CD4+ T-cell depletion is not associated with alterations in survival, bacterial clearance, and inflammation after cecal ligation and puncture.

Our recent studies indicate that mice depleted of T cells that bear the alphabeta T-cell receptor (alphabeta T cells) show less inflammation, less physiological dysfunction, and improved survival after cecal ligation and puncture (CLP) compared with control mice. Classic CD4(+) and CD8(+) T cells comprise most of the alphabeta T-cell population. We previously showed that CD8(+) T cells, in conjunction with natural killer (NK) cells, participate in CLP-induced inflammation. However, the contribution of CD4(+) T cells to the early inflammatory response caused by CLP is largely undefined. In the present study, we evaluated CLP-induced mortality, bacterial clearance, and inflammation in mice that were depleted of CD4(+) T cells. Compared with control mice, CD4 knockout mice and wild-type mice treated with anti-CD4 did not show significant differences in survival, cytokine production, and systemic bacterial counts. The combined depletion of CD4(+) T and NK cells resulted in improved survival and decreased cytokine production compared with mice possessing a full lymphocyte complement, especially when CD4(+) T and NK cell-deficient mice were treated with imipenem. These improvements were nearly identical to those observed in mice depleted only of NK cells. These studies show that CD4(+) T cells do not seem to play a critical role in facilitating the early inflammatory response caused by CLP.

Mice depleted of alphabeta but not gammadelta T cells are resistant to mortality caused by cecal ligation and puncture.

The present study was undertaken to determine whether the mice depleted of alphabeta or gammadelta T cells show resistance to acute polymicrobial sepsis caused by cecal ligation and puncture (CLP). T-cell receptor beta knockout (betaTCRKO) and T-cell receptor delta knockout (deltaTCRKO) mice were used. An additional group of mice was treated with an antibody against the alphabeta T-cell receptor to induce alphabeta T-cell depletion; a subset of alphabeta T cell-deficient mice was also treated with anti-asialoGM1 to deplete natural killer (NK) cells. The mice underwent CLP and were monitored for survival, temperature, acid-base balance, bacterial counts, and cytokine production. The betaTCRKO mice and the wild-type mice treated with anti-beta T-cell receptor (anti-TCRbeta) antibody showed improved survival after CLP compared with wild-type mice. The treatment of alphabeta T cell-deficient mice with anti-asialoGM1further improved survival after CLP, especially when the mice were treated with imipenem. The improved survival observed in alphabeta T cell-deficient mice was associated with less hypothermia, improved acid-base balance, and decreased production of the proinflammatory cytokines interleukin (IL) 6 and macrophage inflammatory protein (MIP) 2. Compared with wild-type controls, the overall survival was not improved in deltaTCRKO mice. The concentrations of IL-6 and MIP-2 in plasma and cytokine mRNA expression in tissues were not significantly different between wild-type and deltaTCRKO mice. These studies indicate that mice depleted of alphabeta but not of gammadelta T cells are resistant to mortality in an acutely lethal model of CLP. The depletion of NK cells caused further survival benefit in alphabeta T cell-deficient mice. These findings suggest that alphabeta T and NK cells mediate or facilitate CLP-induced inflammatory injury.

Differential effect of imipenem treatment on injury caused by cecal ligation and puncture in wild-type and NK cell-deficient beta(2)-microgloblin knockout mice.

Our previous studies showed that beta(2)-microglobulin knockout mice treated with anti-asialoGM1 (beta2MKO/alphaAsGM1 mice) are resistant to injury caused by cecal ligation and puncture (CLP). However, CLP-induced injury is complex. Potential mechanisms of injury include systemic infection, cecal ischemia, and translocation of bacterial toxins such as endotoxin and superantigens. Currently, it is unclear which of these mechanisms of injury contributes to mortality in wild-type mice and whether beta2MKO/alphaAsGM1 mice are resistant to any particular mechanisms of injury. In the present study, we hypothesized that systemic infection is the major cause of injury after CLP in wild-type mice and that beta2MKO/alphaAsGM1 mice are resistant to infection-induced injury. To test this hypothesis, wild-type and beta2MKO/alphaAsGM1 mice were treated with the broad-spectrum antibiotic imipenem immediately after CLP to decrease the impact of systemic infection in our model. Treatment of wild-type and beta2MKO/alphaAsGM1 mice with imipenem decreased bacterial counts by at least two orders of magnitude. However, all wild-type mice, whether treated with saline or imipenem, died by 42 h after CLP and had significant hypothermia, metabolic acidosis, and high plasma concentrations of the cytokines interleukin-6, macrophage inflammatory protein-2, and keratinocyte-derived chemokine. beta2MKO/alphaAsGM1 mice showed 40% long-term survival, which was increased to 90% by imipenem treatment. beta2MKO/alphaAsGM1 mice had less hypothermia, decreased metabolic acidosis, and lower cytokine concentrations at 18 h after CLP compared with wild-type mice. These results suggest that infection is not the major cause of mortality for wild-type mice in our model of CLP. Other mechanisms of injury such as cecal ischemia or translocation of microbial toxins may be more important. beta2MKO/alphaAsGM1 mice appear resistant to these early, non-infection-related causes of CLP-induced injury but showed delayed mortality associated with bacterial dissemination, which was ablated by treatment with imipenem.

Differential effect of imipenem treatment on wild-type and NK cell-deficient CD8 knockout mice during acute intra-abdominal injury.

CD8 knockout mice depleted of natural killer (NK) cells by treatment with anti-asialoGM1 (CD8KO/alphaAsGM1 mice) are resistant to injury caused by cecal ligation and puncture (CLP). However, CLP-induced injury is complex. Potential sources of injury include bacterial dissemination, cecal ischemia, and translocation of bacterial toxins. We treated wild-type and CD8KO/alphaAsGM1 mice with imipenem after CLP to decrease bacterial dissemination. Additional mice were subjected to cecal ligation without puncture of the cecal wall or cecal ligation and removal of cecal contents. Imipenem treatment decreased bacterial counts by at least two orders of magnitude. However, all wild-type mice, whether treated with saline or imipenem, died by 42 h after CLP and exhibited significant hypothermia, metabolic acidosis, and high plasma cytokine concentrations. Wild-type mice subjected to cecal ligation without puncture also died, despite very low bacterial counts in blood, but wild-type mice subjected to cecal ligation and washout of cecal contents survived. In CD8KO/alphaAsGM1 mice subjected to CLP, imipenem treatment increased survival from 50% to 100%. After cecal ligation without puncture, long-term survival was 80-90% in CD8KO/alphaAsGM1 mice. Hypothermia, metabolic acidosis, and cytokine production were attenuated in CD8KO/alphaAsGM1 mice compared with wild-type controls. These results indicate that bacterial dissemination is not a major source of injury in wild-type mice after CLP, but the presence of gut flora in the cecal lumen is required for induction of systemic inflammation after cecal injury. CD8KO/alphaAsGM1 mice are resistant to the systemic manifestations of cecal injury.

Cardiovascular dysfunction caused by cecal ligation and puncture is attenuated in CD8 knockout mice treated with anti-asialoGM1.

The present study was designed to assess hemodynamics and myocardial function at 18 h after injury caused by cecal ligation and puncture (CLP) in CD8-knockout mice treated with anti-asialoGM1 (CD8KO/alphaAsGM1 mice). Arterial pressure was measured by carotid artery cannulation, and left ventricular pressure-volume measurements were obtained by use of a 1.4-Fr conductance catheter. Blood acid-base balance and indexes of hepatic, renal, and pulmonary injury were also measured. CD8KO/alphaAsGM1 mice exhibited higher mean arterial pressure and increased systemic vascular resistance compared with wild-type mice. Cardiac output was significantly decreased in wild-type, but not CD8KO/alphaAsGM1, mice compared with sham controls. Myocardial function was better preserved in CD8KO/alphaAsGM1 mice as indicated by less impairment of left ventricular pressure development over time, time varying maximum elastance, end-systolic pressure-volume relationship, and preload recruitable stroke work. The impairment in myocardial function was associated with induction of proinflammatory cytokine mRNAs in the hearts of wild-type mice. The hemodynamic derangements in wild-type mice were coupled with significant metabolic acidosis and elevated serum creatinine levels. Overall, this study shows that cardiovascular collapse and shock characterized by hypotension, myocardial depression, low systemic vascular resistance, and metabolic acidosis occurs after CLP in wild-type mice but is attenuated in CD8KO/alphaAsGM1 mice. These observations likely explain, in part, the previously observed survival advantage of CD8KO/alphaAsGM1 mice following CLP.

Mice depleted of CD8+ T and NK cells are resistant to injury caused by cecal ligation and puncture.

We previously showed that beta 2 microglobulin knockout mice depleted of NK cells by treatment with anti-asialoGM1 (beta2MKO/alphaAsGM1 mice) are resistant to sepsis caused by cecal ligation and puncture (CLP). beta2MKO mice possess multiple immunological defects including depletion of CD8+ T cells. This study was designed to determine the contribution of CD8+ T and NK cell deficiency to the resistance of beta2MKO/alphaAsGM1 mice to CLP-induced injury. beta2MKO/alphaAsGM1 mice and CD8 knockout mice treated with anti-asialoGM1 (CD8KO/alphaAsGM1 mice) survived significantly longer than wild-type mice following CLP. Improved long-term survival was also observed in wild-type mice rendered CD8+ T/NK cell-deficient by treatment with both anti-CD8alpha and anti-asialoGM1. Blood gas analysis and body temperature measurements showed that CD8+ T and NK cell-deficient mice have significantly reduced metabolic acidosis and less hypothermia compared to control mice at 18 h after CLP. CD8+ T/NK cell-deficient mice also showed an attenuated proinflammatory response as indicated by decreased expression of mRNAs for IL-1, IL-6 and MIP-2 in spleen and heart. IL-6, KC and MIP-2 levels in blood and peritoneal fluid were also significantly decreased CD8+ T/NK cell-deficient mice compared to controls. CD8+ T/NK cell-deficient mice exhibited decreased bacterial concentrations in blood, but not in peritoneal fluid or lung, compared to wild-type controls. These data show that mice depleted of CD8+ T and NK cells exhibit survival benefit, improved physiologic function and an attenuated proinflammatory response following CLP that is comparable to beta2M/alphaAsGM1 mice.