RESUMO
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.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Inflamação/imunologia , Sepse/imunologia , Sepse/microbiologia , Animais , Bactérias/imunologia , Bactérias/isolamento & purificação , Antígenos CD4/genética , Antígenos CD4/metabolismo , Contagem de Colônia Microbiana , Citocinas/biossíntese , Feminino , Células Matadoras Naturais/imunologia , Depleção Linfocítica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
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.
Assuntos
Ceco/lesões , Receptores de Antígenos de Linfócitos T alfa-beta/deficiência , Receptores de Antígenos de Linfócitos T gama-delta/deficiência , Linfócitos T/imunologia , Animais , Antibacterianos/uso terapêutico , Bacteriemia/tratamento farmacológico , Bacteriemia/imunologia , Bacteriemia/mortalidade , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Quimiocina CXCL2 , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Imipenem/uso terapêutico , Interleucina-6/metabolismo , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Ligadura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monocinas/metabolismo , Punções , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Sepse/tratamento farmacológico , Sepse/imunologia , Sepse/mortalidade , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Temperatura , Fatores de TempoRESUMO
These studies evaluated the effects treatment with glucan phosphate, a soluble polysaccharide immunomodulator, on the inflammatory response induced by burn injury and on resistance to Pseudomonas aeruginosa burn wound infection. Mice were exposed to 35% total body surface area burns and were resuscitated with lactated Ringer's (LR) solution alone or LR supplemented with glucan phosphate (40 mg/kg). Glucan phosphate treatment attenuated burn-induced expression of interleukin (IL)-1beta, IL-6, and IL-10 mRNAs in spleen, lung, and heart. Plasma concentrations of IL-1beta, IL-6, macrophage inflammatory protein (MIP)-2, and IL-10 were also decreased in burned mice treated with glucan phosphate compared with vehicle-treated controls. Early postburn mortality was not significantly different between control (20%) and glucan phosphate-treated (10%) mice, but there was a small improvement in acid-base balance in the glucan phosphate-treated group. Mice received a second injection of glucan phosphate or LR on day 4 postburn and were infected by topical application of P. aeruginosa to the burn wound on day 5. Glucan phosphate treatment significantly improved survival in mice exposed to P. aeruginosa burn wound infection. The improved survival correlated with lower bacterial burden in the burn wound, attenuated production of proinflammatory cytokines, and enhanced production of Th1 cytokines. These studies show that glucan phosphate treatment attenuates burn-induced inflammation and increases resistance to P. aeruginosa burn wound infection in an experimental model of burn injury.
Assuntos
Queimaduras/tratamento farmacológico , Glucanos/uso terapêutico , Inflamação/prevenção & controle , Infecções por Pseudomonas/prevenção & controle , Infecção dos Ferimentos/prevenção & controle , Equilíbrio Ácido-Base/efeitos dos fármacos , Animais , Queimaduras/complicações , Queimaduras/genética , Queimaduras/imunologia , Quimiocina CXCL2 , Quimiocinas/sangue , Feminino , Expressão Gênica/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe II/metabolismo , Interferon gama/biossíntese , Interleucina-1/biossíntese , Interleucina-1/genética , Interleucina-10/biossíntese , Interleucina-10/genética , Interleucina-12/biossíntese , Interleucina-6/biossíntese , Interleucina-6/genética , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Thermal injury to 40% or more of the total body surface area poses a significant risk for the development of opportunistic infections that increase complications and mortality. Altered cytokine induction profiles, including suppression of the Th1 cytokines IFN-gamma and IL-12 and elevations in the anti-inflammatory cytokine IL-10, are believed to contribute to burn-associated immunosuppression and the development of sepsis. The specific changes that lead to altered cytokine production following major burns are not known. We examined the effects of burn injuries to 40% of the mouse body surface on IFN-gamma induction in the major IFN-gamma-producing cell types of the spleen. Additionally, effects on key IFN-gamma-regulatory cytokines were examined after bacterial challenge. We report that in vivo induction of IFN-gamma in natural killer lymphocytes is suppressed in burned mice. Splenic IFN-gamma was suppressed at both the mRNA and protein levels. Early suppression was associated with impairments in both the macrophage/dendritic cell and lymphocyte populations, whereas persistent suppression was associated with impaired lymphocyte function and decreased responsiveness to IFN-gamma-inducing factors. IFN-gamma production could be restored by neutralization of the upregulated cytokine IL-10. Induction of the IFN-gamma-inducers IL-15, IL-12, and IL-2 was also impaired after burn injury, whereas IL-18 levels remained unaffected. Exogenous application of these suppressed cytokines to isolated splenocytes did not restore IFN-gamma to sham levels, indicating a loss of responsiveness to these factors. Expression of the IL-2, IL-12, and IL-15 receptors was suppressed after thermal injury. We conclude that burn-associated suppression of IFN-gamma is due to deficient production of inducing factors and their receptors, leading to severe impairments in cellular IFN-gamma induction pathways.
Assuntos
Queimaduras/metabolismo , Citocinas/biossíntese , Interferon gama/biossíntese , Linfócitos/metabolismo , Receptores de Citocinas/metabolismo , Animais , Células Cultivadas , Técnicas de Cocultura , Citocinas/genética , Feminino , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Células Matadoras Naturais/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Pseudomonas aeruginosa/patogenicidade , Baço/citologia , Baço/metabolismoRESUMO
After a major illness or injury, immune status in critically ill patients may fluctuate between a marked proinflammatory response and an immunosuppressed state. Postinflammatory immunosuppression can result in increased susceptibility to infection. Alterations of cytokine production, such as suppression of IFNgamma and elevation of the anti-inflammatory cytokine IL-10, are believed to contribute to postinflammatory immunosuppression. We examined antimicrobial immunity in mice that had previously been subjected to a sublethal cecal ligation and puncture (CLP) as a model of major injury. Mice were challenged with Pseudomonas aeruginosa (5 x 10(7) CFU i.v.) on day 5 after CLP or sham surgery. Bacterial clearance in mice after CLP was impaired and associated with decreased production of IFNgamma and increased production of IL-10 in the early response to the Pseudomonas challenge. Pseudomonas-induced production of the IFNgamma-inducing factor IL-12 was also decreased in post-CLP mice. However, splenocytes from post-CLP mice remained responsive to exogenous stimulation with the IFNgamma-inducing cytokines IL-12, IL-15, and IL-18 as well as T-cell receptor activation. Furthermore, production of the proinflammatory cytokines TNF-alpha, IL-1beta, and IL-6 were as high, or higher, in the post-CLP group compared with sham mice after P. aeruginosa challenge. Blockade of IL-10 did not reverse IL-12 and IFNgamma suppression in splenocytes from post-CLP mice. These studies show that suppressed bacterial clearance in post-CLP mice is associated with decreased production of IFNgamma and IL-12 and with increased production of IL-10 and proinflammatory cytokines.
Assuntos
Terapia de Imunossupressão , Inflamação/patologia , Interferon gama/biossíntese , Interleucina-12/biossíntese , Sepse/patologia , Animais , Ceco/lesões , Ceco/cirurgia , Citocinas/biossíntese , Citocinas/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Tolerância Imunológica , Imunossupressores , Interleucina-1/biossíntese , Interleucina-10/biossíntese , Interleucina-15/biossíntese , Interleucina-18/biossíntese , Interleucina-6/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pseudomonas aeruginosa/metabolismo , Ribonucleases/metabolismo , Sepse/metabolismo , Baço/citologia , Fatores de Tempo , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Evidence suggests that NK and NKT cells contribute to inflammation and mortality during septic shock caused by cecal ligation and puncture (CLP). However, the specific contributions of these cell types to the pathogenesis of CLP-induced septic shock have not been fully defined. The goal of the present study was to determine the mechanisms by which NK and NKT cells mediate the host response to CLP. Control, NK cell-deficient, and NKT cell-deficient mice underwent CLP. Survival, cytokine production, and bacterial clearance were measured. NK cell trafficking and interaction with myeloid cells was also studied. Results show that mice treated with anti-asialoGM1 (NK cell deficient) or anti-NK1.1 (NK/NKT cell deficient) show less systemic inflammation and have improved survival compared with IgG-treated controls. CD1 knockout mice (NKT cell deficient) did not demonstrate decreased cytokine production or improved survival compared with wild type mice. Trafficking studies show migration of NK cells from blood and spleen into the inflamed peritoneal cavity where they appear to facilitate the activation of peritoneal macrophages (F4-80(+)GR-1(-)) and F4-80(+)Gr-1(+) myeloid cells. These findings indicate that NK but not CD1-restricted NKT cells contribute to acute CLP-induced inflammation. NK cells appear to mediate their proinflammatory functions during septic shock, in part, by migration into the peritoneal cavity and amplification of the proinflammatory activities of specific myeloid cell populations. These findings provide new insights into the mechanisms used by NK cells to facilitate acute inflammation during septic shock.
Assuntos
Antígenos CD1/imunologia , Movimento Celular/imunologia , Células Matadoras Naturais/imunologia , Choque Séptico/imunologia , Linfócitos T/imunologia , Abdome/patologia , Animais , Antígenos Ly , Antígenos de Superfície/imunologia , Movimento Celular/efeitos dos fármacos , Gangliosídeo G(M1)/antagonistas & inibidores , Gangliosídeo G(M1)/imunologia , Imunoglobulina G/imunologia , Imunoglobulina G/farmacologia , Inflamação/imunologia , Inflamação/patologia , Células Matadoras Naturais/patologia , Lectinas Tipo C/antagonistas & inibidores , Lectinas Tipo C/imunologia , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Subfamília B de Receptores Semelhantes a Lectina de Células NK , Choque Séptico/patologia , Linfócitos T/patologiaRESUMO
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.
Assuntos
Antibacterianos/farmacologia , Ceco/lesões , Imipenem/farmacologia , Células Matadoras Naturais/fisiologia , Microglobulina beta-2/genética , Equilíbrio Ácido-Base/fisiologia , Animais , Ceco/microbiologia , Ceco/patologia , Contagem de Colônia Microbiana , Citocinas/biossíntese , Ensaio de Imunoadsorção Enzimática , Feminino , Ligadura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Sobrevida , TemperaturaRESUMO
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.
Assuntos
Traumatismos Abdominais/tratamento farmacológico , Traumatismos Abdominais/imunologia , Citocinas/imunologia , Imipenem/administração & dosagem , Células Matadoras Naturais/imunologia , Peritonite/imunologia , Peritonite/prevenção & controle , Traumatismos Abdominais/complicações , Traumatismos Abdominais/microbiologia , Animais , Antibacterianos/administração & dosagem , Infecções Bacterianas , Antígenos CD8/imunologia , Linfócitos T CD8-Positivos/imunologia , Ceco/efeitos dos fármacos , Ceco/imunologia , Ceco/microbiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peritonite/etiologia , Peritonite/microbiologia , Resultado do TratamentoRESUMO
Transforming growth factor-beta (TGF-beta), a cytokine with anti-inflammatory properties, may contribute to postburn immunosuppression. This study was designed to determine whether neutralizing TGF-beta in burned mice could improve resistance to infection. C57BL/6J mice received a 35% TBSA flame burn under isoflurane anesthesia. Four days after injury, mice were treated with TGF-beta antibody or nonspecific IgG. On day 5 after burn injury, mice were inoculated with Pseudomonas aeruginosa at the burn wound site or received intraperitoneal injection with P. aeruginosa. Mice treated with anti-TGF-beta exhibited significantly improved survival compared with mice treated with nonspecific IgG after challenge with P. aeruginosa at the burn wound site or after intraperitoneal injection of P. aeruginosa. In mice with burn wound infections, bacterial counts in burn wounds, blood, and lung were decreased in mice treated with anti-TGF-beta compared with mice treated with control IgG. Bacterial counts in lung and blood after intraperitoneal challenge with P. aeruginosa also were significantly lower in burned mice treated with anti-TGF-beta compared with those treated with nonspecific IgG. Our data suggest that neutralization of TGF-beta at 4 days after burn injury in mice improves local and systemic clearance of P. aeruginosa and enhances survival after P. aeruginosa challenge.
Assuntos
Queimaduras/microbiologia , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/isolamento & purificação , Fator de Crescimento Transformador beta/antagonistas & inibidores , Infecção dos Ferimentos/imunologia , Animais , Anticorpos/farmacologia , Queimaduras/mortalidade , Contagem de Colônia Microbiana , Feminino , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Infecções por Pseudomonas/metabolismo , Infecção dos Ferimentos/microbiologiaRESUMO
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.
Assuntos
Anticorpos/farmacologia , Antígenos CD8/metabolismo , Doenças Cardiovasculares/fisiopatologia , Ceco , Gangliosídeo G(M1)/imunologia , Punções , Equilíbrio Ácido-Base/efeitos dos fármacos , Animais , Antígenos CD8/genética , Doenças Cardiovasculares/etiologia , Citocinas/metabolismo , Feminino , Hemodinâmica , Mediadores da Inflamação/metabolismo , Ligadura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Contração Miocárdica , Miocárdio/metabolismoRESUMO
Endotoxin (lipopolysaccharide [LPS]) tolerance is an altered state of immunity caused by prior exposure to LPS, in which production of many cytokines, including gamma interferon (IFN-gamma) and interleukin-12 (IL-12), are reduced but secretion of the anti-inflammatory cytokine IL-10 is increased in response to a subsequent LPS challenge. This pattern of cytokine production is also characteristic of postinflammatory immunosuppression. Therefore, we hypothesized that LPS-primed mice would exhibit an impaired ability to respond to systemic infection with the opportunistic pathogen Pseudomonas aeruginosa. We further hypothesized that depletion of IL-10 would reverse the endotoxin-tolerant state. To test this hypothesis, systemic clearance of Pseudomonas aeruginosa was measured for LPS-primed wild-type and IL-10-deficient mice. LPS-primed wild-type mice exhibited significant suppression of LPS-induced IFN-gamma and IL-12 but increased IL-10 production in blood and spleen compared to levels exhibited by saline-primed wild-type mice. The suppressed production of IFN-gamma and IL-12 caused by LPS priming was ablated in the spleens, but not blood, of IL-10 knockout mice. LPS-primed wild-type mice cleared Pseudomonas aeruginosa from lungs and blood more effectively than saline-primed mice. LPS-primed IL-10-deficient mice were particularly efficient in clearing Pseudomonas aeruginosa after systemic challenge. These studies show that induction of LPS tolerance enhanced systemic clearance of Pseudomonas aeruginosa and that this effect was augmented by neutralization of IL-10.
Assuntos
Interleucina-10/deficiência , Interleucina-10/metabolismo , Lipopolissacarídeos/farmacologia , Pseudomonas aeruginosa/imunologia , Animais , Biomarcadores , Regulação da Expressão Gênica , Tolerância Imunológica/efeitos dos fármacos , Tolerância Imunológica/imunologia , Interferon gama/biossíntese , Interferon gama/sangue , Interferon gama/genética , Interleucina-10/sangue , Interleucina-10/genética , Interleucina-12/biossíntese , Interleucina-12/sangue , Interleucina-12/genética , Lipopolissacarídeos/imunologia , Pulmão/microbiologia , Camundongos , Camundongos Knockout , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Cloreto de Sódio/farmacologiaRESUMO
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.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Ceco/lesões , Células Matadoras Naturais/imunologia , Depleção Linfocítica , Acidose/imunologia , Acidose/patologia , Animais , Antígenos CD8/genética , Antígenos CD8/fisiologia , Ceco/cirurgia , Citocinas/sangue , Feminino , Hipotermia/imunologia , Hipotermia/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Sepse/genética , Sepse/prevenção & controle , Microglobulina beta-2/deficiência , Microglobulina beta-2/fisiologiaRESUMO
Gamma interferon (IFN-gamma) is an important mediator of endotoxin (lipopolysaccharide [LPS])-induced immune responses. However, the specific cell types that produce IFN-gamma in response to LPS and the cellular factors that regulate LPS-induced IFN-gamma production have not been fully determined. The present studies were undertaken to characterize the cell populations that produce IFN-gamma after LPS challenge in the spleens of mice and to determine the regulatory factors that modulate LPS-induced production of IFN-gamma. Our studies show that the levels of splenic IFN-gamma mRNA and protein production peak at 6 and 8 h, respectively, after systemic LPS challenge. Approximately 60% of IFN-gamma-producing cells are natural killer (NK) cells (CD3(-)DX5(+)) and 25% are NKT cells (CD3(+)DX5(+)). Most of the remaining IFN-gamma-producing cells are T cells (CD3(+)DX5(-)), macrophages, and dendritic cells. Functionally, interleukin-12 (IL-12) is the major IFN-gamma-stimulating factor after LPS challenge, with costimulation provided by IL-15, IL-18, and B7 proteins. IL-10 is a major inhibitor of LPS-induced IFN-gamma production. Unlike intact heat-killed gram-negative and gram-positive bacteria, the class II major histocompatibility complex did not play a functional role in LPS-induced IFN-gamma production. LPS is a potent stimulus for splenic IL-10, IL-12 p40, and IL-15 mRNA expression, whereas IL-12 p35 and IL-18 mRNAs, as well as B7 proteins, are constitutively expressed in the mouse spleen. Of the factors studied, IL-18 serves as the most potent costimulus with IL-12 for IFN-gamma production, followed by IL-15 and B7 proteins. These data demonstrate that NK cells and NKT cells are the most abundant IFN-gamma-producing cells in the mouse spleen after LPS challenge and that IL-10 and IL-12 are key functional regulators of LPS-induced IFN-gamma production.
Assuntos
Antígeno B7-1/imunologia , Interferon gama/biossíntese , Interleucina-12/imunologia , Interleucina-15/imunologia , Interleucina-18/imunologia , Transdução de Sinais/imunologia , Animais , Antígenos CD28/imunologia , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Feminino , Perfilação da Expressão Gênica , Antígenos de Histocompatibilidade Classe II/imunologia , Interferon gama/genética , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/imunologia , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro , Baço/citologia , Baço/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
Patients with large burn injuries are susceptible to opportunistic infections due to impaired functions of multiple effector cells of innate immunity and acquired immunity, including macrophages, dendritic cells (DC), natural killer (NK) cells, and T cells. The ability of a host to produce Th1 cytokines, such as gamma interferon (IFN-gamma) and interleukin-12 (IL-12), upon infectious challenge is also impaired after burn injury. Stimulation of hematopoiesis, to regenerate new immune cells, may be an effective strategy for improving resistance to infections after severe burn trauma. Fms-like tyrosine kinase 3 ligand (Flt3L) is a hematopoietic cytokine that stimulates the expansion and differentiation of NK cells and DC. Using a mouse model, we tested the hypothesis that Flt3L treatments after burn injury stimulate the production of functional effector cells of innate immunity and restore appropriate Th1 cytokine responses to Pseudomonas aeruginosa, a common source of pneumonia and wound infections in burn victims. Flt3L increased splenic cellularity in sham (uninjured) and burned mice and increased the numbers of NK cells (DX5(+)) and DC (CD11c(+)). In response to P. aeruginosa, significant increases in the serum IFN-gamma levels and the numbers of splenic IFN-gamma-producing DC, NK cells, and T cells were observed in Flt3L-treated burned mice compared to the values obtained for untreated burned mice. The splenic levels of IL-12 and IL-15 mRNAs and the IL-12 and IL-15 receptors were also increased. In addition, Flt3L treatment restored the ability of splenic cultures prepared from burned mice to produce IFN-gamma and IL-12 after in vitro challenge with P. aeruginosa. Flt3L may have potential for restoring NK cell and DC functions and improving immunity after burn injury.
Assuntos
Queimaduras/imunologia , Citocinas/biossíntese , Hematopoese/efeitos dos fármacos , Proteínas de Membrana/farmacologia , Pseudomonas aeruginosa/imunologia , Células Th1/imunologia , Animais , Células Dendríticas/fisiologia , Interferon gama/biossíntese , Interleucina-12/biossíntese , Interleucina-15/biossíntese , Células Matadoras Naturais/fisiologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
BACKGROUND: Acute aortic occlusion in vascular surgery patients abruptly increases arterial resistance and blood pressure, which, in turn, makes subsequent volume expansion during cross-clamp application difficult. The use of vasodilatory drugs or volatile anesthetic agents to attenuate this response may have persistent detrimental effects after clamp removal. Another potential therapy that produces rapid effects on myocardial loading conditions is positive end-expiratory pressure (PEEP). In a porcine model of acute aortic clamping, the hemodynamic consequences of 15 cm H(2)O PEEP with and without plasma volume expansion were studied.(2) METHODS: Forty anesthetized pigs underwent 30-min occlusion of the abdominal aorta 1 cm above the origin of the celiac artery. Animals were randomly divided into four treatment groups (n = 10 each) to receive 15 cm H(2)O PEEP or zero end-expiratory pressure (ZEEP) with or without plasma volume expansion using 6% hetastarch (10 ml/kg) during cross-clamp application. Mean aortic pressure was measured with a transducer-tipped catheter placed in the ascending aorta; stroke volume was calculated using thermodilution cardiac output. End-expiratory pressure was discontinued upon aortic declamping, and animals were studied over the ensuing 30-min period. RESULTS: Aortic occlusion doubled systemic vascular resistance in all groups. Mean aortic blood pressure increased significantly in both ZEEP groups at 1 and 5 min but not in animals treated with 15 cm H(2)O PEEP. The application of PEEP with aortic cross-clamping reduced cardiac output and stroke volume by nearly 50%. Cardiac output and stroke volume increased after volume expansion regardless of end-expiratory pressure. After aortic declamping, aortic blood pressure decreased in all groups but was significantly greater in the PEEP + volume group than in either ZEEP group. Similarly, 5 min after declamping, stroke volume was greatest in the PEEP + volume animals. CONCLUSIONS: Fifteen cm H(2)O PEEP reduces the hypertensive response to acute aortic occlusion and allows concomitant volume expansion. Consequently, stroke volume and blood pressure are better maintained after clamp removal in PEEP + volume animals. The use of PEEP during acute aortic occlusion in patients may allow rapid control of loading conditions to attenuate systemic hypertension while permitting simultaneous volume expansion.
Assuntos
Aorta Torácica/fisiologia , Hemodinâmica/fisiologia , Respiração com Pressão Positiva , Animais , Volume Sanguíneo/fisiologia , Débito Cardíaco/fisiologia , Constrição , Feminino , Substitutos do Plasma/uso terapêutico , Mecânica Respiratória/fisiologia , Volume Sistólico/fisiologia , Suínos , Resistência Vascular/fisiologiaRESUMO
beta 2 microglobulin knockout (beta2M-/-) mice lack CD8+ T and natural killer T cells. We hypothesized that beta 2M-/- mice are resistant to lethal intraabdominal sepsis. To test this hypothesis, mortality, cytokine production, and physiologic function were assessed in beta 2M-/- mice during sepsis caused by cecal ligation and puncture (CLP). beta 2M-/- mice survived significantly longer than wild-type mice after CLP but ultimately exhibited 100% mortality. Treatment of beta 2M-/- mice with anti-asialoGM1 to deplete natural killer cells conferred greater than 70% long-term survival. Compared with wild-type mice, beta 2M-/- mice treated with anti-asialoGM1 produced decreased amounts of proinflammatory cytokines and did not exhibit hypothermia or metabolic acidosis after CLP. Adoptive transfer of CD8+ T and natural killer cells into beta 2M-/- mice treated with anti-asialoGM1 re-established CLP-induced mortality. CD8 knockout mice treated with anti-asialoGM1, which are specifically deficient in CD8+ T and natural killer cells, exhibited 40% long-term survival after CLP. Furthermore, treatment of wild-type mice with antibodies to CD8 and asialoGM1 conferred a significant survival benefit compared with wild-type mice treated with nonspecific IgG. These findings demonstrate that beta 2M-/- mice treated with anti-asialoGM1 are resistant to CLP-induced mortality and that depletion of CD8+ T and natural killer cells largely accounts for the survival benefit observed in these mice.