Journal of Leukocyte Biology BioLegend: Treg, Th17, Stem Cell
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published online as doi:10.1189/jlb.0506358 on October 5, 2006

Published online before print October 5, 2006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental figures
Right arrow All Versions of this Article:
jlb.0506358v1
81/1/229    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Albee, L.
Right arrow Articles by Perlman, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Albee, L.
Right arrow Articles by Perlman, H.
(Journal of Leukocyte Biology. 2007;81:229-237.)
© 2007 by Society for Leukocyte Biology

Aspartic protease and caspase 3/7 activation are central for macrophage apoptosis following infection with Escherichia coli

Lee Albee*,1, Bo Shi{dagger} and Harris Perlman*,2

* Saint Louis University, School of Medicine, Department of Molecular Microbiology and Immunology, Saint Louis, Missouri, USA; and
{dagger} Northwestern University, Feinberg School of Medicine, Division of Rheumatology, Chicago, Illinois, USA

2Correspondence: Saint Louis University, School of Medicine, Department of Molecular Microbiology and Immunology, 1402 South Grand Blvd., St. Louis, MO 63104, USA. E-mail: perlmanh{at}slu.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Macrophages are vital for host defense against microbial infections. We have previously shown that infection of macrophages with a nonpathogenic strain of Escherichia coli induces apoptosis rapidly. Here, we demonstrate that infection of macrophages results in the activation of caspases prior to the induction of the intrinsic apoptosis pathway. Caspases 9 and 3 are activated prior to the release of intermembrane mitochondrial protein cytochrome C into the cytosol in infected macrophages. Treatment with an inhibitor to caspase 9 has no effect on the death of macrophages and does not prevent activation of the downstream effector caspase 3/7. In contrast, an inhibitor to caspase 3/7 reduces cell death in E. coli-infected macrophages. Although caspase 9 is not required, activation of aspartic proteases, of which cathepsin D is one of the central members, is essential for activation of caspase 3/7. Treatment with pepstatin A, an inhibitor of aspartic proteases, markedly diminishes the activation of cathepsin D and caspase 3/7 and reduces death in E. coli-infected macrophages. Collectively, these data suggest that cathepsin D activation of caspase 3/7 may be required for inducing one of the death pathways elicited by E. coli.

Key Words: bacteria • lysosomal • mitochondria • Bcl-2


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Macrophages are necessary for the innate immune response to bacterial infections [1 ]. Activation of TLR has been shown to be an early response to bacterial infection (reviewed in ref. [2 ]) and is necessary for phagocytosis and killing of bacteria. Macrophages from mice deficient in TLR4 or the adaptor protein MyD88 have reduced phagocytosis of bacteria and decreased bactericidal activity [3 ]. Depletion of macrophages results in decreased clearance of the pathogenic bacteria Yersinia in blood and increased bacterial load in liver [4 ]. Mice with impaired recruitment of macrophages to sites of Escherichia coli infection have increased bacterial loads in liver and/or increased mortality [5 , 6 ]. Phagocytosis of bacteria, regardless of its pathogenicity, has been shown to induce macrophage apoptosis [7 8 9 ], which results in decreased antigen presentation [10 ], inflammatory response [11 ], and bacterial clearance [11 ]. Mice transgenic for the antiapoptotic protein Bcl-2 in myeloid cells have increased survival and have reduced bacterial loads within the peritoneum in the cecal ligation and puncture model of sepsis [12 ]. These data demonstrate that phagocytosis, recruitment, and survival of macrophages are all necessary to control bacterial infection.

The majority of apoptotic events is initiated by ligand binding to a death receptor on the cell surface ("extrinsic" pathway) or by activation/inactivation of intracellular factors, which induce the release of inner mitochondrial proteins ("intrinsic" pathway). Death receptor binding to its ligand induces the recruitment of Fas-associated death domain and procaspase 8, which results in the autocatalysis/activation of procaspase 8 [13 ]. The intrinsic pathway is regulated by the Bcl-2 protein family, which is divided into antiapoptotic (Bcl-2, Bcl-xL, Mcl-1, A1/Bfl-1, and Bcl-w) and proapoptotic (Bax, Bak, Bad, Bim/Bod, Bok/Mtd, Bik/Blk/Nbk, Bid, Hrk/DP5, Bmf, Noxa, and Puma/Bbc3) members [14 ]. Apoptosis signaling through the intrinsic pathway may be inhibited by overexpression of any Bcl-2-like prosurvival family members [15 16 17 ] or by loss of multi-Bcl-2-homology domain proteins Bak and Bax [18 19 20 21 ]. During intrinsic apoptosis signaling, the integrity of the outer mitochondrial membrane is lost [22 ], and apoptogenic mitochondrial intermembrane proteins, such as cytochrome C, are released into the cytosol [23 , 24 ]. The association of cytochrome C with the adaptor protein Apaf-1 and procaspase 9 leads to the catalysis of the zymogen isoform of caspase 9, which then activates the effector caspases 3, 6, and 7 [25 ], which induce the downstream, degradative events in apoptosis [26 ].

Infection with bacteria, regardless of pathogenicity, may induce macrophages to undergo apoptosis [27 ]. The mechanisms of apoptosis mediated by pathogenic bacteria, including pathogenic species of E. coli, have been investigated extensively and are generally attributed to secretion or injection of toxic protein(s) expressed by the bacteria [28 29 30 31 32 33 34 35 ]. Unlike pathogenic bacteria, few studies have examined the mechanism of apoptosis induced by nonpathogenic E. coli in macrophages [8 , 9 , 11 ]. Although the Bcl-2 family and activation of caspases [8 , 9 , 11 ] have been shown to contribute to the death of E. coli-infected macrophages, here, we show an essential role for caspase 3/7 in the death of macrophages following infection with E. coli. Activation of caspase 3/7 but not 9 is required for apoptosis of macrophages following infection with E. coli. Treatment with an inhibitor to aspartic proteases, which includes cathepsin D, a lysosomal protease that has been shown to induce the activation of caspase 3 [36 37 38 39 40 41 42 43 44 45 ], decreases the activity of caspase 3/7 and reduces the number of apoptotic macrophages following infection with E. coli. These data indicate that in macrophages, aspartic proteases such as cathepsin D but not the cysteine protease caspase 9 are responsible for activation of caspase 3/7, an essential factor for E. coli-induced apoptosis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
E. coli K12 strain DH5{alpha} (Invitrogen, Carlsbad, CA), transformed with an expression construct for enhanced GFP (a gift from Georg Hacker, Technische Universitat Muchen, Munich, Germany) or with pBS-SK–/– (Stratagene, La Jolla, CA), was inoculated from a frozen stock or from an agar plate into liquid Luria-Bertani (LB) medium containing ampicillin. E. coli was incubated at 37°C until reaching an OD between 0.5 and 0.8. E. coli was collected by centrifugation, washed with PBS, and resuspended in RPMI-1640 media. Quantification of E. coli per mL RPMI was determined by dilution of the cultures and counting formation of colonies. Phorbolesters (10 µM, Sigma Chemical Co., St. Louis, MO) were added to human monocytic cells (THP-1) for 18–24 h to induce differentiation into macrophages and cell cycle arrest [46 , 47 ]. Human primary macrophages were derived from peripheral blood monocytes as described previously [48 ]. Macrophages were cultured in RPMI 1640/10% heat-inactivated FBS/50 µM 2-ME (Invitrogen). As a dose response curve revealed that the LD50 for nonpathogenic E. coli is at a ratio of 25:1 bacterial:cell (Supplemental Fig. 1), macrophages were infected with E. coli at 25:1 bacteria:cell in antibiotic-free media containing 10% heat-inactivated FBS as described previously [11 ]. Gentamicin (200 µg/mL, Invitrogen) was added to kill all external bacteria after 2 h of incubation. Benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk; 10 µM), z-Asp-Glu-Val-Asp.fmk (zDEVD.fmk; 20 µM), z-Leu-Glu-His-Asp.fmk (zLEHD.fmk; 20 µM, BD PharMingen, San Diego, CA), pepstatin A (100 µM), or N-acetyl-Leu-Leu-Norleu-al (ALLN; 50 µM, Sigma Chemical Co.) was added to macrophages prior to infection with E. coli to inhibit caspase or cathepsin activity. Macrophages were UV-irradiated at 50 mJ/cm2 using a UV-Stratalinker 1800 (Stratagene Inc.).

Apoptosis assays
Macrophages were removed from plates by incubation with accutase (eBiosciences, San Diego, CA), stained with Annexin V conjugated to allophycocyanin (APC; Caltag, Burlingame, CA) and 7-amino-actinomycin (7-AAD; BD PharMingen), and acquired on FACSCalibur (BD PharMingen) using CellQuest software at the Saint Louis University Core Flow Cytometry Facility (MO). All analysis was performed using FlowJo software (Tree Star Inc., Ashland, OR). To determine the activities of caspases 9 and 3/7 or cathepsin D, whole cell extracts from macrophages were harvested at various time-points following infection, and equal amounts of protein were analyzed for protease activity, as described by the manufacturers (Chemicon, Temecula, CA, and Calbiochem, San Diego, CA). The Cell Death ELISA Plus kit (Roche, Indianapolis, IN) was used to measure DNA fragmentation. Briefly, the cytoplasmic fraction of cell lysates was incubated in antihistone-coated microtiter plates to bind mono- and oligonucleosomal DNA. Histone-bound DNA was detected with a peroxidase-conjugated anti-DNA antibody and quantified by absorbance on a microplate reader (BioRad, Hercules, CA).

Immunoblot analysis
Whole cell extracts were prepared as described previously [49 ] from uninfected and infected cultures. Cytoplasmic and mitochondrial-enriched extracts were prepared by incubating cells in a lysis buffer (220 mM mannitol, 68 mM sucrose, 50 mM PIPES-KOH, pH 7.4, 50 mM KCl, 2 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, and a cocktail of protease inhibitors) for 10 min on ice followed by homogenization with a glass dounce and a B pestle (60–70 strokes). Cell homogenates were spun at 200 g for 5 min, and supernatants were removed and spun at 14,000 g for 15 min. Pellet (mitochondrial) and supernatant (cytoplasmic) fractions were stored at –80°C. Whole cell or enriched extracts (40 µg) and molecular weight markers (Invitrogen) were analyzed by PAGE on 4–12.5% polyacrylamide gels containing SDS and transferred to Immobilon-P (Millipore Corp., Bedford, MA) by semidry blotting. Immunoblots were blocked for 1 h at room temperature in PBS/0.2% Tween 20/5% nonfat dry milk and then incubated with mouse antitubulin (Calbiochem), anticathepsin D (R&D Systems, Minneapolis, MN), or anticytochrome C oxidase subunit IV (Clontech, Mountain View, CA) antibodies or rabbit anti-Bax (BD PharMingen), anti-Bim (BD PharMingen), anticytochrome C (BD PharMingen), anticaspase 9 (Cell Signaling, Danvers, MA), or antiactive caspase 3 (Cell Signaling) antibodies. Immunoblots were washed in PBS/0.2% Tween 20/2% nonfat dry milk and incubated with rabbit antimouse or donkey antirabbit secondary antibodies conjugated to HRP (Amersham Pharmacia Biotech, Piscataway, NJ). Visualization of the immunocomplex was conducted by ECL (ECL Plus, Amersham Pharmacia Biotech).

Colony formation assays
Colony formation assays were performed as described previously [11 ]. Briefly, macrophages infected with GFP-E. coli were washed four times with PBS, incubated with gentamicin (200 µg/mL) for at least 30 min, and then lysed with 1 mL water for 10 min. The lysate was serially diluted, cultured on LB plates containing ampicillin, and incubated at 37°C. Clearance of bacteria was assessed by counting the number of bacterial colonies/plate.

Statistics
Results were expressed as the mean ± SE. Differences between groups were analyzed using a Student’s t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Caspase activation occurs prior to the induction of the intrinsic apoptosis pathway in E. coli-infected macrophages
Previous studies have demonstrated that caspases and the Bcl-2 family of proteins have a role in the apoptosis of murine macrophages following infection with E. coli [8 , 9 ]. Although these studies suggest that the apoptosis is mediated through the mitochondria, they have not determined directly how infection affects the levels of Bax, Bim, or cytochrome C. Therefore, we infected PMA-differentiated THP-1 macrophages with E. coli and determined the levels of Bax, Bim, or cytochrome C in cytoplasm or mitochondria over time. Whereas the levels of Bax in mitochondria and cytoplasm remained constant over time, the levels of the long Bim (BimL) isoform increased in mitochondria and decreased in the cytoplasm at 1.5 h following infection of macrophages with E. coli (Fig. 1 ). Subsequent to BimL translocation to the mitochondria, cytochrome C was released into the cytoplasm at 3 h post-E. coli infection (Fig. 1) . In contrast, irradiation of macrophages with UV light induced a rapid increase in the level of cytochrome C in the cytoplasmic fraction (Supplemental Fig. 2), which preceded activation of caspases 9 and 3/7 (data not shown). As our previous study demonstrated that caspases 9 and 3/7 are activated at 1.5 h following infection [11 ] and as cytochrome C release is not apparent until 3 h postinfection, these data suggest that activation of caspases 9 and 3/7 precedes the induction of the intrinsic apoptotic pathway in E. coli-infected macrophages.


Figure 1
View larger version (53K):
[in this window]
[in a new window]

  		Figure 1. Caspase activation occurs prior to induction of the intrinsic apoptosis pathway in macrophages infected with E. coli. THP-1 macrophages were infected with E. coli at a ratio of 25:1, and cells were harvested at various times following infection. Immunoblot analyses of cytochrome C, Bax, and Bim were performed using mitochondrial or cytoplasmic extracts. {alpha}-Tubulin and subunit IV of cytochrome C oxidase (COX 4) levels were assessed to examine the purity of the extracts. BimS, Short Bim.

 
Caspase 9 activation is not required for macrophage apoptosis following infection with E. coli
Although infection of macrophages with E. coli results in the activation of caspases 9 and 3/7, caspase 9 is not essential for apoptosis mediated by the intrinsic pathway [50 51 52 53 ]. Therefore, we examined the role of caspase 9 in the activation of caspase 3/7 and apoptosis of macrophages following infection. Pretreatment of macrophages with zLEHD.fmk, an inhibitor to caspase 9, blocked the activity of caspase 9 (Fig. 2A ) but only had a minor effect on activation of caspase 3 (28% reduction; Fig. 2B ) and had no effect on the survival of infected macrophages (Fig. 2C) . In contrast, pretreatment with zLEHD.fmk suppressed the activity of caspases 9 and 3/7 and increased survival of macrophages irradiated with UV light (Supplemental Fig. 3), a known inducer of apoptosis, which proceeds through the intrinsic, apoptosis pathway [54 , 55 ]. These data indicate that activity of caspase 9 is dispensable for apoptosis induced by E. coli but is necessary for cell death of macrophages induced by UV. Further, these data suggest that E. coli induces a caspase 9-dependent and -independent activation of caspase 3.


Figure 2
View larger version (14K):
[in this window]
[in a new window]

  		Figure 2. Caspase 9 activity is dispensable for activation of caspase 3/7 and apoptosis in macrophages following infection with E. coli. THP-1 macrophages were infected with E. coli in the presence or absence of zLEHD.fmk. (A) Activities of caspase 9 or (B) caspase 3/7 were assessed at 1.5 h following infection. (C) Percent survival of macrophages was assessed by flow cytometry using Annexin V-APC and 7AAD at 6 h postinfection. Values represent the average ± SEM of three experiments performed in triplicate, which were compared by Student’s t test. *, P < 0.01, as compared with untreated macrophages; **, P < 0.04, as compared with parallel, E. coli-infected macrophages. ns, Not statistically significant.

 
Suppression of caspase 3/7 activity reduces apoptosis and enhances clearance of E. coli by macrophages
Although caspase 3/7 is activated early in E. coli-infected macrophages, we examined the effect of inhibiting caspase 3/7 on apoptosis of macrophages. Pretreatment with zDEVD.fmk increased the survival of macrophages infected with E. coli by 55% (Fig. 3A and 3B ). Further, pretreatment with zDEVD.fmk decreased the number of macrophages that were Annexin V+/7AAD– and Annexin V+/7AAD+ by 35% (P<0.001) and decreased DNA fragmentation by 47% as compared with macrophages infected with E. coli (Fig. 3A , 3C and 3D) . A similar decrease in DNA fragmentation was seen in macrophages derived from human peripheral blood monocytes (see Fig. 6B ). Macrophages pretreated with zVAD.fmk, a general caspase inhibitor, displayed a comparable level of survival as cells pretreated with the caspase 3/7 inhibitor (Fig. 3B 3C 3D) . Pretreatment with zDEVD.fmk enhanced the rate of clearance of viable bacteria over time (Fig. 3E) . These data are consistent with a previous study demonstrating that pretreatment with zVAD.fmk increased the rate of clearance of bacteria [11 ]. Collectively, our data indicate that caspase 3/7 is an essential factor for apoptosis of macrophages infected with E. coli and for enhancing the rate of E. coli clearance.


Figure 3
View larger version (43K):
[in this window]
[in a new window]

  		Figure 3. Activity of caspase 3/7 is required for the death of macrophages induced by infection with E. coli. THP-1 macrophages were untreated or infected with E. coli in the presence or absence of zDEVD.fmk or zVAD.fmk. (A) Representative dot plots of macrophages stained with Annexin V-APC and 7AAD at 6 h following infection. (B) Quantitative analysis of percent live THP-1 macrophages (Annexin V–/7AAD–) at 6 h postinfection. (C) Quantitative analysis of the percent of Annexin V+ THP-1 macrophages at 6 h postinfection. Values represent the average ± SEM of four experiments performed in triplicate, which were compared by Student’s t test. *, P < 0.05, as compared with uninfected control; **, P < 0.01, as compared with parallel, E. coli-infected macrophages. (D) Quantitative analysis of DNA fragmentation in macrophages at 6 h postinfection. DNA fragmentation was determined by cell death ELISA as described in Materials and Methods. Values are the average ± SEM of a representative experiment performed twice in quadruplicate. (E) Quantitative analysis of bacteria colony formation assays in macrophages infected with E. coli. Values were normalized to the number of bacteria recovered from macrophages infected with E. coli in the absence of caspase inhibitors. Values represent the average ± SEM of three experiments performed in triplicate, which were compared by Student’s t test. *, P < 0.05, as compared with 2.5 h postinfection; **, P < 0.01, as compared with parallel, E. coli-infected macrophages.

 

Figure 6
View larger version (36K):
[in this window]
[in a new window]

  		Figure 6. Cathepsin D is required for apoptosis and activation of caspase 3 in macrophages infected with E. coli. THP-1 macrophages (A, C, D) or macrophages derived from peripheral human blood monocytes (B, E) were untreated or infected with E. coli in the presence or absence of pepstatin A, ALLN, or zDEVD.fmk. (A) Quantitative analysis of percent Annexin V+ macrophages at 6 h postinfection. Values represent the average ± SEM of three experiments performed in triplicate, which were compared by Student’s t test. (B) Quantitative analysis of DNA fragmentation in primary macrophages at 6 h postinfection. Values are the average ± SEM of a representative experiment performed twice in quadruplicate. (C) Quantitative analysis of caspase 3/7 activity was assessed in macrophages at various times following infection. Values are the average ± SEM of three experiments performed in triplicate, which were compared by Student’s t test. (D) Immunoblot analysis of active caspase 3 was performed on whole cells extracts of primary macrophages at various times following infection. {alpha}-Tubulin levels were assessed as a loading control. Data are representative of three independent experiments. (E) Immunoblot analysis of cathepsin D was performed on whole cell extracts from macrophages at 1.5 h following infection. {alpha}-Tubulin levels were assessed as a loading control. Data are representative of two independent experiments. *P < 0.1 as compared to untreated control; **P < 0.04 as compared to parallel E. coli-infected macrophages.

 
Caspase 3/7 activates caspase 9 in macrophages infected with E. coli
Our data demonstrate that activity of caspase 3/7 but not caspase 9 is necessary for apoptosis of macrophages following infection. It is interesting that caspase 9 may be activated by caspase 3 [56 , 57 ]. Therefore, we examined the role of caspase 3 in the activation of caspase 9 in macrophages following infection. Pretreatment with zDEVD.fmk completely inhibited the activity of caspases 3/7 and 9 in E. coli-infected macrophages (Fig. 4A and 4B ). Further, zDEVD.fmk reduced the appearance of the active isoform of caspase 3 (p18) and caspase 9 (p35) in E. coli-infected THP-1 macrophages (Fig. 4C) and human monocyte-derived macrophages (data not shown). In contrast, although zDEVD.fmk completely inhibited the activity of caspase 3/7 in macrophages following UV irradiation (Supplemental Fig. 4A), the activity of caspase 9 was only reduced partially (Supplemental Fig. 4B). These data suggest that caspase 9 may be activated by caspase 3 through a feedback loop mechanism as described previously [58 ]. To confirm that zDEVD.fmk does not inhibit caspase 9 directly, addition of zDEVD.fmk to extracts from UV-treated cells revealed a minor reduction of activity of caspase 9 (<9%), and addition of zLEHD.fmk almost completely abolished the activity of caspase 9 activity (>92%; Supplemental Fig. 4C). Collectively, these data indicate that caspase 3 induces the activation of caspase 9 in macrophages following infection with E. coli.


Figure 4
View larger version (28K):
[in this window]
[in a new window]

  		Figure 4. Caspase 3/7 activity is required for activation of caspase 9 following E. coli infection in THP-1 macrophages, which were infected with E. coli at a ratio of 25:1 in the presence or absence of zDEVD.fmk. (A/B) Activity of caspase 3/7 was assessed at 1.5 h following infection with E. coli. Values represent the average ± SEM of five experiments performed in triplicate, which were compared by Student’s t test. *, P < 0.01, as compared with untreated control; **, P < 0.04, as compared with parallel, E. coli-infected macrophages. (C) Immunoblot analysis of active caspase 9 and active caspase 3 was performed on whole cell extracts. {alpha}-Tubulin levels were assessed as a loading control. Data are representative of three independent experiments.

 
The aspartic protease cathepsin D is associated with apoptosis and activation of caspase 3/7 in E. coli-infected macrophages
As our data indicate that caspase 9 is not required for the death of macrophages or for the activation of caspase 3/7 following infection with E. coli and as cathepsins have been shown to induce the activation of caspase 3 and are activated following bacterial infections [36 37 38 39 40 41 ], we examined the role that cathepsins have in the apoptosis of macrophages and in the activation of caspase 3/7 following infection with E. coli. There was a decrease in the inactive zymogen isoform of cathepsin D at 1.5 h postinfection (Fig. 5A ), which corresponded with a 41% (P<0.01) increase in activity of cathepsin D (Fig. 5B) . Pretreatment with pepstatin A, an inhibitor of aspartic protease, including cathepsin D [59 ], blocked the maturation of cathepsin D and markedly suppressed the activity of cathepsin D at 1.5 h postinfection (Fig. 5A and 5B) . Treatment with pepstatin A markedly reduced Annexin V staining by 48% (P<0.01) and DNA fragmentation by 54% (P<0.01) following E. coli infection (Fig. 6A and 6B ). In contrast, pretreatment with ALLN, an inhibitor of cathepsin B and L [60 , 61 ], had no effect on apoptosis in macrophages following infection (Fig. 6A) . Further, the combination of pepstatin A and DEVD.fmk had no additive effect on suppressing E. coli-induced macrophage apoptosis (data not shown). These data suggest that cathepsin D activity but not cathepsin B or L contributes to apoptosis following E. coli infection.


Figure 5
View larger version (30K):
[in this window]
[in a new window]

  		Figure 5. Cathepsin D is activated in macrophages following infection with E. coli. THP-1 macrophages were infected with E. coli in the presence or absence of pepstatin A. (A) Immunoblot analysis of cathepsin D was performed on whole cell extracts. {alpha}-Tubulin levels were assessed as a loading control. Data are representative of three independent experiments. (B) Activity of cathepsin D was assessed using an ELISA-based assay, as described in Materials and Methods. Values represent the average ± SEM of three experiments performed in duplicate, which were compared by Student’s t test.

 
To further investigate the effect of pepstatin A in apoptosis of macrophages following infection with E. coli, we examined the activation of caspase 3/7. Pretreatment with pepstatin A reduced caspase 3/7 activity by 47%, 44%, and 50% (P>0.01), respectively, at 1.5, 3.0, and 6.0 h following E. coli infection (Fig. 6C) . Furthermore, pepstatin A markedly reduced the appearance of the active caspase 3 isoform following infection with E. coli (Fig. 6D) . In contrast, zDEVD.fmk had no effect on the maturation of cathepsin D in macrophages infected with nonpathogenic E. coli (Fig. 6E) . These data indicate that caspase 3 activation is downstream of activation of aspartic proteases including cathepsin D. Taken together, these data indicate that E. coli infection induces the activation of cathepsin D, which leads to activation of caspase 3/7 and the downstream degradative events of apoptosis.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Apoptosis of macrophages following infection with nonpathogenic E. coli involves the Bcl-2 family and the activation of caspases 9 and 3/7 [9 , 11 ]. Here, we show that although infection of macrophages with the nonpathogenic E. coli induces the activation of the intrinsic apoptosis pathway, activation of caspase 3/7 does not require caspase 9. Instead, we demonstrate that activation of caspase 3 and subsequent cell death are likely mediated by cathepsin D. Although pepstatin A may inhibit additional aspartic proteases including cathepsin E, pepsin, or renin [62 ], the majority of the effects of pepstatin A has been attributed to its inhibition of cathepsin D, which is the major intracellular, aspartic protease [63 ]. However, our data do not exclude the possibility of additional aspartic proteases having an effect on the apoptosis of macrophages following infection with E. coli. Future studies will be needed to examine the contribution of other aspartic proteases in addition to cathepsin D in apoptosis of macrophages following infection with E. coli.

Our data are consistent with the fact that caspase 3/7 is activated prior to cytochrome C release and that the inhibition of caspase 3/7 activity blocks the activity of caspase 9. Caspase 3/7 is downstream of cathepsin D, as inhibition of caspase 3/7 has no effect on the activation of cathepsin D. Thus, caspase 9 may function as an enhancer for the activation of caspase 3/7 during infection with E. coli, as there is a minor decrease in the activity of caspase 3/7 following pretreatment with a caspase 9 inhibitor. However, caspase 9 is dispensable for the apoptosis of macrophages following infection with E. coli. This is consistent with previous studies that have shown that activation of caspase 9 is not always required for apoptosis induced through the intrinsic apoptosis pathway, as cells deficient in Apaf-1 or caspase 9 still undergo apoptosis and have activation of the downstream effector caspases [50 51 52 ]. Collectively, our data suggest a novel role for aspartic proteases, including cathepsin D, in the activation of caspase 3 and inducing apoptosis of macrophages following infection with a nonpathogenic species of E. coli.

The apoptotic response of macrophages plays a vital role in the pathogenesis of sepsis [12 , 64 , 65 ]. As inactivation of caspase 3 using caspase 3-deficient mice or caspase 3/7 inhibitors results in reduced development of sepsis [66 ], these data suggest that suppression of caspase 3 and apoptosis is vital for clearance of bacteria during sepsis. Although the role of cathepsin D in the development of sepsis has yet to be elucidated, patients with severe sepsis often exhibit high levels of acid sphingomyelinase (ASMase) [67 ], an upstream activator of cathepsin D [68 69 70 ]. Treatment with inhibitors to ASMase decreases the activation of cathepsin D, improves the survival of mice injected with LPS, and reduces apoptosis of dendritic cells infected with E. coli [67 , 70 ]. Our data demonstrate that aspartic protease inhibitors suppress the level of apoptosis in E. coli-infected macrophages to a similar extent as caspase inhibitors [11 ]. As we can also infer that cathepsin D-mediated activation of caspase 3 may regulate NF-{kappa}B through proteolytic cleavage [11 ], these data indicate that cathepsin D and caspases are linked in the death and inflammation pathways. Further, pepstatin A reduces bacterial clearance to similar levels as an inhibitor to caspase 3/7 (Supplemental Fig. 5). Although initial clearance of E. coli following infection is enhanced markedly in macrophages pretreated with DEVD.fmk, clearance of bacteria at later time-points is less dependent on cell viability. These data suggest that clearance is not solely dependent on cellular survival, but preventing the death of the infected macrophage results in an increased rate of clearance of bacteria. Taken together, these data suggest that the fate of macrophages during a bacterial infection may be a critical determinant for dissemination of the disease and for the proper inflammatory response. Thus, cathepsin D may be a viable target for therapeutic intervention in preventing the development and/or progression of sepsis.


   ACKNOWLEDGEMENTS
 
This work is supported by grants from the National Institutes of Health (AR02147, AR050250) and from the American Heart Association (0510132Z).


   FOOTNOTES
 
1 Current address: Weizmann Institute of Science, Department of Biological Chemistry, 76100 Rehovot, Israel. Back

Received May 26, 2006; revised August 17, 2006; accepted August 30, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Goldstein, E., Bartlema, H. C. (1977) Role of the alveolar macrophage in pulmonary bacterial defense Bull. Eur. Physiopathol. Respir. 13,57-67[Medline]
  2. Underhill, D. M. (2003) Toll-like receptors: networking for success Eur. J. Immunol. 33,1767-1775[CrossRef][Medline]
  3. Blander, J. M., Medzhitov, R. (2004) Regulation of phagosome maturation by signals from Toll-like receptors Science 304,1014-1018[Abstract/Free Full Text]
  4. Burnett, S. H., Kershen, E. J., Zhang, J., Zeng, L., Straley, S. C., Kaplan, A. M., Cohen, D. A. (2004) Conditional macrophage ablation in transgenic mice expressing a Fas-based suicide gene J. Leukoc. Biol. 75,612-623[Abstract/Free Full Text]
  5. Wiktor-Jedrzejczak, W., Dzwigala, B., Szperl, M., Maruszynski, M., Urbanowska, E., Szwech, P. (1996) Colony-stimulating factor 1-dependent resident macrophages play a regulatory role in fighting Escherichia coli fecal peritonitis Infect. Immun. 64,1577-1581[Abstract]
  6. Kurihara, T., Warr, G., Loy, J., Bravo, R. (1997) Defects in macrophage recruitment and host defense in mice lacking the CCR2 chemokine receptor J. Exp. Med. 186,1757-1762[Abstract/Free Full Text]
  7. Baran, J., Guzik, K., Hryniewicz, W., Ernst, M., Flad, H. D., Pryjma, J. (1996) Apoptosis of monocytes and prolonged survival of granulocytes as a result of phagocytosis of bacteria Infect. Immun. 64,4242-4248[Abstract]
  8. Kirschnek, S., Ying, S., Fischer, S. F., Hacker, H., Villunger, A., Hochrein, H., Hacker, G. (2005) Phagocytosis-induced apoptosis in macrophages is mediated by up-regulation and activation of the Bcl-2 homology domain 3-only protein Bim J. Immunol. 174,671-679[Abstract/Free Full Text]
  9. Hacker, H., Furmann, C., Wagner, H., Hacker, G. (2002) Caspase-9/-3 activation and apoptosis are induced in mouse macrophages upon ingestion and digestion of Escherichia coli bacteria J. Immunol. 169,3172-3179[Abstract/Free Full Text]
  10. Pryjma, J., Baran, J., Ernst, M., Woloszyn, M., Flad, H. D. (1994) Altered antigen-presenting capacity of human monocytes after phagocytosis of bacteria Infect. Immun. 62,1961-1967[Abstract/Free Full Text]
  11. Albee, L., Perlman, H. (2006) E. coli infection induces caspase-dependent degradation of NF-{kappa}B and reduces the inflammatory response in macrophages Inflamm. Res. 55,2-9[CrossRef][Medline]
  12. Iwata, A., Stevenson, V. M., Minard, A., Tasch, M., Tupper, J., Lagasse, E., Weissman, I., Harlan, J. M., Winn, R. K. (2003) Over-expression of Bcl-2 provides protection in septic mice by a trans effect J. Immunol. 171,3136-3141[Abstract/Free Full Text]
  13. Du, C., Fang, M., Li, Y., Li, L., Wang, X. (2000) Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition Cell 102,33-42[CrossRef][Medline]
  14. Adams, J. M., Cory, S. (1998) The Bcl-2 protein family: arbiters of cell survival Science 281,1322-1326[Abstract/Free Full Text]
  15. Perlman, H., Georganas, C., Pagliari, L. J., Koch, A. E., Haines, K., Pope, R. M. (2000) Bcl-2 expression in synovial fibroblasts is essential for maintaining mitochondrial homeostasis and cell viability J. Immunol. 164,5227-5235[Abstract/Free Full Text]
  16. Harris, M. H., Thompson, C. B. (2000) The role of the Bcl-2 family in the regulation of outer mitochondrial membrane permeability Cell Death Differ. 7,1182-1191[CrossRef][Medline]
  17. Von Ahsen, O., Waterhouse, N. J., Kuwana, T., Newmeyer, D. D., Green, D. R. (2000) The "harmless" release of cytochrome c Cell Death Differ. 7,1192-1199[CrossRef][Medline]
  18. Rathmell, J. C., Lindsten, T., Zong, W. X., Cinalli, R. M., Thompson, C. B. (2002) Deficiency in Bak and Bax perturbs thymic selection and lymphoid homeostasis Nat. Immunol. 3,932-939[CrossRef][Medline]
  19. Cheng, E. H., Wei, M. C., Weiler, S., Flavell, R. A., Mak, T. W., Lindsten, T., Korsmeyer, S. J. (2001) BCL-2, BCL-X(L) sequester BH3 domain-only molecules preventing BAX- and BAK-mediated mitochondrial apoptosis Mol. Cell 8,705-711[CrossRef][Medline]
  20. Zong, W. X., Lindsten, T., Ross, A. J., MacGregor, G. R., Thompson, C. B. (2001) BH3-only proteins that bind pro-survival Bcl-2 family members fail to induce apoptosis in the absence of Bax and Bak Genes Dev. 15,1481-1486[Abstract/Free Full Text]
  21. Lindsten, T., Ross, A. J., King, A., Zong, W. X., Rathmell, J. C., Shiels, H. A., Ulrich, E., Waymire, K. G., Mahar, P., Frauwirth, K., Chen, Y., Wei, M., Eng, V. M., Adelman, D. M., Simon, M. C., Ma, A., Golden, J. A., Evan, G., Korsmeyer, S. J., MacGregor, G. R., Thompson, C. B. (2000) The combined functions of proapoptotic Bcl-2 family members bak and bax are essential for normal development of multiple tissues Mol. Cell 6,1389-1399[CrossRef][Medline]
  22. Zamzami, N., Brenner, C., Marzo, I., Susin, S. A., Kroemer, G. (1998) Subcellular and submitochondrial mode of action of Bcl-2-like oncoproteins Oncogene 16,2265-2282[CrossRef][Medline]
  23. Yang, J., Liu, X., Bhalla, K., Kim, C. N., Ibrado, A. M., Cai, J., Peng, T-I., Jones, D. P., Wang, X. (1997) Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked Science 275,1129-1132[Abstract/Free Full Text]
  24. Kluck, R. M., Bossy-Wetzel, E., Green, D. R., Newmeyer, D. D. (1997) The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis Science 275,1132-1136[Abstract/Free Full Text]
  25. Kuida, K., Haydar, T. F., Kuan, C., Gu, Y., Taya, C., Karasuyama, H., Su, M. S., Rakic, P., Flavell, R. A. (1998) Reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking caspase 9 Cell 94,325-337[CrossRef][Medline]
  26. Rao, L., White, E. (1997) Bcl-2 and the ICE family of apoptotic regulators: making a connection Curr. Opin. Genet. Dev. 7,52-58[CrossRef][Medline]
  27. DeLeo, F. R. (2004) Modulation of phagocyte apoptosis by bacterial pathogens Apoptosis 9,399-413[CrossRef][Medline]
  28. Orth, K., Palmer, L. E., Bao, Z. Q., Stewart, S., Rudolph, A. E., Bliska, J. B., Dixon, J. E. (1999) Inhibition of the mitogen-activated protein kinase kinase superfamily by a Yersinia effector Science 285,1920-1923[Abstract/Free Full Text]
  29. Ruckdeschel, K., Harb, S., Roggenkamp, A., Hornef, M., Zumbihl, R., Kohler, S., Heesemann, J., Rouot, B. (1998) Yersinia enterocolitica impairs activation of transcription factor NF-{kappa}B: involvement in the induction of programmed cell death and in the suppression of the macrophage tumor necrosis factor {alpha} production J. Exp. Med. 187,1069-1079[Abstract/Free Full Text]
  30. Ruckdeschel, K., Mannel, O., Richter, K., Jacobi, C. A., Trulzsch, K., Rouot, B., Heesemann, J. (2001) Yersinia outer protein P of Yersinia enterocolitica simultaneously blocks the nuclear factor-{kappa} B pathway and exploits lipopolysaccharide signaling to trigger apoptosis in macrophages J. Immunol. 166,1823-1831[Abstract/Free Full Text]
  31. Ruckdeschel, K., Mannel, O., Schrottner, P. (2002) Divergence of apoptosis-inducing and preventing signals in bacteria-faced macrophages through myeloid differentiation factor 88 and IL-1 receptor-associated kinase members J. Immunol. 168,4601-4611[Abstract/Free Full Text]
  32. Schesser, K., Spiik, A. K., Dukuzumuremyi, J. M., Neurath, M. F., Pettersson, S., Wolf-Watz, H. (1998) The yopJ locus is required for Yersinia-mediated inhibition of NF-{kappa}B activation and cytokine expression: YopJ contains a eukaryotic SH2-like domain that is essential for its repressive activity Mol. Microbiol. 28,1067-1079[CrossRef][Medline]
  33. Mills, S. D., Boland, A., Sory, M. P., van der Smissen, P., Kerbourch, C., Finlay, B. B., Cornelis, G. R. (1997) Yersinia enterocolitica induces apoptosis in macrophages by a process requiring functional type III secretion and translocation mechanisms and involving YopP, presumably acting as an effector protein Proc. Natl. Acad. Sci. USA 94,12638-12643[Abstract/Free Full Text]
  34. Monack, D. M., Mecsas, J., Ghori, N., Falkow, S. (1997) Yersinia signals macrophages to undergo apoptosis and YopJ is necessary for this cell death Proc. Natl. Acad. Sci. USA 94,10385-10390[Abstract/Free Full Text]
  35. Denecker, G., Declercq, W., Geuijen, C. A., Boland, A., Benabdillah, R., van Gurp, M., Sory, M. P., Vandenabeele, P., Cornelis, G. R. (2001) Yersinia enterocolitica YopP-induced apoptosis of macrophages involves the apoptotic signaling cascade upstream of bid J. Biol. Chem. 276,19706-19714[Abstract/Free Full Text]
  36. Roberg, K., Johansson, U., Ollinger, K. (1999) Lysosomal release of cathepsin D precedes relocation of cytochrome c and loss of mitochondrial transmembrane potential during apoptosis induced by oxidative stress Free Radic. Biol. Med. 27,1228-1237[CrossRef][Medline]
  37. Broker, L. E., Huisman, C., Span, S. W., Rodriguez, J. A., Kruyt, F. A., Giaccone, G. (2004) Cathepsin B mediates caspase-independent cell death induced by microtubule stabilizing agents in non-small cell lung cancer cells Cancer Res. 64,27-30[Abstract/Free Full Text]
  38. Thibodeau, M., Giardina, C., Hubbard, A. K. (2003) Silica-induced caspase activation in mouse alveolar macrophages is dependent upon mitochondrial integrity and aspartic proteolysis Toxicol. Sci. 76,91-101[Abstract/Free Full Text]
  39. Thibodeau, M. S., Giardina, C., Knecht, D. A., Helble, J., Hubbard, A. K. (2004) Silica-induced apoptosis in mouse alveolar macrophages is initiated by lysosomal enzyme activity Toxicol. Sci. 80,34-48[Abstract/Free Full Text]
  40. Ishisaka, R., Utsumi, T., Kanno, T., Arita, K., Katunuma, N., Akiyama, J., Utsumi, K. (1999) Participation of a cathepsin L-type protease in the activation of caspase-3 Cell Struct. Funct. 24,465-470[CrossRef][Medline]
  41. Roberg, K. (2001) Relocalization of cathepsin D and cytochrome c early in apoptosis revealed by immunoelectron microscopy Lab. Invest. 81,149-158[Medline]
  42. Cooper, P. H., Mayer, P., Baggiolini, M. (1984) Stimulation of phagocytosis in bone marrow-derived mouse macrophages by bacterial lipopolysaccharide: correlation with biochemical and functional parameters J. Immunol. 133,913-922[Abstract]
  43. Machaiah, J. P. (1989) Activation of lysosomal enzymes in chemotactically elicited rat peritoneal macrophages Indian J. Biochem. Biophys. 26,343-347[Medline]
  44. Wasiluk, K. R., Skubitz, K. M., Gray, B. H. (1991) Comparison of granule proteins from human polymorphonuclear leukocytes, which are bactericidal toward Pseudomonas aeruginosa Infect. Immun. 59,4193-4200[Abstract/Free Full Text]
  45. Thorne, K. J., Oliver, R. C., Barrett, A. J. (1976) Lysis and killing of bacteria by lysosomal proteinases Infect. Immun. 14,555-563[Abstract/Free Full Text]
  46. Schwende, H., Fitzke, E., Ambs, P., Dieter, P. (1996) Differences in the state of differentiation of THP-1 cells induced by phorbol ester and 1,25-dihydroxyvitamin D3 J. Leukoc. Biol. 59,555-561[Abstract]
  47. Traore, K., Trush, M. A., George, M., Jr, Spannhake, E. W., Anderson, W., Asseffa, A. (2005) Signal transduction of phorbol 12-myristate 13-acetate (PMA)-induced growth inhibition of human monocytic leukemia THP-1 cells is reactive oxygen dependent Leuk. Res. 29,863-879[CrossRef][Medline]
  48. Perlman, H., Pagliari, L. J., Georganas, C., Mano, T., Walsh, K., Pope, R. M. (1999) FLICE-inhibitory protein expression during macrophage differentiation confers resistance to fas-mediated apoptosis J. Exp. Med. 190,1679-1688[Abstract/Free Full Text]
  49. Smith, R. C., Branellec, D., Gorski, D. H., Guo, K., Perlman, H., Dedieu, J-F., Pastore, C., Mahfoudi, A., Denèfle, P., Isner, J. M., Walsh, K. (1997) p21CIP1-mediated inhibition of cell proliferation by overexpression of the gax homeodomain gene Genes Dev. 11,1674-1689[Abstract/Free Full Text]
  50. Honarpour, N., Tabuchi, K., Stark, J. M., Hammer, R. E., Sudhof, T. C., Parada, L. F., Wang, X., Richardson, J. A., Herz, J. (2001) Embryonic neuronal death due to neurotrophin and neurotransmitter deprivation occurs independent of Apaf-1 Neuroscience 106,263-274[CrossRef][Medline]
  51. Marsden, V. S., O’Connor, L., O’Reilly, L. A., Silke, J., Metcalf, D., Ekert, P. G., Huang, D. C., Cecconi, F., Kuida, K., Tomaselli, K. J., Roy, S., Nicholson, D. W., Vaux, D. L., Bouillet, P., Adams, J. M., Strasser, A. (2002) Apoptosis initiated by Bcl-2-regulated caspase activation independently of the cytochrome c/Apaf-1/caspase-9 apoptosome Nature 419,634-637[CrossRef][Medline]
  52. Marsden, V. S., Ekert, P. G., Van Delft, M., Vaux, D. L., Adams, J. M., Strasser, A. (2004) Bcl-2-regulated apoptosis and cytochrome c release can occur independently of both caspase-2 and caspase-9 J. Cell Biol. 165,775-780[Abstract/Free Full Text]
  53. Goping, I. S., Barry, M., Liston, P., Sawchuk, T., Constantinescu, G., Michalak, K. M., Shostak, I., Roberts, D. L., Hunter, A. M., Korneluk, R., Bleackley, R. C. (2003) Granzyme B-induced apoptosis requires both direct caspase activation and relief of caspase inhibition Immunity 18,355-365[CrossRef][Medline]
  54. Bossy-Wetzel, E., Newmeyer, D. D., Green, D. R. (1998) Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrial transmembrane depolarization EMBO J. 17,37-49[CrossRef][Medline]
  55. Chipuk, J. E., Bouchier-Hayes, L., Kuwana, T., Newmeyer, D. D., Green, D. R. (2005) PUMA couples the nuclear and cytoplasmic proapoptotic function of p53 Science 309,1732-1735[Abstract/Free Full Text]
  56. Srinivasula, S. M., Fernandes-Alnemri, T., Zangrilli, J., Robertson, N., Armstrong, R. C., Wang, L., Trapani, J. A., Tomaselli, K. J., Litwack, G., Alnemri, E. S. (1996) The Ced-3/interleukin 1ß converting enzyme-like homolog Mch6 and the lamin-cleaving enzyme Mch2{alpha} are substrates for the apoptotic mediator CPP32 J. Biol. Chem. 271,27099-27106[Abstract/Free Full Text]
  57. Zou, H., Yang, R., Hao, J., Wang, J., Sun, C., Fesik, S. W., Wu, J. C., Tomaselli, K. J., Armstrong, R. C. (2003) Regulation of the Apaf-1/caspase-9 apoptosome by caspase-3 and XIAP J. Biol. Chem. 278,8091-8098[Abstract/Free Full Text]
  58. Slee, E. A., Harte, M. T., Kluck, R. M., Wolf, B. B., Casiano, C. A., Newmeyer, D. D., Wang, H., Reed, J. C., Nicholson, D. W., Alnemri, E. S., Green, D. R., Martin, S. J. (1999) Ordering the cytochrome c-initiated caspase cascade:hierarchical activation of caspases-2, -3, -6, -7, -8, -10 in a caspase-9-dependent manner J. Cell Biol. 144,281-292[Abstract/Free Full Text]
  59. McAdoo, M. H., Dannenberg, A. M., Jr, Hayes, C. J., James, S. P., Sanner, J. H. (1973) Inhibition of cathepsin D-type proteinase of macrophages by pepstatin, a specific pepsin inhibitor, and other substances Infect. Immun. 7,655-665[Abstract/Free Full Text]
  60. Hiwasa, T., Sawada, T., Sakiyama, S. (1990) Cysteine proteinase inhibitors and ras gene products share the same biological activities including transforming activity toward NIH3T3 mouse fibroblasts and the differentiation-inducing activity toward PC12 rat pheochromocytoma cells Carcinogenesis 11,75-80[Abstract/Free Full Text]
  61. Millest, A. J., Breen, S. A., Loveday, B. E., Clarkson, P. N., Simpson, C. A., Waterton, J. C., Johnstone, D. (1997) Effects of an inhibitor of cathepsin L on bone resorption in thyroparathyroidectomized and ovariectomized rats Bone 20,465-471[Medline]
  62. Marciniszyn, J., Jr, Hartsuck, J. A., Tang, J. (1977) Pepstatin inhibition mechanism Adv. Exp. Med. Biol. 95,199-210[Medline]
  63. Shields, P. P., Gonzales, T. A., Charles, D., Gilligan, J. P., Stern, W. (1991) Accumulation of pepstatin in cultured endothelial cells and its effect on endothelial processing Biochem. Biophys. Res. Commun. 177,1006-1012[CrossRef][Medline]
  64. Simovart, H. E., Poldoja, E., Kokk, K., Tapfer, H., Liigant, A., Talvik, R., Roosaar, P. (2003) Changes of activated macrophages and apoptotic cell count in the organs of rats during experimental sepsis Medicina (Kaunas) 39,932-939
  65. Tapfer, H., Liigant, A., Simovart, H. E., Poldoja, E., Kokk, K., Naaber, P., Talvik, R. (2003) Dissemination of bacteria in multiple organs associated with apoptosis and macrophage activity in different stages of experimental sepsis Scand. J. Surg. 92,163-170[Medline]
  66. Hotchkiss, R. S., Chang, K. C., Swanson, P. E., Tinsley, K. W., Hui, J. J., Klender, P., Xanthoudakis, S., Roy, S., Black, C., Grimm, E., Aspiotis, R., Han, Y., Nicholson, D. W., Karl, I. E. (2000) Caspase inhibitors improve survival in sepsis: a critical role of the lymphocyte Nat. Immunol. 1,496-501[CrossRef][Medline]
  67. Claus, R. A., Bunck, A. C., Bockmeyer, C. L., Brunkhorst, F. M., Losche, W., Kinscherf, R., Deigner, H. P. (2005) Role of increased sphingomyelinase activity in apoptosis and organ failure of patients with severe sepsis FASEB J. 19,1719-1721[Abstract/Free Full Text]
  68. Heinrich, M., Wickel, M., Winoto-Morbach, S., Schneider-Brachert, W., Weber, T., Brunner, J., Saftig, P., Peters, C., Kronke, M., Schutze, S. (2000) Ceramide as an activator lipid of cathepsin D Adv. Exp. Med. Biol. 477,305-315[Medline]
  69. Heinrich, M., Wickel, M., Schneider-Brachert, W., Sandberg, C., Gahr, J., Schwandner, R., Weber, T., Saftig, P., Peters, C., Brunner, J., Kronke, M., Schutze, S. (1999) Cathepsin D targeted by acid sphingomyelinase-derived ceramide EMBO J. 18,5252-5263[CrossRef][Medline]
  70. Falcone, S., Perrotta, C., De Palma, C., Pisconti, A., Sciorati, C., Capobianco, A., Rovere-Querini, P., Manfredi, A. A., Clementi, E. (2004) Activation of acid sphingomyelinase and its inhibition by the nitric oxide/cyclic guanosine 3',5'-monophosphate pathway: key events in Escherichia coli-elicited apoptosis of dendritic cells J. Immunol. 173,4452-4463[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Infect. Immun.Home page
A. R. Cruz, M. W. Moore, C. J. La Vake, C. H. Eggers, J. C. Salazar, and J. D. Radolf
Phagocytosis of Borrelia burgdorferi, the Lyme Disease Spirochete, Potentiates Innate Immune Activation and Induces Apoptosis in Human Monocytes
Infect. Immun., January 1, 2008; 76(1): 56 - 70.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
A. Weigert, N. Tzieply, A. von Knethen, A. M. Johann, H. Schmidt, G. Geisslinger, and B. Brune
Tumor Cell Apoptosis Polarizes Macrophages Role of Sphingosine-1-Phosphate
Mol. Biol. Cell, October 1, 2007; 18(10): 3810 - 3819.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental figures
Right arrow All Versions of this Article:
jlb.0506358v1
81/1/229    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Albee, L.
Right arrow Articles by Perlman, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Albee, L.
Right arrow Articles by Perlman, H.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS