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Departments of
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Anesthesiology,
Medicine,
Surgery,
Radiology, and
¶ Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63110; and
|| Department of Neurology, University of Göttingen, Göttingen, Germany
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Abstract |
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3-fold improvement in survival. TAT-Bcl-xL and TAT-BH4 prevented Escherichia coli-induced human lymphocyte apoptosis ex vivo and markedly decreased lymphocyte apoptosis in an in vivo mouse model of sepsis. In conclusion, TAT-conjugated antiapoptotic Bcl-2-like peptides may offer a novel therapy to prevent apoptosis in sepsis and improve survival. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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The mechanistic importance of apoptosis in sepsis is highlighted by findings from multiple investigative groups showing that therapies that inhibit apoptosis, e.g., overexpression of antiapoptotic proteins, Fas pathway inhibitors, caspase inhibitors, and antiproteases, result in improved survival (11, 12, 13, 14, 15, 16). One strategy that has been used is overexpression of the antiapoptotic protein Bcl-2 (11, 17, 18). Sepsis and endotoxemia are known to result in decreased lymphocyte Bcl-2, and three independent investigative groups have demonstrated that transgenic mice that overexpress the antiapoptotic Bcl-2 in their lymphocytes have improved sepsis survival (11, 17, 18).
Recent remarkable studies demonstrate that large cargoes, proteins, and peptides can be delivered intracellularly if conjugated to permeation peptides derived from HIV-1 TAT basic domain or antennapedia homeodomain (19, 20, 21, 22, 23, 24, 25, 26, 27, 28). Rapid and receptor-independent uptake of TAT-conjugated peptides have been demonstrated to occur in many cell types (24, 26, 27, 28). Although TAT-Bcl-2 is insoluble, another member of the antiapoptotic Bcl-2 family, i.e., Bcl-xL, has been conjugated to TAT and is readily soluble. TAT-Bcl-xL has been shown to prevent ischemia/reperfusion-induced apoptosis in brain tissue (20, 21, 23). Given the reproducible and highly beneficial effects of transgenic overexpression of Bcl-2 in sepsis, we hypothesized that TAT-Bcl-xL would inhibit bacterial-induced apoptosis. Production of TAT-Bcl-xL involves bacterial transfection of Bcl-xL and purification of the bacterial extract (19, 20, 21). A small amount of endotoxin is invariably present in the resultant purified product, a situation that is not ideal for sepsis studies. Consequently, an alternative approach using solid-phase peptide synthesis of the active antiapoptotic BH4 domain of Bcl-xL was also used. Recently, TAT-BH4 has been reported to be efficacious in decreasing apoptosis in a wide range of models, including irradiation, etoposide treatment, and ischemia/reperfusion (22, 25, 26).
In this study, we demonstrate that administration of TAT-Bcl-xL and TAT-BH4 provided highly significant protection both in vitro and in vivo against bacterial-induced lymphocyte apoptosis. We conclude that TAT-conjugated BH4 may offer a novel means to prevent the profound immune cell depletion that is central to the pathophysiology of sepsis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Mice that selectively overexpress Bcl-xL in T lymphocytes using the lck-proximal promoter were provided by C. Thompson (University of Pennsylvania, Philadelphia, PA) (29). These mice had been backcrossed to C57BL/6 mice for >10 generations. Tail snips were obtained to verify the presence of the transgene via PCR analysis. C57BL6/J mice purchased from The Jackson Laboratory were used as controls.
Sepsis model: cecal ligation and puncture (CLP)3
C57BL6/J male mice were housed for at least 1 wk before manipulations. The CLP model was used to induce intra-abdominal peritonitis (30). Previous studies from our laboratory include positive blood cultures for polymicrobial organisms (aerobic and anaerobic bacteria) from CLP, but not sham-operated mice (31). Mice were anesthetized with halothane, and an abdominal incision was performed. The cecum was identified, ligated, and punctured with a 30-gauge needle. The abdomen was closed in two layers, and 1 ml of 0.9% saline was administered s.c. Sham-operated mice were treated identically, except the cecum was not ligated or punctured.
For survival studies, mice received 25 mg/kg imipenem 3 h postoperatively and twice per day for 2 days. Survival was recorded for 7 days.
Quantification of apoptosis
Thymocytes and splenocytes were obtained from CLP and sham-treated mice 20 h postoperatively, and apoptosis was quantified by flow cytometry using Abs to active caspase 3 (Cell Signaling Technology; catalog 9664) and/or TUNEL assay. The APO-BrdU kit (Phoenix Flow Systems) was used for flow cytometric quantitation of TUNEL, and the manufacturer’s instructions were followed without modification. Lymphocyte B and CD3 T cells were identified using fluorescently labeled mAbs directed against their respective CD surface markers (BD Pharmingen): flow cytometric analysis (25,000–50,000 events/sample) was performed on FACScan (BD Biosciences).
Escherichia coli bacterial-induced human lymphocyte apoptosis
Lymphocytes were harvested from peripheral blood obtained from six healthy volunteers using a Ficoll gradient separation technique. Approximately 1 x 106 lymphocytes were plated in individual Transwell containers. E. coli bacteria (strain ATCC 25922), grown overnight in trypticase soy broth, were added to a separate compartment of the Transwell chamber separated from direct contact with the lymphocytes by a 0.02-µm pore size filter (25 µl of bacteria at 3 x 109 CFUs added to 1 ml vol). Bcl-xL, TAT-Bcl-xL, TAT-BH4, or an inactive TAT-BH4(D)2 (see below) were placed in experimental wells within 20 min after addition of bacteria, and the lymphocytes were incubated for 5 h. The inactive TAT-BH4(D)2 was identical with TAT-BH4, except 2 aa that are essential for the antiapoptotic activity of BH4 were replaced by aspartate to render it inactive.
Expression and purification of rTAT-Bcl-xL
The Bcl-xL coding sequence was PCR amplified from C57BL6/J mouse whole-brain cDNA, as previously described (27). Purified PCR fragments were cloned into the XboI/EcoRI sites of the pTAT-HA vector kindly provided by S. Dowdy (University of California School of Medicine, San Diego, CA). All expression cassettes included a sequence encoding six consecutive histidine residues for purification. TAT-Bcl-xL was expressed in E. coli strain BL21(DE3) pLysS (Novagen) and lysed by sonication. E. coli lysates were denatured in 8 M urea before affinity chromatography. Bacterial debris was pelleted, and the supernatant was subjected to metal-affinity chromatography using a Ni-NTA matrix. TAT-Bcl-xL identity was confirmed by Western blotting. Urea and salt were removed by gel filtration using a PD-10 Sephadex G-25M column (Amersham Biosciences).
Peptide synthesis
Amino acid sequences of TAT basic domain and the BH4 peptide used in the present study are similar to those used by other investigators with two exceptions. First, (d)-amino acids were used for synthesis of TAT basic domain because these are more slowly metabolized, and therefore the effective t1/2 of the compound is prolonged (32). Second, previous sequence-activity analysis had shown that substitution of ornithine for glutamine enhanced cell permeation of the TAT peptides by 10-fold (32). The amino acid sequence of TAT-BH4 was the following: (d)-Ac-RKKRR-Orn-RRR, 
-A-(l)-SNRELVVDFLSYKLSQKGYS-COOH, wherein -A represents -alanine, Orn is ornithine, and the N terminus is acetylated.
The peptide that was used as a control for TAT-BH4 was identical with the TAT-BH4, except for 2 aa substitutions (aspartic acid replaced two tyrosines in the BH4 sequence) that rendered the BH4 inactive by simulating the native phosphoprotein domain (22). The amino acid sequence of the inactive TAT-BH4(D)2 was the following: (d)-Ac-RKKRR-Orn- RRR, -A-(l)-SNRELVVDFLSDKLSQKGDS-COOH.
Peptides were generated by solid-phase peptide synthesis using standard Fmoc chemistry by Tufts University Peptide Synthesis Core and purified by HPLC. Identity was confirmed by amino acid analysis and mass spectrometry. Purity was >95%.
In vivo administration of TAT-BH4 via infusion pumps
To evaluate the antiapoptotic efficacy of TAT-BH4 in an in vivo model of sepsis, miniosmotic pumps (Alzet Model 2001D; Durect) were loaded with 1 mg of TAT-BH4 or the TAT-BH4(D)2 inactive analog dissolved in 200 µl of sterile saline and implanted in the s.c. tissues on the dorsum of the mice. The pumps were implanted 3 h before CLP because it requires 3 h for pumps to activate and deliver steady state levels of compound. In addition to the TAT-BH4 peptides that were administered by the Alzet miniosmotic pumps, an additional dose of 0.5 mg of TAT-BH4 or inactive TAT-BH4(D)2 was administered via i.p. injection 2–3 h before sacrifice of the animals, which was 18 h postprocedure.
Laser-scanning confocal microscopy of TAT-BH4-treated human lymphocytes
To confirm that TAT-BH4 was internalized by the cells, freshly isolated human lymphocytes were incubated with a fluorescently labeled TAT-BH4 peptide. To prepare the fluorescent labeled TAT-BH4, (d) Ac-C (FM)RKKRR-Orn-RRR--A-(l)-SNRELVVDFLSYKLSQKGYS-COOH, an N terminus cysteine, was included in the initial solid state peptide synthesis of the peptide and, following HPLC purification, the peptide was thiol conjugated to fluorescein maleimide (1.2 equivalent; Molecular Probes) at ambient temperature in 50% dimethylformamide/water for 2 h. Quantitative yields were analyzed by C18 reverse-phase HPLC. For labeling, cells were suspended for 30 min in modified Earl’s balanced salt solution containing 1 µM fluorescently labeled TAT-BH4 (33). Control cells were treated identically, except no labeled TAT-BH4 was added. Following fixation (10 min) in 4% paraformaldehyde, cells were analyzed for peptide internalization via detection of fluorescence by confocal microscopy using an inverted Zeiss Axiovert 200 laser-scanning confocal microscope coupled to a Zeiss LSM 5 PASCAL fitted with a 488 nm excitation Ar laser and a 520-nm bandpass emission filter. All images were obtained using a water immersion lens (x40) and identical instrument settings.
TAT-BH4-induced gene expression determined by microarray analysis
To determine potential mechanisms of protection afforded by TAT-BH4, microarray analysis was performed. Fresh peripheral blood was obtained from healthy human volunteers (n = 5). Cells were isolated by Ficoll density gradient separation in combination with RosetteSep (StemCell Technologies), which negatively selects for total lymphocytes. Approximately 4 x 106 lymphocytes/well were incubated for 5 h either untreated or treated with E. coli, E. coli + 1 µM TAT-BH4, or E. coli + 1 µM TAT-BH4(D)2. Following incubation, lymphocytes were lysed on QiaShredder spin columns and RNA was isolated using RNeasy Mini Spin Columns (Qiagen). Quality and yield of RNA were determined using 2100 Bioanalyzer (Agilent). Target was prepared from isolated RNA using the Nugen Ovation Biotin System and hybridized to Affymetrix Human Genome U133 Plus 2.0 Genechip Arrays. Arrays were stained and washed using an Affymetrix fluidics station and scanned for signal intensity per manufacturer’s protocol.
Statistical analysis
Data are reported as the mean ± SEM. Data were analyzed using the statistical software program Prism (GraphPad). Data involving two groups only were analyzed by Student’s t test, while data involving more than two groups were analyzed using one-way ANOVA with Tukey’s multiple comparison test. Significance was accepted at p < 0.05.
Statistical analysis for microarray data
Normalized expression values were calculated using Robust Multichip Average software. A two-way ANOVA with a Bonferroni correction for multiple test groups and pairwise comparisons were performed for gene discovery. A false discovery rate (significance = 0.05) was used to identify genes with significantly altered gene expression. Ingenuity Pathways Analysis evaluated potential mechanisms of cell death.
Animal studies were approved by the Animal Studies Committee at Washington University School of Medicine. The use of volunteer blood donors for lymphocyte studies was approved by the Human Studies Committee at Washington University School of Medicine.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Sepsis caused a marked increase in CD3 T cell apoptosis in thymus and in both CD3 T and B cell apoptosis in spleen in wild-type mice as determined by increased active caspase 3 and TUNEL-positive cells (Figs. 1 and 2). In contrast, transgenic overexpression of Bcl-xL totally prevented the increase in CD3 T cell death in both organs (Figs. 1 and 2) (n = 8 each for wild-type and transgenic groups).
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Human lymphocyte apoptosis as determined by active caspase 3 immunohistochemical staining and flow cytometry increased from 6.0 ± 1.1% in control to 30.1 ± 8.2% with E. coli treatment (p < 0.01) (n = 6) (Fig. 4). TAT-Bcl-xL fusion protein at 0.5 and 1.0 µM caused a decrease in E. coli-induced apoptosis to 13.7 ± 2.7% and 9.8 ± 2.6%, respectively (p < 0.05). There was no decrease in E. coli-induced apoptosis by treatment with free Bcl-xL (Bcl-xL that was not conjugated to TAT).
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To verify that the TAT-BH4 peptide was taken up and internalized within the cells, laser-scanning confocal microscopy was performed on freshly isolated human lymphocytes loaded with a fluorescein-conjugated TAT-BH4. Serial sectioning of cells incubated with fluorescein-conjugated TAT-BH4 demonstrated a homogeneous uptake of the compound throughout the cell (Fig. 6, A and B). No fluorescence was demonstrated in cells not incubated with the fluorescein-tagged TAT-BH4.
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To determine whether TAT-BH4 was taken up and internalized within cells when administered in vivo, mice were injected with 1 mg of FITC-conjugated TAT-BH4, and 4 h later PBLs and splenocytes were harvested. Flow cytometry demonstrated uptake of the FITC-labeled TAT-BH4 into the cells compared with cells from mice that had no injection of labeled compound (Fig. 6C).
In vivo administration of TAT-BH4 decreases sepsis-induced lymphocyte apoptosis
Infusion of the active antiapoptotic TAT-BH4 peptide caused a significant decrease in sepsis-induced splenic CD3 T cell (p < 0.05) and B cell apoptosis (p < 0.01) compared with mice infused with inactive TAT-BH4(D)2 peptide (Fig. 7). There was a similar trend toward decreased thymic and blood CD3 T cell apoptosis in septic mice treated with TAT-BH4, but the differences were not statistically significant (n = 6 control, 8 TAT-BH4, and 8 inactive TAT-BH4(D2)).
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Negative selection yields a population of cells that were 90.4 ± 0.6% CD3 T cells by flow cytometric analysis (n = 20). The 330 probe sets showed significantly altered levels of expression >2-fold in E. coli-treated cells compared with control. A complete list of these probe sets that were up- or down-regulated is provided on the following link: 
www.ebi.ac.uk/arrayexpress/
. The accession number is: E-MEXP-555. Cell death, cell-cell interaction/signaling, and cell cycle were three high-level functions identified as significantly altered in E. coli-treated cells compared with control. All 55 of the genes with known involvement in apoptosis were up-regulated. Among these are genes involved in both the receptor and mitochondrial-mediated pathways. Only two genes, the zinc-finger transcription factor egr3 and the NR4A nuclear receptor family member nr4a3 (nor-1), showed a significant change in gene expression in cells treated with E. coli plus TAT-BH4 compared with E. coli plus TAT-BH4(D)2 and E. coli alone. Although the change in expression of these two genes was statistically significant, it was <2-fold. There were no significant changes in gene expression between the E. coli plus TAT-BH4(D)2- and E. coli alone-treated groups.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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An interesting and important point is the contrasting effect of sepsis on lymphocyte vs neutrophil apoptosis. Sepsis accelerates lymphocyte apoptosis, but delays neutrophil apoptosis. The normal t1/2 of the neutrophil is <24 h, but sepsis increases this t1/2 significantly. Although investigators have speculated that the delayed neutrophil apoptosis could be responsible for tissue injury, particularly in the lung, there is currently no convincing data to support this hypothesis. Studies in patients treated with G-CSF, which increased the number of activated neutrophils in patients several-fold and delayed apoptosis in neutrophils, showed no detrimental effects on lung or organ function. Nevertheless, this effect of sepsis to accelerate lymphocyte apoptosis, but delay neutrophil apoptosis, demonstrates the complexity of the cellular response to sepsis and may play a yet unrecognized role in the host response.
In addition to findings in animal models of sepsis, clinical studies have shown decreased bcl-2 gene expression and/or Bcl-2 protein concentration in patients with sepsis (9, 34). Bilbault et al. (34) noted enhanced lymphocyte apoptosis and a 10-fold down-regulation of bcl-2 in patients dying of sepsis vs survivors of sepsis. Our laboratory noted an increase in lymphocyte apoptosis associated with a marked decrease in lymphocyte Bcl-2 protein concentrations in patients with sepsis (9). Furthermore, patients with septic shock had the highest degree of lymphocyte apoptosis and the lowest concentrations of Bcl-2. Overall, the Bcl-xL/Bcl-2 animal sepsis studies together with clinical studies of septic patients demonstrating decreased bcl-2 expression and protein content provide a convincing rationale for efforts to deliver antiapoptotic Bcl-2-like compounds intracellularly to immune cells during sepsis.
The ability to deliver biologically active compounds into the intracellular compartment via conjugation to cell-penetrating peptides creates exciting new therapeutic approaches. Investigators have used cell-penetrating peptide constructs such as TAT basic domain and antennapedia to deliver intracellularly a host of diverse molecules, including proteins, peptides, oligonucleotides, plasmids, radionuclides, and imaging agents (20, 26, 27, 28, 33, 35). TAT basic domain and similar acting peptide constructs are able to cross into cells in a concentration-dependent manner that appears independent of cell surface receptors, but most likely involves the macropinocytosis machinery. The present study is the first to demonstrate the feasibility of delivering Bcl-2-like compounds to prevent sepsis-induced apoptosis, a key component of the pathologic process. Previous groups have shown that TAT-Bcl-xL is protective in animal models of brain ischemia/reperfusion injury (20, 21, 23). Other groups have documented that TAT-BH4 prevented apoptotic cell death using in vivo models of Fas-induced apoptosis, x-ray-induced apoptosis, and ischemia/reperfusion-induced apoptosis (22, 25, 26). Despite the encouraging results with the TAT-conjugated BH4, the protection afforded by these compounds is significantly less than that occurring in the Bcl-xL transgenic mice (see results in Figs. 1 and 2 vs Fig. 7). Hopefully, improvements in the delivery or potency of these compounds will increase their antiapoptotic efficacy. Furthermore, we have not shown that the improvement in survival observed in the Bcl-xL transgenic mice occurs in mice treated with TAT-BH4. These studies will be conducted in future experiments.
Both TAT-Bcl-xL fusion protein and TAT-BH4 peptide were effective in ameliorating the bacterial-induced lymphocyte apoptosis. The in vitro studies showed that TAT-BH4 was at least as potent as TAT-Bcl-xL in preventing apoptosis. Although there are advantages to each of the two agents, TAT-BH4 is superior to TAT-Bcl-xL in regard to the fact that it is readily synthesized in large quantities by solid-phase peptide synthesis and does not necessitate production using bacterial transfection and extraction. Thus, there is no endotoxin contamination in TAT-BH4 peptide. It is important to note that both of these compounds should be delivered to most cells in the body, but penetration across the blood brain barrier may be limited (33, 35, 36). Although lymphocytes are a primary target of sepsis-induced apoptosis, the gastrointestinal epithelial cells also undergo a large acceleration in apoptosis during sepsis (3, 14). Our group has shown that mice that overexpress Bcl-2 in the gastrointestinal epithelial cells have improved survival in a pneumonia model of sepsis (14). Therefore, it is possible that an additional beneficial effect of TAT-BH4 peptides in sepsis may be achieved by decreasing gastrointestinal epithelial cell apoptosis. It should be noted, however, that TAT-BH4 may also prolong the survival of other cells, including granulocytes and monocytes, which could prolong or exacerbate the hyperinflammatory phase of sepsis. Thus, it will be important to determine the effects of TAT-BH4 on survival in sepsis models.
The complex role of Bcl-2 in sepsis has been further elucidated by remarkable work of Iwata et al. (17). These investigators showed that transgenic mice that had overexpression of Bcl-2 in cells of either myeloid origin (neutrophils or monocytes/macrophages) or lymphoid origin had improved survival in sepsis. The survival benefit was preserved in animals that had adoptive transfer of the Bcl-2-overexpressing cells into Rag 1–/– mice. Therefore, Iwata et al. have hypothesized that Bcl-2 may exert a trans effect in which it acts by inducing release of a molecule(s) that has a protective effect against cell death. These investigators have also speculated that Bcl-2 may be released from injured cells and function as an agonist for a yet to be identified cell receptor. There is precedent for this hypothesis as a number of proteins, e.g., heat shock protein 70, have been shown to have such a role (37).
A key question remains: how does TAT-BH4 protect against apoptosis? The findings from the microarray analysis failed to disclose any mechanisms. Microarray analysis was performed on human lymphocytes treated with E. coli with or without TAT-BH4 (Affymetrix gene of chips). The 330 probe sets showed significantly altered levels of expression >2-fold in E. coli-treated cells compared with controls (no E. coli). However, only two genes, egr3 and nor1, showed significant change in gene expression in cells treated with E. coli plus Tat-BH4 compared with E. coli alone, at 5 h. Treatment with TAT-BH4 caused an increase in expression of egr3 and nor1. The significance of the increase in gene expression for these two genes is questionable given the fact that the increase in expression was <2-fold. Also, these two genes serve in proapoptotic function, whereas TAT-BH4 acts to block apoptosis. Given the relative lack of changes in gene expression in E. coli-treated cells incubated with TAT-BH4 vs cells incubated with E. coli alone, we speculate that TAT-BH4 protects in a posttranscriptional manner, possible by a direct protein-protein interaction.
Another interesting aspect of the Bcl-xL transgenic mouse studies that deserves comment is the protection afforded to B cells during sepsis. The lck promoter was used for Bcl-xL expression in transgenic mice in the present study and, therefore, T cells, but not B cells, overexpressed Bcl-xL. However, both T and B cells had decreased sepsis-induced apoptosis in Bcl-xL transgenic mice (Figs. 1 and 2). Previously, our laboratory noted similar findings in which overexpression in either T or B cells provided increased protection against sepsis-induced apoptosis to the other lymphocyte phenotype (11). This phenomenon of cross protection may be explained by a paracrine-like effect. Su et al. (36) demonstrated that paracrine-mediated apoptosis occurred in Jurkat T cells and was mediated by shedding of Fas ligand. Therefore, if T lymphocyte apoptosis is decreased in the spleen, neighboring B cells may also be protected.
In conclusion, TAT-BH4 peptide is readily internalized into human lymphocytes and has potent antiapoptotic activities against bacterial-induced lymphocyte apoptosis both in vitro and in vivo. Given the extensive animal and clinical studies demonstrating an important role for apoptosis in sepsis and the protective effect of Bcl-2 overexpression, these peptides may offer a novel therapy for this highly lethal disorder.
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Disclosures |
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Footnotes |
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1 This work was supported by National Institutes of Health Grants GM44118, GM55194, GM008795, GM66202, CA94056, and CA82841; Grant CMPB of the Deutsche Forschungsgemeinschaft; and the Alan A. and Edith L. Wolff Foundation. 
2 Address correspondence and reprint requests to Dr. Richard S. Hotchkiss, Department of Anesthesiology, 660 South Euclid Avenue, St. Louis, MO 63110. E-mail address: hotchkir{at}msnotes.wustl.edu
3 Abbreviation used in this paper: CLP, cecal ligation and puncture.
Received for publication September 28, 2005. Accepted for publication February 15, 2006.
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