Clinical Significance of Tissue Factor and CD13 Double-Positive Microparticles in Sirs Patients with Trauma and Severe Sepsis.

Activated immune cells such as monocytes are key factors in systemic inflammatory response syndrome (SIRS) following trauma and sepsis. Activated monocytes induce almost all tissue factor (TF) expression contributing to inflammation and coagulation. TF and CD13 double-positive microparticles (TF/CD13MPs) are predominantly released from these activated monocytes. This study aimed to evaluate TF/CD13MPs and assess their usefulness as a biomarker of pathogenesis in early SIRS following trauma and sepsis. This prospective study comprising 24 trauma patients, 25 severe sepsis patients, and 23 healthy controls was conducted from November 2012 to February 2015. Blood samples were collected from patients within 24 h after injury and diagnosis of severe sepsis and from healthy controls. Numbers of TF/CD13MPs were measured by flow cytometry immediately thereafter. Injury Severity Score (ISS) and Acute Physiology and Chronic Health Evaluation (APACHE) II and Sequential Organ Failure Assessment (SOFA) scores were calculated at patient enrollment. APACHE II and SOFA scores and International Society of Thrombosis and Haemostasis (ISTH) overt disseminated intravascular coagulation (DIC) diagnostic criteria algorithm were calculated at the time of enrollment of severe sepsis patients. Numbers of TF/CD13MPs were significantly increased in both trauma and severe sepsis patients versus controls and correlated significantly with ISS and APACHE II score in trauma patients and with APACHE II and ISTH DIC scores in severe sepsis patients. Increased numbers of TF/CD13MPs correlated significantly with severities in the acute phase in trauma and severe sepsis patients, suggesting that TF/CD13MPs are important in the pathogenesis of early SIRS following trauma and sepsis.

Effect of dim and bright light exposure on some immunological parameters measured under thermal neutral conditions.

This study assesses the effects of ambient light conditions, under a thermoneutral environment, on selected immunological parameters of 7 healthy young women (aged 19 to 22 yrs). Subjects entered the bioclimatic chamber at 11: 00 h, controlled at 26 degrees C and 60% relative humidity, a "neutral climate". They lead a well-regulated life in the climatic chamber (pre-condition) while exposed to dim (200 lux) or, on the next day, bright (5000 lux) light between 06 : 00 to 12 : 00 h. Just before the end of each period of light exposure, a blood sample was taken for later immunological assay of white blood cell count (WBC), phagocytosis, interferon-gamma (IFN-gamma), interleukin-4 (IL-4), CD69 T cells (CD69), CD4+CD25+ T cells (CD4+CD25+), and transforming growth factor-beta 1 (TGF-beta1). The results, when compared with the pre-condition, were as follows: 1) CD69 and IFN-gamma increased during normal conditions without thermal stress under dim light; 2) WBC increased and IL-4 decreased under bright light; 3) as shown by the highly significant decrease of TGF-beta1, the immune system was activated under bright light; 4) phagocytosis tended to increase under bright light exposure; 5) CD69 and IFN-gamma were significantly higher, and CD4+CD25+ tended to decrease under bright light; 6) phagocytosis tended to be lower and TGF-beta1 significantly higher under dim light, indicating a decline of immune system function. Taken together, this preliminary single time-point sampling study infers that some parameters are activated (CD69) while others are attenuated (phagocytosis, TGF-beta1) according to the environmental light intensity, dim vs. bright, in women adhering to a standardized routine in the absence of thermal stress. These findings are discussed in terms of inhibition of the sympathetic and excitation of the parasympathetic nervous system under the influence of life-style regularity and daytime bright light exposure.

Enhanced expression of cell-specific surface antigens on endothelial microparticles in sepsis-induced disseminated intravascular coagulation.

Sepsis-induced disseminated intravascular coagulation (DIC) is a major cause of death in patients admitted to intensive care units. Endothelial injury with microparticle production is reported in the pathogenesis of sepsis. Endothelial microparticles (EMPs) present several cell-specific surface antigens with different bioactivities, for example, tissue factor (TF), thrombomodulin (TM), and endothelial protein C receptor (EPCR). We investigated associations between these three different surface antigen-positive EMPs and sepsis-induced DIC. This cross-sectional study composed of 24 patients with sepsis and 23 healthy controls was conducted from November 2012 to September 2013. Blood samples were collected from patients within 24 h of diagnosis of severe sepsis and from healthy controls. Numbers of TF-positive EMPs (TF EMPs), TM-positive EMPs (TM EMPs), and EPCR-positive EMPs (EPCR EMPs) were measured by flow cytometry immediately thereafter. Acute Physiology and Chronic Health Evaluation II and Sequential Organ Failure Assessment scores were assessed in the severe sepsis patients at enrollment. We assessed DIC with the International Society of Thrombosis and Haemostasis (ISTH) overt DIC diagnostic criteria algorithm. Numbers of antigen-positive EMPs were increased significantly in both severe sepsis patients and controls and with the increase in ISTH DIC score. Numbers of TF EMPs and EPCR EMPs correlated significantly with Sequential Organ Failure Assessment score, and numbers of EPCR EMPs correlated significantly with Acute Physiology and Chronic Health Evaluation II score. Numbers of the three antigen-positive EMPs were increased significantly in severe sepsis patients versus those in healthy controls and with the increase of ISTH DIC score, suggesting that the specific bioactivity of each antigen-positive EMP may play a role in the progression of sepsis-induced DIC.

Early activation of gammadelta T lymphocytes in patients with severe systemic inflammatory response syndrome.

Innate immunity plays an important role in host defense after severe insult. gammadelta T lymphocytes are recognized as the first line of defense against microbial invasion. In this study, we evaluated gammadelta T lymphocytes in the peripheral blood of patients with severe systemic inflammatory response syndrome (SIRS), and examined on role of these cells. Thirty-seven patients with severe SIRS (SIRS criteria and serum C-reactive protein > or = 10 mg/dL) and 27 healthy volunteers were studied. Severe SIRS was caused by trauma in 14 patients (Injury Severity Score of 30.1 +/- 10.8) and by sepsis in 23 patients. The counts of gammadelta and alphabeta T lymphocytes were determined by flow cytometry of cells stained with monoclonal antibodies to gammadelta and alphabeta T lymphocyte receptors. The activation of these cells was evaluated by flow cytometry of cells stained with monoclonal antibodies to CD69 and HLA-DR. Serial counts and activation of gammadelta and alphabeta T lymphocytes were also determined in eight trauma patients (Injury Severity Score of 31.0 +/- 13.5) during a 2-week observation period. The count of gammadelta T lymphocytes in the peripheral blood of SIRS patients (30.1 +/- 6.0/microL) was significantly lower (P < 0.05) than that of the healthy volunteers (104.3 +/- 10.9/microL). The expression of CD69, an index of early activation of T lymphocytes, was significantly greater on gammadelta T lymphocytes from SIRS patients (patients 23.9% +/- 3.4%, healthy controls 4.8% +/- 0.6%, P < 0.05). In trauma patients, the expression of CD69 on gammadelta T lymphocytes increased rapidly within 48 h after injuries. In conclusion, gammadelta T lymphocytes are activated and decreased in the peripheral blood of severe SIRS patients. In trauma patients, the activation of gammadelta T lymphocytes occurs in the fairly acute phase after injuries. These results suggest a significant role for gammadelta T lymphocytes as early responders after severe insult.

Long-term enhanced expression of heat shock proteins and decelerated apoptosis in polymorphonuclear leukocytes from major burn patients.

Heat shock proteins (HSPs), as molecular chaperones, have been reported to protect cells against a variety of environmental stresses. The objective of this study was to clarify the serial changes in expression of HSPs, oxidative activity, and apoptosis in polymorphonuclear leukocytes (PMNLs) from burn patients. Eight patients with severe burns (mean burn index 24.0 +/- 6.1) were included. Blood samples were serially obtained at five time points: days 0 to 1, days 2 to 7, days 8 to 14, days 15 to 21, and days 22 to 28. We measured expressions of HSP27, HSP60, HSP70, and HSP90 in permeabilized PMNLs by flow cytometry with the use of a monoclonal antibody against each HSP. The oxidative activity and apoptosis in PMNLs were also measured by flow cytometry. During all five time periods, expressions of HSP27, HSP60, and HSP70 in PMNLs from burn patients were significantly greater than those in PMNLs from healthy volunteers. The expression of HSP90 in PMNLs of burn patients increased between days 2 and 21. Oxidative activity in their PMNLs was significantly enhanced between days 2 and 28, and PMNL apoptosis was markedly inhibited for as long as 4 weeks after thermal injury. In conclusion, major burn causes long-term, enhanced expression of HSPs in PMNLs along with increased oxidative activity and decelerated apoptosis. The enhanced expression of HSPs may regulate the oxidative stress response and life-span of PMNLs in burn patients.

Enhanced expression of aquaporin 9 in activated polymorphonuclear leukocytes in patients with systemic inflammatory response syndrome.

Aquaporins (AQPs) are water channels of cell membranes. All living cells experience osmotic pressure changes in their environment, but the mechanism by which water influx occurs was not known until the discovery of AQPs. AQP9, which is expressed in human polymorphonuclear leukocytes (PMNLs), is reported to relate to morphologic changes of PMNLs in vitro. We examined the expression of AQP9 in PMNLs from patients with systemic inflammatory response syndrome (SIRS) and addressed the role of AQP9 in both morphologic and functional changes of PMNLs in the SIRS condition. Fourteen patients with SIRS were included in our study. Polyclonal antibody was used for the AQP9 assay. F-actin polymerization, oxidative activity, and the expression of AQP9 in PMNLs with and without stimulation by N-formylmethionyl-leucyl-phenylalanine were evaluated by flow cytometry. Expression of AQP9, F-actin polymerization, and oxidative activity in PMNLs were increased significantly in patients with SIRS compared with those in healthy volunteers. The time course of AQP9 fluorescence in PMNLs corresponded to the time course of F-actin polymerization, which showed peak fluorescence at 1 min after N-formylmethionyl-leucyl-phenylalanine stimulation. The expression of AQP9 in PMNLs is increased significantly in SIRS patients. The increased expression of AQP9 in SIRS patients might be associated with F-actin polymerization in PMNLs, which could affect both morphologic and functional changes of PMNLs in the SIRS condition.

Platelet mitochondrial membrane potential correlates with severity in patients with systemic inflammatory response syndrome.

BACKGROUND: The role of mitochondrial dysfunction has not been thoroughly clarified in the pathogenesis of critically ill patients. The objective of this study was to investigate mitochondrial membrane potential (ΔΨm) and apoptosis in circulating platelets in patients with systemic inflammatory response syndrome (SIRS). METHODS: This prospective observational study was conducted from May 2011 to February 2012. Criteria for inclusion were adult patients with SIRS. We used mitochondrial indicator JC-1 in conjunction with flow cytometry to measure ΔΨm and annexin V to evaluate apoptosis in peripheral blood platelets. ΔΨm was expressed as the percentage of platelets with altered ΔΨm. Severity of illness was assessed by SIRS score, Acute Physiology and Chronic Health Evaluation II score, and Sequential Organ Failure Assessment score. RESULTS: This study was composed of 36 patients who met the inclusion criteria and 12 healthy controls. Causes of SIRS were sepsis in 13, trauma in 13, and others in 10 patients. Platelet ΔΨm depolarization was significantly enhanced in patients with SIRS versus that in controls (median [interquartile range], 10.6% [8.1-12.6%] vs. 7.1% [6.1-8.0%]; p < 0.001). The percentage of apoptotic platelets was significantly higher in patients with SIRS than in controls (8.7% [5.5-13.5%] vs. 5.4% [3.9-7.0%]; p = 0.006). Interestingly, ΔΨm depolarization increased significantly with the increase in SIRS scores (p < 0.001). There was a significant correlation between ΔΨm depolarization and severity of illness, as indicated by Acute Physiology and Chronic Health Evaluation II score, Sequential Organ Failure Assessment score, and serum lactate levels (all, p < 0.05). CONCLUSION: We demonstrated that ΔΨm depolarization and apoptosis were enhanced in circulating platelets in patients with SIRS. Our findings suggest that ΔΨm depolarization may be associated with the progression of SIRS. LEVEL OF EVIDENCE: Diagnostic study, level III.

Enhanced expression of intracellular heme oxygenase-1 in deactivated monocytes from patients with severe systemic inflammatory response syndrome.

BACKGROUND: Monocyte deactivation is an important contributor to infectious susceptibility in critically ill patients. However, the mechanism of monocyte deactivation has not been fully elucidated. Recently, intracellular heme oxygenese-1 (HO-1), an anti-inflammatory heat-shock protein, was reported to be activated by Toll-like receptors (TLRs), and to inhibit inflammatory cytokine production such as that of TNF-alpha. In the present study, we evaluated the expression of intracellular HO-1 and TLRs in monocytes from patients with severe systemic inflammatory response syndrome (SIRS) and examined the role of HO-1 in monocyte deactivation. PATIENTS: Twenty-seven patients who fulfilled the criteria for severe SIRS and had a serum C-reactive protein (CRP) level >10 mg/dL were included in this study. The cause of SIRS was sepsis in 16 patients, trauma in 7, and other in 4. Expression of intracellular HO-1, surface TLR2 and TLR4, and intracellular cytokines (TNF-alpha, Interleukin-6) stimulated via TLR activation were measured in circulating monocytes by flow cytometry. Intracellular HO-1 expression was evaluated in normal monocytes stimulated with patient serum. Serum cytokine levels were also measured. Patient data were compared with data from healthy volunteers (n = 16). RESULTS: Cytoplasmic HO-1 was clearly detected by fluorescence microscopy. Expression of HO-1, TLR2, and TLR4 in monocytes was significantly enhanced in patients with severe SIRS compared with that in healthy volunteers, whereas intracellular TNF-alpha expression with peptidoglycan was significantly decreased (p < 0.05) in patients compared with that in healthy volunteers. HO-1 expression was significantly enhanced in normal monocytes stimulated with patient serum. Intracellular HO-1 levels were positively related to serum TNF-alpha levels in patients (r = 0.46). CONCLUSIONS: Expression of intracellular HO-1 and of TLRs was enhanced in deactivated monocytes from patients with SIRS. Increased production of intracellular HO-1 in response to serum factors may play a role in monocyte deactivation after systemic inflammation.

Serial changes in leukocyte deformability and whole blood rheology in patients with sepsis or trauma.

BACKGROUND: The objective of this study was to investigate serial changes in leukocyte deformability and rheologic properties of whole blood in patients with sepsis or trauma. METHODS: Seventeen sepsis patients and 22 trauma patients were enrolled. Leukocyte deformability and rheologic properties of whole blood were determined with the use of a microchannel array etched on a single-crystal silicon tip, simulating the microvasculature. The number of obstructed microchannels (NOM) was used as a measure of leukocyte deformability. Transit time (TT), i.e., the time taken for 100 microL of whole blood to pass through the microchannel array was also used as rheologic measure. Oxidative activity and F-actin content of neutrophils was measured in patients with sepsis. RESULTS: NOM and TT significantly increased in patients when sepsis was diagnosed. In survivors, NOM and TT decreased at the time of recovery from sepsis, but in non-survivors values remained high. Oxidative activity and F-actin content of neutrophils increased significantly as leukocyte deformability decreased. In patients with severe trauma, NOM and TT increased after injury and decreased by the time of recovery from the critical stage. CONCLUSION: We conclude that leukocyte deformability decreases in patients with sepsis or severe trauma and that this change negatively affects rheologic properties of whole blood.

The balance between expression of intranuclear NF-kappaB and glucocorticoid receptor in polymorphonuclear leukocytes in SIRS patients.

BACKGROUND: We previously reported enhanced expression of nuclear factor kappa B (NF-kappaB) in activated polymorphonuclear leukocytes (PMNLs) from patients with systemic inflammatory response syndrome (SIRS). Inflammatory response, however, is not regulated only by stimulatory transcription factors. Glucocorticoid receptor (GR) has been recently reported to play an important role in anti-inflammatory signal transduction. The objective of our study was to evaluate the balance between expression of intranuclear NF-kappaB and GR in PMNLs from SIRS patients. METHODS: In study 1, 29 patients with severe SIRS, who fulfilled the criteria for SIRS and had a serum C-reactive protein level of more than 10 mg/dL, were included. Expression of intranuclear NF-kappaB and GR in PMNLs was measured by flow cytometry with antibodies specific for NF-kappaB subunit p65 and GR. PMNL oxidative activity and serum cytokine levels were also measured. Study 2 included 13 patients with severe trauma (Injury Severity Score 24.6 +/- 12.2). We measured serial changes in expression of intranuclear NF-kappaB and GR in days 0 to 2, 3 to 6, and 7 to 14 after injury. RESULTS: In study 1, expression of both intranuclear NF-kappaB and GR in PMNLs was significantly higher in SIRS patients than in healthy controls. There was a strong correlation between expression of these two transcription factors (r = 0.78). Positive correlations were also found between PMNL oxidative activity and both transcription factors. In study 2, expression of both NF-kappaB and GR in PMNLs was markedly elevated on days 3 to 6 after injury and changed serially with strong mutual correlation. CONCLUSIONS: In activated PMNLs from SIRS patients, levels of both intranuclear NF-kappaB and GR were elevated and strongly correlated. In trauma patients, NF-kappaB and GR in PMNLs changed serially with strong mutual correlation. Further studies are needed to clarify the effect of the balance of NF-kappaB and GR on PMNL activation and systemic inflammatory process.

Activated polymorphonuclear leukocytes enhance production of leukocyte microparticles with increased adhesion molecules in patients with sepsis.

BACKGROUND: Leukocyte microparticles (MPs) derived from polymorphonuclear leukocytes (PMNLs) have been recently found to be activators of vascular endothelium in vitro. The precise role of leukocyte MPs has not been clarified in patients suffering severe insult. The objective of this study was to evaluate production of leukocyte MPs and expression of adhesion molecules on the MP surface in patients with sepsis. METHODS: Twenty-one patients with severe infection (fulfilling the criteria of sepsis with serum C-reactive protein > 10 mg/dL) and 21 healthy volunteers were included as study subjects. Production of leukocyte MPs, expression of CD11b on the MPs, and oxidative activity of PMNLs were measured by flow cytometry in the presence and absence of formyl-methionyl-leucyl-phenylalanine. CD11b expression was evaluated according to the MP size (more than, equal to, or less than 1.0 microm). Soluble E-selectin, thrombomodulin, and PMNL elastase were also measured in blood. RESULTS: Production of leukocyte MPs and superoxide production in PMNLs with and without formyl-methionyl-leucyl-phenylalanine increased significantly in patients with sepsis in comparison with production in normal volunteers. In patients with sepsis, expression of CD11b was also markedly enhanced on MPs less than 1.0 microm in diameter in comparison with expression in control subjects. Levels of soluble E-selectin, thrombomodulin, and PMNL elastase in blood were significantly increased in patients with sepsis. We succeeded in detecting leukocyte MPs visually by fluorescence microscopy. CONCLUSION: Activated PMNLs enhance production of leukocyte MPs with increased adhesion molecules in patients with sepsis. Activated leukocyte MPs may play a role in the pathogenesis of endothelial activation and leukocyte-endothelium interaction in the presence of sepsis.

Increased production of leukocyte microparticles with enhanced expression of adhesion molecules from activated polymorphonuclear leukocytes in severely injured patients.

BACKGROUND: Polymorphonuclear leukocyte (PMNL)-derived microparticles (MPs) have been recently reported as activators of vascular endothelium in vitro. The objectives of the present study were to evaluate the production of MPs in severely injured patients and to clarify the role of these MPs. METHODS: Thirty severely injured patients (mean Injury Severity Score of 27 +/- 11) and 21 healthy volunteers participated in the study. Blood samples were obtained serially at three time points: days 0 to 1, days 2 to 5, and days 6 to 12 after the trauma event. MP production, CD11b and CD62L expression on MPs, and oxidative activity in PMNLs were measured by flow cytometry in both the presence and absence of formylmethionyl-leucyl-phenylalanine. Expressions of CD11b and CD62L were differentially evaluated according to the size of the MPs (>or= or < 1.0 microm). Soluble E-selectin and thrombomodulin levels in blood, variables representative of systemic vascular endothelial damage, were also measured. RESULTS: Production of MPs with and without formylmethionyl-leucyl-phenylalanine and the oxidative activity in PMNLs (O ) were prominently increased on days 2 to 5 after trauma. CD62L expression was enhanced on MPs at all three time points, and CD11b expression was enhanced on MPs < 1.0 microm in diameter at all three time points. Soluble E2-selectin and thrombomodulin in blood did not change significantly between time points. CONCLUSION: Activated PMNLs enhance production of PMNL-derived MPs with increased adhesion molecule expression on days 2 to 5 after severe trauma. This response per se, however, may not progress to systemic vascular endothelial damage.

Enhanced expression of intranuclear NF-kappa B in primed polymorphonuclear leukocytes in systemic inflammatory response syndrome patients.

BACKGROUND: Nuclear factor-kappa B (NF-kappa B) plays a critical role in the cellular response to a variety of stimuli, and it regulates the production of various inflammatory cytokines, adhesion molecules, and enzymes. Polymorphonuclear leukocytes (PMNLs) play a central role in systemic inflammatory response after severe insult. The role of NF-kappa B in activation of PMNLs, however, has not been clear. We developed a simple flow cytometric method for quantifying expression of intranuclear NF-kappa B in PMNLs, and we used it to evaluate NF-kappa B activity in patients with systemic inflammatory response syndrome (SIRS). METHODS: Thirty patients who fulfilled the criteria for SIRS and 24 healthy volunteers were included as study subjects. Expression of intranuclear NF-kappa B with and without stimulation by lipopolysaccharide was quantified by our new method. Oxidative activity in PMNLs with and without formylmethionyl-leucyl-phenylalanine stimulation was measured by flow cytometry. Levels of interleukin-6, interleukin-8, PMNL elastase, and nitric oxide metabolites in blood were also measured. RESULTS: Expression of intranuclear NF-kappa B in PMNLs both with and without LPS stimulation was significantly elevated in SIRS patients in comparison with that of healthy volunteers. PMNL oxidative activity was significantly elevated in SIRS patients. Positive correlation was observed between intranuclear NF-kappa B expression and PMNL oxidative activity, whereas no relation was observed between intranuclear NF-kappa B expression and serum concentrations of chemical mediators. CONCLUSION: Our new flow cytometric method proved useful for quantifying intranuclear NF-kappa B expression in PMNLs. In PMNLs from SIRS patients, intranuclear NF-kappa B expression and oxidative activity were significantly elevated with positive correlation, and enhanced expression of NF-kappa B may play an important role in PMNL activation in SIRS.

Hepatocyte growth factor in polymorphonuclear leukocytes is increased in patients with systemic inflammatory response syndrome.

BACKGROUND: Hepatocyte growth factor (HGF) has a significant effect on the regeneration of epithelial and endothelial cells. Studies have also shown an important role of HGF in wound healing and organ regeneration. Because recent studies indicate that polymorphonuclear leukocytes (PMNLs) store HGF in their specific granules and that HGF can be degranulated in the inflammatory tissue in which activated PMNLs migrate, we evaluated the storage and release of HGF in PMNLs from patients with systemic inflammatory response syndrome (SIRS) and attempted to examine the role of HGF from PMNLs in the systemic inflammatory process. METHODS: Twenty-four patients with SIRS (serum C-reactive protein, 20.2 +/- 12.4 mg/dL [mean +/- SD]) and 18 healthy volunteers were studied. HGF in PMNLs was measured by flow cytometry by using a monoclonal antibody to HGF. The oxidative activity in PMNLs was also measured by flow cytometry. Serum HGF, interleukin (IL)-6, and IL-8 levels in each patient were measured by enzyme-linked immunosorbent assay. HGF degranulation from PMNLs was evaluated in 10 patients. RESULTS: Immunocytochemistry under fluorescence microscopy revealed enhanced expression of HGF in the granules of PMNLs. HGF in PMNLs significantly increased in patients with SIRS compared with PMNLs from healthy volunteers (SIRS, 171.0 +/- 6.6 fluorescence/cell; control, 130.7 +/- 3.8 fluorescence/cell). N-formylmethionyl-leucyl-phenylalanine and lipopolysaccharide stimulation induced further increase of HGF fluorescence in PMNLs from patients. HGF degranulation from PMNLs was also significantly enhanced in patients. Moreover, oxidative activity in PMNLs was significantly enhanced in patients with SIRS. Plasma HGF (pHGF) correlated positively with IL-6 and IL-8 levels in patients (pHGF and IL-6, gamma = 0.635, p < 0.05; pHGF and IL-8, gamma = 0.827, p < 0.01), but these values did not correlate with HGF in PMNLs. CONCLUSION: Activated PMNLs in SIRS patients increased HGF in their granules and demonstrate enhanced degranulation of HGF. The release of HGF from migrated PMNLs in the inflammatory tissue may play an important role in wound healing and organ regeneration under those conditions.

Enhanced production of endothelial microparticles with increased binding to leukocytes in patients with severe systemic inflammatory response syndrome.

BACKGROUND: The vascular endothelium sustains substantial damage after severe insult. Recently, activated endothelial cells have been reported to produce microparticles in vitro. The objective of this study was to evaluate endothelial microparticle formation and microparticle-leukocyte interaction among patients with severe systemic inflammatory response syndrome (SIRS). METHODS: The participants in this study were 28 patients with severe SIRS (SIRS criteria and serum C-reactive protein > 10 mg/dL) and 18 healthy volunteers. Endothelial microparticles in the blood, microparticle-polymorphonuclear leukocyte (PMNL) binding, and PMNL oxidative activity were measured by flow cytometry. Soluble E-selectin, thrombomodulin, and plasminogen activator inhibitor-1 levels in the blood, variables representing systemic vascular endothelial cell activation and damage, and coagulative activity in the blood also were measured. RESULTS: Endothelial microparticle levels in the blood, microparticle binding to PMNLs, and oxidative activity in PMNLs increased significantly in patients with severe SIRS, as compared with the values in healthy volunteers. Soluble E-selectin, thrombomodulin, plasminogen activator inhibitor-1, and procoagulant activity in the blood also increased in these patients. CONCLUSIONS: Activated vascular endothelial cells with increased procoagulant activity enhance production of microparticles with increased binding to leukocytes in patients with severe SIRS. Endothelial microparticles may be involved in the pathogenesis of endothelial injury after severe insult.

Mild hypothermia reduces expression of heat shock protein 60 in leukocytes from severely head-injured patients.

BACKGROUND: Infectious complications are among the most serious problems that occur in severely head-injured patients treated with mild hypothermia. The mechanism underlying the susceptibility to infection has not been clarified. Heat shock protein (HSP) 60 has been reported to play an essential role in innate immunity. Thus, we conducted a study to clarify the impact of mild hypothermia on the expression of HSPs in polymorphonuclear leukocytes (PMNLs) in severely head-injured patients. METHODS: Between September 1997 and November 1999, 17 severely head-injured patients with a Glasgow Coma Scale score of 8 or less at admission in whom intracranial pressure could be maintained below 20 mm Hg by conventional therapy were randomly assigned to two treatment groups: a mild hypothermia group (HT group, nine patients) and a normothermia group (NT group, eight patients). The HT group was subjected to mild hypothermia (intracranial temperature, 34 degrees C) for 48 hours followed by rewarming at a rate of 1 degrees C per day for 3 days, whereas the NT group was subjected to normothermia (intracranial temperature, 37 degrees C) for 5 days. Blood samples were serially obtained at three time points; days 0 to 1, days 2 to 5, and days 6 to 14 after head injury. We measured the expression of HSP27, HSP60, HSP70, and HSP90 by flow cytometry. RESULTS: The two groups were similar with respect to prognostic factors, and there was no difference in clinical outcome. The expression of PMNL HSP60 in the HT group was significantly lower in all three time periods compared with that in the NT group (p < 0.05), whereas expression of the other HSPs did not differ significantly between the groups. The incidence of infectious complications was significantly increased in the HT group over that in the NT group (p < 0.05). In in vitro studies, PMNLs from 10 healthy volunteers were incubated at 37 degrees C, 34 degrees C, or 26 degrees C for 1 hour with sodium arsenite (100 micromol/L), an HSP inducer. The expression of HSP60 at 26 degrees C and 34 degrees C was significantly lower than that at 37 degrees C (p < 0.05), whereas expression of the other HSPs did not differ significantly at 26 degrees C, 34 degrees C, or 37 degrees C. CONCLUSION: Mild hypothermia reduces the expression of HSP60 in PMNLs from severely head-injured patients. Thus, mild hypothermia may suppress innate immunity.

Paradoxical cytoskeleton and microparticle formation changes in monocytes and polymorphonuclear leukocytes in severe systemic inflammatory response syndrome patients.

BACKGROUND: Circulating monocytes and polymorphonuclear leukocytes (PMNLs) are considered as central regulators controlling systemic inflammatory response after severe insults. Recently, activated monocytes and PMNLs have been reported to produce microparticles (MPs) in vitro. The objective of this study was to evaluate production of MPs and changes of cytoskeleton in monocytes from severe systemic inflammatory response syndrome (SIRS) patients, and to compare them with those in PMNLs. METHODS: Twenty severe SIRS patients (SIRS criteria and serum C-reactive protein > 10 mg/dL) and 15 healthy volunteers were included. MP formation and F-actin content in monocytes and PMNLs were measured by flow cytometry in the presence or absence of lipopolysaccharide or formylmethionyl-leucyl-phenylalanine (FMLP). The membrane expression of human leukocyte antigen-DR and CD64 in monocytes and O2- production in PMNLs were also measured by flow cytometry. RESULTS: In severe SIRS patients, MP formation with and without lipopolysaccharide in monocytes significantly decreased in comparison with those in normal controls (p < 0.05), whereas those with and without FMLP in PMNLs increased (p < 0.05). F-actin content with and without FMLP in monocytes also significantly decreased in patients (p < 0.05), whereas those in PMNLs increased as compared with normal controls (p < 0.05). The expression of human leukocyte antigen-DR in monocytes significantly decreased in patients (p < 0.05), which indicated monocyte modulation. The O2- production in PMNLs increased in patients (p < 0.05), which showed PMNL activation. CONCLUSION: The changes of MP formation and cytoskeleton in circulating monocytes and PMNLs were paradoxically different in severe SIRS patients.