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Departments of
*
Surgery and
Medicine, University of Washington, Seattle, WA 98104;
Stem Cell Incorporated, Sunnyvale, CA 94068; and
Department of Pathology, Stanford University, Stanford, CA 94305
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A number of recent studies have reported that sepsis induces apoptosis of lymphocytes, (7, 8, 9), epithelial cells in the intestine and lung (7, 10, 11), and endothelial cells (11). These observations led to experimental studies designed to block apoptosis in various cell types. Hotchkiss et al.(12) showed that over-expression of the anti-apoptotic protein Bcl-2 in either B or T lymphocytes improved survival in experimental sepsis induced by cecal ligation and puncture (CLP) 3 in mice. Moreover, they showed that treatment with a broad-spectrum caspase inhibitor reduced mortality in mice subjected to CLP (13, 14). Both the caspase inhibitor and over-expression of Bcl-2 blocked apoptosis of lymphocytes in the spleen and thymus, and they suggested that the mechanism of action of these interventions was through the protection of lymphocytes from apoptosis. Adoptive transfer of T cells that over-expressed Bcl-2 also offered protection from septic death (13). They proposed that immunodepression resulting from lymphocyte apoptosis is a major contributor to mortality in experimental sepsis. More recently, over-expression of Bcl-2 under an epithelial cell specific promoter was also reported to provide protection from death following CLP suggesting an important role for intestinal epithelial cells in sepsis (15).
In the experiments reported here, we determined the effect of over-expression of Bcl-2 in myeloid cells on survival from sepsis induced by CLP. There was 100% survival of mice over-expressing Bcl-2 in myeloid cells vs 25% in control mice. Moreover, adoptive transfer of myeloid cells from bone marrow of Bcl-2 over-expressing mice into normal C57BL/6 or Rag-1-/- mice also markedly improved survival. Mice over-expressing Bcl-2 in myeloid cells exhibited markedly increased neutrophil accumulation and reduced bacteria counts in peritoneum following CLP, consistent with an increased host response.
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Materials and Methods |
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All protocols were approved by the University of Washington Animal Care and Use Committee (Seattle, WA) and complied with the National Institutes of Health guidelines for animal use.
C57BL/6 mice were purchased from Charles River Breeding Laboratories (Wilmington, MA). Transgenic mice over-expressing human Bcl-2 under the myeloid cells or T lymphocytes were bred in our animal facility. These transgenic mice were on a C57BL/6 background and specific expression was controlled by cell specific promoters. The MRP8 protein is restricted to myeloid cells and the human promoter for this protein was used to drive expression of human Bcl-2 (16). Polymorphonuclear neutrophils (PMNs) and circulating monocytes, but not tissue macrophages, from the human MPR8 (hMRP8)-Bcl-2 mice express the transgene in myeloid cells as expected (16). The Eµ-promoter is active in lymphocytes and mice were produced that over-express human Bcl-2 under control of this promoter. Several strains of transgenic mice have been produced using this promoter resulting in expression of Bcl-2 in only T lymphocytes (17) or only B lymphocytes (18) or both (19). The site of incorporation of the transgene appears to determine where expression will occur. We used the mice expressing Bcl-2 in T lymphocytes. Bcl-2 expression under the Eµ-promoter was restricted to T lymphocytes (17) for experiments reported here. The transgenic mice were genotyped by PCR using genomic DNA from a small portion of the tail. Rag-1-/- mice on a C57BL/6 background were obtained from The Jackson Laboratory (Bar Harbor, ME). These mice are homozygous for the Rag-1-/-Tml Mom mutation and produce no mature T or B cells (20). Mice were maintained at the University of Washington, Department of Comparative Medicine facility (Seattle, WA).
Experimental sepsis induced by CLP
Mice were anesthetized with halothane, an abdominal midline incision was made, and the cecum was gently removed and ligated below the ileocecal valve without obstruction of the ileum or colon. The cecum was then subjected to a single "through and through" perforation with a 20-gauge needle and a small amount of cecal contents was gently expressed through the needle wound. The bowel was carefully returned to its original position, and then the abdominal incision was closed in layers with 4–0 sutures.
Adoptive transfer of bone marrow cells
HMRP8-Bcl-2 and C57BL/6 mice were killed and the long bones were removed from their legs. Bone marrow was removed by flushing with RPMI containing 1% FBS and 7 U/ml heparin, and the marrow cell suspension was filtered through a 70-µm mesh, producing a single cell suspension. Bone marrow cells were washed with PBS containing 0.5% BSA and 2 mM EDTA. CD11b-positive cells were isolated using magnetic beads as described by the manufacturer (Miltenyi Biotec, Auburn, CA). These CD11b-positive cells were >90% PMNs determined by differential cell counts. Cells were resuspended in PBS, and 107 cells/mouse from hMRP8-Bcl-2 mice or control C57BL/6 mice were injected into the peritoneum of C57BL/6 or Rag-1-/- mice 2 h after CLP.
Survival after CLP
Mice were followed for up to 8 day after CLP. Each animal was scored using an assessment form that evaluated each animal’s health as described by Morton and Griffiths (21), and euthanized if they exceeded a predetermined score that suggested irreversible sepsis. All animals received 0.5 mg of Imipenem 2 h after CLP, and the injection of 0.5 mg/mouse of Imipenem in 1.0 ml of D5W was continued every 12 h for 4 days.
PMN and bacteria counts in peritoneal lavage fluid
In a separate set of experiments, mice were killed 24 h after CLP and the peritoneal cavity was washed with sterile saline. Peritoneal lavage was performed by making a small incision in the midline of the abdomen and injecting 1–2 ml of sterile saline. The saline was removed by aspiration and the process was repeated until a total of 5 ml of lavage fluid was obtained. The total number of leukocytes in a 0.1-ml aliquot was determined after methylene blue nuclear staining. A cytospin preparation was made using 0.07 ml of lavage fluid and differential cell counts were determined following Diff-Quick staining (Dade Behring, Deerfield, IL). Aliquots of serial 1–10 dilutions of the peritoneal lavage fluids were plated on tryptic soy agar dishes (PML Microbiologicals, Tualitin, OR), and the number of colonies was counted at 24 and 48 h after CLP.
Thymus cellularity determination
Twenty-four hours after CLP, C57BL/6 and hMRP8-Bcl-2 mice were sacrificed by cervical dislocation. Thymi were harvested and single cell suspensions were prepared by mechanical disruption and maceration between frosted glass slides, as described by Cooke et al. (22). Viable cells, as assessed by trypan blue exclusion, were enumerated by hemocytometer counting.
TUNEL assay
Transgenic and non-transgenic mice were sacrificed 24 h after CLP, and their thymus and entire intestine was removed. The intestine was opened along the length of its cephalocaudal axis and washed in 4% neutral buffered formalin to removed luminal contents. Then the tissue was rolled from the proximal to the distal end and fixed with 10% neutral buffered formalin. Paraffin embedded sections from thymus and intestine were examined for DNA strand breaks TUNEL (In Situ Cell Death Kit; Roche Molecular Biochemicals, Indianapolis, IN) as described by the manufacturer.
Cyokine assay
Whole blood was drawn by cardiac puncture at 12 or 24 h after CLP and plasma prepared. Blood was centrifuged for 10 min at 1200 rpm to separate plasma from cells. Concentrations of IL-1
, IL-1
, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12(p40), IL-12(p70), IL-17, G-CSF, GM-CSF, IFN-
, KC, MIP-1, RANTES, and TNF- were measured by Bio-Plex protein assay system (Bio-Rad, Hercules, CA) and read by Luminex 100 system (Mirai Bio, Alameda, CA) according to manufacturer specifications.
Statistical analysis
The data are presented as the means ± SEM. Statistical analysis was performed using the two-tailed Student t test. Survival was evaluated using Fisher’s exact test. Differences associated with a value of p < 0.05 were considered statistically significant.
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Results |
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The survival curve of hMRP8-Bcl-2 mice compared with C57BL/6 mice is shown in Fig. 1. The hMRP8-Bcl-2 mice showed dramatic improvement in survival compared with C57BL/6 mice. Survival following CLP was 100% in both groups at 24 h but by 72 h survival in the C57BL/6 group decreased to 50% and by 144 h post CLP it was 25%. Survival in the hMRP8-Bcl-2 group was 100% at the end of the experiment (p < 0.02). The experiment was repeated twice for a total of 12 animals in each group.
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The total number of leukocytes and PMNs in peritoneal lavage fluid from hMRP8-Bcl-2 and C57BL/6 mice at 24 h after CLP was determined in separate experiments. Total leukocyte (Fig. 3A) and PMN (Fig. 3B) counts were significantly higher in the hMRP8-Bcl-2 mice compared with C57BL/6 control mice (PMNs: 280 ± 66.6 x 104/ml vs 62.1 ± 32.4 x 104, p = 0.042). Peritoneal leukocytes in the hMRP8-Bcl-2 mice were 86.3% PMNs and in the C57BL/6 mice 51.4% PMNs.
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The number of bacteria in the peritoneal lavage fluid from hMRP8-Bcl-2 and C57BL/6 mice was determined after CLP. Lavage fluid was obtained at 24 h after CLP and colonies were counted after 24 and 48 h of culture. The number of colonies formed in the hMRP8-Bcl-2 mice was significantly lower than in the C57BL/6 mice (Fig. 4).
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We performed adoptive transfer of CD11b-positive bone marrow cells from hMRP8-Bcl-2 mice or C57BL/6 mice into C57BL/6 mice to determine whether the protection seen in the hMRP-Bcl-2 mice could be transferred to normal mice. Transfer of control or Bcl-2 over-expressing bone marrow cells was performed 2 h after CLP. The results of these experiments are shown in Fig. 5. All mice that received CD11b-positive bone marrow cells from hMRP8-Bcl-2 mice survived 6 day following CLP, whereas none of the mice receiving CD11b-positive bone marrow cells from C57BL/6 mice survived past day 4.
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We transferred CD11b-positive bone marrow cells from hMRP8-Bcl-2 or C57BL/6 mice into Rag-1-/- mice 2 h after CLP to determine whether lymphocytes were necessary for the protection observed in the hMRP8-hBcl-2 mice. These Rag-1-/- mice are totally deficient in mature T and B cells. Survival of Rag-1-/- mice that received CD11b-positive bone marrow cells from hMRP8-Bcl-2 mice was 87.5%, whereas Rag-1-/- mice that received CD11b-positive bone marrow cells from C57BL/6 control mice was 12.5% at day 8 (Fig. 6).
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Survival curves for Eµ-Bcl-2 mice compared with C57BL/6 control mice are shown in Fig. 7. Survival in the Eµ-Bcl-2 mice was significantly greater than for the control mice. There was 87.5% survival in transgenic mice compared with 22.2% survival in the control mice.
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DNA strand breaks were determined by TUNEL staining of intestine and thymus taken from mice 24 h after CLP. TUNEL-positive cells were seen in the intestinal villi in the control mice (Fig. 8A). In contrast, hMRP8-Bcl-2 mice had fewer of these TUNEL-positive cells (Fig. 8B). There was no difference in TUNEL staining in the thymus of C57BL/6 control mice and hMRP8-Bcl-2 mice and both showed extensive TUNEL positive staining. (Fig. 8, C and D).
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Plasma from transgenic and nontransgenic littermate animals was measured for 18 different cytokines (IL-1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12(p40), IL-12(p70), IL-17, G-CSF, GM-CSF, IFN-, KC, MIP-1, RANTES, and TNF-). There is no difference between transgenic and control mice at baseline or at 12 h post CLP (transgenic mice: n = 6; littermate control mice: n = 6). At 24 h, IL-1, IL-1, IL-2, IL-3, IL-4, and IL-6 were significantly reduced in hMRP8-Bcl-2 mice compared with littermate controls (transgenic mice: n = 6; littermate control mice: n = 5).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Protection in Rag-1-/- mice, indicates that mature lymphocytes are not necessary for survival, since they are deficient in mature T and B lymphocytes. Also, there was a significant reduction in lymphocyte number in the thymus of both C57BL/6 and hMRP8-Bcl-2 mice following CLP and the reductions in both groups were equivalent. These data suggests that over-expression of Bcl-2 in T lymphocyte under the Eµ-promoter and in myeloid cells under the hMRP8-promoter, may exert their protective effect through a pathway other than by blocking apoptosis of the over-expressing cell (7, 12, 13, 14).
Previous experiments by Hotchkiss and colleagues (12, 13, 14) showed that over-expression of Bcl-2 in T cells or B cells, or adoptive transfer of Bcl-2 over-expressing T lymphocytes, or injection of caspase inhibitors reduced lymphocyte apoptosis and resulted in improved survival in sepsis. Consistent with these studies, we show here that over-expression of Bcl-2 in T cells in a different transgenic line (Eµ-Bcl-2) afforded protection (see Fig. 7). These results would seem to suggest a major role for lymphocytes in eliminating pathogens through an unknown mechanism. Hotchkiss et al. (13) proposed that prevention of lymphocyte apoptosis might result in increased production of cytokines such as IFN- or IL-2 as a first step in a protective pathway since IFN- and IL-2 have been shown to be efficacious in models of sepsis (24). However, our results show that lymphocytes are not necessary since adoptive transfer of myeloid cells over-expressing Bcl-2 protected Rag-1-/- mice, which do not have mature T and B cells. Moreover, the result of TUNEL staining shows that thymocytes of hMRP8-Bcl-2 and C57BL/6 mice had an equivalent amount of apoptosis. In addition, our measurement of cytokines suggests that they did not significantly alter the outcome. There was no specific cytokine increase (or decrease) in transgenic mice that can explain the animal survival.
Furthermore, Coopersmith et al. (15) have shown that over-expression of Bcl-2 in epithelial cells affords protection in CLP. Therefore, we propose an alternative explanation for the protection seen following CLP in mice that over-express Bcl-2 in variouscell lineages. We propose the release of cyto-protective or anti-inflammatory molecule(s) by cells of multiple lineages (i.e., T, B, epithelial, and myeloid) when they over-express Bcl-2.
The mechanism of this protection is not known, however, in vitro studies have shown that over-expressing of Bcl-2 or two of its anti-apoptotic homologues reduces inflammatory signaling in endothelial cells (25, 26) or macrophages (27).
Although the mechanism of protection by the cyto-protective molecules is unknown and under investigation in our lab, our pathological observations indicate that the protection might result from a modification of apoptosis. Others have observed increased intestinal apoptosis in septic animals (7, 8, 28). Consistent with these observations, TUNEL-positive cells were seen in the epithelial villi of control mice, whereas, hMRP8-Bcl-2 mice had a reduced number of these TUNEL-positive cells. This observation is consistent with another report (15) showing intestinal epithelial cell apoptosis peaking at 24 h after CLP.
The increased accumulation of PMNs in the peritoneum of the hMP8-Bcl-2 mice suggests that this could account for protection in the hMRP8-Bcl-2 mice. The importance of PMN accumulation at the site of infection is best shown by patients that suffer from severe leukocyte adhesion deficiency type I (LAD-1) syndrome (reviewed in Refs.29, 30, 31). These patients have a deficiency of 2-integrin receptors, and as a consequence, their PMNs fail to emigrate to the site of infection. This defect in PMN emigration results in early death from recurrent infections unless bone marrow transplantation is performed early in life (30). Clearly, PMNs are required to control infections; however, an excessive neutrophil response might cause damage to the host as a result of undesired release of toxic substances (reviewed in Refs.32, 33, 34).
The potential for inflammatory injury by PMNs raises the question as to whether increasing PMN delivery to the site of inflammation would be helpful or harmful. There have been only a few studies examining the consequences of an increased neutrophil response. We showed that there were more PMNs in peritoneal lavage fluid 24 h after CLP in hMRP8-Bcl-2 mice compared with C57BL/6 control mice. This was accompanied by a reduction in bacteria counts in the hMRP8-Bcl-2 mice compared with C57BL/6 mice at this time-point.
In a recent study, administration of LPS 4 day before infectious challenge also enhanced bacterial clearance and improved survival in septic peritonitis (35). LPS priming resulted in an accumulation of peritoneal PMNs and these cells had a normal ability to produce reactive oxygen metabolite, and they had elevated surface density of Mac-1 and FcR. These factors are thought to act in concert to improve the total anti-microbial activity and the bacterial clearance in the infected peritoneal cavity. In another study of septic peritonitis, the administration of oligodeoxy nucleotides increased the number of PMNs at the site of inflammation and likewise improved outcome (36). These studies and the present study are consistent with a beneficial effect of an increase in PMN number at the site of infection, i.e., PMNs promote host defense and thus increasing their number enhances host defense.
We observed more peritoneal PMNs in hMRP8-Bcl-2 transgenic mice and it is reasonable to ascribe the increase to prolonged survival of PMNs. However, Lagasse and Weissman (37) noted that, although neutrophils from hMRP8-Bcl-2 mice showed prolonged survival ex vivo, PMN accumulation and clearance from the peritoneum following thioglycollate-induced inflammation was not different from controls. In addition, phagocytosis of aged PMNs isolated from hMRP8-Bcl-2 mice was not different from phagocytosis of PMNs from control mice. They suggested that the signal for phagocytosis was not apoptosis, but was instead related to the age of the PMN (16). Thus, the increased number of PMNs may have resulted from increased recruitment to the site of inflammation rather than from decreased apoptosis.
In summary, this study shows that transgenic over-expression of Bcl-2 in myeloid or lymphoid cells, or adoptive transfer of Bcl-2- expressing myeloid cells to Rag-1-/- mice, improves survival in CLP-induced sepsis. This effect might be attributable to PMN survival and/or to the release of a cyto-protective molecule(s) from the over-expressing cells that might alternate the pathway of apoptosis in intestinal epithelium as the mechanisms of protection.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Akiko Iwata, University of Washington, Harborview Medical Center, Department of Surgery, Box 359796, 325 9th Avenue, Seattle, WA 98104. E-mail address: aiwata{at}u.washington.edu
3 Abbreviations used in this paper: CLP, cecal ligation and puncture; PMN, polymorphonuclear neutrophils; hMPR8, human MPR8.
Received for publication November 20, 2002. Accepted for publication July 7, 2003.
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References |
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). There was a significant improvement in the survival of mice that received Bcl-2 expressing PMNs by adoptive transfer compared with control mice.
