SEARCH

SEARCH BY CITATION

Keywords:

  • Cytokine;
  • Sepsis;
  • T cells

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Appendix

To study the role of T cells in gram-negative sepsis, we developed a mouse model in which i.v. injection of Escherichia coli results in severe systemic illness, with high mortality rates after day 5. A large proportion of both CD4+ and CD8+ T cells are activated within 1 day after infection, as evidenced by up-regulation of CD69 and down-regulation of CD62L. Even more surprisingly, T cell-deficient mice exhibit markedly decreased disease severity compared to WT mice, indicating a pathogenic role of T cells. Mice lacking IFN-γ also show diminished disease, and exhibit reduced T cell activation. Therefore, the pathogenic role of T cells may be mediated by IFN-γ. Both T cell- and IFN-γ-deficient mice have reduced serum IL-6 levels compared to WT mice, suggesting that T cells may stimulate innate immune responses, resulting in enhancement of disease. These data indicate an important role for T cells in a mouse model of E. coli sepsis, and reveal an unexpected early and pathogenic T cell response to this bacterial infection.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Appendix

Sepsis continues to be a major burden on health care resources, affecting approximately 700 000 people per year in the USA often requiring extended hospitalization. In addition, mortality remains high at 25–30%, and specific therapies are lacking. This is in part due to incomplete understanding of the pathogenesis of the disease process. The concept of exacerbated inflammatory responses to overwhelming bacterial infection 1 appears to be an over-simplification. It has been established that activation of the innate immune system occurs early in the course of sepsis, resulting in release of a multitude of pro-inflammatory mediators. Although several studies have revealed correlations between the production of mediators and disease severity and/or mortality, clinical trials with anti-inflammatory agents have had disappointing results so far 2, 3. Mortality in humans with sepsis is often delayed and the late phase of sepsis appears to be characterized by immunosuppression rather than overactivation 4. It has been postulated that altered adaptive immune responses may play a role in the outcome of sepsis 2.

In order to study adaptive immune responses, which are traditionally thought to occur at least 3–4 days after antigen exposure, we developed an Escherichia coli mouse model of gram-negative sepsis in which mortality is delayed past the first few days after introduction of the bacteria. We found that mice develop severe systemic illness upon infection with E. coli, with high mortality rates 5–7 days after infection. In this model, T cell activation occurred surprisingly early (within 24 h), and absence of T cells was associated with diminished disease, as was absence of IFN-γ. We conclude that adaptive immune responses play a pathogenic role in this model of E. coli sepsis, possibly mediated by IFN-γ.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Appendix

Intravenous injection of E. coli results in systemic disease in mice

Initial experiments were designed to establish a model of systemic E. coli sepsis in mice. BALB/c mice were injected with either PBS or increasing doses of E. coli, and mortality (Fig. 1A) as well as disease manifestations including weight loss and clinical appearance (quantified as a composite disease score rating the presence of ruffled fur, hunched appearance and lethargy) were monitored daily (Fig. 1B, C).

thumbnail image

Figure 1. WT mice develop a dose-dependent systemic illness upon i.v. injection of E. coli. (A–C) BALB/c mice were injected with either PBS or 1.5×107, 1.5×108 or 5×108 CFU of E. coli and survival (A), weight loss (B) and disease scores (C) were recorded on subsequent days. Data points represent means with standard deviations, n=6–9; *p<0.01 comparing 1.5×108 CFU group to 1.5×107 CFU group, **p<0.001 comparing 1.5×108 CFU group to 1.5×107 CFU group, ##p<0.001 comparing 1.5×108 CFU group to PBS group. (D) C57BL/6 mice were inoculated with 1.5×107 or 1.5×108 CFU of of E. coli and weight loss and disease scores were recorded on subsequent days. Data points represent means with standard deviations, n=4–5; *p<0.05 comparing 1.5×108 CFU group to PBS group, #p<0.05 comparing 1.5×107 CFU group to PBS group, **p<0.05 comparing 1.5×108 CFU group to 1.5×107 CFU group.

Download figure to PowerPoint

Mice inoculated with the highest dose, 5×108 CFU, all died within 2 days after infection, whereas approximately 40% of mice infected with 1.5×108 CFU and all mice inoculated with 1.5×107 CFU survived past day 7. Mice inoculated with 1.5×107 CFU of E. coli exhibited mild weight loss on day 1 after infection (p<0.01 compared to PBS-injected mice), with recovery to baseline weights over subsequent days. Mice infected with 1.5×108 CFU demonstrated progressive weight loss, which was significantly worse in mice inoculated with 1.5×108 CFU of E. coli compared to those given an inoculation dose of 1.5×107 CFU (p<0.01 on day 1 after infection, p<0.001 on subsequent days). In concordance with weight loss, mice inoculated with 1.5×107 CFU of E. coli developed mild and transient outward signs of disease as shown by disease scores (p<0.01 on day 1 after infection compared to PBS group, not significant on subsequent days). Mice inoculated with 1.5×108 CFU had a marked hunched and ruffled appearance as early as day 1 after infection (p<0.01 compared to mice inoculated with 1.5×107 CFU), which became progressively worse with notable lethargy in most animals by day 4 after infection.

In mice infected with 1.5×108 CFU of E. coli, cultures of kidney, liver, lung and spleen homogenates were positive throughout the course of infection, whereas blood cultures were positive during the first 2–3 days only (data not shown). Thus, i.v. injection of E. coli resulted in a dose-dependent systemic disease with evidence of spread of the bacteria to multiple organs. A similar disease pattern was seen in C57BL/6 mice (Fig. 1D).

Activation of T cells in E. coli-infected mice

To examine whether T cells play a role in this mouse model of sepsis, we first assessed whether there was evidence of T cell activation upon systemic E. coli infection. Splenocytes and peripheral lymph nodes from E. coli-infected mice were isolated at various time points after infection and analyzed by staining and flow cytometry for the expression of activation markers.

In comparison with PBS-injected mice, T cells from E. coli-infected mice exhibited increased expression of CD69 and decreased expression of CD62L, indicating T cell activation (Fig. 2). This pattern of expression was noted in both CD4+ and CD8+ cell populations. Increased expression of CD69 was present remarkably early (within 24 h) of E. coli infection on a surprisingly large proportion of T cells (up to 68% of CD4+ cells and up to 82% of CD8+ cells, p<0.05 compared to the percentage of CD69+ cells in PBS-injected mice). Although most pronounced on day 1 after infection, up-regulation of CD69 persisted throughout day 7 of infection (p<0.05 compared to PBS-injected mice). Similarly, down-regulation of CD62L expression was most pronounced on day 1 after infection, but persisted throughout day 7 (p<0.05 compared to the percentage of CD62L+ cells in PBS-injected mice). Of note, there was no significant change in the total number of CD4+ and CD8+ T cells during the course of the disease (Fig. 2D, Table 1).

thumbnail image

Figure 2. Activation of T lymphocytes in E. coli-infected mice. Splenocytes were isolated at different time points after injection with PBS or 1.5×108 CFU of E. coli and analyzed by staining and flow cytometry for CD69 and CD62L expression. (A) FACS blots representative of findings on day 1 in E. coli-infected mice (top panels) and PBS-injected mice (bottom panels). (B, C) Quantification of the percentage of CD69+ cells (B) and CD69+CD62lo cells (C) of total CD4+ (left) and CD8+ T cells (right). (D) Quantification of total number of CD4+ cells (left) and CD8+ cells (right). Data are expressed as means with standard deviations, n=4; *p<0.05 comparing E. coli-infected to PBS-injected mice. Similar results were obtained when analyzing peripheral lymph node cells (data not shown).

Download figure to PowerPoint

Table 1. Leukocyte subpopulations in E .coli- vs. PBS-injected micea)
Total cellsNeutrophils(Gr-1+)B cells(B220+)T cells
  1. a) Splenocytes were isolated at different time points after injection with either PBS or 1.5×108E. coli and analyzed by staining and flow cytometry as described in Fig. 2; data are presented as mean values ± standard deviation.

  2. b)p<0.05 as compared to PBS-injected group on the same day.

CD4+CD8+
TotalCD69+CD69+CD62loTotalCD69+CD69+CD62lo
×107×106×106×106×106×106×106×105×105
Day 1
E. coli5.7 ± 0.912.7 ± 2.8b)16.7 ± 2.720.0 ± 3.512.8 ± 2.6b)3.3 ± 0.4 b)2.8 ± 0.821.6 ± 7.3 b)6.8 ± 1.4b)
PBS6.7 ± 0.77.7 ± 0.318.0 ± 6.218.7 ± 2.91.4 ± 0.20.9 ± 0.13.8 ± 0.91.3 ± 0.30.6 ± 0.2
Day 2
E. coli6.7 ± 1.619.5 ± 1.7b)20.8 ± 1.225.8 ± 1.68.9 ± 5.6b)2.8 ± 1.6b)3.3 ± 1.715.0 ± 7.6b)3.3 ± 0.7b)
PBS5.5 ± 1.96.5 ± 2.518.8 ± 6.518.8 ± 7.91.1 ± 0.50.7 ± 0.43.1 ± 1.10.7 ± 0.40.3 ± 0.2
Day 3
E. coli4.6 ± 1.411.2 ± 2.4b)17.1 ± 6.113.7 ± 9.52.5 ± 1.6b)1.4 ± 0.81.7 ± 1.42.4 ± 2.3b)0.8 ± 0.7b)
PBS4.2 ± 0.25.5 ± 0.813.6 ± 1.414.3 ± 0.71.0 ± 0.20.9 ± 0.21.0 ± 0.10.4 ± 0.100.2 ± 0.1
Day 4
E. coli4.2 ± 2.09.8 ± 4.712.7 ± 5.316.0 ± 7.62.3 ± 0.9b)1.3 ± 0.51.5 ± 0.51.8 ± 0.7b)0.7 ± 0.2b)
PBS4.4 ± 0.75.5 ± 1.214.7 ± 3.417.3 ± 2.91.6 ± 0.41.0 ± 0.32.1 ± 0.20.9 ± 0.30.5 ± 0.1
Day 7
E. coli13.0 ± 6.250.4 ± 33.7 b)21.9 ± 6.715.0 ± 3.52.4 ± 0.5b)1.9 ± 0.5b)1.5 ± 0.22.0 ± 0.2b)1.2 ± 0.5b)
PBS5.1 ± 1.36.0 ± 2.217.2 ± 4.313.4 ± 3.51.3 ± 0.40.8 ± 0.31.8 ± 0.40.9 ± 0.30.4 ± 0.2

To assess whether the observed increase in activation markers was associated with effector cytokine production, splenocytes were stained for the presence of intracellular cytokines after stimulation with PMA and ionomycin, plate-bound anti-CD3 or heat-killed E. coli, respectively. No differences in IL-2 or IFN-γ staining were observed between E. coli- and PBS-injected mice on either day 1 or day 4 after infection (data not shown).

E. coli-infected T cell-deficient mice exhibit less severe disease

Next, we assessed whether absence of T cells had an impact on disease severity. TCRα-knockout mice (TCRα–/–), which lack CD4+, CD8+ and NK T cells due to a deletion in the gene for the TCR α chain, were infected with 1.5×108 CFU of E. coli and disease severity was compared with that in congenic WT mice infected simultaneously with an equal dose. As depicted in Fig. 3A, TCRα–/– mice developed only mild and transient weight loss, which was significantly less severe than that observed in WT mice (p<0.01 after day 1). Similarly, although ruffled fur and hunched appearance were noted in TCRα–/– mice on day 1 after infection, these were less marked than in WT mice (p<0.05 for disease scores on day 1 compared to TCRα–/– mice) and resolved over subsequent days. In contrast, WT mice exhibited progressively worsening signs of disease (p<0.01 compared to TCRα–/– mice on days 2–4; Fig. 3B).

thumbnail image

Figure 3. T cell-deficient mice show reduced disease severity after E. coli infection. (A, B) TCRα–/– mice and age-matched congenic WT mice were inoculated with 1.5×108 CFU of E. coli and weight loss (A) as well as disease scores (B) were recorded on subsequent days. Data points represent means with standard deviations, n=5–6; #p<0.05, *p<0.01 as compared to WT mice. (C) In a separate experiment, E. coli-infected TCRα–/– and WT mice were sacrificed on different days after infection, and bacterial growth in blood and liver was quantified. Data points represent means with standard deviations, n = three to four mice per group per time point.

Download figure to PowerPoint

In a separate experiment, E. coli-infected TCRα–/– mice and WT mice were sacrificed on different days after infection, and bacterial growth in blood and liver was assessed. TCRα–/– mice appeared to clear bacteria from the blood sooner than WT mice (cultures negative at day 2 in TCRα–/– mice vs. days 3–4 in WT mice), but cultures from liver remained positive in both groups up to 7 days after infection, with similar CFU counts through day 4 (Fig. 3C). Taken together, these data indicate a pathogenic role for T cells in this E. coli model of sepsis, possibly related to a paradoxically delayed clearance of bacteria from the blood stream in the presence of T cells.

We attempted to identify the subset of T cells that may be involved in the disease via several approaches. First, we injected antibodies to deplete CD4+ and/or CD8+ T cells in WT mice prior to infection with E. coli. In two separate experiments, there was moderate amelioration of disease in mice depleted of CD8+ T cells, but not in mice depleted of CD4+ T cells (data not shown). Despite repeated doses of depleting antibodies, we were not able to establish complete depletion as tested by flow cytometry of single-cell suspensions obtained from spleen and peripheral lymph nodes. It is therefore difficult to interpret whether lack of a more pronounced effect is due to incomplete depletion, or because other cell populations in addition to CD8+ T cells are involved.

Second, we tried to reconstitute TCRα–/– mice with T cells prior to E. coli infection. Single-cell suspensions of spleens and peripheral lymph nodes obtained from WT mice were enriched for T cells by MACS separation and injected into TCRα–/– mice prior to infection with E. coli. Using this approach, we were unable to detect a difference in disease severity between E. coli-infected, reconstituted TCRα–/– mice and unreconstituted TCRα–/– mice. Reconstitution was, however, incomplete with total number of CD4+ and CD8+ T cell retrieved from spleens less than 50% of the numbers observed in WT mice (data not shown).

IFN-γ-deficient mice exhibit less severe disease and diminished T cell activation

We postulated that the pathogenic effect of T cells in this model is most likely mediated through one or more cytokines. The pathogenic effects of IFN-γ, a cytokine produced by CD4+, CD8+ as well as NK T cells, have been well described in multiple disease models. To test the hypothesis that IFN-γ plays a role in the disease severity of systemic E. coli infection in mice, we performed two separate sets of experiments.

First, IFN-γ-knockout (IFN-γ–/–) mice were infected with 1.5×108 CFU of E. coli and disease severity was compared to that in congenic WT mice as well as TCRα–/– mice, infected simultaneously with an equal dose. As shown in Fig. 4A, weight loss in IFN-γ–/– mice was similar to that seen in TCRα–/– mice, and much less severe than in WT mice (p<0.05 after day 1). Disease symptoms were also much less pronounced in IFN-γ–/– and TCRα–/– mice compared to WT mice (Fig. 4B, p<0.05 for all time points). Although TCRα–/– mice again cleared bacteria from the blood stream by day 2, blood cultures from both WT and IFN-γ–/– mice stayed positive through day 3 with similar CFU counts for both groups. Bacterial growth in liver was similar for all three groups (data not shown).

thumbnail image

Figure 4. IFN-γ-deficient mice show reduced disease severity and decreased T cell activation after infection with E. coli. (A, B) Age-matched IFN-γ–/–, TCRα–/– and WT mice (all on the BALB/c background) were inoculated with 1.5×108 CFU of E. coli and weight loss (A) as well as disease scores (B) were recorded on subsequent days. Data points represent means with standard deviations, n=4–6; *p<0.05 as compared to WT mice. (C) IFN-γ–/– mice and congenic, age-matched WT mice were inoculated with 1.5×108 CFU of E. coli and splenocytes were isolated on subsequent days and analyzed for expression of the activation marker CD69 by staining and flow cytometry. Percentages of CD69+ cells among the CD4+ gated population (left panel) and the CD8+ gated population (right panel) were calculated. Data points represent means with standard deviations, n=3; *p<0.05 comparing IFN-γ–/– to WT mice.

Download figure to PowerPoint

In a separate set of experiments, WT mice were treated with IFN-γ-blocking antibodies prior to infection with E. coli, and disease severity was compared to E. coli-infected, PBS-treated mice. Consistent with the observations in IFN-γ–/– mice, antibody-depleted mice had less severe weight loss than WT mice [weight loss on day 4, –9.3 ± 6.2% (mean ± standard deviation) vs. –17.1 ± 3.9%, p=0.07)] and lower disease scores (disease score on day 4, 1.0 ± 1.0 vs. 3.8 ± 0.5, p<0.01, n=5 mice per group, data not shown).

We then assessed whether IFN-γ–/– mice had similar activation of T cells upon E. coli infection. Fig. 4C shows a quantitative comparison of the results of staining and flow cytometry performed on splenocytes obtained at several time points after infection from E. coli-infected IFN-γ–/– mice and WT mice, respectively. Although IFN-γ–/– mice exhibited increased expression of CD69 on both CD4+ and CD8+ cells after E. coli infection, the percentage of cells expressing this activation marker was significantly reduced when compared to WT mice (mean reduction of 33–64%, p<0.05). Total numbers of CD4+ and CD8+ T cells were not different between IFN-γ–/– mice and WT mice (Table 2).

Table 2. Number of activated T cells in WT vs. IFN-γ–/–E. coli-infected micea)
CD4+CD8+
  1. a) Splenocytes from E. coli-infected WT and IFN-γ–/– mice were isolated at different time points after infection and analyzed for expression of activation markers as described in Fig. 4; data are presented as mean values ± standard deviation.

  2. b)p<0.05 as compared to WT group on the same day.

TotalCD4+CD69+TotalCD8+CD69+
×106×106×106×105
Day 1
WT11.2 ± 2.68.5 ± 4.61.8 ± 0.615.4 ± 5.1
IFN-γ–/–9.2 ± 3.44.7 ± 2.21.1 ± 0.56.5 ± 2.7b)
Day 2
WT13.0 ± 3.16.1 ± 2.31.7 ± 0.37.5 ± 1.0
IFN-γ–/–12.0 ± 3.92.3 ± 0.9b)1.7 ± 0.83.2 ± 1.5b)
Day 3
WT13.5 ± 0.42.8 ± 0.31.5 ± 0.42.1 ± 0.5
IFN-γ–/–14.4 ± 1.21.2 ± 0.3b)1.2 ± 0.50.7 ± 0.2b)

Production of cytokines after E. coli infection

Next, we screened sera obtained from TCRα–/–, IFN-γ–/– and WT mice on day 1 and day 4 after infection with E. coli for the presence of a panel of cytokines using multiplex cytokine detection assay. Surprisingly, there was no difference in IFN-γ levels between TCRα–/– and WT mice on day 1 (Fig. 5A) or day 4 (Fig. 5B). There was, however, significantly less IL-6 in the sera of both TCRα–/– and IFN-γ–/– mice in comparison to WT mice on day 1 (792 ± 318 pg/mL in WT mice vs. 328 ± 118 pg/mL in IFN-γ–/– mice and 126 ± 88 pg/mL in TCRα–/– mice; p<0.05) as well as day 4 (570 ± 288 pg/mL in WT mice vs. 209 ± 69 pg/mL in IFN-γ–/– mice and 211 ± 86 pg/mL in TCRα–/– mice; p<0.05). In addition, on day 1, TNF-α levels were lower in IFN-γ–/– compared to WT mice (758 ± 244 pg/mL in IFN-γ–/– mice vs. 1405 ± 427 in WT mice; p<0.05), whereas levels in TCRα–/– mice were comparable to those in WT mice. TNF-α levels on day 4 were similar for all three groups.

thumbnail image

Figure 5. Serum cytokine levels in E. coli-infected mice. Serum obtained from E. coli-infected IFN-γ–/–, TCRα–/– and congenic WT mice on day 1 (A) and day 4 (B) after infection were analyzed for cytokine expression by multiplex detection assay. All samples were tested in duplicate; circles represent the mean value for each animal tested, lines represent the mean value per group, *p<0.05.

Download figure to PowerPoint

To confirm results from this initial screening assay, additional serum samples from E. coli-infected TCRα–/–, IFN-γ–/– and WT mice sacrificed on days 1, 2, 3 and 4 after infection were assayed by ELISA to quantify IL-6 levels. As summarized in Table 3, increased levels of IL-6 were found in sera from WT mice at all time points tested, whereas IL-6 levels were low (and frequently below level of detection) in all samples obtained from TCRα–/– mice (p<0.05 for all time points). Similarly low levels were measured in sera from IFN-γ–/– mice obtained on days 2–4. Moderately elevated IL-6 levels were measured in samples from some IFN-γ–/– mice on day 1, but both maximum levels and mean levels were lower than those in WT mice on day 1 (p<0.05).

Table 3. Serum IL-6 levels in E. coli-infected micea)
WTTCRα–/–IFN-γ–/–
  1. a) Serum obtained from E. coli-infected WT, TCRα–/– and IFN-γ–/– mice at several time points after infection were analyzed for IL-6 levels by ELISA. Data are presented as mean value ± standard deviation [range of values].

  2. b)p<0.005 as compared to WT group on the same day.

  3. c)p<0.001 as compared to WT group on the same day.

pg/mLPg/mLpg/mL
Day 11690.1 ± 795.7[400.2–3353.0]n=978.3 ± 90.3c)[0–233.4]n=8454.1 ± 247.5b)[21.4–765.2]n=8
Day 21316.1 ± 759.1[362.8–2796.7]n=927.9 ± 57.4c)[0–166.3]n=844.7 ± 80.2b)[0–164.61]n=6
Day 3963.8 ± 682.2[288.7–2159.4]n=755.5 ± 43.4b)[0–117.5]n=6111.4 ± 160.4b)[0–411.9]n=5
Day 41248.5 ± 655.0[349.3–2479.1]n=915.5 ± 18.4c)[0–43.4]n=821.8 ± 27.0c)[0–75.9]n=8

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Appendix

The experiments described here were designed to study the role of T cells in a mouse model of gram-negative sepsis. Surprisingly, activation of a large percentage of T cells occurs very early after i.v. injection of E. coli, and absence of T cells is associated with decreased disease severity, indicating a pathogenic role for T cells in this model. Mice lacking IFN-γ also develop less severe disease than WT mice upon E. coli infection, which appears to be associated with decreased activation of T cells. Amelioration of disease in both IFN-γ–/– mice and TCRα–/– mice is associated with decreased levels of IL-6.

A role for T cells in sepsis was first suggested by Hotchkiss and colleagues 5, who demonstrated rapid apoptosis of CD4+ T cells in the murine colonic ligation and puncture model of sepsis and showed that prevention of apoptosis resulted in improved outcome. These results suggest that T cells are protective, although the exact mechanism behind this phenomenon remains unclear. Although we did not study apoptosis in our model, we did not observe depletion of CD4+ T cells in the spleens of E. coli-infected mice. Rather, our data clearly show that the absence of T cells reduces disease severity, i.e. activated T cells are pathogenic. These differences may reflect difference in the models: colonic ligation and puncture results in peritonitis and polymicrobial bacteremia, whereas in our model sepsis does not arise from a nidus of infection and involves one microbe only.

In a study of humans with severe gram-negative sepsis, a decreased percentage of CD4+ T cells was noted in the peripheral blood of septic patients as compared to healthy controls 6. Patients in this study were selected on criteria that included a known (pulmonary or intra-abdominal) nidus of infection, which, as noted above, may result in different immune responses. In addition, it is unclear whether changes in peripheral blood cell counts reflect those in spleen and lymph nodes; because of small cell numbers, quantification of CD4+ T cells in peripheral blood is difficult in a mouse model. Nevertheless, our results are the first to show a pathogenic role of T cells in gram-negative sepsis.

Activation of T cells occurs in response to antigens presented by antigen-presenting cells together with a co-stimulatory signal; this process is thought to take several days. The percentage of antigen-specific T cells for any given antigen is low; therefore only a small percentage of T cells are expected to become activated in response to a microbial infection. Surprisingly, T cell activation was detected within 24 h after i.v. injection of E. coli, and involved a large percentage of T cells (up to 80%).

It has been postulated that T cells can respond directly to microbial products such as LPS via direct engagement of TLR on T cells 7, 8. E. coli infection of mice lacking MyD88, a central adaptor protein for signal transduction of TLR, resulted in similar early and extensive T cell activation compared to WT mice (data not shown), making this explanation less likely, although signaling through MyD88-independent pathways could still be involved. Early and pronounced up-regulation of CD69 on CD4+ and CD8+ T cells in response to injection of LPS and other microbial mitogens into mice has been demonstrated previously 9, 10. In subsequent studies it was shown that the effect of LPS on T cells is mediated by dendritic cells, in part via secretion of IFN-αβ, which stimulates T cells directly, and in part by stimulating IFN-γ production by NK cells 11. In concordance with these observations, we demonstrated reduced T cell activation in E. coli-infected IFN-γ–/– mice. Of note, we did not find evidence for increased IFN-γ or IL-2 production by T cells, indicating that the observed activation of T cells may be incomplete and not lead to a full effector phenotype with resulting cytokine secretion.

T cell-deficient mice developed significantly less severe disease upon E. coli infection than WT mice, indicating that T cells have a detrimental effect on disease outcome. We were unable to identify with certainty the specific subset of T cells responsible for this effect. Results from antibody depletion experiments pointed in the direction of CD8+ T lymphocytes. In the colonic ligation and puncture model of murine sepsis, mice deficient in CD8+ T cells were found to have longer survival compared to WT mice, although mortality was still 100% after 72 h 12. Considering the observation that both CD4+ and CD8+ T cells show up-regulation of activation markers in our model, we believe it is unlikely that either subset of T cells is solely responsible for the pathogenic effect. T cells involved in modulation of disease severity in our model could have an effector phenotype, or, alternatively, be regulatory cells that have suppressive effects on innate or adaptive immune responses leading to enhanced disease in the presence of these cells.

We initially postulated that the pathogenic effect of T cells was mediated by IFN-γ, since neutralization of IFN-γ has been demonstrated to reduce mortality in mice after LPS injection 13 as well as E. coli infection 14. Indeed, mice lacking IFN-γ demonstrated diminished disease severity after E. coli infection compared to WT mice. Serum IFN-γ levels in WT mice were, however, not different from those in TCRα–/– mice. In addition, we were not able to detect increased IFN-γ production by intracellular staining of lymphocytes isolated from spleen or peripheral lymph nodes after restimulation with either plate-bound anti-CD3, PMA and ionomycin, or heat-killed E. coli lysates (data not shown).

Although this still leaves the possibility of the presence of IFN-γ-secreting T cells in other tissues and a direct pathogenic role for IFN-γ in this disease, it seems more likely that in the absence of IFN-γ, a T cell-mediated pathogenic process is not fully activated. As noted above, T cells are known to express TLR, but the function of these TLR is far from clear 15. In addition, other, yet to be identified pathogen recognition receptors may be implicated. Activation of such receptors and/or the downstream signaling events may be dependent on IFN-γ.

Comparison of serum cytokine levels in E. coli-infected TCRα–/–, IFN-γ–/– and WT mice revealed a marked difference in IL-6 levels, with much higher levels noted in WT mice at all time points studied. High serum levels of IL-6 have been demonstrated in human subjects with sepsis, and correlate with an increased risk of mortality 1619. It is unclear whether IL-6 is merely a marker of disease severity or whether it is involved in the development of the disease. Increased levels of IL-6 have also been detected in several mouse models of sepsis, and neutralization of IL-6 with monoclonal antibody treatment improved survival 20. Studies in IL-6-knockout mice subjected to colonic ligation and puncture, however, have found no difference in survival 21, 22, whereas mortality in IL-6-deficient mice compared to WT mice was increased upon systemic E. coli infection 23.

IL-6 is synthesized by both immune cells (including activated T cells) and non-immune cells, and has a wide range of biological effects. These include stimulation of acute-phase protein production by hepatocytes, activation of T cells, regulation of neutrophil and T cell trafficking 24, and stimulation of IL-17 production 25. In particular, IL-6 is thought to decrease neutrophil recruitment and induce neutrophil apoptosis while increasing T cell trafficking to inflamed tissues and rescuing T cells from apoptosis, thereby shifting the immune response from innate to acquired immunity. In addition, IL-6 has been shown to block the suppressor activity of CD4+CD25+ regulatory T cells in the context of TLR signaling 26. The results of our studies imply that in the presence of T cells, IL-6 secretion in response to E. coli infection is enhanced. This suggests the presence of a positive feedback mechanism that results in ongoing activation of T cells.

The precise nature of this postulated feedback mechanism remains to be elucidated. It may be cytokine-mediated, and dependent on T cell activation. A possible candidate is IL-17, which is produced by both CD4+ and CD8+ T cells in response to microbial infection and IL-6 secretion, and augments neutrophil chemoattraction as well as granulopoiesis 27. Serum IL-17 levels were modestly higher in WT mice than in IFN-γ–/– or TCRα–/– mice on day 4 after infection; however, this difference did not reach statistical significance (p=0.08). Although this observation fits with increased IL-6 levels in WT mice, it is at odds with findings in other mouse models, in which IFN-γ–/– mice were noted to have increased numbers of IL-17-producing T cells 28, 29. These reports describe findings in models of chronic disease, which may not extrapolate to an acute infectious process. The possible involvement of IL-17 in sepsis deserves further study.

Of note, E. coli-infected BALB/c mice exhibited an increase in neutrophilic granulocytes among splenocytes as compared to PBS-treated animals, which was remarkably pronounced on day 7 after infection. Only 40% of E. coli-infected WT mice where still alive on day 7; it is therefore possible that only mice capable of mounting an extensive neutrophil response are able to survive until this time point, possibly through control of further bacterial proliferation. On the other hand, neutrophils have been implicated in sepsis-associated multi-organ dysfunction, both in humans with the disease as well as in animal models, although the exact role of neutrophils in sepsis is far from clear 30. The role of neutrophils in our model of E. coli sepsis warrants further investigation in the future.

In IFN-γ–/– mice, T cell activation after E. coli infection was significantly decreased compared to WT mice, but still pronounced compared to PBS-injected mice. In addition, IFN-γ–/– mice, but not TCRα–/– mice, had significantly decreased levels of TNF-α on day 1 after E. coli infection. This suggests that an alternate mechanism may be (in part) responsible for the decreased levels of IL-6 observed in E. coli-infected IFN-γ–/– mice. Others have shown that IFN-γ–/– mice have delayed recruitment and diminished clearance of polymorphonuclear cells in response to an inflammatory stimulus, which is associated with decreased IL-6 levels 31.

Results of our studies do not demonstrate a clear relationship between disease severity and bacterial elimination. Both TCRα–/– and IFN-γ–/– mice had markedly less severe disease than WT mice, but only TCR-α–/– mice cleared bacteria faster from the blood stream whereas bacterial load in the liver was not different between the different strains at any of the time points analyzed. Bacteria could not be cultured from the blood of any mouse past day 3, regardless of disease severity. This is not unlike the situation in humans with sepsis, in whom negative blood cultures, even without prior antibiotic therapy, are not uncommon 32. Conversely, many animals that appeared to have recovered completely from disease in terms of weight loss and disease score were found to have significant bacterial counts in the liver. Taken together we believe these data to indicate that it is not the extent of the bacterial infection itself, but rather the nature of the hosts’ response that determines disease severity.

The pathogenesis of sepsis is complex, and our understanding of the immune mechanisms involved remains incomplete. The development of therapeutic modalities for this highly lethal disease is impeded by this lack of insight. The experiments described in this report add to a growing body of evidence that pathogenic immune responses are not limited to the innate immune system. T cells do get activated early after a systemic bacterial infection, and this appears to be detrimental to disease outcome. Our results challenge the idea that adaptive immune responses are slow and protective against infection, and emphasize the need for further delineation of the interplay between innate and adaptive immune cells and the mediators involved.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Appendix

Mice, bacteria, and infection protocol

The experiments were approved by The Committee on Animal Research of the University of California, San Francisco, CA. BALB/c mice and C57BL/6 mice were purchased from Charles River Laboratories (Wilmington, MA). T cell-deficient (TCRα–/–) mice on the BALB/c background were kindly provided by Dr. R. Locksley, University of California, San Francisco, CA. IFN-γ-knockout (IFN-γ–/–) mice, also on the BALB/c, background were purchased from Jackson Laboratory (Bar Harbor, ME). Experimental mice were used at 5–7 wk of age, and age- as well as sex-matched for each experiment.

A commensal strain of E. coli was isolated from feces of mice maintained in the pathogen-free facility of the Laboratory Animal Resource Center at the University of California, San Francisco, CA. Bacteria were cultured in Luria–Bertani broth (Fisher Scientific, Fair Lawn, NJ) at 37°C, harvested at mid-log phase, and washed twice in sterile PBS before injection into the lateral tail vein of experimental mice. Control mice were injected with an equal volume (500 μL) of PBS. To enumerate the presence of bacteria after infection, serial dilutions of whole blood or organ homogenates were plated on Luria-Bertani plates and incubated at 37°C overnight.

All mice were bred and maintained in the pathogen-free facility of the Laboratory Animal Resource Center at the University of California, San Francisco, CA, in accordance with University guidelines. After infection with E. coli, mice were weighed daily and monitored for signs of disease. A composite disease score was developed to quantify the degree of ruffled fur, hunched appearance and lethargy. Each item was scored 0 (none present), 1 (mild) or 2 (severe), and scores for each item were added to obtain the composite disease score, with a maximum score of 6. Reproducibility was confirmed and an assistant blinded to the experimental design assigned similar disease scores as the primary investigators

Antibodies

Monoclonal depleting antibodies against CD4 (clone GK1.5), CD8 (YTS-169) and IFN-γ (clone R46A2) were a generous gift from Dr. J. Bluestone, University of California, San Francisco. Mice were injected i.p. with 0.5 mg of anti-CD4 antibody, anti-CD8 antibody, or both, 2 days prior to infection with E. coli. IFN-γ-depleting antibody was injected at 1 mg/dose i.p. 1 day prior to and 1 day after infection with E. coli.

FACS analysis

Single-cell suspensions were prepared from spleen and peripheral lymph nodes, and stained with anti-CD4 (GK1.5, H129.19, RM4-5), anti-CD8 (53-6.7), anti-CD69 (H1.2F3), anti-CD62L (MEL-14), anti-Gr1 (RB8-8C5), anti-B220 (RA36B2) conjugated to either FITC, PE, PerCP or allophycocyanin. All antibodies were purchased from BD Pharmingen. Flow cytometric analysis was performed on a FACSCalibur with CellQuest software (BD Biosciences).

Cytokine analysis in serum

Cytokine levels in serum were analyzed with a bead-based assay for simultaneous detection of multiple cytokines (BioPlex Cytokine Assay, Bio-Rad Laboratories) as per the manufacturer's instructions. In addition, serum levels of IL-6 were quantified with a standard ELISA protocol, utilizing a purified rat anti-mouse antibody for capture (MP5-20F3) and a biotinylated rat anti-mouse antibody for detection (MP5–32C11), both purchased from eBioscience. All samples were tested in duplicate; normal mouse serum served as control.

Statistical analysis

Data were expressed as mean values and standard deviations. Because of lack of normal distribution for some data and fairly small sample size, statistical analysis was performed using the non-parametric Mann–Whitney U-test for comparisons between groups. All experimental findings were confirmed in a minimum of two independent experiments.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Appendix

Supported in part by NICHD Institutional Training for Pediatricians Grant T32HD044331 (recipient Sandrijn van Schaik).

  • 1

    WILEY-VCH

  • 2

    WILEY-VCH

  • 3

    WILEY-VCH

  • 4

    WILEY-VCH

  • 5

    WILEY-VCH

Appendix

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Appendix

Conflict of interest: These authors declare no financial or commercial conflicts of interest.

  • 1
    Jacobs, R. F. and Tabor, D. R., Immune cellular interactions during sepsis and septic injury. Crit. Care Clin. 1989. 5: 926.
  • 2
    Hotchkiss, R. S. and Karl, I. E., The pathophysiology and treatment of sepsis. N. Engl. J. Med. 2003. 348: 138150.
  • 3
    Riedemann, N. C., Guo, R. F. and Ward, P. A., Novel strategies for the treatment of sepsis. Nat. Med. 2003. 9: 517524.
  • 4
    Bone, R. C., Sir Isaac Newton, sepsis, SIRS, and CARS. Crit. Care Med. 1996. 24: 11251128.
  • 5
    Hotchkiss, R. S., Tinsley, K. W., Swanson, P. E., Chang, K. C., Cobb, J. P., Buchman, T. G., Korsmeyer, S. J. et al., Prevention of lymphocyte cell death in sepsis improves survival in mice. Proc. Natl. Acad. Sci. USA 1999. 96: 1454114546.
  • 6
    Giamarellos-Bourboulis, E. J., Tsaganos, T., Spyridaki, E., Mouktaroudi, M., Plachouras, D., Vaki, I., Karagianni, V. et al., Early changes of CD4-positive lymphocytes and NK cells in patients with severe gram-negative sepsis. Crit. Care 2006. 10: R166.
  • 7
    Gelman, A. E., Zhang, J., Choi, Y. and Turka, L. A., Toll-like receptor ligands directly promote activated CD4+ T cell survival. J. Immunol. 2004. 172: 60656073.
  • 8
    Caramalho, I., Lopes-Carvalho, T., Ostler, D., Zelenay, S., Haury, M. and Demengeot, J., Regulatory T cells selectively express Toll-like receptors and are activated by lipopolysaccharide. J. Exp. Med. 2003. 197: 403411.
  • 9
    Tough, D. F., Sun, S. and Sprent, J., T cell stimulation in vivo by lipopolysaccharide (LPS). J. Exp. Med. 1997. 185: 20892094.
  • 10
    Vilanova, M., Tavares, D., Ferreira, P., Oliveira, L., Nobrega, A., Appelberg, R. and Arala-Chaves, M., Role of monocytes in the up-regulation of the early activation marker CD69 on B and T murine lymphocytes induced by microbial mitogens. Scand. J. Immunol. 1996. 43: 155163.
  • 11
    Kamath, A. T., Sheasby, C. E. and Tough, D. F., Dendritic cells and NK cells stimulate bystander T cell activation in response to TLR agonists through secretion of IFN-alpha beta and IFN-gamma. J. Immunol. 2005. 174: 767776.
  • 12
    Sherwood, E. R., Enoh, V. T., Murphey, E. D. and Lin, C. Y., Mice depleted of CD8+ T and NK cells are resistant to injury caused by cecal ligation and puncture. Lab. Invest. 2004. 84: 16551665.
  • 13
    Heinzel, F. P., The role of IFN-gamma in the pathology of experimental endotoxemia. J. Immunol. 1990. 145: 29202924.
  • 14
    Silva, A. T. and Cohen, J., Role of interferon-gamma in experimental gram-negative sepsis. J. Infect. Dis. 1992. 166: 331335.
  • 15
    Kabelitz, D., Expression and function of Toll-like receptors in T lymphocytes. Curr. Opin. Immunol. 2007. 19: 3945.
  • 16
    Calandra, T., Gerain, J., Heumann, D., Baumgartner, J. D. and Glauser, M. P., High circulating levels of interleukin-6 in patients with septic shock: Evolution during sepsis, prognostic value, and interplay with other cytokines. The Swiss-Dutch J5 Immunoglobulin Study Group. Am. J. Med. 1991. 91: 2329.
  • 17
    Damas, P., Ledoux, D., Nys, M., Vrindts, Y., De Groote, D., Franchimont, P. and Lamy, M., Cytokine serum level during severe sepsis in human IL-6 as a marker of severity. Ann. Surg. 1992. 215: 356362.
  • 18
    Casey, L. C., Balk, R. A. and Bone, R. C., Plasma cytokine and endotoxin levels correlate with survival in patients with the sepsis syndrome. Ann. Intern. Med. 1993. 119: 771778.
  • 19
    Gardlund, B., Sjolin, J., Nilsson, A., Roll, M., Wickerts, C. J. and Wretlind, B., Plasma levels of cytokines in primary septic shock in humans: Correlation with disease severity. J. Infect. Dis. 1995. 172: 296301.
  • 20
    Riedemann, N. C., Neff, T. A., Guo, R. F., Bernacki, K. D., Laudes, I. J., Sarma, J. V., Lambris, J. D. et al., Protective effects of IL-6 blockade in sepsis are linked to reduced C5a receptor expression. J. Immunol. 2003. 170: 503507.
  • 21
    Leon, L. R., White, A. A. and Kluger, M. J., Role of IL-6 and TNF in thermoregulation and survival during sepsis in mice. Am. J. Physiol. 1998. 275: R269–R277.
  • 22
    Remick, D. G., Bolgos, G., Copeland, S. and Siddiqui, J., Role of interleukin-6 in mortality from and physiologic response to sepsis. Infect. Immun. 2005. 73: 27512757.
  • 23
    Dalrymple, S. A., Slattery, R., Aud, D. M., Krishna, M., Lucian, L. A. and Murray, R., Interleukin-6 is required for a protective immune response to systemic Escherichia coli infection. Infect. Immun. 1996. 64: 32313235.
  • 24
    Jones, S. A., Directing transition from innate to acquired immunity: Defining a role for IL-6. J. Immunol. 2005. 175: 34633468.
  • 25
    Veldhoen, M., Hocking, R. J., Atkins, C. J., Locksley, R. M. and Stockinger, B., TGFbeta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells. Immunity 2006. 24: 179189.
  • 26
    Pasare, C. and Medzhitov, R., Toll pathway-dependent blockade of CD4+CD25+ T cell-mediated suppression by dendritic cells. Science 2003. 299: 10331036.
  • 27
    Happel, K. I., Zheng, M., Young, E., Quinton, L. J., Lockhart, E., Ramsay, A. J., Shellito, J. E. et al., Cutting edge: Roles of Toll-like receptor 4 and IL-23 in IL-17 expression in response to Klebsiella pneumoniae infection. J. Immunol. 2003. 170: 44324436.
  • 28
    Komiyama, Y., Nakae, S., Matsuki, T., Nambu, A., Ishigame, H., Kakuta, S., Sudo, K. et al., IL-17 plays an important role in the development of experimental autoimmune encephalomyelitis. J. Immunol. 2006. 177: 566573.
  • 29
    Cruz, A., Khader, S. A., Torrado, E., Fraga, A., Pearl, J. E., Pedrosa, J., Cooper, A. M. et al., Cutting edge: IFN-gamma regulates the induction and expansion of IL-17-producing CD4 T cells during mycobacterial infection. J. Immunol. 2006. 177: 14161420.
  • 30
    Brown, K. A., Brain, S. D., Pearson, J. D., Edgeworth, J. D., Lewis, S. M. and Treacher, D. F., Neutrophils in development of multiple organ failure in sepsis. Lancet 2006. 368: 157169.
  • 31
    McLoughlin, R. M., Witowski, J., Robson, R. L., Wilkinson, T. S., Hurst, S. M., Williams, A. S., Williams, J. D. et al., Interplay between IFN-gamma and IL-6 signaling governs neutrophil trafficking and apoptosis during acute inflammation. J. Clin. Invest. 2003. 112: 598607.
  • 32
    Annane, D., Bellissant, E. and Cavaillon, J. M., Septic shock. Lancet 2005. 365: 6378.