Douglas Y. Tamura

p38 mitogen-activated protein kinase inhibition attenuates intercellular adhesion molecule-1 up-regulation on human pulmonary microvascular endothelial cells.

BACKGROUND Increased expression of pulmonary endothelial intercellular adhesion molecule-1 (ICAM-1) is obligatory to neutrophil adherence culminating in adult respiratory distress syndrome (ARDS). The p38 mitogen-activated protein kinases (MAPKs) have been established as crucial in leukocyte proinflammatory signaling, but their role in the endothelial cells remains ill defined. We hypothesized that p38 MAPK activity is integral to ICAM-1 up-regulation on pulmonary endothelium. METHODS Human pulmonary microvascular endothelial cells (HMVECs) were grown to confluence and pretreated with either the tyrosine phosphorylation inhibitor herbimycin A (1 mumol/L or the p38 MAPK inhibitor SB 203580 (10(-7) to 10(-5) mol /L) for 6 hours. ICAM-1 expression was quantified by flow cytometry. Data expressed as mean fluorescence intensity. Western blotting was used to show p38 MAPK activity after stimulation with lipopolysaccharide (LOS) or tumor necrosis factor-alpha (TNF-alpha). RESULTS Tyrosine phosphorylation inhibition with herbimycin A attenuated both LPS and TNF-alpha stimulated ICAM-1 up-regulation. Similarly, specific inhibition of p38 MAPK attenuated both LPS (10(-6) to 10(-5) mol/L SB203580) and TNF-alpha (10(-7) to 10(-5) mol/L SB203580) stimulated expression of ICAM-1 on HMVECs. Both LPS and TNF-alpha induced activation of p38 in HMVECs. CONCLUSIONS Signaling through p 38 MAPKs contributes to LP and TNF-alpha stimulated ICAM-1 surface expression on HMVECs. Thus p38 MAPKs appear integral to both neutrophil and endothelial cell proinflammatory signaling and may be a potential therapeutic target in the treatment of ARDS.

Resuscitation with a blood substitute abrogates pathologic postinjury neutrophil cytotoxic function

BACKGROUND Resuscitation with oxygen-carrying fluids is critically important in the patient with hemorrhagic shock caused by trauma. However, it is clear that a number of biologic mediators present in stored blood (packed red blood cells [PRBCs]) have the potential to exacerbate early postinjury hyperinflammation and multiple organ failure through priming of circulating neutrophils (PMNs). PolyHeme (Northfield Laboratories, Evanston, IL), a hemoglobin-based substitute that is free of priming agents, provides an alternative. We hypothesized that PMN priming would be attenuated in patients resuscitated with PolyHeme in lieu of stored blood. METHODS Injured patients requiring urgent transfusion were given either PolyHeme (up to 20 units) or PRBCs. Early postinjury PMN priming was measured via beta-2 integrin expression, superoxide production, and elastase release. RESULTS Treatment groups were comparable with respect to extent of injury and early physiologic compromise. PMNs from patients resuscitated with PRBCs showed priming in the early postinjury period by all three measures. No such priming was evident in patients resuscitated with PolyHeme. CONCLUSION The use of a blood substitute in the early postinjury period avoids PMN priming and may thereby provide an avenue to decrease the incidence or severity of postinjury multiple organ failure.

Circulating Postinjury neutrophils are primed for the release of proinflammatory cytokines

BACKGROUND Postinjury neutrophil (PMN) priming identifies the injured patient at risk for the subsequent development of multiple organ failure (MOF). PMN priming has previously been shown to cause enhanced release of proteases and superoxide. PMNs, however, are a rich source of proinflammatory cytokines, such as interleukin (IL)-8 and tumor necrosis factor (TNF), which have been implicated in the development of MOF. PMNs also make IL-1ra, which is an anti-inflammatory cytokine that inhibits IL-1. It is our hypothesis that postinjury PMNs are primed for increased stimulated release of the proinflammatory cytokines IL-8 and TNF but not the anti-inflammatory cytokine IL-1ra. METHODS Twelve trauma patients with a mean Injury Severity Score of 24 (+/-4.6) and 10 elective surgical patients were studied. Postinjury PMNs were isolated from blood obtained at presentation (within 2 hours after injury) and 24 hours after trauma. PMNs from elective surgical patients were obtained preoperatively, immediately postoperatively, and at 24 hours. The PMNs were stimulated with platelet-activating factor (200 nM)/N-formyl-methionyl-leucyl-phenylalanine (1 micromol/L) or lipopolysaccharide (100 ng/mL) incubated for 24 hours in RPMI-1640, and release of IL-8, TNF, and IL-1ra were measured. RESULTS Postinjury PMNs were primed for both platelet-activating factor/N-formyl-methionyl-leucyl-phenylalanine-stimulated and lipopolysaccharide-stimulated IL-8 and TNF release at 2 hours after injury (fourfold increase of IL-8 release and fivefold increase of TNF release), whereas elective surgical patients demonstrated no priming. In contrast, postinjury patients were not primed for increased release of the counterinflammatory cytokine IL-1ra, suggesting a specific postinjury up-regulation of IL-8 and TNF. CONCLUSION After injury, PMNs are primed for proinflammatory cytokine release in addition to superoxide and elastase. This augmented release of IL-8 and TNF may be involved in the subsequent development of organ dysfunction and ultimately MOF.

Acute hypoxemia in humans enhances the neutrophil inflammatory response

The neutrophil (PMN) is regarded as a key component in the hyperinflammatory response known as the systemic inflammatory response syndrome. Acute respiratory distress syndrome (ARDS) and subsequent multiple organ failure (MOF) are related to the severity of this hyperinflammation. ICU patients who are at highest risk of developing MOF may have acute hypoxic events that complicate their hospital course. This study was undertaken to evaluate the effects of acute hypoxia and subsequent hypoxemia on circulating PMNs in human volunteers. Healthy subjects were exposed to a changing 02/N2 mixture until their 02 saturation (Sa02) reached a level of 68% saturation. These subjects were then exposed to room air and then returned to their baseline Sa02. PMNs were isolated from pre- and post-hypoxemic arterial blood samples and were then either stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) or PMA alone, or they were primed with L-&agr;-phosphatidylcholine, &bgr;-acetyl-&ggr;-O-alkyl (PAF) followed by fMLP activation. Reactive oxygen species generation as measured by superoxide anion production was enhanced in primed PMNs after hypoxemia. Protease degranulation as measured by elastase release was enhanced in both quiescent PMNs and primed PMNs after fMLP activation following the hypoxemic event. Adhesion molecule upregulation as measured by CD11b/CD18, however, was not significantly changed after hypoxemia. Apoptosis of quiescent PMNs was delayed after the hypoxemic event. TNF&agr;, IL-1, IL-6, and IL-8 cytokine levels were unchanged following hypoxemia. These results indicate that relevant acute hypoxemic events observed in the clinical setting enhance several PMN cytotoxic functions and suggest that a transient hypoxemic insult may promote hyperinflammation.

Hypertonic saline resuscitation abrogates neutrophil priming by mesenteric lymph

OBJECTIVE Neutrophil (PMN) priming after hemorrhagic shock is predictive of the subsequent development of multiple organ failure, but the mechanism remains unknown. Recently, we and others have demonstrated that mesenteric lymph from shock animals resuscitated with lactated Ringers solution (LR) is not only a potent PMN priming agent but also causes lung injury. Work by others has shown that resuscitation with hypertonic saline (HTS) protects animals from lung injury after hemorrhagic shock. Therefore, we hypothesize that resuscitation with HTS will abolish PMN priming by postshock mesenteric lymph. METHODS After mesenteric lymph duct catheterization, male rats underwent hemorrhagic shock (mean arterial pressure of 40 mm Hg for 90 minutes) and resuscitation with shed blood plus either LR (2x volume of shed blood) or 4 mL/kg of 7% HTS (isonatremic). Priming for superoxide by PMN was measured after fMLP (1 microM) activation. RESULTS Shock significantly decreased mesenteric lymph flow from preshock levels in both groups. LR resuscitation produced significantly more mesenteric lymph than HTS resuscitation. Mesenteric lymph from LR animals primed PMN for superoxide production, whereas, HTS eliminated this priming. CONCLUSION HTS not only decreases postshock mesenteric lymph production, it eliminates PMN priming by mesenteric lymph, suggesting a mechanism for the beneficial effects of HTS resuscitation.

Resuscitation of the injured patient with polymerized stroma-free hemoglobin does not produce systemic or pulmonary hypertension

BACKGROUND Hemoglobin-based blood substitutes appear poised to deliver the promise of a universally compatible, disease-free alternative to banked blood. However, vasoconstriction following administration of tetrameric hemoglobins has been problematic, likely because of nitric oxide binding. Polymerized hemoglobin is effectively excluded from the abluminal space because of its size, and is thus less likely to perturb vasorelaxation. We therefore hypothesized that hemodynamic responses would be no different in injured patients receiving polymerized hemoglobin versus banked blood. METHODS Injured patients requiring urgent transfusion were randomized to receive either polymerized hemoglobin or banked blood. Systemic arterial pressure, pulmonary arterial pressure, cardiac index, pulmonary capillary wedge pressure, systemic vascular resistance, and pulmonary vascular resistance were measured serially. RESULTS There was no difference in any of the measured hemodynamic parameters between patients resuscitated with polymerized hemoglobin versus blood. CONCLUSIONS Polymerized hemoglobin given in large doses to injured patients lacks the vasoconstrictive effects reported in the use of other hemoglobin-based blood substitutes. This supports the continued investigation of polymerized hemoglobin in injured patients requiring urgent transfusion.

Clinically Relevant Concentrations of Ethanol Attenuate Primed Neutrophil Bactericidal Activity

BACKGROUND Acute alcohol intoxication is associated with an increased risk of infection in the injured patient. The impact of clinically relevant levels of ethanol (ETOH) on neutrophil (PMN) bactericidal activity remains ill-defined. PMN priming optimizes microbicidal activity by enhancing oxygen radical production, degranulation, and adhesion molecule up-regulation. We hypothesized that clinically relevant levels of ETOH attenuate these primed PMN responses critical to eradicate infection. METHODS After incubation with ETOH (0-1.0%), isolated human PMNs were primed with beta-acetyl-gamma-O-alkyl and activated with N-formyl-methionyl-leucyl-phenylalanine. Superoxide generation was measured by cytochrome c reduction, elastase release was measured by cleavage of methoxysuccinyl-ala-ala-pro-val-p-nitroanilide, and CD11b was measured by fluorescent monoclonal antibody staining. Bactericidal activity was assessed by Staphylococcus aureus killing. RESULTS ETOH attenuated superoxide production dose-dependently with significance at 0.3% ETOH. Elastase release was attenuated starting at 0.2% ETOH, and CD11b expression was reduced starting at 0.4% ETOH. S. aureus killing was impaired dose-dependently with significance at 0.3% ETOH. CONCLUSION Clinically relevant concentrations of ETOH attenuate PMN functions critical in host defense against invading pathogens. These results provide direct in vitro evidence consistent with previous in vivo data that acute alcohol intoxication is important in the pathogenesis of trauma-related infections.

Disparities in the respiratory burst between human and rat neutrophils.

The importance of reactive oxygen species (ROS) in neutrophil (PMN)‐mediated injury to host tissues has been strongly implicated in a number of animal models. Peculiarities of the laboratory rat PMN, including an apparent paucity of superoxide release, prompted us to examine disparities in the respiratory burst between human and rat PMNs. Using isolated PMNs, we examined oxygen consumption, superoxide release, nitrate/nitrite release, and dihydrorhodamine (DHR) oxidation in response to an array of soluble stimuli. Our findings confirm that intact rat PMNs release little superoxide in comparison to human PMNs when primed and activated by soluble stimuli. For example, PMA‐activated human PMNs released superoxide at 10.1 ± 2.7 times the rate of rat PMNs (P < 0.01). However, measurements of oxygen consumption, cell‐associated oxidant production (by DHR oxidation) and release of superoxide from electroporated cells suggests that rat PMNs generate oxidants at rates equivalent to human PMNs but preferentially release them in an intracellular compartment. Implications for the study of PMN‐ mediated oxidant injury in animal models are discussed. J. Leukoc. Biol. 65: 211–216; 1999.

Prostaglandin E1 attenuates cytotoxic mechanisms of primed neutrophils

In a recent clinical trial, liposomal prostaglandin E1 (PGE1) improved oxygenation, increased compliance, and decreased ventilator dependency in patients with adult respiratory distress syndrome (ARDS), thus renewing interest in PGE1 as a potential modulator of inflammation. The neutrophil (PMN) is believed to play a key role in the development of ARDS. Consequently, we investigated the effects of PGE1 on three components of the neutrophil inflammatory response: reactive oxygen species (ROS) generation, protease release, and surface expression of adhesion molecules. Human neutrophils were incubated with PGE1 and then primed with platelet-activating factor (PAF) and activation with N-formyl-methionyl-leucylphenylalanine (fMLP). PGE1 at a dose range of (10-8 to 10-5 M) attenuated primed/activated (PAF/fMLP) PMN superoxide anion generation and elastase release. In contrast, PGE1 doses ≤ 10-5 M were required to attenuate PAF-stimulated neutrophil upregulation of CD11b/CD18 adhesion molecules. PGE1 also diminished the duration of the PAF-induced cytosolic calcium (Ca2+) flux. Our results suggest that plasma levels of PGE1 attained in patients with ARDS may attenuate ROS and protease neutrophil cytotoxicity but may not effectively block PMN-endothelial cell (EC) adhesion. This attenuation may occur through abrogation of the Ca2+ influx.

Postinjury suppression of human neutrophil cytokine production results from the stabilization of inhibitory kappaB.

UNLABELLED Postinjury neutrophil (PMN) dysfunction is a well recognized event that may be responsible for increased infections. PMN cytokine production is an important component of their bactericidal capacity. When PMNs are stimulated, inhibitory factor kappaB (IkappaB) is degraded, allowing nuclear factor kappaB (NFkappaB) to translocate to the nucleus and promotes genes for the transcription of the interleukin-8 (IL-8) and tumor necrosis factor (TNF) genes. We hypothesize that similar to their late postinjury depressed superoxide production, postinjury PMNs manifest suppressed cytokine production, which is mediated by stabilization of IkappaB levels. METHODS Twelve severely injured patients with an injury severity score (ISS) of 24 (+/-4.6) were studied as well as 10 elective surgical patients as a control. PMNs were isolated and incubated for 24 h in RPMI. PMNs were stimulated with lipopolysaccharide (LPS; 100 ng) or PAF (200 nm) and fMLP (1 microM) and release of IL-8, TNF, and interleukin-1 receptor antagonist (IL-1ra) were measured. Postinjury PMNs were also stimulated with LPS (100 ng), and IkappaB breakdown was measured at 0, 30, and 60 min using gel electrophoresis. RESULTS Postinjury PMNs displayed a significant suppression of both IL-8 and TNF on postinjury Days 1-3, while the release of IL-1ra was preserved throughout the entire study period. In contrast, elective surgical patients demonstrated no decrease in IL-8 or TNF. Furthermore, IkappaB levels were preserved in the postinjury PMNs as compared with normal control PMNs. CONCLUSION Postinjury PMNs have a suppressed release of both IL-8 and TNF following injury that did not occur in elective surgical patients. Furthermore, the NFkappaB/IkappaB-independent IL-1ra did not show suppression of release. In addition, stabilization of IkappaB following severe injury leads to decreased PMN IL-8 and TNF production. This genetic reprogramming may help explain PMN dysfunction and subsequent infections seen in severely injured patients.