described (8, 12). These data suggest that, in wild-type neutrophils, the
basal proapoptotic action of caspase-1 is independent of IL-1
production, but that this proapoptotic action is overridden following
LPS stimulation by IL-1 production and its resulting antiapoptotic
effect. The inability of LPS to extend peripheral blood neutrophil
survival in caspase-1-deficient mice, as well as greatly reduced
IL-1 production, may well contribute to the resistance of these mice
to endotoxic shock induced by i.p. administration of high-dose LPS
(17).
Examination of an experimental model of LPS-induced acute lung injury
showed no delay in neutrophil influx into the lung in
caspase-1-deficient mice, with equivalent neutrophil numbers to
wild-type at 6-h post instillation. Our data, together with the
findings of Parsey et al. (40), who showed normal
neutrophil influx to the lung in IL-1-deficient mice, suggest
neither IL-1 production nor processing in response to LPS is
required for neutrophil emigration to the lung. The inability of LPS to
extend the lifespan of caspase-1-deficient neutrophils did not result
in reduced numbers of inflammatory neutrophils at any time point.
Rather, both cell counts and neutrophil numbers at 12 and 24 h
were significantly increased in caspase-1-deficient compared with
wild-type. The decline in numbers in the caspase-1-deficient mice at
72 h, to equivalent levels to those seen in wild-type, was
accompanied by a significant wave of neutrophil apoptosis in these
mice. This suggests the increased cell numbers seen in
caspase-1-deficient mice were due, at least in part, to a delay in
neutrophil apoptosis, as observed in peripheral blood neutrophils from
these animals.
Measurements of IL-1 concentrations in BAL fluid showed that IL-1
was detected in caspase-1-deficient mice, but at relatively low levels
and with no significant increase with time. The IL-1 production was
independent of caspase-1. Candidate enzymes for IL-1 processing in
the inflamed lung would be proteases, e.g., proteinase-3
(37) or gelatinase B (38), derived from
inflammatory neutrophils and macrophages. Of interest, gelatinase
B-deficient mice show normal neutrophil numbers but reduced lung injury
in an acute lung injury model (41). In contrast, wild-type
mice showed similar levels to those in caspase-1-deficient mice at
6 h, but IL-1 levels increased significantly in the wild-type
cells at 12 h, presumably as a result of caspase-1-dependent
processing following LPS stimulation. Therefore, differences in IL-1
production do not explain the delayed apoptosis of inflammatory
neutrophils in caspase-1-deficient mice.
The studies of Fantuzzi et al. (42) in caspase-1-deficient
mice examined two different models of local inflammation. Following a
s.c. injection of turpentine, there were no differences either in the
development of a systemic acute phase response, nor in levels of mature
IL-1, in caspase-1-deficient compared with wild-type mice. In
zymosan-induced peritonitis, in contrast, levels of mature IL-1 were
significantly lower and there was reduced cellular infiltrate into the
peritoneum in caspase-1-deficient mice at 6 h, although this
effect was lost by 12 h following zymosan administration. Similar
differences in the cellular infiltrate were seen in the
IL-1-deficient mice, suggesting inflammatory cell recruitment was at
least partially IL-1-dependent. However, in the lung we did not
detect any reduction in inflammatory cell recruitment in
caspase-1-deficient mice. This could relate either to differences in
the inflammatory stimulus used or the organ studied. Therefore, it is
of interest that Parsey et al. (40) found no diminution of
neutrophil migration into the lung in IL-1-deficient mice after
hemorrhage or endotoxemia and Kawasaki et al. (43) showed
that a broad-spectrum caspase inhibitor, zVAD.fmk, did not reduce
pulmonary inflammation 24 h after administration of i.v. LPS. All
these data suggest that caspase-1 activity and levels of IL-1
production are not critical determinants of neutrophil influx to the
lung. The previously described inability of caspase-1-deficient mice to
respond to systemically administered LPS appears to be due to absence
of IL-18 or IFN-
, rather than IL-1 (17, 44).
In conclusion, studies of neutrophil apoptosis in caspase-1-deficient
mice provide evidence for a proapoptotic role for caspase-1 in
apoptosis of unstimulated neutrophils. This proapoptotic function of
caspase-1 is reversed in LPS-treated cells by the antiapoptotic
effects of processing of IL-1. Neutrophil migration to the lung in
response to intratracheal LPS is independent of caspase-1, but
caspase-1-deficient mice showed delayed resolution of pulmonary
inflammation, presumably a consequence of delayed apoptosis of
inflammatory neutrophils. These studies have revealed a role for
caspase-1 in the regulation of neutrophil apoptosis in addition to its
well-recognized roles in cytokine processing.
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Acknowledgments |
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Footnotes |
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2 S.J.R., L.A., and V.C.R. contributed equally to this work and are joint first authors.
3 Address correspondence and reprint requests to Dr. Moira K. B. Whyte, Respiratory Medicine Unit, Division of Genomic Medicine, University of Sheffield Medical School, Royal Hallamshire Hospital, Sheffield S10 2JF, U.K. E-mail address: m.k.whyte{at}sheffield.ac.uk
4 Abbreviations used in this paper: BAL, bronchoalveolar lavage; RPA, RNase protection assay.
Received for publication February 5, 2002. Accepted for publication September 20, 2002.
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References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-converting enzyme. Cell 75:641.[Medline]
converting enzyme. Science 256:97. (IL-1) in IL-1-converting enzyme-mediated apoptosis. J. Exp. Med. 184:717. and IL-18 are essential for Shigella flexneri-induced inflammation. Immunity 12:581.[Medline]
-converting enzyme on acinar cell necrosis and apoptosis in severe acute experimental pancreatitis. Lab. Invest. 81:1001.[Medline]
-converting enzyme (caspase-1) inhibits apoptosis of inflammatory neutrophils through activation of IL-1. J. Immunol. 161:957.-converting enzyme are defective in production of mature IL-1 and resistant to endotoxic shock. Cell 80:401.[Medline]
synthesis. J. Immunol. Methods 148:255.[Medline]
and caspases defines life versus death of polymorphonuclear cells. Eur. Cytokine Network 12:177.[Medline]
release and inflammation induced by the apoptosis inducer Fas ligand. Nat. Med. 4:1287.[Medline]
converting enzyme (ICE)/Ced-3 family and an upstream regulator of ICE. J. Biol. Chem. 271:20580.-inducing factor and regulates LPS-induced IFN- production. Nature 386:619.[Medline]
converting enzyme. Science 267:2000. and genes by human blood polymorphonuclear leukocytes. J. Clin. Invest. 87:1312.
and IL-1 from a stimulated human monocytic cell line in the presence of activated neutrophils or purified proteinase 3. Proc. Natl. Acad. Sci. USA 96:6261. by matrix metalloproteinases: a novel caspase-1-independent pathway of IL-1 processing. J. Immunol. 161:3340. and IL-18 requires priming by lipopolysaccharide and is independent of caspase-1 cleavage. J. Biol. Chem. 276:3820.. Chest 116:67.S..
-converting enzyme-deficient mice. J. Immunol. 158:1818.[Abstract]
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 S. M. Bianchi, L. R. Prince, K. McPhillips, L. Allen, H. M. Marriott, G. W. Taylor, P. G. Hellewell, I. Sabroe, D. H. Dockrell, P. W. Henson, et al. Impairment of Apoptotic Cell Engulfment by Pyocyanin, a Toxic Metabolite of Pseudomonas aeruginosa Am. J. Respir. Crit. Care Med., January 1, 2008; 177(1): 35 - 43. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Sarai, D. Hartung, A. Petrov, J. Zhou, N. Narula, L. Hofstra, F. Kolodgie, S. Isobe, S. Fujimoto, J.-L. Vanderheyden, et al. Broad and Specific Caspase Inhibitor-Induced Acute Repression of Apoptosis in Atherosclerotic Lesions Evaluated by Radiolabeled Annexin A5 Imaging J. Am. Coll. Cardiol., December 11, 2007; 50(24): 2305 - 2312. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Tsuruta, Y.-J. Park, G. P. Siegal, G. Liu, and E. Abraham Involvement of Vitronectin in Lipopolysaccaride-Induced Acute Lung Injury J. Immunol., November 15, 2007; 179(10): 7079 - 7086. [Abstract] [Full Text] [PDF]
|
|
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H. M. Marriott, P. G. Hellewell, S. S. Cross, P. G. Ince, M. K. B. Whyte, and D. H. Dockrell Decreased Alveolar Macrophage Apoptosis Is Associated with Increased Pulmonary Inflammation in a Murine Model of Pneumococcal Pneumonia J. Immunol., November 1, 2006; 177(9): 6480 - 6488. [Abstract] [Full Text] [PDF]
|
|
|
|
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X.-Q. Wang, K. Bdeir, S. Yarovoi, D. B. Cines, W. Fang, and E. Abraham Involvement of the Urokinase Kringle Domain in Lipopolysaccharide-Induced Acute Lung Injury J. Immunol., October 15, 2006; 177(8): 5550 - 5557. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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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|
J. M. Kahlenberg, K. C. Lundberg, S. B. Kertesy, Y. Qu, and G. R. Dubyak Potentiation of Caspase-1 Activation by the P2X7 Receptor Is Dependent on TLR Signals and Requires NF-{kappa}B-Driven Protein Synthesis J. Immunol., December 1, 2005; 175(11): 7611 - 7622. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Pei, C. Li, Y. Adereth, T. Hsu, D. K. Watson, and R. Li Caspase-1 Is a Direct Target Gene of ETS1 and Plays a Role in ETS1-Induced Apoptosis Cancer Res., August 15, 2005; 65(16): 7205 - 7213. [Abstract] [Full Text] [PDF]
|
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L. Allen, D. H. Dockrell, T. Pattery, D. G. Lee, P. Cornelis, P. G. Hellewell, and M. K. B. Whyte Pyocyanin Production by Pseudomonas aeruginosa Induces Neutrophil Apoptosis and Impairs Neutrophil-Mediated Host Defenses In Vivo J. Immunol., March 15, 2005; 174(6): 3643 - 3649. [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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)
and caspase-1-deficient (
) is plotted against time (h). At
t = 0 h apoptosis was <1.5%. *,
Statistically significant difference between wild-type and
caspase-1-deficient neutrophils (p < 0.05). Trypan
blue-positive cells were <5% at 6 and 12 h, but at 18 h
were 10–20%.
