| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
* Institute of Virology and Immunobiology, University of Würzburg, Würzburg, Germany; Departments of
Anesthesiology and
Medicine, Washington University School of Medicine, St. Louis, MO 63110; and
Department of Pathology, University of Washington, Seattle, WA 98195
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
production, while increasing IL-4 production >2-fold. In contrast, T cells from Akt transgenic mice had an elevated production of IFN- at baseline that was maintained during sepsis, while IL-4 had little change. Akt overexpression also decreased sepsis-induced lymphocyte apoptosis via a non-Bcl-2 mechanism. In conclusion, Akt overexpression in lymphocytes prevents sepsis-induced apoptosis, causes a Th1 cytokine propensity, and improves survival. Findings from this study strengthen the concept that a major defect in sepsis is impairment of the adaptive immune system, and suggest that strategies to prevent lymphocyte apoptosis represent a potential important new therapy. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Although Akt has been shown to prevent apoptotic cell death, the precise mechanism by which Akt exerts this protective effect is not known. Potential downstream targets of Akt include forkhead proteins (10), Bcl-xL (11), NF-
B (17), Bad (18), caspase 8 (19), and glycogen synthase kinase (20). Importantly, lymphocytes that overexpress Bcl-2, a close relative of Bcl-xL, are resistant to sepsis-induced apoptosis and confer a survival advantage in transgenic mice (7).
Previously, we have shown that transgenic expression of a constitutively active form of Akt1 (myr Akt) influences thymocyte selection and enhances peripheral T cell survival (15). In addition, myr Akt was found to increase T cell reactivity, cell cycle progression, and production of Th1 as well as Th2 cytokines after in vitro stimulation (21). The focus of the present study was to determine whether lymphocytes from mice that express a constitutively active Akt would be protected against sepsis-induced lymphocyte apoptosis and have improved survival in the disorder. T cells that overexpress Akt have been shown to have increased expression of activation markers and promote inflammation (14, 15). We also investigated whether Akt could modulate the impaired T cell activation and cytokine production that are hallmarks of sepsis.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Construction of human CD2-myr Akt1 (myr Akt) transgenic mice and expression of myr Akt in thymocytes and peripheral T cells by Western blot analysis have been described previously (15). Myr Akt expression in T cells was also verified via flow cytometry using an anti-phospho-Akt Ab from Cell Signaling (Beverly, MA), catalog 9277. Mice transgenic for Akt1 were identified using PCR of tail snips, as previously described (15). Mice used in the experimental protocols were backcrossed more than five generations to C57BL/6 mice (B6) and were 6–12 wk old. Preliminary studies demonstrated no difference in the effect of heterozygous or homozygous Akt overexpression on apoptosis or cytokine production (data not shown). Therefore, mice that were either heterozygous or homozygous for Akt overexpression and their negative wild-type littermates were used for studies.
Cecal ligation and puncture (CLP) 3 model of sepsis
The CLP model of sepsis is a widely used, clinically relevant model of sepsis (peritonitis) that has been validated in many laboratories (2, 3, 4). Multiple Gram-negative and Gram-positive organisms are obtained on blood culture from CLP mice (4). Mice weighing 17–25 g were housed for at least 3 days before manipulations. In anesthetized mice, the cecum is isolated, ligated with 4-0 silk, and punctured once with a 26-gauge needle. Sham-operated mice had cecal manipulation only. The abdomen was closed in two layers, and the mice were injected with 1.0 ml of 0.9% saline s.c. At 20–22 h postsurgery, CLP and sham mice were killed, and thymi and spleens were removed for study.
Survival studies in sepsis
Additional groups of mice underwent CLP and were included in survival studies. The methods for the survival studies in the mouse CLP model have been described previously (7). Briefly, an investigator blinded to the identity of the mice performed CLP in the various groups of mice. Approximately 1 h after CLP, the mice received imipenem (1 mg/mouse), and the antibiotics were repeated every 12 h for 48 h and then discontinued. The mice were allowed free access to food and water, and survival was recorded for 7 days.
Cytokine analysis
ELISPOT (Cellular Technology, Cleveland, OH) was performed, as per the manufacturer’s instructions, to determine the effects of overexpression of Akt on stimulated T cell Th1 and Th2 cytokine production in splenocytes. Approximately 24 h after CLP or sham surgery, splenocytes were isolated by gently grinding the moistened organ between the ends of two frosted microscope slides, followed by filtering through sterile mesh wire, as described previously (4). Because the Akt transgenic mice have a larger percentage of CD4 T cells in the spleen compared with wild-type mice, magnetic cell sorting (positive selection) of CD4 T cells (Miltenyi Biotec, Auburn, CA) was used to isolate these cells (
90% purity via flow cytometry). Equal numbers of CD4 T cells (0.5 x 106) from the transgenic and wild-type mice were used for ELISPOT determinations. Cells were loaded on plates precoated with capture Ab (BD PharMingen, San Diego, CA) for IFN- or IL-4 and stimulated with anti-CD3 and anti-CD28 mAbs, as described previously (22). Cell culture medium was RPMI 1640 with 50 µM 2-ME, 2 mM glutamine, supplemented with 10% FCS. Cell incubation was maintained for 24 or 48 h for IFN- and IL-4, respectively. A secondary HRP-labeled detecting Ab was added and detected with 3-amino-9-ethyl-carbazole as a substrate. Images of the individual wells were obtained, and comparison between the intensities of the individual wells was performed using a software program from the manufacturer (Cellular Technology) that counted the number of spots on the ELISPOT plate. The spots are proportional to the number of cytokine-producing cells.
Flow cytometry detection of apoptosis
Briefly, splenocytes were obtained from the different treatment groups 24 h after sham or CLP surgery, and apoptosis was quantified using a commercially available fluorescein-labeled annexin V/7-aminoactinomycin D (7-AAD) kit (apoptosis detection kit; R&D Systems, Minneapolis, MN), as described previously (4, 7). The lymphocyte phenotypes were identified using fluorescently labeled mAbs directed against lymphocyte surface markers (BD PharMingen): B cells, CD4 T cells, CD8 T cells, and CD3 T cells. Flow cytometric analysis (10,000–50,000 events/sample) was performed on FACSCalibur (BD Biosciences, San Jose, CA).
Annexin V is an accepted marker for acute detection of apoptosis in cells that have not undergone fixation, but it cannot be used in cells that have been fixed for intracellular protein detection. To relate intracellular expression of Akt (vide infra) and apoptosis, i.e., to determine whether cells that expressed Akt were resistant to apoptosis, the CaspACE FITC-VAD-FMK in situ marker kit (Promega, Madison, WI; catalog G7461) was used, as per the manufacturer’s instructions.
Flow cytometry detection of Akt and active caspase 3
Intracellular staining of splenocytes for active Akt was performed using the protocol recommended by Cell Signaling, and by Tazzari et al. (23). Briefly, dissociated splenocytes were fixed in 1% paraformaldehyde, permeabilized in ice-cold methanol, and incubated with an anti-phospho-Akt Ab (Ser473) (Cell Signaling; catalog 9271). The secondary Ab used to identify the primary Ab was a donkey anti-rabbit (Jackson ImmunoResearch Laboratories, West Grove, PA). For detection of active caspase 3, an Ab specific to the cleaved short fragment of caspase 3 was used, per the manufacturer’s instructions (Cell Signaling; catalog 9664). The cells were fixed in 1% paraformaldehyde, followed by permeabilization with methanol. The secondary Ab was a donkey anti-rabbit Ab (Jackson ImmunoResearch Laboratories).
Western blot analysis of Bcl-2
Spleens from Akt transgenic mice or wild-type littermates were removed, and the cells were dissociated by gentle grinding between the frosted ends of two microscope slides. To have equal numbers of T cells for analysis of Bcl-2, CD3 T cells were isolated using the Miltenyi Biotec magnetic bead protocol, as per the manufacturer’s instructions. Approximately 5 x 106 CD3-positive T cells were obtained from each animal, and protein extractions were prepared. A total of 20 µg of protein/well was loaded onto a 12% SDS-PAGE gel, and electrophoresis was performed. The protein was transferred to a nitrocellulose membrane and blocked for 2 h at room temperature with skim milk. Next, the primary Ab was incubated overnight at 4°C with skim milk. The primary Ab against mouse Bcl-2 (BD PharMingen) was detected by HRP-conjugated Ab (BD PharMingen) and the SuperSignal West Fento (Pierce, Rockford, IL).
Western blot analysis of B cells for myr Akt
For Western blot analysis of B cells, splenic cells from wild-type and Akt transgenic mice were treated with anti-CD4 (RL174.2) and anti-CD8 (31.68.1) hybridoma supernatant and low toxin baby rabbit complement (Cedarlane, Hornby, Ontario, Canada). Purity of B cells was verified by staining for CD3 expression (145.2C11; BD PharMingen) and routinely was greater than 90%. Isolated B cells were left untreated or were stimulated with anti-IgM F(ab')2 Ab (10 µg/ml; Sigma-Aldrich, Taufkirchen, Germany) for the indicated time points. A total of 2 x 106 B cells was lysed in buffer containing 20 mM HEPES, 2 mM EGTA, 50 mM 
-glycerophosphate, 1% Triton X-100, 10% glycerol, 50 mM NaF, 0.04% azide, 1 mM DTT, 1 mM orthovanadate, 2 µM leupeptin, and 0.4 mM PMSF for 30 min on ice. Protein extracts were separated on 8% SDS-PAGE, electroblotted to nitrocellulose membrane, and analyzed for expression of active Akt using anti-phospho-Akt (Ser473) Ab (Cell Signaling). The blot was reprobed with anti-actin Ab (Santa Cruz Biotechnology, Santa Cruz, CA) to control protein loading. Primary Abs were detected by goat anti-rabbit or rabbit anti-goat Ab coupled with HRP (both Jackson ImmunoResearch Laboratories) and SuperSignal West Pico (Pierce).
Immunohistochemical staining for phospho-Akt and phospho-forkhead
For immunohistochemical staining of phospho-Akt, a rabbit anti-phospho-Akt Ab from Cell Signaling (catalog 9277) was used, as per the manufacturer’s instructions on frozen spleen sections. The secondary Ab was a goat anti-rabbit Ab labeled with either Alexa 488 or Alexa 594 (Molecular Probes, Eugene, OR).
For immunohistochemical staining of phospho-forkhead, an anti-phospho-forkhead Ab (Fox03; catalog 9464; Cell Signaling) was used, as per the manufacturer’s protocol on paraffin-embedded spleen sections. For Ag unmasking, slides were heated in citrate buffer. The slides were incubated overnight in primary Ab at 4°C. A secondary goat anti-rabbit HRP-labeled Ab was then used.
Statistical analysis
Data are reported as the mean ± SEM. Data were analyzed using a statistical software program Prism (GraphPad Software, San Diego, CA). Data for the percentage of apoptosis determined via annexin V labeling were analyzed using one-way ANOVA. For comparison of protection against sepsis-induced apoptosis by Akt-expressing vs non-Akt-expressing cells within the same animal, a paired t test was used. Differences in group survival were determined using Fischer’s exact p test. Values of p <0.05 were accepted as significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Transgenic mice overexpressing active Akt had a marked improvement in sepsis survival compared with wild-type matched littermates (Fig. 1). After 7 days, survival was 94 and 47% for Akt transgenic mice and matched wild-type littermates, respectively (p < 0.01).
|
ELISPOT examination of anti-CD3- and anti-CD28-stimulated CD4 T cells demonstrated that compared with sham wild-type littermates, cells from sham-operated Akt transgenic mice had increased production of IFN- that was at the upper limit of detection due to saturation of the microplate (Fig. 2; Table I). The most marked difference was the production of IFN- in splenocytes from wild-type littermates vs Akt transgenic mice following sepsis (Fig. 2; Table I). Compared with septic wild-type littermates, Akt transgenic mice have a 3-fold increase in IFN- (Fig. 2; Table I). IL-4 production in sham-operated Akt transgenics was slightly increased compared with wild-type littermate sham mice. Interestingly, the response to the septic insult was markedly different in the wild-type littermates vs Akt transgenic mice. IL-4 production increased >2-fold in the wild-type littermate septic mice, but no increase occurred in Akt transgenic mice. This indicates that Akt transgenic CD4 T cells react to the induced sepsis with a Th1-biased cytokine profile, whereas wild-type littermate CD4 T cells show a shift toward a Th2 cytokine immune response.
|
|
As noted previously (15), flow cytometry revealed an increase in the percentage of CD4 T cells in splenocytes from the Akt transgenics vs wild-type littermate mice (Figs. 3A and 4). In spleens of sham-operated wild-type littermate mice, the percentage of CD4 T cells was 24.2 ± 1.3%, whereas the percentage of CD4 T cells in Akt transgenic mice was 46.6 ± 1.8%, p < 0.05 (Fig. 3A). In contrast, the percentage of B cells (CD19+) was decreased in the sham-operated Akt transgenics (30.1 ± 2.5%) compared with sham-operated wild-type littermate controls (48.0 ± 3.7%), and this most likely represents a compensatory effect of the increased percentage of CD4 T cells (Fig. 3B). The percentage of CD8 T cells in sham-operated Akt transgenic vs wild-type littermate mice was not different (Fig. 3C).
|
|
|
5–12% of CD3 T cells were positive for phospho-Akt, whereas the percentage of CD3 T cells in wild-type littermate mice that were positive for phospho-Akt was negligible (Fig. 6). To determine whether overexpression of Akt protected against sepsis-induced apoptosis, the percentage of apoptotic CD3-positive T cells that expressed Akt was compared with the percentage of apoptotic CD3-positive T cells that did not express Akt from the same mouse. This value was determined by comparing the ratio of the percentage of cells in the upper right quadrant (apoptotic cells that express Akt) with the percentage of cells in the upper left quadrant (nonapoptotic cells that express Akt) vs the ratio of the percentage of cells in the lower right quadrant (apoptotic non-Akt-expressing cells) to the lower left quadrant (nonapoptotic non-Akt-expressing cells). In sepsis, the percentage of Akt-expressing CD3 T cells that were apoptotic was 4.7 ± 3.7%, whereas the percentage of apoptotic non-Akt-expressing CD3 T cells was 7.3 ± 4.1%, and this difference was statistically significant (n = 10), p < 0.05. This represented a 36% reduction in sepsis-induced lymphocyte apoptosis in cells expressing Akt.
|
The percentage of T cells determined to be positive for phospho-Akt by flow cytometry was compared with conventional immunohistochemical staining for phospho-Akt of paraffin-embedded tissues. Although not quantified, immunohistochemical staining for phospho-Akt in formalin-fixed spleen slices showed an obviously larger percentage of T cells positive for phospho-Akt as compared with the percentage of splenocytes positive for phospho-Akt, as determined by flow cytometry (Fig. 7). In the T cell regions of the spleen, i.e., periarteriolar lymphoid sheaths, the majority of cells were positive for phospho-Akt in the Akt-overexpressing mice (Fig. 7). Thus, flow cytometry seems to underestimate the percentage of cells that overexpressed Akt. Immunohistochemical staining for phospho-Akt was negligible in wild-type littermate mice (data not shown).
|
Western blots for Bcl-2 in splenic CD3 T cells from Akt transgenic and wild-type littermates showed abundant Bcl-2 in all spleens, but there was no difference in the amount of Bcl-2 in CD3 splenic T cells between the two groups (Fig. 8).
|
The CD2 minigene cassette was used in construction of the Akt transgene. Although the use of this construct would induce expression of Akt primarily in T cells, additional expression has been reported to occur in B cells as well (24). Myr Akt expression in T cells, as previously shown by Western blot (15), correlates with the reduced apoptosis of Akt transgenic T cells after sepsis induction compared with wild-type T cells. To determine whether the protection against sepsis-induced B cell apoptosis observed in the Akt transgenic mice (Fig. 5) might be due to myr Akt expression in B cells, Western blotting of B cells was performed (see Materials and Methods). B cells were stimulated with anti-IgM F(ab')2 Abs or LPS. As demonstrated in Fig. 9, myr Akt was present only in the B cells from the transgenic mice, while endogenous active Akt was present in B cells from wild-type littermates and transgenic mice.
|
Immunohistochemical staining of spleens showed a marked increase in cells positive for phospho-forkhead (Fox 03) in Akt transgenics vs wild-type littermate mice (Fig. 10). Only rare positive cells were present in wild-type littermate mice, whereas Akt transgenic mice had foci of positive cells that were located in both T cell regions (periarteriolar sheaths) and to a lesser degree in splenic follicles. The phospho-forkhead staining was confined to the cytoplasm of the cell with little to absent nuclear staining. Clusters of cells demonstrating the typical features of apoptosis, i.e., compacted and fragmented nuclei, were present in both Akt transgenic and wild-type littermate mice. Although not systematically quantitated, few cells that were positive for phospho-forkhead were observed to be undergoing apoptosis.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
compared with wild-type septic mice (Table I). IFN- has been shown to improve survival in a variety of infectious models, including sepsis (22). A small clinical study in patients with sepsis also demonstrated improved survival in patients treated with IFN- (30).
Previous studies from many groups, including our own, have shown that Akt has complex effects on cell survival and prevention of apoptosis (10, 11, 12, 13, 14, 15). Pancreatic cells expressing myr Akt1 had an increase in basal apoptosis, but were resistant to streptozotocin-induced apoptosis (13). The increase in baseline apoptosis in Akt-overexpressing cells is unknown, but may be related to the effect of Akt on cell cycle and cell size (31, 32). Further evidence that the antiapoptotic effect of Akt is cell type and context specific has been provided by previous studies in which we documented that myr Akt did not prevent thymocyte apoptosis secondary to a variety of apoptogenic stimuli, but did prevent splenocyte apoptosis under these same conditions (15). This failure of myr Akt to protect thymocytes may be related to the fact that Akt provides stronger survival signals for mature peripheral T cells than for thymocytes that might be related to the subcellular localization of transgenic Akt (15, 33). Similarly, myr Akt did not confer protection against sepsis-induced apoptosis in thymocytes, even though these cells expressed myr Akt (our unpublished data), but myr Akt did protect splenocytes.
In the present study, three independent methods of evaluation of apoptosis were used, i.e., annexin V labeling, fluorescent labeling of caspase-cleaved substrates, and active caspase 3 staining. There was an increase in lymphocyte apoptosis at baseline in Akt-overexpressing mice, as noted by both annexin V and active caspase 3 staining. However, both methods also showed that sepsis did not increase lymphocyte apoptosis in Akt-overexpressing mice (Fig. 5, A–C, and results for active caspase 3). The increase in lymphocyte apoptosis in the sham-operated Akt transgenic mice was somewhat unexpected, but, as noted previously, a similar increase in baseline apoptotic rate was noted in Akt-expressing pancreatic cells (13). The increased rate of apoptosis in lymphocytes from the Akt transgenic mice may be related to their activation state; activated lymphocytes are more susceptible to apoptosis in certain situations (34). T cells from the Akt transgenic mice were more reactive compared with T cells from wild-type mice, as evidenced by the increased production of IFN- in the former compared with the latter. Previous studies from our laboratory confirmed that Akt increased T cell responsiveness by enhancing Lck and Erk activities (15). Of note, blinded evaluation of spleens from septic and sham-operated Akt transgenics and wild-type littermate mice, including immunohistochemical staining for Akt and TUNEL, confirmed the protective effect of Akt against sepsis-induced apoptosis in the spleen (data not presented).
Despite the increase in baseline apoptosis in the Akt transgenics vs wild-type littermate mice, there was no increase in apoptosis in CD4 T cells during sepsis, and therefore, the Akt transgenics most likely had an effective increase in the numbers of lymphocytes that were resistant to sepsis-induced apoptosis. Importantly, studies from our laboratory have shown that adoptive transfer of normal lymphocytes (lymphocytes that are not resistant to sepsis-induced apoptosis) does not confer resistance to mortality in sepsis (our unpublished findings). Therefore, one possible factor in the improved sepsis survival in the Akt transgenic mice may have been the increase in the number of lymphocytes that were resistant to cell death during sepsis. Most likely, the improved survival in the Akt transgenic mice was multifactorial and included increased numbers of activated apoptosis-resistant lymphocytes with a Th1 cytokine profile propensity.
The mechanism for the protective effect of Akt in preventing sepsis-induced lymphocyte apoptosis is unknown. Akt has a number of downstream targets, e.g., forkhead proteins, pro- and antiapoptotic Bcl-2 family members, NF-B, etc., that potentially could be involved (16, 17, 18, 19). Previous studies have shown that mice that overexpress the antiapoptotic protein Bcl-2 in T or B cells are resistant to sepsis-induced lymphocyte apoptosis and have improved survival (7, 20). In the present study, no increase in expression of Bcl-2 was found in lymphocytes from Akt transgenic vs wild-type mice. This finding does not rule out the possibility that Akt may modulate other Bcl-2 family members. In this regard, Jones et al. (11) showed that lymphocytes that overexpress gag Akt had increased expression of the antiapoptotic protein Bcl-xL.
In a previous report, we showed that myr Akt transgenic T cells stimulated in vitro show a reduced nuclear translocation of NF-AT and NF-B p65/RelA and RelB proteins (21). Because both of these transcription factors have been shown to be involved in regulating apoptosis (35, 36), it is possible that the reduced apoptosis of Akt transgenic T cells in sepsis is connected to reduced expression of apoptosis-mediating ligands such as Fas ligand or inflammatory proteins that are regulated by NF-AT and NF-B proteins, respectively. In gag Akt transgenic T cells, Fas-mediated cell death was strongly reduced due to a failure of recruitment of caspase 8 to the fas-associated death domain signaling complex (19). Because myr Akt can replace CD28 costimulatory signals in T cell activation and proliferation (21), it is also possible that the apoptotic signals induced in sepsis are counteracted by the positive signals provided by myr Akt on T cell activation and cell cycle progression. As demonstrated in the present study, another potential mechanism for the antiapoptotic effect of Akt is increased expression of the phosphorylated forkhead family member FOX03 (37, 38). Immunohistochemical staining showed a large increase in the percentage of lymphocytes expressing cytosolic phospho-forkhead in Akt transgenic mice vs wild-type mice (Fig. 7). FOX03 can act as a proapoptotic transcription factor, and Akt-mediated phosphorylation of FOX03 is thought to prevent its translocation to the nucleus, thereby preventing transcription of proapoptotic genes, including Bim and Fas (37, 38).
The appropriate response of the host to invading pathogens is clonal expansion of T and B cells to combat the microorganisms (2, 3, 4, 5). Numerous studies have shown that in sepsis there is a marked apoptosis-induced depletion of lymphocytes rather than the expected lymphocyte proliferation. Additional studies demonstrate that prevention of lymphocyte apoptosis by overexpression of Bcl-2 improves survival, and thus have provided support for the concept that loss of lymphocytes in sepsis is a key pathogenic mechanism in the lethal disorder. Recent work by Iwata et al. (39) using transgenic mice that overexpress Bcl-2 in a number of cell lines other than lymphocytes has also shown improved survival in sepsis. Therefore, these investigators postulated that the protective effect of Bcl-2 may be independent of its ability to prevent lymphocyte death and instead may be related to a secretion product that is released by cells that overexpress Bcl-2. The current findings demonstrating that overexpression of a second antiapoptotic protein, i.e., Akt, also prevents sepsis-induced lymphocyte apoptosis, does not increase Bcl-2 and improves sepsis survival strengthen the hypothesis that the loss of lymphocytes is critical in sepsis and that prevention of lymphocyte apoptosis may represent an effective new therapy in sepsis.
In summary, overexpression of Akt decreased sepsis-induced lymphocyte apoptosis, increased production of the Th1 cytokine IFN-, increased cytosolic localization of phospho-forkhead, and improved survival in sepsis. The results bolster the important role of the lymphocyte in sepsis and open new possible targets for therapy of sepsis.
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Richard S. Hotchkiss, Washington University School of Medicine, 660 South Euclid Avenue, Box 8054, St. Louis, MO 63110. E-mail address: hotchkir{at}msnotes.wustl.edu
3 Abbreviations used in this paper: CLP, cecal ligation and puncture; 7-AAD, 7-amino- actinomycin D.
Received for publication October 3, 2003. Accepted for publication April 1, 2004.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B activation, and Bcl-xL levels in vivo. J. Exp. Med. 191:1721.
- and Fas-mediated apoptosis through NK-B activation. Am. J. Physiol. Gastrointest. Liver Physiol. 281:G1357.B. Mol. Cell. Biol. 20:1626.B proteins. J. Immunol. 172:4812. treatment. Nat. Med. 3:678.[Medline]
1 regulates lymphocyte homeostasis by preventing activation and subsequent apoptosis of peripheral lymphocytes. J. Immunol. 170:4612.B regulation in the immune system. Nat. Rev. Immunol. 2:725.[Medline]
This article has been cited by other articles:
|
|
 |
|
  I. Bekeredjian-Ding, A. Doster, M. Schiller, P. Heyder, H.-M. Lorenz, B. Schraven, U. Bommhardt, and K. Heeg TLR9-Activating DNA Up-Regulates ZAP70 via Sustained PKB Induction in IgM+ B Cells J. Immunol., December 15, 2008; 181(12): 8267 - 8277. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. O. Alpdogan, S. X. Lu, N. Patel, S. McGoldrick, D. Suh, T. Budak-Alpdogan, O. M. Smith, J. Grubin, C. King, G. L. Goldberg, et al. Rapidly proliferating CD44hi peripheral T cells undergo apoptosis and delay posttransplantation T-cell reconstitution after allogeneic bone marrow transplantation Blood, December 1, 2008; 112(12): 4755 - 4764. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. L. Gautier, T. Huby, F. Saint-Charles, B. Ouzilleau, M. J. Chapman, and P. Lesnik Enhanced Dendritic Cell Survival Attenuates Lipopolysaccharide-Induced Immunosuppression and Increases Resistance to Lethal Endotoxic Shock J. Immunol., May 15, 2008; 180(10): 6941 - 6946. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Yasuda, A. Leelahavanichkul, S. Tsunoda, J. W. Dear, Y. Takahashi, S. Ito, X. Hu, H. Zhou, K. Doi, R. Childs, et al. Chloroquine and inhibition of Toll-like receptor 9 protect from sepsis-induced acute kidney injury Am J Physiol Renal Physiol, May 1, 2008; 294(5): F1050 - F1058. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Rodriguez-Manzanet, J. H. Meyers, S. Balasubramanian, J. Slavik, N. Kassam, V. Dardalhon, E. A. Greenfield, A. C. Anderson, R. A. Sobel, D. A. Hafler, et al. TIM-4 Expressed on APCs Induces T Cell Expansion and Survival J. Immunol., April 1, 2008; 180(7): 4706 - 4713. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. E. McDunn, J. T. Muenzer, L. Rachdi, K. C. Chang, C. G. Davis, W. M. Dunne, D. Piwnica-Worms, E. Bernal-Mizrachi, and R. S. Hotchkiss Peptide-mediated activation of Akt and extracellular regulated kinase signaling prevents lymphocyte apoptosis FASEB J, February 1, 2008; 22(2): 561 - 568. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M Busse, T Traeger, C Potschke, A Billing, A Dummer, E Friebe, C Kiank, U Grunwald, R S Jack, C Schutt, et al. Detrimental role for CD4+ T lymphocytes in murine diffuse peritonitis due to inhibition of local bacterial elimination Gut, February 1, 2008; 57(2): 188 - 195. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. K. Polumuri, V. Y. Toshchakov, and S. N. Vogel Role of Phosphatidylinositol-3 Kinase in Transcriptional Regulation of TLR-Induced IL-12 and IL-10 by Fc{gamma} Receptor Ligation in Murine Macrophages J. Immunol., July 1, 2007; 179(1): 236 - 246. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. D. Wrann, N. A. Tabriz, T. Barkhausen, A. Klos, M. van Griensven, H. C. Pape, D. O. Kendoff, R. Guo, P. A. Ward, C. Krettek, et al. The Phosphatidylinositol 3-Kinase Signaling Pathway Exerts Protective Effects during Sepsis by Controlling C5a-Mediated Activation of Innate Immune Functions J. Immunol., May 1, 2007; 178(9): 5940 - 5948. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W.-J. Zhang, H. Wei, T. Hagen, and B. Frei {alpha}-Lipoic acid attenuates LPS-induced inflammatory responses by activating the phosphoinositide 3-kinase/Akt signaling pathway PNAS, March 6, 2007; 104(10): 4077 - 4082. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. C. McGown and Z. L. S. Brookes Beneficial effects of statins on the microcirculation during sepsis: the role of nitric oxide Br. J. Anaesth., February 1, 2007; 98(2): 163 - 175. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Matsuda, Y. Hayashi, Y. Takahashi, and Y. Hattori Phosphorylation of Endothelial Nitric-Oxide Synthase Is Diminished in Mesenteric Arteries from Septic Rabbits Depending on the Altered Phosphatidylinositol 3-Kinase/Akt Pathway: Reversal Effect of Fluvastatin Therapy J. Pharmacol. Exp. Ther., December 1, 2006; 319(3): 1348 - 1354. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. E. Thomas, C. L. Galligan, R. D. Newman, E. N. Fish, and S. N. Vogel Contribution of Interferon-beta to the Murine Macrophage Response to the Toll-like Receptor 4 Agonist, Lipopolysaccharide J. Biol. Chem., October 13, 2006; 281(41): 31119 - 31130. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Ha, F. Hua, D. Grant, Y. Xia, J. Ma, X. Gao, J. Kelley, D. L. Williams, J. Kalbfleisch, I. W. Browder, et al. Glucan phosphate attenuates cardiac dysfunction and inhibits cardiac MIF expression and apoptosis in septic mice Am J Physiol Heart Circ Physiol, October 1, 2006; 291(4): H1910 - H1918. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. J. Schwulst, M. H. Grayson, P. J. DiPasco, C. G. Davis, T. S. Brahmbhatt, T. A. Ferguson, and R. S. Hotchkiss Agonistic Monoclonal Antibody Against CD40 Receptor Decreases Lymphocyte Apoptosis and Improves Survival in Sepsis J. Immunol., July 1, 2006; 177(1): 557 - 565. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Ashare, M. M. Monick, L. S. Powers, T. Yarovinsky, and G. W. Hunninghake Severe Bacteremia Results in a Loss of Hepatic Bacterial Clearance Am. J. Respir. Crit. Care Med., March 15, 2006; 173(6): 644 - 652. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. E. Wesche-Soldato, J. L. Lomas-Neira, M. Perl, L. Jones, C.-S. Chung, and A. Ayala The role and regulation of apoptosis in sepsis Innate Immunity, December 1, 2005; 11(6): 375 - 382. [Abstract] [PDF]
|
|
|
|
 |
|
 D. E. Wesche, J. L. Lomas-Neira, M. Perl, C.-S. Chung, and A. Ayala Leukocyte apoptosis and its significance in sepsis and shock J. Leukoc. Biol., August 1, 2005; 78(2): 325 - 337. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
