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The Journal of Immunology

Caspase-1-Deficient Mice Have Delayed Neutrophil Apoptosis and a Prolonged Inflammatory Response to Lipopolysaccharide-Induced Acute Lung Injury1 Sarah J. Rowe,2* Lucy Allen,2† Victoria C. Ridger,2† Paul G. Hellewell,† and Moira K. B. Whyte3* Caspase-1, the prototypic caspase, is known to process the cytokines IL-1␤ 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. The Journal of Immunology, 2002, 169: 6401– 6407. he prototypic caspase, caspase-1 or IL-1␤-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 –11). IL-1␤ exerts po-

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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 Received for publication February 5, 2002. Accepted for publication September 20, 2002. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by a Wellcome Trust Project Grant (Ref. No. 053841), a British Heart Foundation PhD Studentship (Ref. No. FS/99031 to S.J.R.), and a grant from the Sheffield Hospitals Charitable Trust (Ref. No. 87792). 2

S.J.R., L.A., and V.C.R. contributed equally to this work and are joint first authors.

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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: [email protected] Copyright © 2002 by The American Association of Immunologists, Inc.

tent 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.

Materials and Methods Animals 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 0022-1767/02/$02.00

6402 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 lowendotoxin 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 ⫻ 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 ⫻ 106/ml in RPMI 1640 with 10% FCS and penicillin and streptomycin (100 U/L). Aliquots (100 ␮l) 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. 1A). In addition, necrosis was assessed at all time points by exclusion of the vital dye trypan blue and was ⬍5% unless otherwise indicated.

Model of LPS-mediated acute lung injury 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 ␮g), 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 4 Abbreviations used in this paper: BAL, bronchoalveolar lavage; RPA, RNase protection assay.

NEUTROPHIL APOPTOSIS IN CASPASE-1-DEFICIENT MICE previously used to instill LPS or PBS. The catheter was tied in place, and heparinized saline (10U/ml) was instilled in 4 ⫻ 1-ml aliquots. Lavage fluid was recovered and placed on ice. Ten microliters of each BAL sample was diluted in 90 ␮l of 3% acetic acid and a total cell count was conducted. Cytocentrifuge preparations were made from each BAL sample (100 ␮l) 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 ⫻ 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 ␮g) 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.

Results Delayed constitutive apoptosis of peripheral blood neutrophils from caspase-1-deficient mice 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. 1A). 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-1deficient 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 ␮g/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 ␮M 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 ⫾

The Journal of Immunology

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FIGURE 3. Caspase-1-deficient neutrophils are susceptible to Fas-mediated induction of apoptosis. Percentage apoptosis of wild-type (䡺) and caspase-1-deficient neutrophils (f) was determined in the presence of isotype control Ab (5 ␮g/ml) (unhatched bars) or a Fas-activating Ab, Jo-2 (5 ␮g/ml) (corresponding hatched bars) after 6 and 12 h in culture (mean ⫾ SEM of five independent experiments at 6 h and 3 at 12 h). Fas ligation significantly induced apoptosis of both wild-type and caspase-1-deficient neutrophils at both time points. Significant differences between untreated and Fas-treated cells are indicated by ⴱⴱⴱ (p ⬍ 0.001) and ⴱⴱ (p ⬍ 0.01).

Neutrophils from caspase-1⫺/⫺ mice are susceptible to Fasmediated apoptosis FIGURE 1. Delayed constitutive apoptosis in caspase-1-deficient neutrophils compared with wild-type controls. A, Photomicrograph of a cytocentrifuge preparation of purified peripheral blood neutrophils from caspase-1-deficient mice shows an apoptotic neutrophil (arrow) readily distinguishable from neutrophils of nonapoptotic morphology. B, Neutrophils were purified from peripheral blood as described in Materials and Methods and sampled after 6, 12, and 18 h in culture. Apoptosis was assessed by cytospin morphology as described. Percent apoptosis (mean ⫾ SE of five independent experiments at 6 h, 3 at 12 h, and 3 at 18 h) of neutrophils from control (䡺) and caspase-1-deficient (f) is plotted against time (h). At t ⫽ 0 h apoptosis was ⬍1.5%. ⴱ, Statistically significant difference between wild-type and caspase-1-deficient neutrophils (p ⬍ 0.05). Trypan bluepositive cells were ⬍5% at 6 and 12 h, but at 18 h were 10 –20%.

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).

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-2treated cultures (up to 15%) but with no difference between wildtype and caspase-1-deficient mice. The fold increases in apoptosis following Fas treatment in wild-type compared with caspase-1deficient 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). Production of IL-1␤ is impaired in neutrophils from caspase-1deficient mice Circulating plasma levels of IL-1␤ were ⬍5 pg/ml in both wildtype and caspase-1-deficient mice as measured by ELISA (data not

FIGURE 2. LPS-mediated inhibition of constitutive neutrophil apoptosis is absent in caspase-1-deficient neutrophils. Percentage apoptosis of wild-type (䡺) and caspase-1-deficient neutrophils (f) was determined in the presence (corresponding hatched bars) or absence of LPS (1 ␮g/ml) after 6 and 12 h in culture (mean ⫾ SEM of five independent experiments at 6 h and 3 at 12 h). LPS significantly inhibited apoptosis of wild-type neutrophils at both time points. Caspase-1-deficient neutrophils showed delayed constitutive apoptosis that was not further delayed following LPS treatment. Significant differences between untreated and LPS-treated cells are indicated by ⴱⴱⴱ (p ⬍ 0.001) and ⴱⴱ (p ⬍ 0.01).

FIGURE 4. IL-1␤ levels in cell culture supernatants from wild-type and caspase-1-deficient neutrophils. IL-1␤ levels, measured by ELISA, in cell culture supernatant from both wild-type (䡺) and caspase-1-deficient (f) neutrophils cultured for 6 h in the presence or absence of LPS (1 ␮g/ml) or Jo-2 (5 ␮g/ml). LPS significantly increased IL-1␤ production in wild-type but not caspase-1-deficient mice (mean ⫾ SEM of four independent experiments; ⴱⴱⴱ, p ⬍ 0.001).

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NEUTROPHIL APOPTOSIS IN CASPASE-1-DEFICIENT MICE

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. LPS-mediated pulmonary inflammation is increased in caspase1-deficient mice 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 ⫻ 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 wildtype 17.9 ⫾ 32.4%, p ⬍ 0.001). Local production of IL-1␤ in the inflamed lung in caspase-1deficient 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

FIGURE 5. Neutrophil counts in BAL fluid following intratracheal administration of LPS in wild-type and caspase-1-deficient mice. Differential neutrophil counts were obtained from cytocentrifuge preparations of BAL fluid lavaged from the lungs at time points up to 72 h. A, Total neutrophil counts (mean ⫾ SEM of at least four independent experiments) for each time point were obtained by multiplying the differential count by the total leukocyte number obtained from hemocytometer counts. At 24 and 48 h, there were significantly more neutrophils in the caspase-1-deficient mice BAL (solid line; 24 h p ⬍ 0.001, 48 h p ⬍ 0.05), when compared with wild-type BAL (dashed line). B, The percentage of neutrophils (mean ⫾ SEM) in caspase-1-deficient mice (solid line) was significantly increased at both 24 (p ⬍ 0.01) and 48 h (p ⬍ 0.05) after LPS instillation when compared with wild-type mice (dashed line). C, The percentage of BAL fluid neutrophils with morphological appearances of apoptosis was assessed at 6, 12, 24, 48, and 72 h after LPS instillation. At time points up to 48 h apoptosis was ⬍5% in both wild-type (dashed line) and caspase-1-deficient mice (solid line). At 72 h, the percentage of apoptotic neutrophils was increased in both populations, but there was a significantly higher proportion of apoptotic neutrophils in the caspase-1-deficient mice (ⴱⴱⴱ, p ⬍ 0.001). D, IL-1␤ levels, measured by ELISA, in BAL fluid lavaged from the lungs at 6, 12, 24, 48, and 72 h following intratracheal LPS. Wild-type mice (dashed line) showed a significant rise in IL-1␤ levels 12 h after LPS instillation, which rapidly drops off at 24 h (mean ⫾ SEM of at least three independent experiments), ⴱ, Statistically significant difference (p ⬍ 0.05). In caspase-1-deficient mice (solid line) IL-1␤ levels in BAL fluid do not alter significantly with LPS instillation. At 72 h, IL-1␤ levels were below the detection limit in caspase-1-deficient mice.

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. Expression of IL-1␤ 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 Although the central role of caspases in the regulation of apoptosis is well established (10, 30), there has been considerable debate regarding whether the role of caspase-1 (IL-1␤-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

The Journal of Immunology

FIGURE 6. RPAs in lung tissue from wild-type and caspase-1-deficient mice. A, RPA assays were performed on total lung tissue harvested at 0 or 6 h following intratracheal administration of LPS. A caspase multiple expression gel shows absence of caspase-11 expression in caspase-1-deficient mice, together with a very faint band for caspase-1 itself (see Results). Expression of other caspases does not differ between wild-type and caspase-1-deficient samples. Samples shown include two housekeeping genes (L32 and GAPDH) as loading controls. B, Expression of mRNAs for IL-1␣ and IL-1␤ was also examined by RPA at 0 or 6 h following LPS stimulation. Induction of both cytokines was seen in lung tissue from both wild-type and caspase-1-deficient mice but was particularly marked in the caspase-1-deficient tissue. Samples shown include two housekeeping genes (L32 and GAPDH) as loading controls. These results are each from a single experiment but are representative of three independent experiments.

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

6405 is evidence that human neutrophils express caspase-1 which is upregulated 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 (37–39) 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

6406 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, wildtype mice showed similar levels to those in caspase-1-deficient mice at 6 h, but IL-1␤ levels increased significantly in the wildtype 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

NEUTROPHIL APOPTOSIS IN CASPASE-1-DEFICIENT MICE for caspase-1 in the regulation of neutrophil apoptosis in addition to its well-recognized roles in cytokine processing.

Acknowledgments We thank Dr. Colin Bingle for helpful advice and discussion and Matthew Cotter and Sarah Bond for their invaluable technical assistance.

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