Published online before print June 3, 2003
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* Surgical Research, Department of Surgery, Brown University School of Medicine and Rhode Island Hospital, Providence;
Department of Surgery, North Shore University Hospital, Manhasset, New York; and
Department of Surgery, University of Alabama at Birmingham School of Medicine
1Correspondence: Surgical Research, Aldrich 2, Rhode Island Hospital, 593 Eddy St., Providence, RI 02903. E-mail: aayala{at}lifespan.org
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Key Words: cecal ligation and puncture • mice • Kupffer cells • peritoneal macrophage • splenic macrophage
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Studies have suggested that cytokines such as tumor necrosis factor (TNF), interleukin (IL)-1, or IL-6 may be involved in the initiation of organ dysfunction associated with sepsis and multiple organ failure [4 ]. Activated macrophages are thought to be one of the major producers of cytokines and growth factors, which not only regulate innate- and acquired-immune responses but also play a key role in the clearance and killing of invading pathogens during septic insult. We have previously demonstrated that macrophages isolated from septic mice show a marked loss of important immune functional capacity (i.e., decreased inducible TNF, IL-1, and IL-6 production), which is associated with a significant increase in macrophage apoptosis when subsequently challenged with lipopolysaccharide (LPS) [5 6 7 ]. However, the mechanism responsible for these changes as well as the interactions between cytokine production and apoptotic processes in macrophages are poorly understood. Apoptotic cell death plays a critical role in normal physiologic processes and pathologic conditions [8 ]. Studies have demonstrated that sepsis induces an increase in apoptosis in lymphocytes, monocytes, and epithelial cells in patients and experimental animals [9 , 10 ]. It has been proposed that loss of immune (T and B cells and monocytes) and nonimmune (epithelial, cardiac myocytes, hepatocytes, and vascular endothelial) cells through the apoptotic process may contribute to the immunosuppression seen in sepsis, which in turn, may lead to the subsequent multiple organ dysfunction [2 , 11 , 12 ]. Furthermore, studies in animal models of sepsis show that prevention of lymphocyte apoptosis by administration of caspase inhibitors or through overexpression of Bcl-2, an antiapoptotic protein, improves survival during sepsis [13 ]. However, the mechanisms by which apoptosis is regulated during sepsis as well as its contribution to organ dysfunction remain to be established.
The Fas/Fas ligand (FasL) system has been identified as a major pathway involved in the induction of apoptotic cell death in many cell types, including lymphocytes and other leukocytes [14 15 16 ]. Fas is widely expressed in many cell types, and FasL expression is restricted and usually requires activation [17 , 18 ]. The expression of Fas and FasL has been reported in monocytes and macrophages in low but detectable levels [19 ]. In a previous study, we demonstrated that FasL deficiency improves the ability of mice to survive septic challenge [20 ]. However, the role of the Fas/FasL system in the regulation of macrophage apoptosis during sepsis has not been established.
In the present study, we attempted to determine whether inhibition of the activation of Fas by FasL through the administration of Fas fusion protein (FasFP) in septic mice could improve survival as well as alter the apoptotic response and functional capacity of various macrophage populations in response to LPS stimulation.
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Model of polymicrobial sepsis
Polymicrobial sepsis was induced in mice using the method described in our previous study [5
]. In brief, mice were lightly anesthetized with Metofane (Methoxyflurane, Pitman-Moore, Mundelein, IL), and the abdomen was shaved and scrubbed with betadine. A midline incision (1.5–2.0 cm) was made below the diaphragm to expose the cecum, which was isolated, ligated, and punctured twice with a 22-gauge needle and gently compressed to extrude a small amount of cecal contents through the punctured holes. The cecum was returned to the abdomen, and the incision was closed in layers with a 6-0 Ethilon suture (Ethicon, Somerville, NJ). The animals were then resuscitated with 1.0 mL lactated Ringer’s solution by subcutaneous (s.c.) injection. For sham controls, the animals were subjected to the same surgical procedure, i.e., laparotomy and cecal isolation, but the cecum was neither ligated nor punctured.
Reagent
FasFP (kindly provided by Dr. Carl Edwards, Amgen, Thousand Oaks, CA) is a chimeric fusion protein consisting of the extracellular domain of murine Fas covalently linked to the Fc region and hinge protein of a human immunoglobulin G (IgG) [21
]. For the survival study, mice were subjected to cecal ligation and puncture (CLP), received a single dose of 200 µg/kg FasFP or saline at 0 h or 12 h after surgery, and were monitored for mortality over a 10-day period. In all subsequent in vivo studies, animals subjected to CLP or sham procedures were randomized into four groups (sham-saline vehicle, sham-FasFP, CLP-saline vehicle, and CLP-FasFP), receiving FasFP (200 µg/kg body weight) or vehicle s.c. 12 h postsurgery. It should be noted that in prior studies, we have not seen vasoactive effects of this agent on mean arterial pressure [22
].
Cell preparations
Animals were killed 24 h after CLP or sham CLP operations by Metofane overdose, and splenic macrophages were obtained as described previously [23
]. The spleens were removed and gently glass ground, and erythrocytes were lysed using hypotonic buffers; isolated splenocytes were then washed, counted, plated at 1 x 107 cells per mL in plastic tissue-culture plates, and incubated at 37°C for 2 h. The adherent splenic macrophages were then washed with fresh Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL, Grand Island, NY).
Peritoneal macrophage monolayers were established by harvesting peritoneal exudate cells [24 ] via lavage of the peritoneal cavity with 2 x 5 mL cold DMEM. The cells were centrifuged (800 g at 4°C for 15 min), resuspended in fresh DMEM at 2 x 106 cells per mL, plated onto plastic tissue-culture plates, and incubated at 37°C for 2 h. The nonadherent cells were removed by washing three times with fresh DMEM. These protocols provided adherent cells that were greater than 95% positive by nonspecific esterase staining and exhibited typical macrophage morphology.
Kupffer cells were harvested as described previously [25 ]. In brief, the liver was retrograde perfused with 25 mL ice-cold Hanks’ balanced salt solution (HBSS; Gibco-BRL) through the portal vein and immediately followed by perfusion with 10 mL prewarmed 0.05% collagenase IV (Sigma Chemical Co., St. Louis, MO) in HBSS. The liver was then removed, minced finely in a Petri dish containing 5 mL warm collagenase, and incubated at 37°C for 20 min. The cell suspension was passed through a steel screen, centrifuged, resuspended in DMEM, layered onto 16% Metrizamide (Accurate Chemical, Westbury, NJ) in HBSS, and centrifuged at 3000 g, 4°C for 45 min. The cells from the interface were collected, washed, resuspended in DMEM medium containing 10% fetal bovine serum (FBS) at 4 x 106 cells per mL, plated onto plastic tissue-culture plates, and incubated at 37°C overnight. The nonadherent cells were removed by washing three times with fresh DMEM.
Adherent macrophage monolayers were stimulated with 10 µg LPS per mL DMEM medium supplemented with 10% FBS for 24 h (37°C, 5% CO2; 95% humidity). At the end of the incubation period, cells and the cultured supernatants were collected for analysis.
Analysis of apoptosis and Fas expression
Apoptosis of macrophages was determined by DNA fragmentation, morphological changes, and caspase-3 activity. The percent of apoptotic-positive cells was detected by the DNA-binding agent propidium iodide (PI; Sigma Chemical Co.) according to the methods of Telford et al. [26
]. Macrophages were collected after culture, resuspended in 0.5 mL PI-staining reagent (0.1% Nonidet P-40, 0.1% sodium citrate, and 50 µg PI per mL), and immediately analyzed using a fluorescein-activated cell sorter flow cytometer (Becton Dickinson, San Jose, CA).
To assess apoptosis by morphology, macrophages were cultured in Laboratory-Tek II eight-chamber slides (Nalge NUNC, Naperville, IL), fixed with ethanol, stained with Giemsa stain (Gibco-BRL), and counted. Apoptotic cells were characterized by the presence of nuclear condensation and loss of cytoplasm. Two hundred cells were counted per slide, three slides per group.
Fas expression in Kupffer cells was analyzed by immunofluorescent staining. Cells were cultured in Laboratory-Tek II slides (Nalge NUNC), fixed with 2% paraformaldehyde (Sigma Chemical Co.), and blocked in phosphate-buffered saline (PBS) containing 2% normal goat serum (Pierce, Rockford, IL) for 30 min at room temperature. Phycoerythrin (PE)-labeled anti-Fas or hamster IgG in PBS with 2% goat serum was added to cells and incubated overnight at 4°C. Cells were washed and mounted in Vectashield (Vector Laboratories, Burlingame, CA) and viewed by fluorescent microscopy.
Caspase-3 activity assays
Caspase activity was determined by fluorogenic assay according to Fukuzuka et al. [27
] with minor modifications. LPS-stimulated macrophages were collected and lysed with lysis buffer {50 mM HEPES, 0.1% 3-[(3-cholamidopropyl) dimethylammonio]-1-propansulfonate (CHAPS), 1 mM dithiothreitol (DTT), 0.1 mM EDTA, 0.4% Triton X-100, pH 7.4} at 4°C for 15 min. The cell lysate was collected after centrifugation at 13,000 g for 10 min and incubated with the fluorescent caspase-3 substrate, Ac-DEVD-AFC (N-acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin; Enzyme Systems Products, Livermore, CA), at 100 µM in the assay buffer (50 mM HEPES, 1% sucrose, 0.1% CHAPS, 10 mM DTT, pH 7.4). The fluorescence of the cleaved substrates was followed spectrofluorometrically (excitation of 400 nm and emission of 505 nm) with a Bio-Tek FL500 microplate fluorescence reader (Winooski, VT). The protein content of the cell lysate was determined with a Bio-Rad (Hercules, CA) protein assay reagent, and the data are presented as picomole-cleaved AFC per mg protein per min, which is calculated from a standard curve plot with free AFC.
Determination of macrophage cytokine production by enzyme-linked immunosorbent assay (ELISA)
IL-1ß, IL-6, IL-10, and IL-12 produced from macrophages upon LPS stimulation were measured in culture supernatants by the "sandwich ELISA" technique as described previously [28
] with monoclonal antibody pairs and the appropriate cytokine standards obtained from R&D Systems (Minneapolis, MN; IL-1ß) and BD-Pharmingen (San Diego, CA; IL-6, IL-10, and IL-12), respectively.
Plasma levels of liver enzymes and soluble FasL
Blood was collected in a syringe containing 2 units heparin, transferred to a microtube, and centrifuged immediately at 10,000 g for 10 min at 4°C. Plasma samples were stored at -70°C until analyzed. Plasma levels of glutamate-pyruvate-transaminase (GPT) and lactate dehydrogenase (LDH) were determined by using a Sigma assay kit (Sigma Chemical Co.), according to the manufacturer’s instruction. Soluble FasL concentration in plasma was determined with a commercially available coated ELISA kit (R&D Systems).
Presentation of data and statistical analysis
The data are presented as mean ± SE of n = 5–7 animals for each group. Differences were considered to be significant if P < 0.05, as determined using the one-way ANOVA and Tukey’s test. The survival data were compared using the Fisher exact test and were considered significant at P< 0.05.
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Figure 1. Percent survival of mice following sepsis induced by CLP. Administration of FasFP to animals immediately (0 h) after CLP had no effect on survival compared with vehicle (veh)-treated mice. However, FasFP treatment at the beginning of the hypodynamic stage (12 h) of sepsis significantly increased survival compared with vehicle and 0 h-treated mice. *, P < 0.05, versus vehicle-treated mice; Fisher Exact test; n = 19–20 mice/group.
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Figure 2. Apoptosis in LPS-stimulated peritoneal macrophages from four groups of mice. Cells were harvested 24 h after surgery and stimulated with 10 µg LPS per mL for another 24 h. PI cell-cycle analysis was used to determine the frequency of apoptosis. Peritoneal macrophages from vehicle-treated CLP mice show an increase in apoptosis in response to stimulation. Cells from FasFP-treated (12 h) CLP mice did not show changes in their apoptotic frequency. *, P < 0.05, versus sham. One-way ANOVA and Tukey’s test. Mean ± SE; n = 5–7 mice/group.
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Figure 3. Giemsa staining of representative LPS-stimulated peritoneal macrophages (x400 original magnification). Cells from Sham (A) and CLP (B) were harvested 24 h after surgery and stimulated with 10 µg LPS per mL for another 24 h. Cells at arrows show feature characteristics of apoptosis, i.e., nuclear condensation, pyknosis, and shrinking cytoplasm.
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Figure 4. Apoptosis in LPS-stimulated Kupffer cells from four groups of mice. Cells were harvested 24 h after surgery and stimulated with 10 µg LPS per mL for another 24 h. PI cell-cycle analysis was used to determine the frequency of apoptosis. Kupffer cells from vehicle-treated CLP mice show an increase in apoptosis in response to stimulation. FasFP-treated (12 h) CLP mice show a decrease in their apoptotic frequency. *, P < 0.05, versus sham; #, P < 0.05, versus CLP. One-way ANOVA and Tukey’s test. Mean ± SE; n = 5–7 mice/group.
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Figure 5. Giemsa staining of representative LPS-stimulated Kupffer cells (x400 original magnification). Cells from Sham (A), CLP (B), and CLP-FasFP (12 h; C) were harvested 24 h after surgery and stimulated with 10 µg LPS per mL for another 24 h. Cells at arrows show feature characteristics of apoptosis, i.e., nuclear condensation, pyknosis, and shrinking cytoplasm.
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Effects of FasFP treatment on caspase-3 activity in LPS-stimulated Kupffer cells after sepsis
In a previous study [7
], we demonstrated that caspases-3 activity is significantly increased in LPS-stimulated peritoneal macrophages of septic mice that were associated with increased apoptosis in these cells. Here, we determined whether the induction of caspase-3 activity in Kupffer cells from CLP mice was affected by FasFP treatment. As shown in Figure 6
, caspase-3 activity in LPS-stimulated Kupffer cells from septic mice is up-regulated significantly when compared with cells from sham mice. Consistent with the morphological data/apoptosis rates, FasFP treatment decreased caspase-3 activity in these cells.
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Figure 6. Caspase-3 activity in LPS-stimulated Kupffer cells from four groups of mice. Enzyme activity was measured by the cleavage of the caspase-3 substrate Ac-DEVD-AFC. Increased caspase-3 activity was observed in Kupffer cells after sepsis. FasFP treatment (12 h) significantly decreased caspase-3 activity in Kupffer cells. *, P < 0.05, versus sham; #, P < 0.05, versus CLP. One-way ANOVA and Tukey’s test. Mean ± SE; n = 5–7 mice/group.
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Figure 7. Cytokine release in LPS-stimulated peritoneal macrophages (A), splenic macrophages (B), and Kupffer cells (C). Cells were harvested 24 h after surgery and stimulated with 10 µg LPS per mL for another 24 h. Cytokine release in cultured supernatants was determined by ELISA. Although a marked decrease in IL-1ß (only detected in peritoneal macrophages), IL-6, and IL-12 levels in vehicle-treated CLP mice compared with sham and sham-vehicle groups, IL-10 release is increased in all three macrophages from septic mice. FasFP treatment (12 h) only attenuated IL-10 but not the proinflammatory cytokine release in Kupffer cells. *, P < 0.05, versus sham; #, P < 0.05, versus CLP. One-way ANOVA and Tukey’s test. Mean ± SE; n = 5–7 mice/group. ND, Not detectable.
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Kupffer cell IL-6 (IL-1ß and IL-12 were not detectable) release upon LPS stimulation was significantly depressed in septic mice (Fig. 7C) . Like peritoneal and splenic macrophages, treatment of CLP animals with FasFP did not restore IL-6 release. However, although Kupffer cell IL-10 release increased in vehicle-treated animals after sepsis, the administration of FasFP significantly attenuated the rise of IL-10 release.
Effects of FasFP treatment on plasma levels of liver enzymes, GPT and LDH, and soluble FasL after sepsis
To investigate whether the changes in Kupffer cell apoptosis or function might be associated with the onset of liver damage, we determined the plasma levels of GPT and LDH in these mice. GPT (Fig. 8A
) and LDH (Fig. 8B)
were increased significantly at 24 h after the onset of sepsis; however, these enzyme levels were significantly attenuated in FasFP-treated animals.
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Figure 8. Plasma levels of GPT (A) and LDH (B) at 24 h after CLP. Both enzymes were significantly increased in vehicle-treated CLP mice; treatment of FasFP (12 h) significantly attenuated the production of these enzymes. *, P < 0.05, versus sham; #, P< 0.05, versus CLP. One-way ANOVA and Tukey’s test. Mean ± SE; n = 5–7 mice/group.
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The role of the Fas/FasL system in the regulation of lymphocyte apoptosis is well described [29 30 31 ]; however, less is known about its effect on specific macrophage populations. In vivo studies have demonstrated that various agents, such as LPS, TNF, IL-1, IL-10, IL-4, nitric oxide, and FasL [19 , 32 , 33 ], can regulate macrophage apoptosis. We have previously observed that macrophages isolated from septic mice that are not subjected to further stimulation or culture exhibited variable degrees of apoptosis, with an increase in peritoneal macrophage basal apoptosis, a small but significant decline in Kupffer cell apoptosis, and no change in splenic macrophage apoptosis [6 ]. In contrast, when cells from septic mice are stimulated in vitro with LPS, peritoneal macrophages and Kupffer cells exhibit a consistent increase beyond their own base level of apoptosis compared with sham animal cells [6 ]. It is interesting that the apoptotic frequency in splenic macrophages from septic mice remains unchanged in the presence or absence of LPS [5 , 6 ]. Here, we have continued to expand our understanding of this divergence and the changes in susceptibility to the onset of apoptosis in macrophages by inhibiting the Fas/FasL system during sepsis. The results indicate that blockade of Fas/FasL signaling at 12 h after sepsis has a differential effect on the apoptotic frequency in macrophages harvested from various tissue sites. FasFP treatment attenuated the elevation in Kupffer cell apoptosis, which was seen in vehicle-treated CLP mice. However, the increase in peritoneal macrophage apoptosis was not affected by FasFP treatment, and similarly, it had no effect on splenic macrophage apoptosis.
Macrophages express Fas and FasL constitutively; however, studies demonstrate that treatment with LPS did not alter the expression of either protein [19 , 34 ]. Consistent with these findings, our results show that there was no difference in Fas expression on Kupffer cells taken from sham or CLP mice (data not shown). It has been proposed that monocyte FasL is not used in the autocrine induction of apoptosis but may be involved in effector (paracrine/endocrine) functions when released from the cell surface [29 ]. It is possible that in peritoneal and liver macrophages, the Fas/FasL system is not the central apoptotic pathway activated during sepsis but may be serving as a regulatory mechanism. This may in part explain why blocking Fas activation did not decrease peritoneal macrophage apoptosis. It appears that other factors besides Fas and FasL are induced during LPS stimulation, which may contribute to the sensitivity of Kupffer cells to apoptosis. Additionally, it has been suggested that soluble FasL in circulation may also play some role in a paracrine regulation of apoptosis [35 ]. However, soluble FasL concentration in plasma was nearly undetectable in our animals regardless of treatment. Thus, the mechanism by which FasFP mediates its divergent effects on apoptosis and function of macrophages from various tissue sites remains to be further elucidated.
In keeping with our previous studies [5 , 36 ], macrophages from mice subjected to polymicrobial sepsis exhibited a depression of proinflammatory cytokine release in response to LPS. This macrophage hyporesponsiveness is closely associated with enhanced macrophage apoptosis, particularly in peritoneal macrophages and Kupffer cells. In addition, anti-inflammatory cytokine IL-10 release is significantly increased in septic macrophages following LPS stimulation. In vivo administration of FasFP at 12 h post-CLP had no effect on the decline in proinflammatory cytokine release from each macrophage population tested here. Similarly, it did not affect the release of IL-10 from peritoneal and splenic macrophages, but it did significantly attenuate IL-10 release from Kupffer cells.
The liver is one potential target organ of Fas-mediated cell death, as injection of an agonistic anti-Fas antibody induces extensive cell death of hepatocytes, corresponding with a high level of liver enzymes and lethality in mice [37 ]. We have demonstrated that the changes in apoptosis and IL-10 release of Kupffer cells were associated with a reduction in liver damage (attenuated plasma levels of liver enzymes) subsequent to septic insult when FasFP was administered in vivo. Therefore, protecting Kupffer cells from apoptosis with FasFP treatment may directly or indirectly provide a benefit for the liver by preventing bystander cellular damage and dysfunction, thereby improving survival to septic challenge. Alternatively, attenuating IL-10 production by FasFP treatment may play some role in the decrease in inducible Kupffer cell apoptosis seen during sepsis, as studies have shown that the T helper cell type 2 cytokines IL-10 and IL-4 can enhance monocyte apoptosis [38 ].
In summary, our data indicate that in vivo blockade of the Fas/FasL pathway by the administration of FasFP 12 h after the onset of sepsis improves survival and has a divergent effect on macrophage apoptosis and function, which is dependent on the tissue of origin. FasFP treatment attenuates liver damage, suppresses Kupffer cell apoptosis, and blocks the increased IL-10 release seen in response to LPS stimulation in septic mice. However, FasFP neither restores function nor decreases apoptosis in peritoneal macrophages. Although the precise mechanism is not known, we speculate that blocking Fas-mediated apoptosis may preserve specific/key organ and immune functions, contributing to the host’s defense against septic challenge and thus, may provide a novel target for therapeutic intervention.
Received August 20, 2002; accepted August 20, 2002.
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