Naoyuki Hashiguchi

ATP release guides neutrophil chemotaxis via P2Y2 and A3 receptors.

Cells must amplify external signals to orient and migrate in chemotactic gradient fields. We find that human neutrophils release adenosine triphosphate (ATP) from the leading edge of the cell surface to amplify chemotactic signals and direct cell orientation by feedback through P2Y2 nucleotide receptors. Neutrophils rapidly hydrolyze released ATP to adenosine that then acts via A3-type adenosine receptors, which are recruited to the leading edge, to promote cell migration. Thus, ATP release and autocrine feedback through P2Y2 and A3 receptors provide signal amplification, controlling gradient sensing and migration of neutrophils.

Priming, second-hit priming, and apoptosis in leukocytes from trauma patients

BACKGROUND Polymorphonuclear leukocytes (PMNL) play important roles in both host defenses and systemic inflammatory responses after insults. The objectives of this study are to examine the serial changes in PMNL priming and apoptosis in severely injured patients and to evaluate the impact of second hits on primed PMNL function and systemic vascular endothelial damage. METHODS Twenty-four severely injured patients (mean Injury Severity Score, 31.1 +/- 9.7) were included. Infections were seen as second hits after trauma in seven patients. Oxidative activity, phagocytosis, and apoptosis of PMNL from serial blood samples were measured by flow cytometry. Oxidative activity with no stimulus and with formylmethionyl-leucyl-phenylalanine (FMLP) were analyzed as the priming index and FMLP response, respectively. Interleukin (IL)-6, IL-10, PMNL elastase, and thrombomodulin concentrations in blood were also measured before and after the second hit. RESULTS The PMNL priming index was elevated from days 2 to 13, especially days 2 to 5 after injury. FMLP response was enhanced from days 2 to 21 after injury. Apoptosis of PMNL was inhibited for as long as 3 weeks after injury. Infections as second hits after trauma enhanced both the priming index and the FMLP response within 24 hours after diagnosis of infection and increased serum IL-6 concentrations. However, serum thrombomodulin levels were not affected by second hits. All patients with second hits survived. CONCLUSION Severe trauma stimulated acute-phase priming in PMNL and inhibited apoptosis. Infections after trauma induced second-hit priming in PMNL, but the unchanged serum levels of thrombomodulin suggest that priming per se may not cause systemic vascular endothelial damage.

Osmotic regulation of cell function and possible clinical applications.

Inflammation and immunosuppression can cause acute respiratory distress syndrome, multiple organ failure, and sepsis, all of which are lethal posttraumatic complications in trauma patients. Prevention of the inflammation and immunosuppression has been a main focus of trauma researcher for many years. Recently, hypertonic resuscitation has attracted attention as a possible therapeutic approach to counteract such deleterious immune responses in trauma patients. We have begun to understand how hypertonic fluids affect immune cell signaling, and a number of experimental and clinical studies have started to reveal valuable information on the clinical efficacy and the limitations of hypertonic resuscitation fluids. Knowledge of how osmotic cues regulate immune cell function will enable us to fully exploit the clinical potential of hypertonic resuscitation to reduce inflammatory and anergic complications in trauma patients.

Enhanced expression of heat shock proteins in activated polymorphonuclear leukocytes in patients with sepsis.

BACKGROUND Heat shock proteins (HSPs) in cells, as molecular chaperons, have been reported to regulate cell functions. The objective of this study was to investigate the HSP expression in polymorphonuclear leukocytes (PMNLs) from severe septic patients and the relation between the expression of HSPs and PMNL function. METHODS In blood samples from 21 patients with sepsis and serum C-reactive protein levels more than 10 mg/dL, we used flow cytometry to measure expressions of HSP27, HSP60, HSP70, and HSP90; oxidative activity; and levels of apoptosis in PMNLs during sepsis. In in vitro studies, we used cells from 14 healthy volunteers to examine the relation between the expression of HSP70 and PMNL function. Quercetin (30 microM), a suppressor of HSP, and sodium arsenite (100 microM), an inducer of HSP, were used to regulate the expression of HSP70 in PMNLs, and oxidative activity and apoptosis in these cells were measured. RESULTS In patients with sepsis, the expressions of HSP27, HSP60, HSP70, and HSP90 and oxidative activity in PMNLs were significantly increased. Apoptosis of these PMNLs was markedly inhibited. In the in vitro studies, administration of sodium arsenite enhanced the expression of HSP70, significantly increased oxidative activity, and inhibited apoptosis. Administration of quercetin before sodium arsenite prevented the expression of HSP70, the increase in oxidative activity, and the inhibition of apoptosis. CONCLUSION Sepsis causes the enhanced expression of HSPs in activated PMNLs. In PMNLs with enhanced expression of HSP70, oxidative activity is increased and apoptosis is inhibited. The enhanced expression of HSPs may play a role in regulating PMNL function in patients with sepsis.

Surface expression of HSP72 by LPS-stimulated neutrophils facilitates γδT cell-mediated killing

During inflammation and sepsis, accumulation of activated neutrophils causes lung tissue damage and organ failure. Effective clearance of neutrophils reduces the risk of organ failure; however, its mechanisms are poorly understood. Because lungs are rich in γδT cells, we investigated the physiological role of these cells in the protection of lung tissue from infiltrating neutrophils. In a mouse model of sepsis, we found that the lungs of survivors contained significantly higher numbers of γδT cells than those of mice that died from sepsis. The number of γδT cells correlated inversely with the number of neutrophils in the lungs and with the degree of lung tissue damage. LPS rapidly elicited the expression of heat shock protein (HSP) 72 on the surface of human neutrophils. Inhibitors of transcription, protein synthesis, and intracellular protein transport blocked HSP72 expression, indicating that de novo synthesis is required. γδT cells targeted and rapidly killed LPS‐treated neutrophils through direct cell‐to‐cell contact. Pre‐treatment with neutralizing antibodies to HSP72 diminished neutrophil killing. Our data indicate that HSP72 expression on the cell surface predisposes inflamed neutrophils to killing by γδT cells. This intercellular exchange may allow γδT cells to resolve inflammation and limit host tissue damage during sepsis.

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.

Neutrophil cell death in response to infection and its relation to coagulation

Neutrophil is a major player in the pathophysiology of severe sepsis. Recent studies have revealed that the cell death mechanism of neutrophils directly relates to the development of organ dysfunction during sepsis. Here we discuss about the different types of neutrophil cell death such as necrosis, apoptosis, autophagy, and the unique cell death style dubbed NETosis. NETosis cells release neutrophil extracellular traps (NETs), which are composed of chromatin bound to granular and nucleic proteins. The primary purpose of NET release is thought to be the control of microbial infections; however, it acts as a danger signal for the host as well. The harmful substances such as DNA, histones, and high-mobility group box 1 (HMGB1) and many other danger-associated molecular patterns (DAMPs) released along with NETosis or from necrotic neutrophils also contribute to the pathogenesis of sepsis. At the same time, the coagulation system, which is closely tied to these neutrophil cell death mechanisms, is often over-activated. It is well known that individual bacterial pathogens express virulence factors that modulate cell death pathways and influence the coagulation disorder during sepsis. Moreover, extensive cross talk exists between these two phenomena, whereby inflammation leads to activation of coagulation and coagulation considerably affects inflammatory activity. A greater knowledge of cell death pathways in sepsis informs the potential for future therapies designed to ameliorate excessive immune responses during sepsis.

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 heat shock proteins in leukocytes from trauma patients.

BACKGROUND Heat shock proteins (HSPs) play essential roles as molecular chaperones in cells to assist in the repair of degenerated proteins. The expression of HSPs in polymorphonuclear leukocytes (PMNLs) following insult has not been delineated. The objective of this study was to clarify the serial changes in HSP expression in PMNLs from trauma patients. METHODS Fifty severely injured patients (mean Injury Severity Score of 31.8 +/- 10.8) and 17 healthy volunteers were included as study subjects. Blood samples were serially obtained at three time points: days 0 to 1, days 2 to 5, and days 6 to 14 after the trauma event. We measured expressions of HSP27, HSP60, HSP70, and HSP90 in permeabilized PMNLs by flow cytometry using a monoclonal antibody generated against each HSP and fluorescein isothiocyanate-conjugated antimouse immunoglobulins as secondary reagents. We also evaluated the expression of HSP70 mRNA in PMNLs by Northern blot hybridization and the expression of HSP70 in PMNLs by fluorescence microscopy. RESULTS Expressions of HSP27, HSP70, and HSP90 in PMNLs from trauma patients were significantly greater than in PMNLs from healthy volunteers in all three periods (days 0-1, days 2-5, and days 6-14). The expression of HSP60 in PMNLs from trauma patients was significantly greater than normal expression on days 2 to 5 and days 6 to 14. The values for HSP27, HSP60, and HSP70 on days 2 to 5 were significantly higher than those on days 0 to 1. The expression of HSP70 mRNA in PMNLs was significantly enhanced for as long as 2 weeks after trauma compared with that in normal volunteers. CONCLUSION Severe trauma causes demonstrated enhanced expression of HSPs in PMNLs during the acute phase. This enhanced expression of HSPs may regulate PMNL functions.

Combination of antithrombin and recombinant thrombomodulin modulates neutrophil cell-death and decreases circulating DAMPs levels in endotoxemic rats

INTRODUCTION The activation of coagulation is recognized as a universal event in severe sepsis. Both antithrombin and thrombomodulin play pivotal roles as suppressors of coagulation. Since the levels of both anticoagulants decrease significantly, we hypothesized that a combination therapy would be beneficial. METHODS A sepsis model was established using the intravenous infusion of lipopolysaccharide (LPS). Either 125 IU/kg of antithrombin, 0.25mg/kg of recombinant thrombomodulin, or a combination of both agents was injected immediately after LPS infusion (n=7 each), while only a physiological saline was injected in the control group (n=7). Blood samples were obtained at eight hours after LPS infusion, and organ damage markers and the plasma levels of damage-associated molecular patterns (DAMPs), such as histone H3 and cell-free DNA (cf-DNA), were measured. In another series, the leukocytes harvested from normal rats were cultured in plasma obtained from each group (n=7). Eight hours later, the leukocytes were stained with green fluorescent protein, Annexin V and 7-AAD, and the proportion of alive+apoptic/necrotic cells was calculated. RESULTS Organ damage markers such as ALT and BUN were maintained best in the combination group (P<0.05). The circulating levels of histone H3 and cf-DNA were both significantly lower in the combination therapy group (P<0.01, 0.05, respectively). The proportion of alive+apoptic/necrotic cells was significantly higher in the combination therapy group (P<0.05). CONCLUSION The coadministration of antithrombin and recombinant thrombomodulin can modulate cell death and decrease the circulating levels of histone H3 and cf-DNA, leading to protection against organ damage in a rat model of sepsis.