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The Journal of Immunology, 2006, 177: 8650-8657.
Copyright © 2006 by The American Association of Immunologists, Inc.

Overexpression of Suppressor of Cytokine Signaling-5 in T Cells Augments Innate Immunity during Septic Peritonitis1

Hiroyuki Watanabe*,{dagger}, Masato Kubo{ddagger}, Kosuke Numata*,{dagger}, Katsumasa Takagi{dagger}, Hiroshi Mizuta{dagger}, Seiji Okada§, Takaaki Ito* and Akihiro Matsukawa2,*,¶

* Department of Pathology and Experimental Medicine, {dagger} Department of Orthopaedic and Neuro-Musculoskeletal Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan; {ddagger} Laboratory for Signal Network, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan; § Division of Hematopoiesis, Center for AIDS Research, Kumamoto University, Kumamoto, Japan; and Department of Pathology and Experimental Medicine, Graduate School of Medical, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Suppressors of cytokine signaling (SOCS) proteins are negative regulators of cytokine signaling by inhibiting the JAK-STAT signal transduction pathway, but their role in innate immunity remains to be investigated. In the present study, we demonstrate that overexpression of SOCS5 in T cells augments innate immunity during septic peritonitis induced by cecal ligation and puncture (CLP). Mice with a cell-specific overexpression of SOCS5 in T cells (SOCS5 transgenic (Tg)) were resistant to the lethality relative to the wild-type (WT) mice. This was most likely due to the enhanced innate immunity in SOCS5Tg mice, as bacterial burden in SOCS5Tg mice was significantly lower than WT mice. Accumulation of neutrophils and macrophages was augmented in SOCS5Tg mice, an event that was accompanied by increased peritoneal levels of IL-12, IFN-{gamma}, and TNF-{alpha}. In vitro bactericidal activities of macrophages and neutrophils were enhanced in SOCS5Tg mice. Both neutrophils and macrophages from WT mice adopted enhanced bacterial killing activity when cocultured with CD4+ T cells from SOCS5Tg mice, relative to CD4+ T cells from WT mice. Adoptive transfer of SOCS5Tg-CD4+ T cells into T- and B cell-deficient RAG-2–/– mice resulted in augmented leukocyte infiltration and increased peritoneal levels of IL-12, IFN-{gamma}, and TNF-{alpha} after CLP, as compared with the controls. Furthermore, CLP-induced bacterial burden in RAG-2–/– mice harboring SOCS5Tg-CD4+ T cells was significantly reduced relative to the controls. These findings provide evidence that intervention of SOCS5 expression in T cells affects innate immunity, which highlight a novel role of T cells during sepsis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Sepsis is a severe illness caused by overwhelming infection of the bloodstream by toxin-producing bacteria that can originate anywhere in the body, the frequent sites of which are lung, urinary tract, and abdomen (1, 2). Sepsis may be initialized by intense local inflammation to inactivate and clear the invading pathogens (3, 4). Evidence indicates that cytokines that include IL-10, IL-12, and IL-13 control a wide variety of inflammatory responses during sepsis (5, 6, 7, 8).

Many of these cytokines exert their biological functions through the JAK/STAT pathway (9, 10). STAT3/4/6 are transcription factors that mediate IL-10/IL-12/IL-13 cytokine signaling, respectively. We have heretofore shown in a murine model of septic peritonitis that mice with targeted disruption of the Stat3 gene in macrophages and neutrophils exhibit increased lethality by exaggerated local and systemic inflammation through overzealous production of cytokines (11). STAT4- and STAT6-deficient mice are resistant to the lethality by balancing local type 1 and systemic type 2 cytokine responses (12). Thus, Stat proteins are crucial in innate immunity during septic peritonitis.

Balanced cytokine responses are important in immune system. Several mechanisms for negative regulation of cytokine signaling have been recently identified. Suppressor of cytokine signaling (SOCS)3 proteins are a family of Src homology 2 domain-containing cytoplasmic proteins that complete a negative feedback loop to attenuate signal transduction from cytokines that act through the JAK/STAT pathway (13, 14). SOCS5, a member of SOCS proteins, is predominantly expressed in Th1 cells and inhibits the Th2 immune responses, indicating a regulatory role of SOCS5 in Th1 and Th2 balance (15). However, little is known about the role of SOCS5 in innate immunity. Because T cells are present in the peritoneal cavity even on a steady basis (Thy1.2-positive cells, 11–16%, our own data by flow cytometry), SOCS5 in T cells may affect innate immunity during septic peritonitis, in which a type 1 response is important.

In this study, we have extended our previous work and attempted to explore the role of SOCS5 in innate immunity during sepsis. For this purpose, we here used mice with a cell-specific overexpression of SOCS5 in T cells (SOCS5 transgenic (Tg)) (15). The mice underwent a well-established murine septic peritonitis model, cecal ligation and puncture (CLP), a clinically relevant model of intra-abdominal sepsis (16). We here provide evidence that SOCS5 in T cells serves a beneficial role in host defense during septic peritonitis by augmenting the innate immune responses. Thus, innate immunity can be controlled under T cells, particularly by SOCS5.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

The Flag-tagged SOCS5 was expressed under the control of the lck proximal promoter Eµ enhancer and backcrossed with C57BL/6J mice over 10 generations, as described (15). In the mice, SOCS5 was overexpressed in T cells, but not in B cells (data not shown). Recombination activating gene (RAG)-2 knockout mice (RAG-2–/–, lacking T and B cells, C57BL/6J strain) were a gift from Dr. T. Taniguchi (University of Tokyo, Tokyo, Japan). C57BL/6J mice were used as the wild-type (WT) mice. These mice were transferred to the Center for Animal Resources and Development, Kumamoto University and then bred at the facility. Female mice (6–8 wk) were used in this study under specific pathogen-free conditions. The animal use committee at Kumamoto University approved all experiments conducted in this study.

Induction of sepsis

Sepsis was induced by CLP, as described elsewhere (17, 18). Briefly, mice were anesthetized and the cecum was exposed, ligated with a 3-0 silk suture, and punctured through and through once with an 18-gauge needle. The cecum was replaced in the peritoneal cavity and the incision was closed with surgical staples. All mice were received a s.c. injection of 1 ml of sterile saline to avoid dehydration and were placed on a heating pad until they recovered from anesthesia.

To determine mice survival, CLP mice were monitored for 7 days after CLP. In a different set of experiments, CLP mice were anesthetized, bled, and euthanized at indicated time points after CLP. The peritoneal cavities were washed with 2 ml of sterile saline, and the lavage fluids were harvested. A 10-µl aliquot of lavage fluids was used for assessment of bacteria CFU. The remaining fluids were centrifuged, and cell-free peritoneal fluids were stored at –80°C for measurements of cytokines. Cell pellets were resuspended in saline and the cell numbers were counted in a hemocytometer. Differential cell analysis was made after Diff-Quik staining of cytospin preparations. The kidney was excised, snap-frozen in liquid nitrogen, and stored at –80°C. A part of tissues was fixed in 4% paraformaldehyde, embedded in paraffin, and the tissue sections were stained with H&E.

In other experiments, CD4+ T cells from SOCS5Tg or WT mice (3 x 106/mouse) were transferred into the peritoneum of nontreated RAG-2–/– mice. Immediately after the injection, the mice underwent CLP as describe above.

Determination of CFU

Ten microliters of peritoneal lavage fluids and peripheral blood from each mouse was serially diluted with sterile saline. Ten microliters of each dilution was plated on tryptose soy agar (TSA)-blood plates and incubated overnight at 37°C, after which the number of aerobic colonies was counted. Data were expressed as CFU per 10 µl.

Cell preparation

Peritoneal cells were harvested from nontreated mice, cultured for 1 h at 37°C in RPMI 1640 containing 10% FCS, and the adherent cells were used as macrophages. Neutrophils (>94%) were isolated from infiltrating leukocytes by Ficoll gradient centrifugation that were harvested at 4 h after i.p. injection of 1 ml of 4% thioglycolate. Most of the cells were alive as assessed by trypan blue exclusion (>97%).

CD4+ T cells were isolated from spleen cells. In brief, spleens were collected from nontreated SOCS5Tg and WT mice and dispersed into single-cell suspensions. RBC were lysed with 0.1 N NH4Cl, and the spleen cells were washed three times with RPMI 1640, after which CD4+ T cells were positively purified with anti-mouse CD4 microbeads using Midi MACS system (Miltenyi Biotec). Most of the purified cells were alive (>97%) and routinely >95% CD4+ T cells, as assessed by flow cytometry (data not shown).

In vitro bactericidal activities of leukocytes

Macrophages and neutrophils, prepared as described above, were suspended in antibiotics-free RPMI 1640 containing 10% FCS, and placed in 24-well culture plates. In vitro bactericidal activities of the cells were determined by a classical CFU assay with minor modifications (11, 19). In brief, cells (3 x 106/well) were infected with 1 x 106 CFU of live bacteria recovered from mice undergone CLP. Control wells contained only bacteria. After a 4-h culture in a 5% CO2 incubator, plates were placed at –80°C for 30 min and cells were lysed by thawing. This did not affect bacteria viability, as determined in preliminary experiments. The lysates were serially diluted, plated on TSA-blood plates, incubated overnight at 37°C, and the colonies were counted. Bactericidal activity was expressed as the percentage of bacteria death = (CFU from control wells (without cells) – CFU from experimental wells)/(CFU from control wells (without cells)) x 100. In other experiments, bactericidal activities of cells were assessed in the presence of CD4+ T cells (1 x 106/well) from nontreated WT mice or SOCS5Tg mice. CD4+ T cells were purified from spleens as described above.

Cell culture

Macrophages (1 x 106/ml) and CD4+ T cells (0.5 x 106/ml) were cocultured in RPMI 1640 supplemented with 5% FCS, glutamine, and antibiotics in a 5% CO2 incubator for 24 h with a TLR4 ligand, Escherichia coli LPS (100 ng/ml, 0111, B4; Difco Laboratories) and in the presence of rabbit anti-murine IFN-{gamma} IgG or control IgG (10 µg/ml). The culture supernatants were used for measurements of cytokines. In some experiments, cells were cultured in a dual chamber transwell culture apparatus (Corning Scientific Products), in which macrophages were cultured in a lower chamber and CD4+ T cells were done in an upper chamber.

Superoxide production

The production of superoxide was measured using a highly reactive oxygen species detection reagent, aminophenyl fluorescein (APF; Daiichi Pure Chemicals), as described (20). In brief, macrophages and neutrophils (2 x 106 cells/ml), prepared as described above, were stimulated with LPS (100 ng/ml) for 24 h in phenol red-free RPMI 1640 in the presence of APF (1 mM/L), after which the culture supernatants were harvested. Superoxide production was measured spectrophotometrically using FLx800 Fluorescence Microplate Reader (Bio-Tek Instruments). The excitation wavelength was 490 nm and the emission was 515 nm.

Measurements of cytokines and myeloperoxidase (MPO)

Murine cytokines were quantitated using a standard method of sandwich ELISA as previously described (6, 18). The captured Abs, detection Abs, and the recombinant cytokines were purchased from R&D Systems. The ELISAs used in this study did not cross-react with other murine cytokines available. MPO levels in the kidney were measured by ELISA kit (Calbiochem). The kidneys were homogenized in PBS containing 0.1% Triton X-100 and complete protease inhibitor (Roche), centrifuged, and the cleared supernatants were obtained. Protein concentrations in the extracts were measured by protein dye-binding assay (Bio-Rad).

Clinical chemistry

Serum levels of blood urea nitrogen (BUN) and creatinine were measured using standardized techniques.

Western blotting

Leukocytes were dissolved in Laemmli buffer (1 x 106/50 µl), sonicated, boiled, fractionated on SDS-polyacrylamide gel (10 µl), and transferred to a nitrocellulose membrane. After blocking with TBST containing 5% skim milk for 1 h at room temperature, the membrane was incubated with Abs to STAT6 or tyrosine-phosphorylated STAT6 (Cell Signaling) overnight at 4°C. After washing with TBST, the membrane was incubated with anti-HRP-linked Ab for 1 h at room temperature and visualized with an ECL system (Cell Signaling), photographed, digitized, and the band densities were measured with NIH image.

Immunocytochemistry

Cytospin preparations of peritoneal exudates at 24 h after CLP were immediately fixed in 100% methanol. After blocking endogenous peroxidase using 0.3% H2O2 in methanol, the slides were rehydrated in TBS and blocked with 10% normal goat serum for 1 h at room temperature. The slides were incubated with anti-tyrosine-phosphorylated STAT6 (Cell Signaling) or control rabbit IgG at 5 µg/ml in TBS containing 1% BSA overnight at 4°C. After washing with TBST, the slides were incubated with anti-rabbit IgG conjugated to HRP-labeled dextran polymer (EnVision plus, Peroxidase; DakoCytomation) for 30 min at room temperature. After washing, the reaction was developed with diaminobenzidine (Sigma- Aldrich). Counterstaining was done with hematoxylin.

Flow cytometry analysis

Peritoneal cells were harvested from nontreated WT and SOCS5Tg mice and the cells (1 x 106 cells/ml) were suspended in PBS supplemented with 2% FCS and 0.1% sodium azide. Cells were stained with mAb specific for mouse CD4, CD8, CD44, CD62L, and CD69 (BD Pharmingen). Stained cells were analyzed using a FACSCalibur (BD Biosciences).

Statistics

Statistical significance was determined by ANOVA. In case of survival curve and CFU count, the data were analyzed by the log-rank test and Mann-Whitney U test, respectively. A p value <0.05 was regarded as statistically significant. All data were expressed as mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Improved survival to CLP in SOCS5Tg mice

To understand whether overexpression of SOCS5 in T cells would affect host defense during CLP, initial studies were conducted to determine mice survival in SOCS5Tg and WT mice after CLP. As shown in Fig. 1, survival rate in SOCS5Tg mice was significantly higher than that in WT mice. Of the 29 SOCS5Tg mice, 15 survived for 7 days (51.7%); whereas only 7 of 30 WT mice survived for 7 days (23.3%) (p < 0.001). Thus, SOCS5Tg mice were resistant to the lethality induced by CLP, thereby indicating that overexpression of SOCS5 in T cells was beneficial in host defense during septic peritonitis.


Figure 1
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  		FIGURE 1. Improved survival after CLP in SOCS5Tg mice. The survival rates in SOCS5Tg (29 mice) and WT mice (30 mice) were monitored for 7 days after CLP. Three different experiments were conducted and the data were pooled. The mortality rates were very similar in individual experiments. §, p < 0.001 vs WT mice.

 
Augmented bacterial clearance in SOCS5Tg mice

CLP-induced lethality is firmly linked with bacterial burden (17, 18). In an attempt to identify the basis whereby SOCS5Tg mice were resistant to CLP, we first examined the bacterial load in the peritoneum after CLP. Although bacterial burden at 6 h post-CLP was unchanged between SOCS5Tg and WT mice (data not shown), SOCS5Tg mice exhibited an enhanced bacterial clearance at 24 h post-CLP, as indicated by smaller numbers of recovered CFU counts in the peritoneum (Fig. 2). At this time point, the bacterial load recovered from peripheral blood in SOCS5Tg mice was 23-fold lower than that in WT mice, although it was not statistically significant (p = 0.107, Fig. 2).


Figure 2
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  		FIGURE 2. Enhanced bacterial clearance in SOCS5Tg mice. At 24 h after CLP (WT, 14 mice; SOCS5Tg mice, 10 mice), mice were killed and the peritoneal fluids and peripheral blood were harvested. Ten microliters of peritoneal fluids and blood was serially diluted and plated on TSA-blood plates. Line represents mean CFU count. {ddagger}, p < 0.05, vs WT mice.

 
Phagocytes, such as neutrophils and macrophages, are cells responsible for clearing bacteria (21, 22, 23). Experiments were next conducted to examine bactericidal activities of neutrophils and macrophages. For this purpose, neutrophils and macrophages from WT and SOCS5Tg mice were infected with live bacteria recovered from the peritoneum of WT mice that had undergone CLP. As shown in Fig. 3A, bactericidal activities of macrophages from SOCS5Tg mice were higher than those from WT mice. There was a trend toward increase in SOCS5Tg neutrophils. Superoxide generation, an effector molecule for bacterial killing (21), was subsequently examined in the culture supernatants of LPS-stimulated neutrophils and macrophages, which demonstrated that cells from SOCS5Tg mice produced higher levels of superoxide generation than those from WT mice (Fig. 3B). Thus, SOCS5Tg mice cleared bacteria more effectively than WT mice, possibly due to enhanced bactericidal activities of phagocytes.


Figure 3
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  		FIGURE 3. Bactericidal activities of leukocytes in SOCS5Tg mice. Neutrophils and macrophages were collected from WT and SOCS5Tg mice, as described in Materials and Methods. A, Cells (3 x 106/ml) were infected with bacteria (1 x 106 CFU) and the killing activity was examined, as described in Materials and Methods. B, Cells (2 x 106/ml) were stimulated with LPS (100 ng/ml) for 24 h in the presence of APF. Superoxide production in the supernatants were measured spectrophotometrically. Shown are representative data from three independent experiments. {ddagger}, p < 0.05; {dagger}, p < 0.01 vs WT mice.

 
Enhanced local inflammation in SOC5Tg mice

The leukocyte infiltration in the peritoneum after CLP was next investigated, which demonstrated that the numbers of infiltrating neutrophils and macrophages were significantly augmented in SOCS5Tg mice, resulting in a 1.5- (at 24 h) and 2.1-fold (at 6 h) increase, respectively, relative to the WT mice (Fig. 4A). Consequently, cytokine levels in the peritoneum after CLP were measured. The data in Fig. 4B demonstrated that levels of IL-12 and IFN-{gamma} were elevated relative to the WT mice. In addition, TNF-{alpha} level in SOCS5Tg mice was significantly higher than that in WT mice (Fig. 4B). These cytokines are known to enhance bacterial clearance in a variety of infectious models including septic peritonitis (8, 24, 25). Anti-inflammatory cytokines were also measured, which showed that no appreciable levels of IL-4 and IL-13 were detected in the peritoneum from WT and SOCS5Tg mice after CLP. There were no statistical differences in the peritoneal IL-10 level between WT and SOCS5Tg mice (6 h; 1.0 ± 0.3 vs 1.3 ± 0.3 ng/cavity, 24 h; 2.1 ± 0.5 vs 1.1 ± 0.3 ng/cavity, respectively, eight mice each, not significant). Upon stimulation with LPS, peritoneal macrophages from SOCS5Tg mice produced higher levels of TNF-{alpha} and IL-12, but not IFN-{gamma}, relative to the controls (Table I). No substantial IL-4 and IL-13 were detected (data not shown). IL-10 level was not significant between WT and SOCS5Tg macrophages (Table I). STAT6 activation in peritoneal exudates at 24 h post-CLP were subsequently examined, which showed that the expression of phosphorylated STAT6 (pSTAT6) was reduced in SOCS5Tg mice compared with that in WT mice (Fig. 5A), despite no alteration in STAT4 activation (data not shown). Cells with STAT6 activation were infiltrating neutrophils and macrophages, as these cells were positively stained with anti-pSTAT6 IgG (Fig. 5B). Thus, SOCS5Tg mice exhibited an altered cytokine balance in favor of type 1 cytokine, possibly contributing to the enhanced bacterial killing in SOCS5Tg mice.


Figure 4
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  		FIGURE 4. Leukocyte infiltration and production of cytokines in SOCS5Tg mice. At 6 and 24 h after CLP, mice were killed, bled, and the peritoneal fluids were harvested. A, The numbers of infiltrating leukocytes in the peritoneum were counted. {circ}, WT mice, •, SOCS5Tg mice. B, Peritoneal levels of IL-12, IFN-{gamma}, and TNF-{alpha} were measured by ELISA. {square}, WT mice; {blacksquare}, SOCS5Tg mice. The data represent the mean ± SEM of 8–11 estimations from separate mice. {ddagger}, p < 0.05; {dagger}, p < 0.01 vs WT mice.

 

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  		Table I. Cytokine production from peritoneal macrophagesa

 

Figure 5
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  		FIGURE 5. STAT6 activation in SOCS5Tg mice. A, Infiltrating leukocytes were harvested at 24 h after CLP and the lysates were immunoblotted with anti-STAT6 or anti-tyrosine-phosphorylated STAT6. Upper, Representative photos. Lower, Blots were photographed, digitized, and the densities were measured with NIH image. The pSTAT6/STAT6 ratio was calculated and the data were expressed as relative index from four independent experiments. {ddagger}, p < 0.05 vs WT controls. B, Peritoneal exudate cells at 24 h post-CLP were harvested from WT mice and the cells were stained with anti-pSTAT6 IgG. A representative photograph. In addition to cytoplasm, nuclei were faintly stained. Original magnification, x400.

 
Adoptive transfer of resident CD4+ T cells augments innate immune response

These results suggest that overexpression of SOCS5 in T cells affects innate immune response during septic peritonitis. We then asked whether CD4+ T cells could be responsible for the enhanced innate immune response in SOCS5Tg mice. For this, phagocytes (neutrophils and macrophages) from WT mice were cocultured with CD4+ T cells from WT or SOCS5Tg mice and the cells were infected with live bacteria recovered from CLP mice, after which the bacterial killing activities of phagocytes were examined. The data in Fig. 6A showed that both neutrophils and macrophages exhibited enhanced bacterial killing activities in the presence of CD4+ T cells from SOCS5Tg mice, as compared with the controls. To further investigate the role of CD4+ T cells during CLP, CD4+ T cells from WT or SOCS5Tg mice were transferred into RAG-2–/– mice, T- and B cell-deficient mice, and the mice underwent CLP. As a result, mice harboring SOCS5Tg-CD4+ T cells cleared bacteria more effectively than those with WT-CD4+ T cells (Fig. 6B). The numbers of infiltrating leukocytes were increased in mice with SOCS5Tg-CD4+ T cells (Fig. 6C). RAG-2–/– mice received SOCS5Tg-CD4+ T cells produced significantly higher levels of IL-12 and IFN-{gamma} in the peritoneum relative to the mice with control CD4+ T cells. TNF-{alpha} level was also increased by ~150%, albeit they were not statistically significant (p = 0.12) (Fig. 6D). No apparent IL-4 and IL-13 was detected in the peritoneum (data not shown). There was no statistical differences in the peritoneal IL-10 level between mice with WT- and SOCS5Tg-CD4+ T cells (0.62 ± 0.14 vs 0.87 ± 0.16 ng/cavity, respectively, 10 mice each, not significant). Accordingly, these data clearly indicate that SOCS5 in T cells is responsible for the skewed innate immune response in this particular model.


Figure 6
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  		FIGURE 6. CD4+ T cells from SOCS5Tg augment innate immune responses. A, Phagocytes (neutrophils and macrophages, 3 x 106/ml) cocultured with CD4+ T cells (1 x 106/ml) from either WT mice or SOCS5Tg mice were infected with 1 x 106 CFU of live bacteria recovered from CLP mice and the killing activities of phagocytes were examined. Shown are representative data from three independent experiments. B–D, CD4+ T cells from WT or SOCS5Tg mice were transferred into RAG-2–/– mice (3 x 106 cells/peritoneum, 10 mice each) and the mice underwent CLP. At 24 h post-CLP, mice were killed and the peritoneal fluids and peripheral blood were harvested. B, Ten microliters of peritoneal fluids and blood was serially diluted and plated on TSA-blood plates. Line represents mean CFU count. C, The numbers of neutrophils and macrophages were counted. D, Peritoneal cytokine levels were measured. E, WT macrophages (1 x 106/ml) were coincubated with WT-CD4+ T cells ({square}) or SOCS5Tg-CD4+ T cells ({blacksquare}) (0.5 x 106/ml), and the cells were stimulated with LPS (100 ng/ml) in the presence of control IgG or neutralizing anti-IFN-{gamma} IgG (10 µg/ml). Twenty-four hours later, the culture supernatants were harvested and TNF-{alpha} level was measured. Shown are representative data from two independent experiments. {ddagger}, p < 0.05; {dagger}, p < 0.01; §, p < 0.001; ¶, p < 0.0001vs controls.

 
To understand the mechanism by which SOCS5Tg-CD4+ T cells influence the innate immune response, WT macrophages were stimulated with LPS in the presence of WT- or SOCS5Tg-CD4+ T cells, after which a proinflammatory cytokine TNF-{alpha} level in the culture supernatants was measured. As shown in Fig. 6E, TNF-{alpha} level was augmented when WT macrophages were cocultured with SOCS5Tg-CD4+ T cells as compared with WT-CD4+ T cells, suggesting that SOCS5Tg-CD4+ T cells might augment TNF-{alpha} production. The augmented TNF-{alpha} level was not found when cells were cultured using transwell apparatus (WT-CD4+ T cells vs SOCS5Tg-CD4+ T cells = 1.06 ± 0.03 vs 1.09 ± 0.03 ng/ml, respectively, n = 5, not significant). In addition, there was no difference in TNF-{alpha} level when macrophages were cultured with LPS supernatants of WT- or SOCS5Tg-CD4+ T cells (0.99 ± 0.01 vs 0.95 ± 0.04 ng/ml, n = 5, not significant). To examine the molecular mechanism behind the augmented TNF-{alpha}, cocultured cells were stimulated with LPS in the presence of neutralizing anti-IFN-{gamma} IgG. As a result, the augmentation in TNF-{alpha} level by SOCS5Tg-CD4+ T cells was completely abrogated. Thus, SOCS5Tg-CD4+ T cells appear to influence TNF-{alpha} production through IFN-{gamma} production.

The nature of peripheral T cells in SOCS5Tg mice

The different response of SOCS5Tg-CD4+ T cells may result from different nature of peritoneal T cells. We then examined the surface marker expressions of peritoneal T cells in these nontreated mice. Representative data are shown in Fig. 7, which demonstrated that CD4+- and CD8+-positive cells were similar between WT- and SOCS5Tg-CD4+ T cells. An early activation marker CD69+ was not increased both in WT- and SOCS5Tg-CD4+ T cells. Interestingly, CD4+CD44+ double-positive T cells (memory T cells) were increased in SOCS5Tg mice whereas CD4+CD62L+ double-positive cells (naive T cells) were decreased (Fig. 7). Thus, SOCS5Tg mice showed distinct phenotype of peritoneal CD4+ T cells as compared with the controls.


Figure 7
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  		FIGURE 7. Representative flow cytometry data of peritoneal T cells. Peritoneal cells were harvested from nontreated WT and SOCS5Tg mice, and the cells were stained with CD4, CD8, CD44, CD62L, and CD69. Percentages for individual cell types are shown. Data are from two to three independent experiments with similar results.

 
Attenuated renal injury in SOCS5Tg mice

Sepsis frequently causes multiple organ failure. Kidney is a major target organ during sepsis, and the dysfunction can be fatal to the host (1, 6, 22). Effective bacteria killing in SOCS5Tg mice may attenuate systemic inflammatory response and tissue damage induced by CLP. As shown in Fig. 8A, renal injury was reduced in SOCS5Tg mice, as evidenced by decreased serum levels of BUN and creatinine at 24 h after CLP relative to the WT mice. RAG-2–/– mice transferred with SOCS5Tg-CD4+ T cells were also exhibited decreases in the BUN and creatinine level at 24 h after CLP as compared with the mice received WT-CD4+ T cells (BUN: 42.9 ± 4.2 vs 62.5 ± 6.6 mg/dl, creatinine: 0.21 ± 0.03 vs 0.38 ± 0.06 mg/dl, respectively, 10 mice each, p < 0.05). In WT mice underwent CLP, epithelial cells in proximal tubules demonstrated karyorrhexis and grainy pink cytoplasm of epithelial cells in proximal tubules, indicative of acute tubular necrosis. In contrast, CLP caused little histological change in the SOCS5Tg mice (Fig. 8B). MPO level in the kidney, a marker of neutrophil infiltration, was decreased, and renal levels of TNF-{alpha}, MIP-2, and KC were significantly reduced in SOCS5Tg mice, compared with WT mice (Fig. 9). Thus, SOCS5Tg mice evaded renal injury induced by CLP, probably resulting in the improved mice survival post-CLP.


Figure 8
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  		FIGURE 8. Ameliorated renal injury in SOCS5Tg mice. At 24 h after CLP, mice were killed, bled, and the sera were harvested. A, BUN and creatinine level in sera was measured (WT, 11 mice; SOCS5Tg, 13 mice). {ddagger}, p < 0.05; {dagger}, p < 0.01 vs WT mice. B, Shown are representative histological sections of the kidney (H&E) at 24 h after CLP. Original magnification, x200.

 

Figure 9
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  		FIGURE 9. MPO and cytokine level in the kidney in SOCS5Tg mice. At 24 h after CLP, mice were bled and euthanized. MPO (A) and cytokines/chemokines levels (B) in kidney extracts were quantitated by ELISA. The data represent the mean ± SEM (10 mice, each). {ddagger}, p < 0.05; {dagger}, p < 0.01 vs WT mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
SOCS5 is a Th1-specific protein that inhibits Th2 differentiation (15), and is highly expressed in lymphoid organs in human tissues (26). Allergic conjunctivitis, a Th2-polarized inflammation model, was attenuated in SOCS5Tg mice (27). Thus, SOCS5 appears to skew acquired immunity in favor of Th1 response. In the present study, we have attempted to explore the role of SOCS5 in T cells in host defense during sepsis. Given our recent data showing that a balanced Th1/Th2 response controlled by STAT proteins is critical in host defense during sepsis (6, 11, 12), we had hypothesized that SOCS5 would play a beneficial role during sepsis. As expected, mice with a cell-specific overexpression of SOCS5 in T cells were resistant to CLP-induced lethality.

The innate immune response is the first line of host defense in infection, in which neutrophils and macrophages are cells in charge, enabling the host to achieve efficient removal of invading microbes (4, 28). In contrast, evidence indicates that T cells are also involved in host defense during infection. Mice genetically deficient in lymphocytes (nude and RAG–/– mice) were susceptible to various types of bacteria infection (29, 30, 31, 32, 33). Hotchkiss et al. (34) has demonstrated that RAG-1–/– mice were vulnerable to CLP; an event that was accompanied by increased blood bacteria counts. Immunocompromised hosts whose T cell function can be impaired are at high risk for opportunistic infections (35, 36). Thus, T cells appear to play a protective role in innate immunity during infection. Our present data highlight a new aspect that T cells, particularly CD4+ T cells, are capable of enhancing the innate immunity when SOCS5 is overexpressed in the cells, allowing the host to decrease bacteria burden, leading to subsequent alleviation of renal injury, a major cause of death during sepsis.

A question arises how SOCS5 in T cells affects innate immune response during septic peritonitis. Several mechanisms might contribute to the enhanced innate immunity in SOCS5Tg mice. Seki et al. (15) have demonstrated that overexpression of SOCS5 in T cells negatively regulates IL-4-dependent STAT6 activation without inhibiting IL-12-mediated STAT4 activation and produces higher IFN-{gamma}. IFN-{gamma} primes/stimulates macrophages to increase their responsiveness to microbial products such as bacterial LPS that include up-regulation of TLR4 expression, induction of antibacterial mediator NO, and production of IL-12 and TNF-{alpha} (37, 38, 39). In the present study, we have shown that phagocytes from SOCS5Tg mice exhibited augmented bactericidal activities and increased production of IL-12 and TNF-{alpha} by nature. WT phagocytes adopted enhanced bactericidal activities when cocultured with SOCS5Tg-CD4+ T cells. Considering that T cells are present in the peritoneum under physiological conditions (Thy1.2-positive cells, 11–16%, our own data), these findings suggest that peritoneal resident T cells in SOCS5Tg mice might influence innate immune responses. In the current study, we have shown that LPS-induced TNF-{alpha} production from macrophages was increased when macrophages were cultured with SOCS5Tg-CD4+ T cells relative to the WT-CD4+ T cells. This appeared to need cell-cell contacts, as no augmentation was found when macrophages and CD4+ T cells were separately cultured using transwell apparatus, and culture supernatants of CD4+ T cells failed to transfer the augmentation. More importantly, enhanced TNF-{alpha} production by SOCS5Tg-CD4+ T cells was diminished by neutralization of IFN-{gamma}. Considering the current data showing that macrophages are capable of producing TNF-{alpha} but not IFN-{gamma}, enhanced IFN-{gamma} production by SOCS5Tg-CD4+ T cells appear to be responsible for the augmented production of TNF-{alpha} by macrophages. In addition, we have demonstrated in the current study that SOCS5Tg-CD4+ T cells bear an increased memory cell-like phenotype. Acquisition of the ability to produce IFN-{gamma} is a fundamental property of memory T cells (40). These results suggest that peritoneal SOCS5Tg-CD4+ T cells may rapidly become activated and produce higher IFN-{gamma} in an initial phase of infection and then influences the innate immune response of phagocytes. Interestingly, STAT6 activation in peritoneal neutrophils and macrophages was impaired in SOCS5Tg mice. We have demonstrated that STAT6–/– mice are resistant to septic peritonitis by exerting an improved bacterial clearance via an altered cytokine profile in the peritoneum in favor of bacterial clearance (12). Attenuated STAT6 activation in phagocytes possibly through type 1-polarized peritoneal cytokine response also appears to contribute to the skewed immune response in SOCS5Tg mice.

Other mechanism(s) might be involved in the increased survival in SOCS5Tg mice. Studies in experimental animals and critically ill patients have demonstrated that increased apoptosis of lymphoid organs may provoke a marked immune suppression and organ system dysfunction (41). Prevention of thymic apoptosis protects mice from CLP-induced lethality (42, 43). Given the observation that SOCS5 is expressed in thymus of nontreated SOCS5Tg mice (15), the thymic T cells may fulfill a protective role during CLP. Alternatively, overexpression of SOCS5 in T cells may affect the recruitment of T cells into the thymus through decreased IL-4 signaling. The contribution of SOCS5 in lymphoid organs during sepsis is under investigation.

An attractive question is whether endogenous SOCS5 could regulate host defense during sepsis. Could SOCS5 deletion interfere with innate immune responses? A recent study by Brender et al. (44) demonstrated that SOCS5-deficient CD4+ T cells differentiated in vitro into either Th1 or Th2 cells with the same efficiency as WT cells. Furthermore, the mice mounted a protective Th1 response when infected with the intracellular parasite Leishmania major. Thus, endogenous SOCS5 seems not to be essential for Th1 cell generation. Endogenous SOCS5 in T cells may not play a role in innate immunity, however, the findings here and elsewhere (15) provide evidence that overexpression of SOCS5 in T cells appears to bias innate immunity in favor of a type 1 response. Another interest is whether SOCS5Tg mice are sensitive to LPS-induced shock due to the skewed immune response. Our preliminary data suggest that this is not likely as there were no significant differences in mice survival between WT and SOCS5Tg mice after i.p. injection of 100 mg/kg LPS (data not shown). This may result from discrepancies between infectious (CLP) and noninfectious (endotoxin) models of sepsis, which differ in their complexity and in their pathogenesis (45).

Our present data challenge a current concept that lymphocytes do not play a principal role in an initial phase of infection (46). We have provided evidence in the present study that the T cell response appears rapidly during infection, activating phagocytes to enhance their bactericidal activities and cytokine production within hours. Sepsis is a fatal disease and its mortality rate has not improved over the past three decades (1, 2). The treatment of sepsis and septic shock remains a clinical conundrum and recent prospective trials with biological response modifiers aimed at the inflammatory response have shown only modest clinical benefit (47, 48). The present findings raise the new prospect that up-regulation of SOCS5 in T cells may provide a novel therapeutic strategy in the management of sepsis.


   Acknowledgments
 
We thank Shinji Kudo, Motoko Kagayama, and Takako Maeda for their technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by grants from the Ministry of Education, Science, Sports and Culture, and the Ministry of Health and Welfare, Japan. Back

2 Address correspondence and reprint requests to Dr. Akihiro Matsukawa, Department of Pathology and Experimental Medicine, Graduate School of Medical, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan, 2-5-1, Shikata, Okayama 700-8558, Japan. E-mail address: amatsu{at}md.okayama-u.ac.jp Back

3 Abbreviations used in this paper: SOCS, suppressor of cytokine signaling; CLP, cecal ligation and puncture; WT, wild type; TSA, tryptose soy agar; APF, aminophenyl fluorescein; MPO, myeloperoxidase; BUN, blood urea nitrogen; MOF, multiple organ failure; Tg, transgenic. Back

Received for publication July 20, 2006. Accepted for publication October 10, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Astiz, M. E., E. C. Rackow. 1998. Septic shock. Lancet 351: 1501-1505. [Medline]
  2. Bone, R., R. Balk, F. Cerra, R. Dellinger, A. Fein, W. Knaus, R. Schein, W. Sibbald. 1992. Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. Chest 101: 1644-1655. [Medline]
  3. Fearon, D. T., R. M. Locksley. 1996. The instructive role of innate immunity in the acquired immune response. Science 272: 50-53. [Abstract]
  4. Hoffmann, J. A., F. C. Kafatos, C. A. Janeway, R. A. Ezekowitz. 1999. Phylogenetic perspectives in innate immunity. Science 284: 1313-1318. [Abstract/Free Full Text]
  5. Hack, C. E., L. A. Aarden, L. G. Thijs. 1997. Role of cytokines in sepsis. Adv. Immunol. 66: 101-195. [Medline]
  6. Matsukawa, A., C. M. Hogaboam, N. W. Lukacs, P. M. Lincoln, H. L. Evanoff, R. M. Strieter, S. L. Kunkel. 2000. Expression and contribution of endogenous IL-13 in an experimental model of sepsis. J. Immunol. 164: 2738-2744. [Abstract/Free Full Text]
  7. Sewnath, M. E., D. P. Olszyna, R. Birjmohun, F. J. ten Kate, D. J. Gouma, T. van Der Pollqq. 2001. IL-10-deficient mice demonstrate multiple organ failure and increased mortality during Escherichia coli peritonitis despite an accelerated bacterial clearance. J. Immunol. 166: 6323-6331. [Abstract/Free Full Text]
  8. Steinhauser, M. L., C. M. Hogaboam, N. W. Lukacs, R. M. Strieter, S. L. Kunkel. 1999. Multiple roles for IL-12 in a model of acute septic peritonitis. J. Immunol. 162: 5437-5443. [Abstract/Free Full Text]
  9. Levy, D. E., J. E. Darnell, Jr. 2002. Stats: transcriptional control and biological impact. Nat. Rev. Mol. Cell Biol. 3: 651-662. [Medline]
  10. O’Shea, J. J., M. Gadina, R. D. Schreiber. 2002. Cytokine signaling in 2002: new surprises in the Jak/Stat pathway. Cell 109: (Suppl.):S121-S131. [Medline]
  11. Matsukawa, A., K. Takeda, S. Kudo, T. Maeda, M. Kagayama, S. Akira. 2003. Aberrant inflammation and lethality to septic peritonitis in mice lacking STAT3 in macrophages and neutrophils. J. Immunol. 171: 6198-6205. [Abstract/Free Full Text]
  12. Matsukawa, A., M. H. Kaplan, C. M. Hogaboam, N. W. Lukacs, S. L. Kunkel. 2001. Pivotal role of signal transducer and activator of transcription (Stat)4 and Stat6 in the innate immune response during sepsis. J. Exp. Med. 193: 679-688. [Abstract/Free Full Text]
  13. Alexander, W. S.. 2002. Suppressors of cytokine signalling (SOCS) in the immune system. Nat. Rev. Immunol. 2: 410-416. [Medline]
  14. Yasukawa, H., A. Sasaki, A. Yoshimura. 2000. Negative regulation of cytokine signaling pathways. Annu. Rev. Immunol. 18: 143-164. [Medline]
  15. Seki, Y., K. Hayashi, A. Matsumoto, N. Seki, J. Tsukada, J. Ransom, T. Naka, T. Kishimoto, A. Yoshimura, M. Kubo. 2002. Expression of the suppressor of cytokine signaling-5 (SOCS5) negatively regulates IL-4-dependent STAT6 activation and Th2 differentiation. Proc. Natl. Acad. Sci. USA 99: 13003-13008. [Abstract/Free Full Text]
  16. Fink, M. P., S. O. Heard. 1990. Laboratory models of sepsis and septic shock. J. Surg. Res. 49: 186-196. [Medline]
  17. Matsukawa, A., C. M. Hogaboam, N. W. Lukacs, P. M. Lincoln, H. L. Evanoff, S. L. Kunkel. 2000. Pivotal role of the CC chemokine, macrophage-derived chemokine, in the innate immune response. J. Immunol. 164: 5362-5368. [Abstract/Free Full Text]
  18. Matsukawa, A., C. M. Hogaboam, N. W. Lukacs, P. M. Lincoln, R. M. Strieter, S. L. Kunkel. 1999. Endogenous monocyte chemoattractant protein-1 (MCP-1) protects mice in a model of acute septic peritonitis: cross-talk between MCP-1 and leukotriene B4. J. Immunol. 163: 6148-6154. [Abstract/Free Full Text]
  19. Leijh, P. C., M. T. van den Barselaar, R. van Furth. 1977. Kinetics of phagocytosis and intracellular killing of Candida albicans by human granulocytes and monocytes. Infect. Immun. 17: 313-318. [Abstract/Free Full Text]
  20. Setsukinai, K., Y. Urano, K. Kakinuma, H. J. Majima, T. Nagano. 2003. Development of novel fluorescence probes that can reliably detect reactive oxygen species and distinguish specific species. J. Biol. Chem. 278: 3170-3175. [Abstract/Free Full Text]
  21. Babior, B. M., C. R. Hoyal, R. I. Lehrer, T. Ganz. 2001. Antimicrobial activity of leukocytes. K. Ley, Jr, ed. Physiology of inflammation 189-203. Oxford University Press, New York.
  22. Matsukawa, A., S. Kudoh, G. Sano, T. Maeda, T. Ito, N. W. Lukacs, C. M. Hogaboam, S. L. Kunkel, S. A. Lira. 2006. Absence of CC chemokine receptor 8 enhances innate immunity during septic peritonitis. FASEB J. 20: 302-304. [Abstract/Free Full Text]
  23. Watanabe, H., K. Numata, T. Ito, K. Takagi, A. Matsukawa. 2004. Innate immune response in Th1- and Th2-dominant mouse strains. Shock 22: 460-466. [Medline]
  24. Boehm, U., T. Klamp, M. Groot, J. C. Howard. 1997. Cellular responses to interferon-{gamma}. Annu. Rev. Immunol. 15: 749-795. [Medline]
  25. Malaviya, R., T. Ikeda, E. Ross, S. N. Abraham. 1996. Mast cell modulation of neutrophil influx and bacterial clearance at sites of infection through TNF-{alpha}. Nature 381: 77-80. [Medline]
  26. Magrangeas, F., F. Apiou, S. Denis, U. Weidle, Y. Jacques, S. Minvielle. 2000. Cloning and expression of CIS6, chromosome assignment to 3p22 and 2p21 by in situ hybridization. Cytogenet. Cell Genet. 88: 78-81. [Medline]
  27. Ozaki, A., Y. Seki, A. Fukushima, M. Kubo. 2005. The control of allergic conjunctivitis by suppressor of cytokine signaling (SOCS)3 and SOCS5 in a murine model. J. Immunol. 175: 5489-5497. [Abstract/Free Full Text]
  28. Matsukawa, A., C. M. Hogaboam, N. W. Lukacs, S. L. Kunkel. 2000. Chemokines and innate immunity. Rev. Immunogenet. 2: 339-358. [Medline]
  29. Black, C. A., F. M. Eyers, A. Russell, M. L. Dunkley, R. L. Clancy, K. W. Beagley. 1999. Increased severity of Candida vaginitis in BALB/c nu/nu mice versus the parent strain is not abrogated by adoptive transfer of T cell enriched lymphocytes. J. Reprod. Immunol. 45: 1-18. [Medline]
  30. Le-Barillec, K., J. G. Magalhaes, E. Corcuff, A. Thuizat, P. J. Sansonetti, A. Phalipon, J. P. Di Santo. 2005. Roles for T and NK cells in the innate immune response to Shigella flexneri. J. Immunol. 175: 1735-1740. [Abstract/Free Full Text]
  31. Teles, R., C. Y. Wang, P. Stashenko. 1997. Increased susceptibility of RAG-2 SCID mice to dissemination of endodontic infections. Infect. Immun. 65: 3781-3787. [Abstract]
  32. Ueda, K., S. Yamazaki, J. Saegusa, S. Someya. 1977. Septicemic infection in nude mice caused by a virulent strain of Mycobacterium bovis. Jpn. J. Exp. Med. 47: 467-474. [Medline]
  33. Vallance, B. A., W. Deng, L. A. Knodler, B. B. Finlay. 2002. Mice lacking T and B lymphocytes develop transient colitis and crypt hyperplasia yet suffer impaired bacterial clearance during Citrobacter rodentium infection. Infect. Immun. 70: 2070-2081. [Abstract/Free Full Text]
  34. Hotchkiss, R. S., K. C. Chang, P. E. Swanson, K. W. Tinsley, J. J. Hui, P. Klender, S. Xanthoudakis, S. Roy, C. Black, E. Grimm, et al 2000. Caspase inhibitors improve survival in sepsis: a critical role of the lymphocyte. Nat. Immunol. 1: 496-501. [Medline]
  35. Waite, S., J. Jeudy, C. S. White. 2006. Acute lung infections in normal and immunocompromised hosts. Radiol. Clin. North Am. 44: 295-315. [Medline]
  36. Beck, J. M.. 2005. The immunocompromised host: HIV infection. Proc. Am. Thorac. Soc. 2: 423-427. [Abstract/Free Full Text]
  37. Bosisio, D., N. Polentarutti, M. Sironi, S. Bernasconi, K. Miyake, G. R. Webb, M. U. Martin, A. Mantovani, M. Muzio. 2002. Stimulation of Toll-like receptor 4 expression in human mononuclear phagocytes by interferon-{gamma}: a molecular basis for priming and synergism with bacterial lipopolysaccharide. Blood 99: 3427-3431. [Abstract/Free Full Text]
  38. Philip, R., L. B. Epstein. 1986. Tumour necrosis factor as immunomodulator and mediator of monocyte cytotoxicity induced by itself, {gamma}-interferon and interleukin-1. Nature 323: 86-89. [Medline]
  39. Totemeyer, S., M. Sheppard, A. Lloyd, D. Roper, C. Dowson, D. Underhill, P. Murray, D. Maskell, C. Bryant. 2006. IFN-{gamma} enhances production of nitric oxide from macrophages via a mechanism that depends on nucleotide oligomerization domain-2. J. Immunol. 176: 4804-4810. [Abstract/Free Full Text]
  40. Murphy, K. M., S. L. Reiner. 2002. The lineage decisions of helper T cells. Nat. Rev. Immunol. 2: 933-944. [Medline]
  41. Oberholzer, C., A. Oberholzer, M. Clare-Salzler, L. L. Moldawer. 2001. Apoptosis in sepsis: a new target for therapeutic exploration. FASEB J. 15: 879-892. [Abstract/Free Full Text]
  42. Iwata, A., V. M. Stevenson, A. Minard, M. Tasch, J. Tupper, E. Lagasse, I. Weissman, J. M. Harlan, R. K. Winn. 2003. Over-expression of Bcl-2 provides protection in septic mice by a trans effect. J. Immunol. 171: 3136-3141. [Abstract/Free Full Text]
  43. Guo, R. F., M. Huber-Lang, X. Wang, V. Sarma, V. A. Padgaonkar, R. A. Craig, N. C. Riedemann, S. D. McClintock, T. Hlaing, M. M. Shi, P. A. Ward. 2000. Protective effects of anti-C5a in sepsis-induced thymocyte apoptosis. J. Clin. Invest. 106: 1271-1280. [Medline]
  44. Brender, C., R. Columbus, D. Metcalf, E. Handman, R. Starr, N. Huntington, D. Tarlinton, N. Odum, S. E. Nicholson, N. A. Nicola, et al 2004. SOCS5 is expressed in primary B and T lymphoid cells but is dispensable for lymphocyte production and function. Mol. Cell Biol. 24: 6094-6103. [Abstract/Free Full Text]
  45. Remick, D. G., D. E. Newcomb, G. L. Bolgos, D. R. Call. 2000. Comparison of the mortality and inflammatory response of two models of sepsis: lipopolysaccharide vs. cecal ligation and puncture. Shock 13: 110-116. [Medline]
  46. Medzhitov, R., C. Janeway, Jr. 2000. Innate immunity. N. Engl. J. Med. 343: 338-344. [Free Full Text]
  47. Patel, G. P., D. P. Gurka, R. A. Balk. 2003. New treatment strategies for severe sepsis and septic shock. Curr. Opin. Crit. Care 9: 390-396. [Medline]
  48. Rice, T. W., G. R. Bernard. 2005. Therapeutic intervention and targets for sepsis. Annu. Rev. Med. 56: 225-248. [Medline]



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