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Published ahead of print on March 5, 2003, doi:10.1164/rccm.200208-950OC
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American Journal of Respiratory and Critical Care Medicine Vol 167. pp. 1641-1649, (2003)
© 2003 American Thoracic Society

Original Article

ß2 Microglobulin Knockout Mice Are Resistant to Lethal Intraabdominal Sepsis

Edward R. Sherwood, Cheng Y. Lin, Weike Tao, Christopher A. Hartmann, Jay E. Dujon, Andrew J. French and Tushar K. Varma

Department of Anesthesiology, University of Texas Medical Branch, Shriners Hospital for Children, Galveston, Texas

Correspondence and requests for reprints should be addressed to Edward R. Sherwood, M.D., Department of Anesthesiology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0591. E-mail: ersherwo{at}utmb.edu


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ß2 microglobulin knockout (ß2M-/-) mice lack CD8+ T and natural killer T cells. We hypothesized that ß2M-/- mice are resistant to lethal intraabdominal sepsis. To test this hypothesis, mortality, cytokine production, and physiologic function were assessed in ß2M-/- mice during sepsis caused by cecal ligation and puncture (CLP). ß2M-/- mice survived significantly longer than wild-type mice after CLP but ultimately exhibited 100% mortality. Treatment of ß2M-/- mice with anti-asialoGM1 to deplete natural killer cells conferred greater than 70% long-term survival. Compared with wild-type mice, ß2M-/- mice treated with anti-asialoGM1 produced decreased amounts of proinflammatory cytokines and did not exhibit hypothermia or metabolic acidosis after CLP. Adoptive transfer of CD8+ T and natural killer cells into ß2M-/- mice treated with anti-asialoGM1 re-established CLP-induced mortality. CD8 knockout mice treated with anti-asialoGM1, which are specifically deficient in CD8+ T and natural killer cells, exhibited 40% long-term survival after CLP. Furthermore, treatment of wild-type mice with antibodies to CD8 and asialoGM1 conferred a significant survival benefit compared with wild-type mice treated with nonspecific IgG. These findings demonstrate that ß2M-/- mice treated with anti-asialoGM1 are resistant to CLP-induced mortality and that depletion of CD8+ T and natural killer cells largely accounts for the survival benefit observed in these mice.

Key Words: CD8+ T lymphocytes • natural killer cells • septic shock

More than 750,000 cases of sepsis occur each year in the United States (1). Mortality rates range from 3 to 43%, depending on the patient population, and average annual costs of caring for patients with sepsis are estimated to be $10 to 16 billion (1, 2). Epidemiologists predict that the incidence of sepsis will increase as the American population ages and as medical technology advances (3). Survival during septic shock is dependent on cardiovascular and pulmonary support, treatment of infection with antibiotics, and elimination of initiating sources such as abscesses and necrotic tissue. However, much of the morbidity and mortality associated with sepsis is due to systemic inflammation. The sepsis syndrome is caused, in part, by release of macrophage-derived proinflammatory cytokines and chemokines such as tumor necrosis factor {alpha} (), interleukin (IL)-1, and IL-8 as well as noncytokine mediators of inflammation such as complement components, eicosanoids, and platelet-activating factor (4, 5). Systemic release of these factors activates pathophysiologic processes that can result in diffuse vascular injury, multiorgan dysfunction, and death (6). Numerous approaches aimed at attenuating the acute inflammatory response have been studied in clinical sepsis (7, 8). Blockade or inhibition of tumor necrosis factor {alpha}, IL-1, platelet-activating factor, and prostaglandins, among others, have been extensively studied and have not shown clinical benefit (7). More recent clinical trials have shown that agents such as activated protein C that inhibit both the proinflammatory and procoagulant components of sepsis confer significant survival benefit for patients with severe sepsis (8). However, in the studies published to date, less than 10% improvement in survival has been reported. The relative ineffectiveness of antiinflammatory treatment approaches for sepsis is likely due to several factors. Specifically, the complexity of the inflammatory response and the redundancy of inflammatory mediators make blockade of a single factor ineffective. In addition, many of the targeted mediators are produced early in sepsis and blockade of these factors outside of a critical window of opportunity may decrease the effectiveness of treatment. A final consideration is that we do not fully understand the mechanisms causing vascular injury, multiorgan dysfunction, and death in sepsis.

Much attention has been focused on macrophage function during sepsis. However, few studies have assessed the roles of CD8+ T, natural killer, and natural killer T cells in the pathogenesis of septic shock. Recent reports indicate that ß2 microglobulin knockout (ß2M-/-) mice are resistant to the systemic Shwartzman reaction, a lethal shock syndrome caused by endotoxin challenge in mice that are primed with endotoxin or IL-12 (9). ß2M-/- mice are deficient in CD8+ T and natural killer T cells that require the ß2 microglobulin–associated Class I major histocompatability complex and CD1 molecules, respectively, for their maturation and differentiation (10–13). In the present study, we studied the response of ß2M-/- mice to septic peritonitis caused by cecal ligation and puncture (CLP). We hypothesized that ß2M-/- mice are resistant to mortality caused by CLP. To test this hypothesis, we measured mortality, proinflammatory cytokine production, and physiologic function in ß2M-/- mice after CLP. In further studies, we assessed the specific contributions of CD8+ T, natural killer, and natural killer T cells to the pathogenesis of lethal intraabdominal sepsis.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
Female, 6- to 8-week-old C57BL/6J (ß2M-/-, strain B6.129P-B2mtm1 Unc) and CD8{alpha} knockout (CD8-/-, strain B6.129S2-Cd8atm1Mac) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Natural killer cells were depleted by treatment of mice with anti-asialoGM1 (50 µg, intraperitoneal; Cedarlane Laboratories, Hornby, ON, Canada) 24 hours before CLP. CD8+ T cells were depleted by injection of anti-CD8{alpha} (50 µg, intraperitoneal, Cedarlane Laboratories) 24 hours before CLP. Control mice were treated with nonspecific IgG (0.2 ml, intraperitoneal; Sigma Chemical, St. Louis, MO). The Institutional Animal Care and Use Committee at the University of Texas Medical Branch approved all studies.

Cecal Ligation and Puncture
Mice were anesthetized with 2% isoflurane in oxygen via face mask. A 1- to 2-cm midline incision was made through the abdominal wall; the cecum was identified and ligated with a 3-0 silk tie, 1 cm from the tip. Care was taken not to cause bowel obstruction. A single puncture of the cecal wall was performed with a 20-gauge needle. The cecum was lightly squeezed to express a small amount of stool from the puncture site to assure a full thickness perforation. Mice did not receive fluid resuscitation. The cecum was returned to the abdominal cavity and the incision was closed with surgiclips. All mice being compared in survival studies underwent CLP in the same sitting, and mice from different cages were randomly passed to the surgeon in a blinded fashion to minimize experimental bias.

Isolation of Mononuclear Cells from Spleen and Liver
Spleens were harvested from mice killed by cervical dislocation, placed in 35-mm dishes containing RPMI-1640 media with 10% fetal bovine serum, and homogenized by smashing with the plunger from a 1-ml syringe. The dispersed spleens were passed through a 100-µm nylon mesh, erythrocytes were lysed (Erythrocyte Lysis Kit; R&D Systems, Minneapolis, MN), and cells were counted using a hemacytometer. Livers were harvested, pressed through a 200-gauge stainless steel wire mesh, and collected in media. The dispersed liver suspension was transferred to a 50-ml conical centrifuge tube and centrifuged at 50 x g for 5 minutes to pellet debris and hepatocytes. The supernatant was harvested, mixed with isotonic 35% percoll, and centrifuged (750 x g for 15 minutes). The supernatant was discarded, erythrocytes were lysed, and the resulting mononuclear cells were counted using a hemacytometer. Viability of cells was greater than 95% in all cases as determined by trypan blue exclusion.

Flow Cytometry
Fluorescein isothiocyanate–conjugated anti–{alpha}ß T cell receptor, phycoerythrin-conjugated anti-NK1.1, fluorescein-conjugated anti-CD8, phycoerythrin-conjugated anti-CD3, phycoerythrin-conjugated anti-CD4, and isotype control animals were purchased from Caltag Laboratories (Burlingame, CA). Mononuclear cells were isolated as outlined previously: cells (1 x 106/tube) were placed in polystyrene tubes containing labeling antibodies (0.5–1 µg of antibody per tube) and incubated (4°C) for 30 minutes. Cells were washed with 2 ml of cold phosphate-buffered saline and fixed with 250 µl of 1% paraformaldehyde. Samples were analyzed with a FACScan flow cytometer (Becton-Dickinson, San Diego, CA).

Adoptive Transfer of CD8+ T, Natural Killer, and Natural Killer T Cells
Splenic leukocytes were harvested as described previously. The leukocyte preparation was enriched using a CD8 enrichment column (R&D Systems). Briefly, cells were incubated with an antibody mixture that binds macrophages/monocytes, B cells, and CD4+ T cells. The cell suspension was passed through a column containing anti-IgG–coated glass beads that bind antibody-laden cells. Because CD8+ T cells, natural killer cells, and most natural killer T cells do not bind the supplied antibodies, these cells were washed through the column and collected. The enriched cell preparation was resuspended in lactated Ringer's solution followed by intraperitoneal injection (1.5 x 107 cells/mouse) into recipient mice. Mice were used in studies 24 to 48 hours after adoptive cell transfer.

RNAse Protection Assay
Spleen, heart, lung, and liver were harvested and flash frozen in liquid nitrogen. Samples were stored at -80°C until use. Total RNA was isolated using Tri-Reagent (Molecular Research Center, Cincinnati, OH). The RNAse protection assay was performed using the Riboquant system (B-D Pharmingen, San Diego, CA) as per the manufacturer's instructions. Briefly, radiolabeled RNA probes were synthesized from DNA template sets using T7 RNA polymerase, 32P-uridyl triphosphate, and pooled nonradiolabeled nucleotides. Isolated total RNAs (20 µg/sample) were hybridized with the purified riboprobes and subjected to RNAse digestion. Protected RNA species were separated on 5% polyacrylamide sequencing gels using 0.5x Tris–borate–ethylenediaminetetraacetic acid running buffer. Gels were run at 50 W constant power for 70 minutes and dried under vacuum, and the protected fragments were visualized using autoradiography.

ELISA
Peritoneal fluid was harvested from mice by peritoneal lavage with 5 ml of sterile saline. Cytokine levels in peritoneal fluid were determined using an ELISA according to the manufacturer's protocol (eBioscience, San Diego, CA). Briefly, standards or experimental samples were added to microtiter plates coated with capture antibody to the cytokine of interest and incubated for 2 hours. After washing, horseradish peroxidase–conjugated, cytokine-specific antibody was added to each well, incubated for 2 hours, and washed. Substrate solution was added and incubated for 30 minutes, and the reaction was terminated by the addition of stop solution. Cytokine levels were determined by measuring optical density at 450 nM using a microtiter plate reader (Dynatech Laboratories, Chantilly, VA).

Measurement of Temperature, Acid–Base Balance, Organ Injury, and Bacterial Counts in Septic Mice
Blood samples for chemistry, bacteriology, and gas measurements were obtained from separate groups of mice anesthetized with 1.5% isoflurane in 100% oxygen via face mask. The carotid artery was isolated and lacerated under direct visualization using a surgical microscope. For blood gas measurements, blood was harvested into a heparinized syringe. Blood gas measurements were performed using iStat cartridges (iStat Corporation, East Windsor, NJ).

Lung injury was determined by measuring wet/dry weight and PO2/FIO2 ratios. Wet/dry weight ratios were determined by harvesting lung tissue from killed mice. All extraneous tissue was removed; lungs were blotted dry and weighed. Lungs were then desiccated by heating (55°C) in a drying oven for 5 days and weighed. The wet/dry weight ratio was calculated as an index of increased lung water. PO2/FIO2 ratios were calculated from arterial PO2 measurements obtained from mice breathing 100% oxygen. Plasma alanine aminotransferase/aspartate aminotransferase and creatinine levels were measured as indices of acute liver and renal injuries, respectively. Blood was obtained by carotid artery laceration. Alanine aminotransferase, aspartate aminotransferase, and creatinine levels were measured in the clinical chemistry laboratory at the Shriners Burns Hospital, Galveston. Bacterial counts were performed on aseptically harvested blood and peritoneal lavage fluid. Samples were serially diluted in sterile saline and cultured in tryptic soy agar pour plates. Plates were incubated for 48 to 72 hours and colony counts were performed.

Body temperature was measured by insertion of a rectal temperature probe in mice that were anesthetized with 1.5% isoflurane.

Statistical Analysis
All data were analyzed using GraphPad Prism software (GraphPad Software, San Diego, CA). Survival curves were compared using the log rank test. The mean and SEM were calculated in experiments with multiple data points. Data from multiple group experiments were analyzed using one-way analysis of variance followed by a post hoc Tukey test to compare pairs of data.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
ß2M-/- Mice Treated with Anti-asialoGM1 Are Depleted of Cd8+ T, Natural Killer, and Natural Killer T Cells
The relative proportions of CD8+ T, natural killer, and natural killer T cells in the spleen and liver of wild-type and ß2M-/- mice were determined using flow cytometry (Figure 1) . Injection of anti-asialoGM1 significantly (p < 0.05) decreased splenic and hepatic natural killer cells by 90 and 97%, respectively, in control mice and by 96 and 95%, respectively, in ß2M-/- mice (Figure 1). Wild-type and ß2M-/- mice possess similar numbers of natural killer cells. Splenic and hepatic natural killer T cell numbers were significantly (p < 0.05) decreased by approximately 60% in ß2M-/- mice compared with wild-type control animals. Treatment of mice with anti-asialoGM1 did not decrease natural killer T cell numbers in liver or spleen. ß2M-/- mice exhibited significantly (p < 0.05) decreased numbers of splenic and hepatic CD8+ T cells compared with wild-type control animals. Specifically, the CD8+ T cell populations in spleen and liver were decreased by 91 and 94%, respectively. Anti-asialoGM1 treatment did not alter CD8+ T cell numbers (Figure 1).



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  		Figure 1. Overview of splenic and hepatic CTL, NK, and NKT cell populations in wild-type mice treated with nonspecific IgG (WT) or anti-asialoGM1 ({alpha}AsGM1) and ß2 microglobulin knockout (ß2M-/-) mice treated with nonspecific IgG or anti-asialoGM1 (ß2M-/-/{alpha}AsGM1). The percentage of each cell population in spleen and liver is presented. The total number of each cell population in spleen and liver was determined by multiplying the percent positive for each cell type by the average mononuclear cell yield from spleen (8 x 107) and liver (4 x 106). Results were calculated from at least three different mice for each group. *Significantly (p < 0.05) less than that for wild-type control animals. CTL = CD8+ T cells; NK = natural killer; NKT = natural killer T cells; WT = wild type.

 
In addition to the percentage of CD8+ T, natural killer, and natural killer T cells in the spleen and liver of mice, data are also presented on the total number of each cell type in each organ (Figure 1). Although the percentage of natural killer and natural killer T cells in the spleen is less than in the liver, the total number of natural killer and natural killer T cells in the spleen is fourfold to eightfold greater than in the liver. This is due to the larger average number of mononuclear cells isolated from the spleen (8 x 107 cells/spleen) compared with liver (4 x 106 cells/liver). The same proportional changes in total numbers of each cell type were observed as reported for average cell numbers previously.

ß2M-/- Mice Treated with Anti-asialoGM1 Are Resistant to Mortality after CLP
Survival analysis showed that control mice exhibited a median survival time of 18 hours with 100% mortality occurring by 24 hours after CLP (Figure 2) . A median survival time of 18 hours was also observed in wild-type mice treated with anti-asialoGM1. Although the time to 100% mortality was 78 hours in wild-type mice treated with anti-asialoGM1, overall survival was not statistically different from control mice (Figure 2). ß2M-/- mice exhibited a median survival time of 42 hours, and 100% mortality occurred at 90 hours after CLP (Figure 2). Overall survival was significantly (p < 0.05) prolonged in ß2M-/- mice compared with control mice. ß2M-/- mice treated with anti-asialoGM1 exhibited 100% survival out to 2 weeks after CLP, which was significantly (p < 0.05) greater than that for control mice or ß2M-/- mice treated with nonspecific IgG (Figure 2).



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  		Figure 2. ß2M-/- mice treated with anti-asialoGM1 are resistant to lethal intraabdominal sepsis. Wild-type mice treated with nonspecific IgG (WT) or anti-asialoGM1 ({alpha}As-GM1, NK cell depleted) and ß2M-/- mice treated with nonspecific IgG (ß2M-/-, CD8+ T,- NKT cell depleted) or anti-asialoGM1 (ß2M-/-/{alpha}AsGM1, CD8+ T, NK, NKT cell depleted) underwent CLP and were observed for mortality. n = 10 to 15 mice/group. CLP = cecal ligation and puncture.

 
Proinflammatory Cytokine Production Is Attenuated in ß2M-/- Mice Treated with Anti-asialoGM1
Studies were performed to assess production of proinflammatory cytokine and chemokine messenger RNAs in the spleen, heart, lung, and liver of wild-type mice and ß2M-/- mice treated with anti-asialoGM1 (Figure 3) . Messenger RNAs for IL-1ß, IL-6, and macrophage inflammatory protein-2 (mouse IL-8 homolog) were strongly induced in the spleen, heart, liver, and lung of wild-type mice at 16 hours after CLP. Compared with wild-type mice, expression of IL-1ß, IL-6, and macrophage inflammatory protein-2 was markedly attenuated in all tissues obtained from ß2M-/- mice treated with anti-asialoGM1. IFN-{gamma} messenger RNA expression was induced in the spleen and liver of wild-type mice and was decreased in liver and spleen obtained from ß2M-/- mice treated with anti-asialoGM1. IFN-{gamma} messenger RNA expression was weakly induced or not observed in heart and lung from both groups (Figure 3).



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  		Figure 3. Cytokine and chemokine messenger RNA expression is decreased in ß2M-/- mice treated with anti-asialoGM1 after CLP. Spleen, liver, lung, and heart were harvested 0, 8, and 16 hours after CLP from wild-type mice (WT) and ß2M-/- mice treated with anti-asialoGM1 (ß2M-/-/{alpha}AsGM1). Cytokine and chemokine messenger RNA expression was determined using an RNAse protection assay. Results are typical of at least three different experiments. The L32 housekeeping gene product served as a loading control.

 
We performed additional studies to determine whether the observed differences in messenger RNA expression translated into differences in secreted cytokine protein levels. IL-1ß, IL-6, macrophage inflammatory protein-2, and IFN-{gamma} were detected in peritoneal fluid after CLP and levels of these cytokines were significantly less in ß2M-/- mice treated with anti-asialoGM1 compared with wild-type mice (Figure 4) .



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  		Figure 4. Cytokine and chemokine secretion into peritoneal fluid is decreased in ß2M-/- mice treated with anti-asialoGM1 (ß2M-/-/{alpha}AsGM1). WT and ß2M-/-/{alpha}AsGM1 mice underwent CLP and after 18 hours peritoneal lavage. Cytokine levels in peritoneal fluid were determined using an ELISA. n = 5 mice per group. *Significantly (p < 0.05) less than that for wild-type mice.

 
Wild-type Mice, but Not ß2M-/- Mice Treated with Anti-asialoGM1, Exhibit Hypothermia and Metabolic Acidosis after CLP
We next sought to assess mechanisms of mortality caused by CLP in wild-type mice and ß2M-/- mice treated with anti-asialoGM1. Initially, we measured bacterial counts in blood and peritoneal fluid at 18 hours after CLP (Figure 5) . Bacterial colony counts in blood and peritoneal fluid were not significantly different between wild-type mice and ß2M-/- mice treated with anti-asialoGM1. We also performed temperature measurements in both groups of mice. The mean body temperature was significantly (p < 0.05) lower at 18 hours after CLP in wild-type mice compared with nonseptic control animals and ß2M-/- mice treated with anti-asialoGM1 (Figure 5).



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  		Figure 5. Bacterial burden and temperature in WT and ß2M-/- mice treated with anti-asialoGM1 (ß2M-/-/{alpha}AsGM1) after cecal ligation and puncture. Nonseptic mice served as control animals. Blood and peritoneal fluid were aseptically harvested 18 hours after CLP. Bacterial colony counts were performed on serially diluted samples using tryptic soy broth pour plates. Rectal temperature measurements were made at 18 hours after CLP. *Significantly (p < 0.05) less than that for control animals. n = 4 to 6 mice per group.

 
We performed blood gas analysis to assess acid–base balance at 18 hours after CLP. Nonseptic wild-type mice served as a baseline control. We observed significantly lower pH in the blood of wild-type mice compared with control animals. The arterial pH of ß2M-/- mice treated with anti-asialoGM1 was not significantly different from that of control mice (Figure 6) . Although a trend toward decreased PCO2 was observed in the wild-type group after CLP, arterial PCO2 levels were not significantly different between groups. Blood bicarbonate levels were significantly lower in wild-type mice undergoing CLP compared with both ß2M-/- mice treated with anti-asialoGM1 and control mice. Likewise, the base deficit in wild-type mice was significantly higher compared with our other experimental groups.



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  		Figure 6. Acid–base balance in WT and ß2M-/- mice treated with anti-asialoGM1 (ß2M-/-/{alpha}AsGM1) after CLP. Nonseptic mice served as control animals. Arterial blood was harvested 18 hours after CLP and blood gas measurements were performed. *Significantly (p < 0.05) different from that for control animals. n = 4 to 6 mice per group.

 
Indices of liver, kidney, and lung injuries were also assessed. Acute hepatocellular injury was assessed by measuring plasma aspartate transaminase and alanine transaminase levels (Figure 7) . Compared with control nonseptic mice, both wild-type mice and ß2M-/- mice treated with anti-asialoGM1 exhibited significantly (p < 0.05) increased levels of aspartate aminotransferase and alanine aminotransferase in plasma. Plasma creatinine levels did not differ among any of the groups studied. We evaluated the presence of acute lung injury by measuring PO2/FIO2 and lung wet/dry weight ratios (Figure 7). PO2/FIO2 ratio was not different between groups and all were greater than 400. Differences in lung wet/dry ratio were also not observed when comparing groups.



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  		Figure 7. Assessment of organ injury in mice after CLP. Blood and tissues were harvested 18 hours after CLP from WT and ß2M-/- mice treated with anti-asialoGM1 (ß2M-/-/{alpha}AsGM1). Nonseptic mice served as control animals. Acute liver injury was determined by measurement of plasma aspartate transaminase and alanine transaminase levels. Renal function was determined by measurement of plasma creatinine. Lung PO2/FIO2 ratios were calculated on the basis of arterial PO2 measurements performed on mice breathing 100% oxygen. Lung wet to dry weight ratios were measured as an index of acute lung injury. n = 4 to 6 mice per group. *Significantly (p < 0.05) different from that for control animals.

 
Adoptive Transfer of CD8+ T, Natural Killer, and Natural Killer T Cells into ß2M-/- Mice Treated with Anti-asialoGM1 Restores Mortality Caused by CLP
Adoptive transfer experiments were undertaken to further define the role of CD8+ T, natural killer, and natural killer T cells in the pathogenesis of lethal intraabdominal sepsis (Figure 8) . CD8+ T cells comprised a mean of 20% of the total splenocyte population before the enrichment procedure. Natural killer and natural killer T cells each accounted for approximately 7 and 6%, respectively, of the total splenocyte population. The percentage of CD8+ T cells was increased to a mean of 85% after passage through CD8 enrichment columns (Figure 9) . Natural killer and natural killer T cells accounted for approximately 7 and 3%, respectively, of the enriched splenocyte population.



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  		Figure 8. Characterization of cells for adoptive transfer. Splenocytes were harvested from wild-type mice and passed through a CD8 enrichment column. CTL, NK, and NKT cell content was analyzed using flow cytometry. CTL were analyzed after staining with anti-CD8 and anti-CD3. NK and NKT cells were evaluated after staining with anti–{alpha}ß T cell receptor (TCR) and anti-NK1.1. The numbers indicate the mean ± SE for the percentage of each cell population in whole spleen and in the enriched cell preparation for five different enrichment procedures. The dot plot is representative of data obtained from five different enrichment procedures.

 


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  		Figure 9. Adoptive transfer of CTL, NK, and NKT cells into ß2M-/- mice treated with anti-asialoGM1 (ß2M-/-/{alpha}AsGM1) restores mortality caused by CLP. ß2M-/- mice were treated with {alpha}AsGM1 on Day 0 followed by adoptive transfer of enriched splenic CD8+ T, NK, and NKT cells on Day 1. On Day 2, mice underwent CLP. WT mice treated with nonspecific IgG served as control animals. n = 8 to 10 mice/group.

 
The enriched cell preparation was adoptively transferred into ß2M-/- mice treated with anti-asialoGM1, and mortality was measured after CLP (Figure 9). Wild-type mice exhibited a median survival time of 24 hours and 100% mortality by 30 hours. Eighty percent long-term survival was observed in ß2M-/- mice treated with anti-asialoGM1 (Figure 9). Anti-asialoGM1–treated ß2M-/- mice that received adoptive cell transfer exhibited a median survival time of 30 hours and 100% mortality by 80 hours. This survival difference was significantly (p < 0.05) longer than that for wild-type mice but significantly (p < 0.05) less than that for anti-asialoGM1–treated ß2M-/- mice that did not receive adoptive cell transfer.

CD8-deficient Mice Exhibit Improved Survival during Acute Intraabdominal Sepsis
CD8 knockout (CD8-/-) mice were used as a model to study the effect of CD8+ T cell depletion on mortality caused by CLP (Figure 10A) . Wild-type mice treated with anti-asialoGM1 did not exhibit significantly different survival compared with wild-type mice treated with nonspecific IgG. CD8-/- mice showed a significantly (p < 0.05) increased median survival time (36 vs. 18 hours) and time to 100% mortality (72 vs. 36 hours) compared with wild-type mice. CD8-/- mice treated with anti-asialoGM1 exhibited a long-term survival rate of 40%, which was significantly (p < 0.05) better than that for CD8-/- mice treated with nonspecific IgG (Figure 10A). CD8-/- mice treated with anti-NK1.1 to deplete both natural killer and natural killer T cells exhibited 37% long-term survival, which was significantly greater than that for wild-type mice treated with nonspecific IgG but not significantly different than that for CD8-/- mice treated anti-asialoGM1 (data not shown).



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  		Figure 10. Mice depleted of CD8+ T cells exhibit improved survival after CLP. (A) WT or CD8 knockout (CD8-/-) mice were treated with nonspecific IgG or anti-asialoGM1 ({alpha}AsGM1) before cecal ligation and puncture. (B) Wild-type mice were treated with nonspecific IgG, anti-CD8, or anti-asialoGM1 before cecal ligation and puncture. n = 5 to 10 mice/group.

 
In further studies, survival studies were performed in wild-type mice treated with anti-CD8. The ability of the CD8 antibody to deplete CD8+ cells was determined by evaluating splenic CD8+ cell populations in control and antibody-treated mice using flow cytometry. The spleens of wild-type mice treated with nonspecific IgG contained 21 ± 1.3% CD8+ cells compared with 2.3 ± 1.9% in wild-type mice treated with anti-CD8{alpha}. Most of the CD8 staining observed in splenocytes from mice treated with anti-CD8 was due to nonspecific binding as determined by isotype control staining. Therefore, treatment of wild-type mice with anti-CD8{alpha} resulted in depletion of greater than 95% of CD8+ cells. Analysis of survival data showed that mice treated with anti-CD8 exhibited a significantly (p < 0.05) longer time to 100% mortality compared with wild-type mice treated with nonspecific IgG (Figure 10B). In addition, treatment of wild-type mice with both anti-CD8 and anti-asialoGM1 resulted in 40% long-term survival, a significant (p < 0.05) improvement compared with wild-type mice treated with nonspecific IgG. Wild-type mice treated with anti-asialoGM1 did not exhibit significantly improved survival compared with wild-type mice treated with nonspecific IgG (Figure 10B).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we observed that ß2M-/- mice treated with anti-asialoGM1 exhibit resistance to lethal intraabdominal sepsis. ß2M-/- mice possess multiple immunologic defects including deficiencies in expression of the Class I major histocompatability complex and CD1 as well as depletion of CD8+ T and natural killer T cells. Treatment of ß2M-/- mice with anti-asialoGM1 causes elimination of natural killer cells. Therefore, the resistance of anti-asialoGM1–treated ß2M-/- mice to lethal peritonitis may be due to multiple mechanisms. We focused on determining the importance of CD8+ T, natural killer, and natural killer T cell deficiencies. It is likely that the loss of these cell populations is a significant factor in conferring the resistance of anti-asialoGM1–treated ß2M-/- mice to lethal intraabdominal sepsis. Adoptive transfer of CD8+ T, natural killer, and natural killer T cells into ß2M-/- mice treated with anti-asialoGM1 re-established CLP-induced mortality. The specific contributions of CD8+ T, natural killer, and natural killer T cells to CLP-induced mortality were further demonstrated through the use of CD8-/- mice. CD8-/- mice are deficient in CD8+ T cells but possess natural killer and natural killer T cells as well as intact Class I major histocompatability complex and CD1 molecules (14). CD8-/- mice exhibited improved survival after CLP compared with wild-type mice and treatment of CD8-/- mice with anti-asialoGM1 further improved survival. In further studies, treatment of wild-type mice with anti-CD8{alpha} to deplete CD8+ T cells resulted in significant improvement in the time to 100% mortality and 40% long-term survival was observed after treatment of wild-type mice with both anti-CD8{alpha} and anti-asialoGM1. These findings provide evidence that CD8+ T cells play a functional role in the pathogenesis of lethal intraabdominal sepsis. Depletion of natural killer cells alone did not improve survival after CLP, but natural killer cell-depletion potentiated the survival benefit observed in mice that were depleted of CD8+ T cells.

Chang and coworkers (15) showed that CD8+ T cells contribute to P. berghei–induced shock in mice. In another study, Badgwell and colleagues (16) demonstrated that natural killer cell depletion imparts resistance to Escherichia coli–induced sepsis. Our study shows that depletion of both cell populations is required for full survival benefit after CLP. The improved survival observed after depletion of both CD8+ T and natural killer cells in our model may be due to the shared cellular functions of these two cell populations. CD8+ T and natural killer cells both have the ability to mediate direct cellular cytotoxicity and secrete proinflammatory cytokines (17). Whether or not direct cytotoxic mechanisms play a role in the pathogenesis of septic shock caused by CLP remains to be determined. Our studies did not show differences in indices of acute liver, kidney, and lung injury when comparing wild-type and ß2M-/- mice treated with anti-asialoGM1. Mice in both groups did not show evidence of renal or pulmonary injury. However, both groups showed elevated liver enzymes that were likely affected by the manipulation of intraabdominal organs during the CLP procedure. Because of the acuity of our model, there may not have been adequate time for significant organ injury to develop. Clearly, organ dysfunction is a common event in patients with sepsis. The importance of direct cytotoxicity in causing organ dysfunction is controversial because some investigators have been unable to demonstrate significant direct parenchymal cell injury in clinical and experimental sepsis (18, 19). Another potential cellular target during sepsis is the vascular endothelium. CD8+ T and natural killer cells can cause endothelial injury during acute transplant rejection (20, 21). Endothelial injury is likely to account for many of the alterations in coagulation and microvascular function observed during sepsis (22, 23). However, whether CD8+ T and natural killer cells cause direct endothelial injury during sepsis has not been determined.

In the present study, hypothermia and metabolic acidosis developed in wild-type mice exposed to CLP but not in ß2M-/- mice treated with anti-asialoGM1. Metabolic acidosis is a common feature of sepsis that develops due to inadequate tissue oxygen delivery and utilization (24, 25). The ischemic cecum may contribute to the development of metabolic acidosis in our model. However, CLP was performed using identical techniques in all groups, yet wild-type mice developed metabolic acidosis whereas anti-asialoGM1–treated ß2M-/- mice did not. The similarity of the CLP procedures is demonstrated by our observation that blood and intraperitoneal bacterial counts were not different between groups. Another potential factor in the development of metabolic acidosis is organ hypoperfusion. Chung and colleagues (26) have demonstrated decreased blood flow to liver, kidneys, spleen, gut, and heart at 24 hours after CLP in mice. Therefore, regional hypoperfusion is a possible contributor to the metabolic acidosis observed in wild-type mice in our study.

Some investigators have implicated a role for natural killer and natural killer T cells in the pathogenesis of experimental sepsis. Ogasawara and colleagues (9) showed that ß2M-/- mice are resistant to the generalized Shwartzman reaction, an endotoxin-induced shock syndrome. Additional studies show that CD1 knockout mice and J{alpha}281 knockout mice, both of which are natural killer T cell–deficient, are resistant to the Shwartzman reaction (27, 28). Emoto and colleagues (29) recently showed that natural killer cell–deficient mice are resistant to endotoxin-induced death. Badgwell and colleagues (16) reported that natural killer cell depletion imparts resistance of mice to E. coli–induced sepsis. A common observation in all of these studies was decreased production of IFN-{gamma}. Natural killer and natural killer T cells are major sources of IFN-{gamma} after endotoxin challenge (30–32). The main function of IFN-{gamma} is to amplify macrophage functions including the secretion of proinflammatory cytokines such as tumor necrosis factor {alpha}, IL-1, and IL-8. Therefore, it has been postulated that the resistance of natural killer and natural killer T cell–deficient mice to endotoxin-induced sepsis is due, in part, to decreased production of IFN-{gamma} with a subsequent decrease in production of macrophage-derived proinflammatory cytokines. However, the role of IFN-{gamma} in the pathogenesis of sepsis caused by CLP is controversial. Seki and colleagues (33) showed that IFN-{gamma} is induced after CLP and that mice depleted of natural killer and natural killer T cells produce lower levels of IFN-{gamma}. Steinhauser and colleagues (34) demonstrated secretion of IFN-{gamma} into peritoneal fluid in mice undergoing CLP but not in sham mice. Furthermore, Yin and coworkers (35) showed that IFN-{gamma} is an important contributor to pulmonary injury in mice exposed to CLP. In a more recent study, Godshall and colleagues (36) showed that CLP induced intraperitoneal production of IL-12 and that natural killer cell depletion attenuated the production of this key IFN-{gamma}–inducing factor. However, Echtenacher and colleagues (37) showed that IFN-{gamma} receptor–deficient mice or mice receiving neutralizing antibodies to IFN-{gamma} do not exhibit improved survival after CLP. In the same study, administration of exogenous IFN-{gamma} to wild-type mice undergoing CLP markedly increased mortality and essentially converted a low mortality model of CLP into a high mortality model. Our studies show that ß2M-/- mice treated with anti-asialoGM1 exhibit decreased production of IL-1ß, IL-6 and macrophage inflammatory protein-2. We also demonstrated that IFN-{gamma} production is induced in liver, spleen, and peritoneum of wild-type mice after CLP. IFN-{gamma} levels were significantly lower in ß2M-/- mice treated with anti-asialoGM1 compared with wild-type mice. On the basis of these results, studies are underway in our laboratory to fully assess the importance of IFN-{gamma} in regulating the proinflammatory response after CLP and determine the contribution of IFN-{gamma} deficiency to improved outcome in our model.

In conclusion, our studies demonstrate that ß2M-/- mice depleted of natural killer cells by treatment with anti-asialoGM1 exhibit near-complete resistance to mortality caused by CLP. These mice exhibit decreased systemic inflammation as well as improved cardiovascular function and acid–base balance compared with wild-type mice. The resistance of anti-asialoGM1–treated ß2M-/- mice to sepsis-induced mortality is due largely to depletion of CD8+ T cells and natural killer cells. Overall, these studies demonstrate an important role of CD8+ T cells and natural killer cells during the acute inflammatory phase associated with systemic infection. The importance of these cell populations in regulating the proinflammatory response during early sepsis has not been previously recognized and our findings provide new insights into the mechanisms facilitating acute inflammation during sepsis. Many patients can be supported through this acute inflammatory phase with fluid resuscitation, inotropic support, and mechanical ventilation. However, it is likely that the early inflammatory response initiates downstream mechanisms that ultimately result in the demise of the patient with sepsis. By better understanding these early mechanisms, new treatment approaches may be developed to limit inflammatory injury and the pathophysiologic alterations precipitated by this response.


   FOOTNOTES
 
Supported by grants K08 GM61243 from the National Institutes of Health and 8650/8780 from the Shriners of North America.

Received in original form August 27, 2002; accepted in final form March 3, 2003


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Angus DC, Linde-Zwirble WT, Lidicker J, Clermont G, Carcillo J, Pinsky MR. Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care. Crit Care Med 2001; 29:1303–1310.[CrossRef][Medline]
  2. Mirzanejad Y, Roman S, Talbot J, Nicolle L. Pneumococcal bacteremia in two tertiary care hospitals in Winnipeg, Canada: Pneumococcal Bacteremia Study Group. Chest 1996;109:173–178.[Abstract/Free Full Text]
  3. Rangel-Frausto MS. The epidemiology of bacterial sepsis. Infect Dis Clin North Am 1999;13:299–312.[CrossRef][Medline]
  4. Beutler B, Poltorak A. Sepsis and evolution of the innate immune response. Crit Care Med 2001;29(Suppl 7):S2–S6.[CrossRef][Medline]
  5. Oberholzer A, Oberholzer C, Moldawer LL. Sepsis syndromes: understanding the role of innate and acquired immunity. Shock 2001;16:83–96.[Medline]
  6. Marshall JC. Inflammation, coagulopathy, and the pathogenesis of multiple organ dysfunction syndrome. Crit Care Med 2001;29(Suppl 7):S99–S106.[CrossRef][Medline]
  7. Natanson C, Esposito CJ, Banks SM. The sirens' songs of confirmatory sepsis trials: selection bias and sampling error. Crit Care Med 1998;26:1927–1928.[CrossRef][Medline]
  8. Bernard GR, Vincent JL, Laterre PF, LaRosa SP, Dhainaut JF, Lopez-Rodriguez A, Steingrub JS, Garber GE, Helterbrand JD, Ely EW, et al. Efficacy and safety of recombinant human activated protein C for severe sepsis. N Engl J Med 2001;344:699–709.[Abstract/Free Full Text]
  9. Ogasawara K, Takeda K, Hashimoto W, Satoh M, Okuyama R, Yanai N, Obinata M, Kumagai K, Takada H, Hiraide H, et al. Involvement of NK1+ T cells and their IFN-gamma production in the generalized Shwartzman reaction. J Immunol 1998;160:3522–3530.[Abstract/Free Full Text]
  10. Dang Y, Heyborne KD. Cutting edge: regulation of uterine NKT cells by a fetal class I molecule other than CD1. J Immunol 2001;166:3641–3646.[Abstract/Free Full Text]
  11. Rolph MS, Raupach B, Kobernick HH, Collins HL, Perarnau B, Lemonnier FA, Kaufmann SH. MHC class Ia-restricted T cells partially account for beta2-microglobulin-dependent resistance to Mycobacterium tuberculosis. Eur J Immunol 2001;31:1944–1953.[CrossRef][Medline]
  12. Osman Y, Kawamura T, Naito T, Takeda K, Van Kaer L, Okumura K, Abo T. Activation of hepatic NKT cells and subsequent liver injury following administration of alpha-galactosylceramide. Eur J Immunol 2000;30:1919–1930.[CrossRef][Medline]
  13. Quinn DG, Zajac AJ, Hioe CE, Frelinger JA. Virus-specific, CD8+ major histocompatibility complex class I-restricted cytotoxic T lymphocytes in lymphocytic choriomeningitis virus-infected beta2-microglobulin-deficient mice. J Virol 1997;71:8392–8400.[Abstract]
  14. McGargill MA, Mayerova D, Stefanski HE, Koehn B, Parke EA, Jameson SC, Panoskaltsis-Mortari A, Hogquist KA. A spontaneous CD8 T cell-dependent autoimmune disease to an antigen expressed under the human keratin 14 promoter. J Immunol 2002;169:2141–2147.[Abstract/Free Full Text]
  15. Chang W, Jones S, Lefer D, Welbourne T, Sun G, Yin L, Suzuki H, Huang J, Granger DN, van der Heyde H. CD8+-T-cell depletion emeliorates circulatory shock in Plasmodium berghei-infected mice. Infect Immun 2001;69:7341–7348.[Abstract/Free Full Text]
  16. Badgwell B, Parihar R, Magro C, Dierksheide J, Russo T, Carson WE III. Natural killer cells contribute to the lethality of a murine model of Escherichia coli infection. Surgery 2002;132:205–212.[CrossRef][Medline]
  17. Smyth MJ, Snook MB. Perforin-dependent cytolytic responses in beta 2-microglobulin-deficient mice. Cell Immunol 1999;196:51–60.[CrossRef][Medline]
  18. Hiramatsu M, Hotchkiss RS, Karl IE, Buchman TG. Cecal ligation and puncture (CLP) induces apoptosis in thymus, spleen, lung, and gut by an endotoxin and TNF-independent pathway. Shock 1997;7:247–253.[Medline]
  19. Oberholzer C, Oberholzer A, Clare-Salzler M, Moldawer LL. Apoptosis in sepsis: a new target for therapeutic exploration. FASEB J 2001;15:879–892.[Abstract/Free Full Text]
  20. Yoneda O, Imai T, Goda S, Inoue H, Yamauchi A, Okazaki T, Imai H, Yoshie O, Bloom ET, Domae N, et al. Fractalkine-mediated endothelial cell injury by NK cells. J Immunol 2000;164:4055–4062.[Abstract/Free Full Text]
  21. Nakajima T, Schulte S, Warrington KJ, Kopecky SL, Frye RL, Goronzy JJ, Weyand CM. T-cell-mediated lysis of endothelial cells in acute coronary syndromes. Circulation 2002;105:570–575.[Abstract/Free Full Text]
  22. Vallet B, Wiel E. Endothelial cell dysfunction and coagulation. Crit Care Med 2001;29:S36–S41.[CrossRef][Medline]
  23. Munshi N, Fernandis AZ, Cherla RP, Park IW, Ganju RK. Lipopolysaccharide-induced apoptosis of endothelial cells and its inhibition by vascular endothelial growth factor. J Immunol 2002;168:5860–5866.[Abstract/Free Full Text]
  24. Treggiari MM, Romand JA, Burgener D, Suter PM, Aneman A. Effect of increasing norepinephrine dosage on regional blood flow in a porcinemodel of endotoxin shock. Crit Care Med 2002;30:1334–1339.[CrossRef][Medline]
  25. Gando S, Kameue T, Morimoto Y, Matsuda N, Hayakawa M, Kemmotsu O. Tissue factor production not balanced by tissue factor pathway inhibitor in sepsis promotes poor prognosis. Crit Care Med 2002;30:1729–1734.[CrossRef][Medline]
  26. Chung CS, Yang S, Song GY, Lomas J, Wang P, Simms HH, Chaudry IH, Ayala A. Inhibition of Fas signaling prevents hepatic injury and improves organ blood flow during sepsis. Surgery 2001;130:339–345.[CrossRef][Medline]
  27. Kawamura T, Takeda K, Mendiratta SK, Kawamura H, Van Kaer L, Yagita H, Abo T, Okumura K. Critical role of NK1+ T cells in IL-12-induced immune responses in vivo. J Immunol 1998;160:16–26.[Abstract/Free Full Text]
  28. Dieli F, Sireci G, Russo D, Taniguchi M, Ivanyi J, Fernandez C, Troye-Blomberg M, De Leo G, Salerno A. Resistance of NKT cell-deficient mice to systemic Shwartzman reaction. J Exp Med 2000;192:1645–1652.[Abstract/Free Full Text]
  29. Emoto M, Miyamoto M, Yoshizawa I, Emoto Y, Schaible U, Kita E, Kaufmann H. Critical role of NK cells rather than V{alpha}14+NKT cells in lipopolysaccharide-induced lethal shock in mice. J Immunol 2002;169:1426–1432.[Abstract/Free Full Text]
  30. Carnaud C, Lee D, Donnars O, Park SH, Beavis A, Koezuka Y, Bendelac A. Cutting edge: cross-talk between cells of the innate immune system: NKT cells rapidly activate NK cells. J Immunol 1999;163:4647–4651.[Abstract/Free Full Text]
  31. Varma TK, Lin CY, Toliver-Kinsky TE, Sherwood ER. Endotoxin-induced gamma interferon production: contributing cell types and key regulatory factors. Clin Diagn Lab Immunol 2002;9:530–541.[Abstract/Free Full Text]
  32. Varma TK, Toliver-Kinsky TE, Lin CY, Koutrouvelis AP, Nichols JE, Sherwood ER. Cellular mechanisms that cause suppressed gamma interferon secretion in endotoxin-tolerant mice. Infect Immun 2001;69:5249–5261.[Abstract/Free Full Text]
  33. Seki S, Habu Y, Kawamura T, Takeda K, Dobashi H, Ohkawa T, Hiraide H. The liver as a crucial organ in the first line of host defense: the roles of Kupffer cells, natural killer (NK) cells and NK1.1 Ag+ T cells in T helper 1 immune responses. Immunol Rev 2000;174:35–46.[CrossRef][Medline]
  34. Steinhauser ML, Hogaboam CM, Lukacs NW, Strieter RM, Kunkel SL. Multiple roles for IL-12 in a model of acute septic peritonitis. J Immunol 1999;162:5437–5443.[Abstract/Free Full Text]
  35. Yin K, Hock CE, Lai PS, Ross JT, Yue G. Role of interferon-gamma in lung inflammation following cecal ligation and puncture in rats. Shock 1999;12:215–221.[Medline]
  36. Godshall CJ, Scott MJ, Burch PT, Peyton JC, Cheadle WG. Natural killer cells participate in bacterial clearance during septic peritonitis through interactions with macrophages. Shock 2003;19:144–149.[Medline]
  37. Echtenacher B, Freudenberg MA, Jack RS, Mannel DN. Differences in innate defense mechanisms in endotoxemia and polymicrobial septic peritonitis. Infect Immun 2001;69:7271–7278.[Abstract/Free Full Text]



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