RESUMO
BACKGROUND: Leukocyte apoptosis allows safe removal of potentially harmful cells and facilitates resolution of inflammation. We hypothesized that the number of apoptotic cells changes in a disproportionate fashion in parenchymal organs in response to intra-abdominal infection. MATERIALS AND METHODS: The percentage of apoptotic cells in the liver, spleen, lung, and peripheral blood was evaluated following cecal ligation and puncture (CLP) in mice. Tissue myeloperoxidase (MPO) levels were measured as an index of neutrophil extravasation. RESULTS: Liver & spleen MPO continually increased, while lung MPO remained low after CLP. In parallel to the increase in MPO, liver & spleen apoptosis continually increased throughout the 9-day follow-up period, whereas lung apoptosis remained unchanged. CONCLUSIONS: The distribution of apoptotic cells during intraperitoneal infection occurs in an organ specific manner, with significant increases in the spleen and liver. This distribution likely reflected the clearance of apoptotic cells as the inflammatory focus became contained.
Assuntos
Apoptose , Infecções Bacterianas/microbiologia , Infecções Bacterianas/patologia , Doenças Peritoneais/microbiologia , Doenças Peritoneais/patologia , Abscesso Abdominal/microbiologia , Abscesso Abdominal/patologia , Animais , Infecções Bacterianas/enzimologia , Contagem de Leucócitos , Fígado/patologia , Pulmão/patologia , Masculino , Camundongos , Especificidade de Órgãos , Doenças Peritoneais/enzimologia , Peroxidase/metabolismo , Baço/patologiaRESUMO
Natural killer (NK) cell interactions with macrophages have been shown to be important during bacterial sepsis in activating macrophages to improve bacterial clearance. The mechanism for this increased activation, however, is unclear. This study determines the relative roles of interferon (IFN)-gamma and CD40/CD154 direct cell interactions on macrophage and NK cell activation in an experimental model of sepsis. Splenic NK cells and peritoneal macrophages were isolated and cultured alone or in coculture, with and without LPS. CD69 expression on NK cells, phagocytosis ability of macrophages, and cell cytokine production was assessed at 24 and 48 h. Coculture of NK cells and macrophages significantly increased activation levels of both cell types, and through experiments culturing NK cells with supernatants from stimulated macrophages and macrophages with supernatants from stimulated NK cells, this activation was determined to be cell-contact-dependent. Similar experiments were conducted using NK cells from IFN-gamma deficient (-/-) mice, as well as anti-IFN-gamma neutralizing antibody. These experiments determined that IFN-gamma is not required for NK or macrophage activation, although it did augment activation levels. Experiments were again repeated using peritoneal macrophages from CD40-/- mice or splenic NK cells from CD154-/- mice. CD40/CD154 interactions were important in the ingestion of bacteria by macrophages, but did not affect NK cell activation at 24 h. There was, however, a protective effect of CD40/CD154 interactions on NK cell activation-induced cell death that occurred at 48 h. CD40/CD154 interactions between macrophages and NK cells are therefore important in macrophage phagocytosis, and are not dependent on IFN-gamma.
Assuntos
Antígenos CD40/imunologia , Ligante de CD40/imunologia , Interferon gama/imunologia , Células Matadoras Naturais/imunologia , Macrófagos Peritoneais/imunologia , Sepse/imunologia , Animais , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos CD40/genética , Ligante de CD40/genética , Comunicação Celular , Células Cultivadas , Citocinas/imunologia , Interferon gama/genética , Lectinas Tipo C , Lipopolissacarídeos , Ativação de Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos AnimaisRESUMO
The signal transducer and activator of transcription factor 4 (STAT4) pathway mediates the intracellular effects of interleukin-12 (IL-12), leading to the production of gamma interferon, induction of a T helper type 1 response, and increased natural killer cell cytotoxicity. The purpose of this study was to determine the role of the STAT4 pathway during polymicrobial peritonitis in the cecal ligation and puncture (CLP) model. CLP was performed on STAT4-deficient (STAT4(-/-)) and wild-type control (BALB/c) mice. At 4 h after CLP, STAT4(-/-) mice had significantly higher bacterial counts in the peritoneal lavage fluid, liver, and blood. This difference persisted for 18 h in the peritoneal lavage fluid and blood. Neutrophil migration to the site of infection and into remote tissues was unaffected. Despite higher bacterial counts locally and systemically, STAT4(-/-) mice had a lower mortality rate than BALB/c controls. In contrast, blockade of IL-12 in BALB/c mice was detrimental to host survival. A blunted serum IL-12 response at 18 h after CLP was exhibited in STAT4(-/-) mice. These results suggest several critical roles for the STAT4 pathway in the resolution of polymicrobial infections. Additionally, the disparate effects observed with IL-12 blockade and STAT4 deficiency on host survival suggest that IL-12 may activate alternate pathways promoting survival.
Assuntos
Infecções Bacterianas/imunologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Sepse/imunologia , Transativadores/genética , Transativadores/imunologia , Animais , Anticorpos/farmacologia , Infecções Bacterianas/mortalidade , Ceco/microbiologia , Contagem de Colônia Microbiana , Interleucina-12/imunologia , Ligadura , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Neutrófilos/imunologia , Peritonite/imunologia , Peritonite/mortalidade , Fator de Transcrição STAT4 , Sepse/mortalidade , Taxa de Sobrevida , Ferimentos Perfurantes/microbiologiaRESUMO
Leukocyte apoptosis is an energy-dependent process that facilitates resolution of the cellular inflammatory response. Levels of apoptosis can be accelerated or inhibited after exposure to various stimuli. To compare apoptosis in transmigrated leukocytes, two models of peritonitis in mice were used that both cause leukocyte influx into the peritoneal cavity: (1) intraperitoneal thioglycollate administration producing a sterile peritonitis and (2) cecal ligation and puncture (CLP) producing a polymicrobial bacterial peritonitis. Samples of blood and peritoneal exudate cells (PEC) were collected at multiple time points after induction of peritonitis. Leukocytes were either fixed immediately to determine an immediate apoptosis level or cultured for 24 h to determine a delayed apoptosis level. Apoptosis was assessed using terminal uridine-triphosphate nick-end labeling (TUNEL) assay, flow cytometry, and confocal microscopy. Leukocyte influx into the peritoneal cavity was confirmed in both models. At all time points, and in both models, there was increased immediate apoptosis in PEC compared with unmanipulated controls and this increase was maximal in CLP after 18 h, although it appeared to remain at a stable level in the sterile peritonitis model by 3 h. There was also an increase in PEC delayed apoptosis at early time points in both models, again maximal at 18 h for CLP, with the levels being significantly higher than the thioglycollate model at 6 h and 18 h. The mice had a relative peripheral neutropenia at 6 h after CLP, but not post thioglycollate injection, and this persisted until 42 h. Lung and liver MPO levels were elevated in CLP but did not increase after thioglycollate. There was no increase in immediate peripheral leukocyte apoptosis in either model, but an increase in delayed peripheral leukocyte apoptosis was observed by 18 h in both models. Peripheral leukocyte CD1lb expression, which is a marker of activation, was also persistently elevated in the CLP model, but not in sterile peritonitis. In conclusion, CLP is a more potent stimulus for apoptosis of leukocytes than their migration to the site of inflammation alone, as occurs in the thioglycollate model. Blood leukocyte apoptosis also appears not to be dependent on CD11b expression, and therefore activation status.
Assuntos
Apoptose , Modelos Animais de Doenças , Leucócitos/patologia , Peritonite/patologia , Animais , Antígeno CD11b/metabolismo , Ceco/lesões , Quimiotaxia de Leucócito , Camundongos , Ativação de Neutrófilo , Neutrófilos/citologia , Neutrófilos/metabolismo , Neutrófilos/patologia , Peritônio/microbiologia , Peritônio/patologia , Peritonite/etiologia , Tioglicolatos , Fatores de TempoRESUMO
BACKGROUND AND OBJECTIVE: CAP37, also known as heparin-binding protein (HBP), is neutrophil-derived protein with multifunctional properties that include monocyte chemotaxis and the enhancement of LPS-induced tumor necrosis factor (TNF-alpha), IL-1, IL-6, and PGE2production from isolated monocytes, which suggest a generalized effect on LPS-induced monocyte activation. In this study, we tested whether HBP amplifies the release of other LPS-responsive cytokines from isolated human monocytes. METHODS: Freshly isolated monocytes from 5 healthy donors were stimulated for 24 h with saline, LPS (10 ng/ml), HBP (10 microg/ml), or a combination of LPS + HBP. Cytokine levels in the supernate were measured with ELISA. ANOVA and Fisher's posthoc test were used to determine significance (p < 0.05). Differential display was used to assess cellular mRNA levels. RESULTS: HBP alone induced the production of IL-8, macrophage inhibitory protein MIP-1alpha, and TNF-alpha. HBP increased the LPS-induced production of IL-8, MIP-1alpha, TNF-alpha, IL-1beta, but HBP did not increase the significant LPS-induced release of IL-10, monocyte chemoattractant protein MCP-1, and IL- 12. Differential display demonstrated that HBP induced an mRNA pattern that was different from the mRNA pattern induced by saline, LPS, or HBP + LPS, indicating multiple and different gene activation. CONCLUSIONS: We conclude that HBP is not a general amplificator of LPS-induced monocyte activation but rather a molecule that targets the production of a distinct set of mediators including pro-inflammatory cytokines such as TNF-alpha and IL-1beta, but not the anti-inflammatory cytokine IL-10, nor IL-12 and MCP-1. The exact intracellular signaling pathways remain unknown but include mechanisms that alter gene transcription.
Assuntos
Proteínas Sanguíneas/farmacologia , Proteínas de Transporte/farmacologia , Citocinas/biossíntese , Citocinas/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Monócitos/efeitos dos fármacos , Análise de Variância , Peptídeos Catiônicos Antimicrobianos , Células Cultivadas , Quimiocina CCL2/biossíntese , Quimiocina CCL3 , Quimiocina CCL4 , Humanos , Interleucina-1/biossíntese , Interleucina-8/biossíntese , Proteínas Inflamatórias de Macrófagos/biossíntese , Monócitos/imunologia , Monócitos/metabolismo , Reação em Cadeia da Polimerase , Probabilidade , RNA Mensageiro/análise , Valores de Referência , Sensibilidade e Especificidade , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/efeitos dos fármacosRESUMO
OBJECTIVE: To test the effectiveness of recombinant heparin-binding protein (HBP), a neutrophil-derived multifunctional protein with monocytic-specific properties, in fecal peritonitis and polymicrobial sepsis. DESIGN: Prospective, controlled animal trial. SETTING: Animal research laboratory. SUBJECTS: Swiss Webster mice challenged with cecal ligation and puncture (CLP) and treated with recombinant HBP and 60 mg/kg cefoxitin twice a day. INTERVENTIONS: HBP was administered to mice at different concentrations and different intervals before and after CLP. Rat albumin (1%) was administered to control animals. MEASUREMENTS AND MAIN RESULTS: MORTALITY RATE: Survival was increased in mice pretreated intraperitoneally 24 hrs before CLP with 10 microg or 100 microg of HBP without cefoxitin (p = .01, Cox-Mantel log-rank test). Compared with control animals, survival was increased significantly (from 5% to 47%, p = .014) in mice that received cefoxitin and 50 microg ip HBP immediately after CLP, followed by continuous administration of HBP (12 microg/24 hrs). Intravenous administration of HBP (0.1, 1, and 10 microg) at the time of CLP showed an opposite dose effect; low doses (0.1 microg) prolonged early survival, whereas high dose (10 microg) shortened survival (p = .036). Compared with control animals, overall survival was not different. CHEMOTAXIS: Cytospin preparations from peritoneal exudate cells (PECs) 48 hrs after administration of 10 microg and 100 microg ip HBP demonstrated a 1.7-fold increase in the total number of macrophages compared with carrier control (p < .05). PHAGOCYTOSIS: A flow cytometric in vitro assay demonstrated that administration of 10 microg ip HBP alone did not enhance phagocytosis of fluorescent Escherichia coli in PECs. However, 24-hr pretreatment with 10 microg of HBP followed by CLP increased phagocytosis in PECs 1.8-fold compared with the control CLP group (p = .04). RECEPTOR EXPRESSION: CD16/CD32w expression in PECs did not change after HBP or CLP. CD11b and CD18 expression in PECs was increased significantly after CLP compared with PECs from non-CLP-challenged animals (p < .05). Pretreatment with 10 microg of HBP did not further enhance CD11b/CD18 expression in PECs. CONCLUSIONS: Recombinant HBP increases survival in murine fecal peritonitis. The mechanisms by which HBP reduces septic death are not fully understood, but they include monocyte chemotaxis and increased phagocytosis of E. coli by PECs. Our data suggest that the inflammatory response induced by CLP is important for the effect of HBP to enhance phagocytosis.
Assuntos
Proteínas de Transporte/uso terapêutico , Glicoproteínas/uso terapêutico , Chaperonas Moleculares/uso terapêutico , Peritonite/tratamento farmacológico , Peritonite/mortalidade , Proteínas Recombinantes/uso terapêutico , Animais , Fezes , Proteína Associada a Proteínas Relacionadas a Receptor de LDL , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Monócitos/efeitos dos fármacos , Peritonite/etiologia , Fagocitose/efeitos dos fármacos , Taxa de SobrevidaRESUMO
OBJECTIVE: To compare the safety and efficacy of intravenous (IV) ciprofloxacin plus IV metronidazole (CIP+MET) with that of IV piperacillin/tazobactam (PIP/TAZO) in adults with complicated intraabdominal infections, and to compare the efficacy of sequential IV-to-oral CIP+MET therapy with that of the IV CIP-only regimen. SUMMARY BACKGROUND DATA: Treatment of intraabdominal infections remains a challenge, mainly because of their polymicrobial etiology and attendant death and complications. Antimicrobial regimens using sequential IV-to-oral therapy may reduce the length of hospital stay. METHODS: In this multicenter, randomized, double-blind trial involving 459 patients, clinically improved IV-treated patients were switched to oral therapy after 48 hours. Overall clinical response was the primary efficacy measurement. RESULTS: A total of 282 patients (151 CIP+MET, 131 PIP/TAZO) were valid for efficacy. Of these patients, 64% CIP+MET and 57% PIP/TAZO patients were considered candidates for oral therapy. Patients had a mean APACHE II score of 9.6. The most common diagnoses were appendicitis (33%), other intraabdominal infection (29%), and abscess (25%). Overall clinical resolution rates were statistically superior for CIP+MET (74%) compared with PIP/TAZO (63%). Corresponding rates in the subgroup suitable for oral therapy were 85% for CIP+MET and 70% for PIP/TAZO. Postsurgical wound infection rates were significantly lower in CIP+MET (11%) versus PIP/TAZO patients (19%). Mean length of stay was 14 days for CIP+MET and 17 days for PIP/TAZO patients. CONCLUSION: CIP+MET, initially administered IV and followed by CIP+MET oral therapy, was clinically more effective than IV PIP/TAZO for the treatment of patients with complicated intraabdominal infections.
Assuntos
Abdome , Anti-Infecciosos/administração & dosagem , Infecções Bacterianas/tratamento farmacológico , Ciprofloxacina/administração & dosagem , Quimioterapia Combinada/administração & dosagem , Metronidazol/administração & dosagem , Abscesso Abdominal/etiologia , Administração Oral , Apendicite/tratamento farmacológico , Apendicite/microbiologia , Método Duplo-Cego , Feminino , Humanos , Injeções Intravenosas , Masculino , Pessoa de Meia-Idade , Ácido Penicilânico/administração & dosagem , Ácido Penicilânico/análogos & derivados , Piperacilina/administração & dosagem , Combinação Piperacilina e Tazobactam , Estudos ProspectivosRESUMO
Nosocomial pneumonia is a leading cause of morbidity and mortality in the surgical and trauma patient. Inadequate treatment can lead to the complications of acute respiratory distress syndrome (ARDS), empyema, and lung abscess. The prevention and treatment of these complications revolve around several key principles. Complete treatment of pneumonia requires appropriate antimicrobial therapy, as well as mechanical pulmonary hygiene and proper airway management. Despite advances in treatment of pneumonia, complications arise necessitating treatment. This article reviews the treatment of ARDS, empyema, and lung abscesses. In particular, the many options for treatment of empyema are discussed in detail. Additionally, the treatment of pulmonary contusion and hemopneumothorax in the trauma patient is discussed. The understanding of sound treatment principles in the critically ill postsurgical patient helps prevent complicated or recurrent pneumonia and allows the surgeon to intervene effectively when such complications occur.
Assuntos
Infecção Hospitalar/complicações , Empiema/etiologia , Abscesso Pulmonar/etiologia , Pneumonia/complicações , Síndrome do Desconforto Respiratório/etiologia , Antibacterianos/uso terapêutico , Estado Terminal , Empiema/prevenção & controle , Humanos , Controle de Infecções/métodos , Unidades de Terapia Intensiva , Abscesso Pulmonar/prevenção & controle , Pneumonia/etiologia , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/prevenção & controle , Síndrome do Desconforto Respiratório/prevenção & controleRESUMO
Nosocomial pneumonia is a frequent complication in hospitalized patients. Gram-positive pathogens, particularly Staphylococcus aureus, are responsible for the increasing frequency of nosocomial pneumonia. To evaluate the efficacy and safety of intravenous quinupristin/dalfopristin (Synercid) in the treatment of nosocomial pneumonia caused by gram-positive pathogens we conducted a prospective, randomized, open-label, international, multicenter, comparative clinical trial. Two hundred ninety-eight patients with nosocomial pneumonia were enrolled in 74 active centers in five countries: a subgroup of 171 (87 quinupristin/dalfopristin-treated and 84 vancomycin-treated patients) were evaluable for the major efficacy end points. One hundred fifty received 7.5 mg/kg of quinupristin/dalfopristin every 8 h; 148 patients received 1 g of vancomycin every 12 h. Aztreonam at a dose of 2 g every 8 h could be administered in both groups for coverage of gram-negative organisms, and tobramycin was added for coverage against Pseudomonas aeruginosa. The primary efficacy end point was the clinical response between the seventh and the thirteenth day after the end of treatment in clinically evaluable patients with documented causative pathogen(s) at baseline (bacteriologically evaluable population). Therapy was clinically successful (cure or improvement) in 49 (56.3%) of patients receiving quinupristin/dalfopristin and 49 (58.3%) patients receiving vancomycin (difference, -2.0% [95% CI, -16.8% to 12.8%]) in the bacteriologically evaluable population. Equivalent clinical success rates were also observed in the all-treated population (n = 298), and in the bacteriologically evaluable patients intubated in baseline (39/72 [54%] compared with 36/67 [54%]). The by-pathogen bacteriologic response was similar in both treatment groups, with equivalent clinical success rates for Streptococcus pneumoniae, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus. Adverse events (venous and nonvenous) were equally distributed between groups; 15.3% of quinupristin/dalfopristin patients and 9.5% of vancomycin patients discontinued therapy because of an adverse clinical event. In this study quinupristin/dalfopristin was shown to be equivalent to vancomycin in the treatment of nosocomial pneumonia caused by gram-positive pathogens. Quinupristin/dalfopristin merits further evaluation for the treatment of nosocomial pneumonia caused by methicillin-resistant S. aureus.
Assuntos
Antibacterianos/uso terapêutico , Infecção Hospitalar/tratamento farmacológico , Quimioterapia Combinada/uso terapêutico , Pneumonia Pneumocócica/tratamento farmacológico , Pneumonia Estafilocócica/tratamento farmacológico , Vancomicina/uso terapêutico , Virginiamicina/uso terapêutico , Adulto , Idoso , Antibacterianos/efeitos adversos , Quimioterapia Combinada/efeitos adversos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Resultado do Tratamento , Vancomicina/efeitos adversos , Virginiamicina/efeitos adversosRESUMO
BACKGROUND: Heparin-binding protein (HBP), a serine protease without any known proteolytic activity, is found in human polymorphonuclear leukocyte (PMN) granules, but not in mice. HBP potentiates the endotoxin-induced release of tumor necrosis factor (TNF) alpha, interleukin (IL)-1, and IL-6 from isolated monocytes. HBP has also been shown to increase the survival of cultured monocytes and protect them from oxidative stress. However, whether HBP affects PMNs themselves is not known. MATERIALS AND METHODS: Based on our work with cultured monocytes and the survival benefit noted in experimental peritonitis, we hypothesized that HBP would have a beneficial effect on the survival of neutrophils. We evaluated the effect of HBP on apoptosis in murine peritoneal exudative cells elicited by intraperitoneal thioglycollate administration and in normal human neutrophils from volunteers. Leukocytes were isolated from the peritoneal cavity and blood of mice that underwent intraperitoneal thioglycollate instillation. The mouse peritoneal exudate cells and normal human neutrophils isolated from peripheral blood were used to study the effect of HBP on survival and apoptosis. RESULTS: HBP decreased percentage apoptosis of mouse cells in both serum-enriched (from 24.8 to 4.5%) and serum-deprived (from 23.1 to 8.2%) cultures. In human PMNs, the protective effect of HBP was seen only in the serum-deprived group, with a decrease in percentage apoptosis from 69.1 to 43.3%. CONCLUSIONS: For the first time, we have shown that HBP, in addition to its known augmentation of the proinflammatory response of monocytes, also acts as a prosurvival protein for neutrophils themselves, and thereby enhances local host defense.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas de Transporte/farmacologia , Glicoproteínas/farmacologia , Neutrófilos/fisiologia , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Proteína Associada a Proteínas Relacionadas a Receptor de LDL , Leucócitos/fisiologia , Camundongos , Microscopia Confocal , Neutrófilos/efeitos dos fármacos , Peritônio/citologia , Peritônio/fisiologia , Peritonite/induzido quimicamente , Peritonite/patologia , Peritonite/fisiopatologia , Tioglicolatos , Fatores de TempoRESUMO
Using a zymosan-induced mouse model of multiple organ dysfunction syndrome (MODS), it has been shown that the absence of MIP-1alpha increased mortality fourfold, whereas the absence of C5 decreased mortality fourfold. The purpose of the present study was to determine the early events following zymosan injection in MIP-1alpha knockout and C5-deficient mice. B10.D2/nSnJ (C5-sufficient) and B10.D2/0SnJ (C5-deficient) and genetically matched MIP-1alpha +/+ and MIP-1alpha -/- mice were divided into 3 groups: Group1 received no injection, Group 2 received intraperitoneal saline injection (1.0 mL), and Group 3 were given intraperitoneal zymosan (1 mg/gm, 1.0 mL). Two hours, 24 h, and 48 h after injection, peritoneal exudate leukocyte counts, total WBC count, lung MPO levels, and organ histology were examined for signs of changes in cellular infiltration. An acute local and systemic inflammatory response characterized by an increase in the peritoneal leukocyte count, total WBC counts, and circulating neutrophil levels was observed within 2-48 h of zymosan injection. Lack of MIP-1alpha attenuated local recruitment of phagocytes into the peritoneal cavity, and absence of MIP-1alpha or C5 caused a decrease in circulating neutrophil levels. The presence or absence of either C5 or MIP-1alpha did not affect early pulmonary neutrophil sequestration. Organ histopathology suggested early neutrophil infiltration in the lung and spleen within 48 h. These studies indicate that MIP-1alpha and C5 play a critical role in modulating cellular changes associated with lethality in a zymosan model of MODS.
Assuntos
Complemento C3/genética , Complemento C5/metabolismo , Proteínas Inflamatórias de Macrófagos/metabolismo , Insuficiência de Múltiplos Órgãos/metabolismo , Insuficiência de Múltiplos Órgãos/patologia , Animais , Quimiocina CCL3 , Quimiocina CCL4 , Ativação do Complemento , Complemento C3/metabolismo , Complemento C5/genética , Modelos Animais de Doenças , Exsudatos e Transudatos/citologia , Exsudatos e Transudatos/metabolismo , Injeções Intraperitoneais , Rim/patologia , Contagem de Leucócitos , Pulmão/metabolismo , Pulmão/patologia , Proteínas Inflamatórias de Macrófagos/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Knockout , Camundongos Mutantes , Insuficiência de Múltiplos Órgãos/induzido quimicamente , Miocárdio/patologia , Infiltração de Neutrófilos , Peritonite/metabolismo , Peritonite/patologia , Baço/patologia , Transaminases/metabolismo , ZimosanRESUMO
Hepatic ischemia and reperfusion causes neutrophil-dependent liver injury. Although the mechanisms of ischemia/reperfusion-induced liver neutrophil recruitment are somewhat understood, less is known regarding the early events that initiate the inflammatory injury. Using a murine model of partial hepatic ischemia and reperfusion, we evaluated the role of endogenous interleukin (IL)-12 in this inflammatory response. Hepatic ischemia for 90 minutes and reperfusion for up to 4 hours resulted in hepatocyte expression of IL-12. By 8 hours of reperfusion there were large increases in serum levels of interferon-gamma (IFNgamma) and tumor necrosis factor-alpha (TNFalpha). In addition, hepatic ischemia/reperfusion caused significant increases in liver neutrophil recruitment, hepatocellular injury, and liver edema, as defined by liver myeloperoxidase content, serum alanine aminotransferase, and liver wet to dry weight ratios, respectively. In mice treated with neutralizing antibody to IL-12 and in mice deficient in the IL-12 p40 gene, ischemia/reperfusion-induced increases in IFNgamma and TNFalpha were greatly diminished. These conditions also caused significant reductions in liver myeloperoxidase content and attenuated the parameters of liver injury. The data suggest that IL-12 is required for the full induction of injury after hepatic ischemia and reperfusion.
Assuntos
Citocinas/sangue , Interleucina-12/fisiologia , Hepatopatias/fisiopatologia , Fígado/fisiopatologia , Infiltração de Neutrófilos , Traumatismo por Reperfusão/fisiopatologia , Alanina Transaminase/sangue , Animais , Anticorpos , Deleção de Genes , Temperatura Alta , Interferon gama/sangue , Interferon gama/fisiologia , Interleucina-12/genética , Fígado/irrigação sanguínea , Fígado/imunologia , Fígado/patologia , Hepatopatias/imunologia , Hepatopatias/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Traumatismo por Reperfusão/imunologia , Traumatismo por Reperfusão/patologia , Fatores de Tempo , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/metabolismo , Regulação para CimaRESUMO
C-X-C chemokines play an important role in the migration and activation of neutrophils (PMNs) during an inflammatory event. We measured mRNA and protein expression of the murine C-X-C chemokines macrophage inflammatory protein-2 (MIP-2) and KC in the lungs, liver, blood, and peritoneal cavity of Swiss Webster mice after cecal ligation and puncture (CLP). Neutralizing antibodies to MIP-2 and KC were also used to determine the biological effects of these chemokines on neutrophil sequestration and organ injury in the CLP model. The data showed that early after CLP, MIP-2 mRNA and protein were expressed predominantly by the lung, whereas KC mRNA and protein were expressed by the liver. Inhibition of MIP-2 reduced both lung neutrophil sequestration and peritoneal neutrophil migration. Inhibition of KC had no effect on overall neutrophil sequestration in liver but reduced injury as measured by serum transaminases. An early survival benefit was found with anti-KC treatment, although overall survival was not different. Our study showed a differential expression by organs of C-X-C chemokines during sepsis and suggested that such chemokine effects are tissue-specific.
Assuntos
Movimento Celular/imunologia , Quimiocinas CXC/fisiologia , Neutrófilos/fisiologia , Peritonite/imunologia , Animais , Ceco/fisiologia , Quimiocina CXCL1 , Quimiocina CXCL2 , Quimiocinas , Fatores Quimiotáticos/biossíntese , Citocinas/biossíntese , Citocinas/genética , Citocinas/imunologia , Soros Imunes/farmacologia , Ligadura , Masculino , Camundongos , Monocinas/biossíntese , Monocinas/genética , Monocinas/imunologia , Neutrófilos/imunologia , Peritonite/patologia , PunçõesRESUMO
Neutrophil (PMN) migration into the peritoneal cavity in response to fecal peritonitis is an important mechanism of host defense against bacterial invasion. We show that the murine C-X-C (PMN-specific) chemokine, macrophage inflammatory protein-2 (MIP-2), on intraperitoneal injection in mice, causes PMN migration into the peritoneum. MIP-2 mRNA and protein were expressed by peritoneal leukocytes after cecal ligation and puncture (CLP) in mice and neutralization of MIP-2 reduced peritoneal PMN migration. A prerequisite for neutrophil-endothelial adhesion and subsequent migration from the circulation is selectin-mediated rolling. Pretreatment of mice with an anti-P-selectin antibody before intraperitoneal injection of MIP-2 significantly reduced peritoneal PMN migration. However, there are no reports that a C-X-C chemokine can up-regulate endothelial selectins. We postulated that MIP-2, when injected intraperitoneally, interacts with a cell that is known to release factors that up-regulate endothelial selectins. A likely candidate is the mast cell, which contains histamine and tumor necrosis factor alpha (TNF-alpha), and both of these factors induce selectins. Intraperitoneally injected MIP-2 caused an early significant increase in peritoneal TNF-alpha, whereas histamine levels were unaffected. In a subsequent experiment, mast cell-deficient mice and their normal controls were then injected intraperitoneally with MIP-2 or underwent CLP. Significantly fewer PMNs migrated into the peritoneal cavity in the mast cell-deficient mice after MIP-2 injection or CLP. Thus, our findings indicate that mast cells and MIP-2 are necessary for PMN migration into the peritoneum in response to intra-abdominal infection, and that MIP-2 appears to facilitate this through an increase in TNF-alpha release.
Assuntos
Fatores Quimiotáticos/fisiologia , Mastócitos/imunologia , Monocinas/fisiologia , Neutrófilos/fisiologia , Peritonite/imunologia , Animais , Ceco , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Quimiocina CXCL2 , Fatores Quimiotáticos/farmacologia , Fezes/microbiologia , Ligadura , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Masculino , Camundongos , Monocinas/farmacologia , Neutrófilos/efeitos dos fármacos , Selectina-P/metabolismo , Peritonite/microbiologia , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Regulação para CimaRESUMO
BACKGROUND: Hepatic ischemia/reperfusion injury during both hepatic resection and transplantation may lead to local and systemic organ dysfunction. Proinflammatory mediators released by Kupffer cells during the initial phase of ischemia/reperfusion are thought to be involved in the development of neutrophil-mediated lung injury. However, the precise factors involved in lung recruitment of neutrophils are unclear. The objective of current study was to determine whether the CXC chemokines, macrophage inflammatory protein-2 (MIP-2) and KC, contribute to pulmonary neutrophil recruitment and injury following hepatic ischemia/reperfusion. METHODS: C57BL/6 mice were subjected to 90 min of partial hepatic ischemia and 3, 6, and 9 h of reperfusion. Neutrophil accumulation in lung was assessed by lung content of myeloperoxidase (MPO). MIP-2 and KC mRNA were measured using RT-PCR. Lung edema was quantified by wet to dry weight ratios. RESULTS: Three hours after hepatic reperfusion, serum levels of tumor necrosis factor-alpha were increased. Lung expression of both MIP-2 and KC mRNA was also increased at this time. Both MIP-2 and KC mRNA expression remained elevated 9 h after reperfusion, although levels of MIP-2 mRNA were significantly lower than at 3 h. Pulmonary recruitment of neutrophils was increased within 3 h after reperfusion, but returned to baseline levels by 9 h. Lung edema was increased 3 and 9 h after reperfusion. Neutralization of MIP-2 or KC with antibody significantly decreased lung edema 9 h after reperfusion. CONCLUSIONS: These data suggest that mediators released during hepatic ischemia/reperfusion induce the expression of MIP-2 and KC in the lung. In addition, it appears that MIP-2 and KC contribute to lung neutrophil accumulation and the associated pulmonary injury following hepatic ischemia/reperfusion.
Assuntos
Quimiocinas/genética , Fígado/irrigação sanguínea , Pneumopatias/metabolismo , Pulmão/metabolismo , Traumatismo por Reperfusão/complicações , Animais , Quimiocina CXCL1 , Quimiocina CXCL2 , Quimiocinas CXC , Citocinas/genética , Expressão Gênica , Mediadores da Inflamação , Pulmão/patologia , Pneumopatias/etiologia , Pneumopatias/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monocinas/genética , Neutrófilos/patologia , Edema Pulmonar/etiologia , Edema Pulmonar/metabolismo , Edema Pulmonar/patologia , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Neutrophil (PMN) influx into the peritoneal cavity in response to bacterial peritonitis is an indispensable aspect of host defense. PMNs also are responsible for the remote organ injury observed after major abdominal infection. The aim of this study was to examine the effect of selectin blockade on PMN migration into the peritoneum and on PMN sequestration in the lungs, early in the course of peritonitis. Cecal ligation and puncture (CLP) was performed on P-selectin-deficient (P-def) mice and their genetic controls (C57). Both groups were treated with anti-E-selectin antibody, anti-L-selectin, or isotypic control immunoglobulin G at the time of CLP. 6 h after CLP, mice were sacrificed. Peritoneal PMN migration decreased in P-def mice compared with C57 controls after CLP. Blockade of E- or L-selectin alone in controls did not alter peritoneal PMN influx or circulating PMNs after CLP. In the P-def mice, treatment with anti-E-antibody or anti-L-antibody nearly eliminated PMN influx into the peritoneum. In contrast, circulating PMNs markedly increased after CLP in P-def mice when compared with baseline values. Lung myeloperoxidase increased in all groups of mice following CLP. Blockade of P-selectin with anti-P-selectin antibody elicited a response similar to that observed in the P-def mice. In conclusion, P-selectin mediates PMN influx into the peritoneal cavity, while E- and L-selectins do not appear to play any significant role in the 6 h time period following CLP. Lung PMN sequestration, after CLP, occurred independent of P-, E-, or L-selectin expression. Blockade of P-selectin during peritonitis appears to be potentially deleterious by preventing early PMN influx into the compartment containing the septic focus.
Assuntos
Pulmão/fisiologia , Neutrófilos/fisiologia , Selectina-P/metabolismo , Peritônio/citologia , Peritonite/metabolismo , Animais , Anticorpos/farmacologia , Bacteriemia/tratamento farmacológico , Bacteriemia/metabolismo , Ceco/cirurgia , Movimento Celular , Selectina E/imunologia , Selectina E/metabolismo , Selectina L/imunologia , Selectina L/metabolismo , Ligadura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Selectina-P/genética , Peritônio/metabolismo , Peritonite/microbiologia , Peroxidase/análise , Peroxidase/metabolismoRESUMO
Hepatic injury induced by ischemia and reperfusion is an important clinical problem after liver resection or transplantation. Neutrophils are known to mediate the organ injury, but the precise mechanisms leading to hepatic neutrophil recruitment are undefined. Two CXC chemokines, macrophage inflammatory protein-2 (MIP-2) and KC, are potently chemotactic for neutrophils in vitro and have been reported to be involved in neutrophil-dependent inflammatory tissue injury. The objective of the present study was to determine the roles of MIP-2 and KC in the induction of hepatic ischemia/reperfusion injury. C57BL/6 mice were subjected to 90 minutes of partial hepatic ischemia followed by reperfusion. Hepatic injury was associated with neutrophil sequestration, edema, and elevated serum levels of hepatic transaminases. The expression of MIP-2 messenger RNA (mRNA) was induced within 3 hours after reperfusion, before any detectable increase in neutrophil accumulation, and was also increased to a greater extent in the ischemic lobe after 9 hours of reperfusion. These data suggest that MIP-2 may be involved in the initial recruitment of neutrophils to the ischemic lobe. In contrast, KC mRNA expression was not increased after 3 hours of reperfusion but after 9 hours increased equivalently in both ischemic and non-ischemic lobes, suggesting a more generalized role in neutrophil recruitment. Neutralization of MIP-2 or KC resulted in significant decreases in hepatic neutrophil accumulation, edema, and hepatocellular injury. These data suggest that the local expression of MIP-2 and KC are important mediators involved in neutrophil-dependent hepatic injury induced by ischemia and reperfusion in mice.
Assuntos
Citocinas/fisiologia , Isquemia/metabolismo , Fígado/irrigação sanguínea , Monocinas/fisiologia , Traumatismo por Reperfusão/etiologia , Animais , Quimiocina CXCL1 , Quimiocina CXCL2 , Quimiocinas , Quimiocinas CXC , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/fisiologiaRESUMO
BACKGROUND: Tumour necrosis factor (TNF) alpha and interleukin (IL) 1 beta are produced in the lung after peritonitis and may contribute to neutrophil-mediated organ injury. It was hypothesized that, during experimental peritonitis, continuous rather than intermittent antibiotic therapy would reduce lung expression of TNF-alpha and IL-1 beta messenger RNA (mRNA) and neutrophil sequestration. METHODS: After caecal ligation and puncture, mice received either intermittent or continuous cefoxitin, or continuous metronidazole or aztreonam. Cytokine mRNAs were determined by reverse transcription differential polymerase chain reaction and lung neutrophil content by myeloperoxidase (MPO) assay. RESULTS: Continuous cefoxitin reduced median (interquartile range (i.q.r.)) lung IL-1 beta mRNA expression ((ratio to beta-actin): continuous 0.18 (0.14-0.34), intermittent 0.46 (0.44-0.49), saline 0.43 (0.38-0.53), P < 0.05) and median (i.q.r.) lung MPO content (continuous 22.5 (9.7-40), intermittent 65 (57.5-76), saline 47 (41-64), P < 0.05) compared with intermittent therapy and saline controls. Continuous infusion was also associated with reduced bacteraemia (P < 0.05) but not serum TNF-alpha or endotoxin levels. Both continuous metronidazole and aztreonam reduced lung MPO concentration (P < 0.05) and TNF-alpha and IL-1 beta mRNA expression (P < 0.05) compared with those in saline controls. These effects were dependent on a reduction in the number of susceptible bacteria rather than serum TNF-alpha or endotoxin levels. CONCLUSION: The stimulus for organ inflammatory cytokine production and neutrophil sequestration during peritonitis is the level of bacteraemia present, which is more effectively controlled with continuous antibiotic therapy.
Assuntos
Antibacterianos/uso terapêutico , Citocinas/metabolismo , Sepse/tratamento farmacológico , Sepse/metabolismo , Animais , Aztreonam/uso terapêutico , Cefoxitina/uso terapêutico , Cefamicinas/uso terapêutico , Endotoxinas/metabolismo , Interleucina-1/metabolismo , Metronidazol/uso terapêutico , Camundongos , Peroxidase/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Hepatic injury induced by ischemia and reperfusion is an important clinical problem after liver resection or transplantation. Neutrophils are known to mediate the organ injury, but the precise mechanisms leading to hepatic neutrophil recruitment are undefined. Two CXC chemokines, macrophage inflammatory protein-2 (MIP-2) and KC, are potently chemotactic for neutrophils in vitro and have been reported to be involved in neutrophil-dependent inflammatory tissue injury. The objective of the present study was to determine the roles of MIP-2 and KC in the induction of hepatic ischemia/reperfusion injury. C57BL/6 mice were subjected to 90 minutes of partial hepatic ischemia followed by reperfusion. Hepatic injury was associated with neutrophil sequestration, edema, and elevated serum levels of hepatic transaminases. The expression of MIP-2 messenger RNA (mRNA) was induced within 3 hours after reperfusion, before any detectable increase in neutrophil accumulation, and was also increased to a greater extent in the ischemic lobe after 9 hours of reperfusion. These data suggest that MIP-2 may be involved in the initial recruitment of neutrophils to the ischemic lobe. In contrast, KC mRNA expression was not increased after 3 hours of reperfusion but after 9 hours increased equivalently in both ischemic and nonischemic lobes, suggesting a more generalized role in neutrophil recruitment. Neutralization of MIP-2 or KC resulted in significant decreases in hepatic neutrophil accumulation, edema, and hepatocellular injury. These data suggest that the local expression of MIP-2 and KC are important mediators involved in neutrophil-dependent hepatic injury induced by ischemia and reperfusion in mice.
Assuntos
Citocinas/metabolismo , Células de Kupffer/metabolismo , Fígado/metabolismo , Monocinas/metabolismo , Traumatismo por Reperfusão/metabolismo , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Quimiocina CXCL1 , Quimiocina CXCL2 , Quimiocinas , Quimiocinas CXC , Fatores Quimiotáticos/metabolismo , Citocinas/imunologia , Imunização Passiva , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Células de Kupffer/imunologia , Fígado/irrigação sanguínea , Fígado/patologia , Masculino , Camundongos , Monocinas/imunologia , Neutrófilos/citologia , RNA Mensageiro/análise , Traumatismo por Reperfusão/prevenção & controle , Fatores de TempoRESUMO
OBJECTIVES: To determine the efficacy and magnitude of associated adverse effects of 2 different antibiotic regimens for the treatment of pneumonia in intubated surgical patients and to assay and compare blood samples and bronchoalveolar lavage fluid with respect to some host-defense parameters, especially in patients with unilateral pneumonia. DESIGN: Randomized, prospective, unblinded clinical comparison of 2 treatment arms with respect to intent to treat and clinical and microbiologically evaluable patients. SETTING: Six university surgical services in teaching hospitals with modern and well-staffed intensive care units. INTERVENTIONS: The consistency and objectively of the diagnosis of pneumonia was improved by the use of a grid of diagnostic parameters. Aggressive mechanical approaches to pneumonia in intubated surgical patients were supplemented by therapeutic use of aztreonam and vancomycin hydrochloride or combined imipenem and cilastatin sodium. RESULTS: Patients randomized to the aztreonam-vancomycin group were somewhat more ill, fared slightly better, and had fewer serious drug-related side effects than did those treated with imipenem-cilastatin (all P > .05). Immunologic parameters assessed by evaluation of bronchoalveolar lavage fluid showed differences between infected pulmonary lobes and noninfected ones; some changes were also noted in patients who recovered compared with those whose pneumonia persisted or recurred. CONCLUSIONS: Clinical studies of pneumonia in surgical patients need to be stratified to assure comparability, to identify patients in whom treatment is likely to fail, and to display differences between more and less effective therapies. Studies of blood and bronchoalveolar lavage samples showed that certain local and systemic immunologic parameters correlate with clinical status and outcome.