Pro-inflammatory alterations and status of blood plasma iron in a model of blast-induced lung trauma.

Impact of blast shock waves (SW) with the body wall produces blast lung injuries characterized by bilateral traumatic hemorrhages. Such injuries often have no external signs, are difficult to diagnose, and therefore, are frequently underestimated. Predictive assessment of acute respiratory distress syndrome outcome in SW-related accidents should be based on experimental data from appropriate animal models. Blood plasma transferrin is a major carrier of blood iron essential for proliferative "emergency" response of hematopoietic and immune systems as well as injured tissue in major trauma. Iron-transferrin complexes (Fe3+ TRF) can be quantitatively analyzed in blood and tissue samples with low-temperature EPR techniques. We hypothesized that use of EPR techniques in combination with assays for pro-inflammatory cytokines and granulocytes in the peripheral blood and BAL would reveal a pattern of systemic sequestration of (Fe3+)TRF that could be useful for development of biomarkers of the systemic inflammatory response to lung injury. With this goal we (i) analyzed time-dependent dynamics of (Fe3+)TRF in the peripheral blood of rats after impacts of SW generated in a laboratory shock-tube and (ii) assayed the fluctuation of granulocyte (PMN) counts and expression of CD11b adhesion molecules on the surface of PMNs during the first 24 h after SW induced injury. Sham-treated animals were used as control. Exposure to SW led to a significant decrease in the amount of blood (Fe3+)TRF that correlated with the extent of lung injury and developed gradually during the first 24 h. Thus, sequestration of (Fe3+)TRF occurred as early as 3 h post-exposure. At that time, the steady state concentration of (Fe3+)TRF in blood samples decreased from 19.7+/-0.6 microM in controls to 7.5+/-1.3 microM in exposed animals. The levels of (Fe3+)TRF remained decreased throughout the entire study period. PMN counts increased 5-fold and 3.5-fold over controls respectively, at 3 and 6 h postexposure. These effects were accompanied by an increase in expression of CD11b on the surface membrane of PMNs. Extensive release of cytokines IL-1, IL-6, MCP-1, and MIP-2 was observed in BAL fluid and blood plasma during 24 h postexposure. We conclude that EPR monitoring of blood (Fe3+)TRF can be a useful approach for assessment of systemic pro-inflammatory alterations due to SW-induced lung injury.

White particulate matter found in blood collection bags consist of platelets and leukocytes.

BACKGROUND: In late January 2003, some blood centers and hospitals throughout the US voluntarily sus-pended the use of some RBC and plasma units for trans-fusion due to the presence of unknown white particulate matter (WPM) in these units. To better understand the WPM phenomena, a number of technologies were used to establish the nature of the particulates observed in Terumo Collection sets. STUDY DESIGN AND METHODS: All AS-5 nonleuko-reduced RBCs and plasma units were visually inspected for WPM by placing the bags on a flat counter, undisturbed, for approximately 10 minutes and then perform-ing a visual examination for particles. Particles were isolated and placed on microscope slides or in plastic tubes for further analysis. Electron microscopy, bright field microscopy, differential interference contrast microscopy, infrared spectroscopy, and flow cytometry procedures were performed to establish the nature of the particulate matter. In addition, leukoreduction filters and blood transfusion sets were used on RBCs units with WPM. RESULTS: The particles were mostly composed of PLTs and WBCs, and fragments of these cells. All macroscopic WPM was removed from RBCs with leukoeduction and transfusion filters. CONCLUSIONS: WPM originated from PLTs and WBCs. Foreign matter (e.g., plastic) was not observed in any of the units. Leukoreduction and transfusion filters can be used to remove macroscopic WPM.

Hemoglobin stimulates the release of proinflammatory cytokines from leukocytes in whole blood.

Isolated mononuclear leukocytes, when incubated with purified hemoglobin Ao (HbAo), release the proinflammatory cytokines interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-alpha). In this study we examined whether leukocytes in whole blood, when incubated with HbAo, release IL-8, TNF-alpha, and IL-6. Leukocytes in whole blood incubated with HbAo for 4 hours at 37 degrees C, 5% CO2, and 95% humidity released 187, 1313, and 50 pg/mL of IL-6, IL-8, and TNF-alpha, respectively, as compared with 6, 192, and 2 pg/mL released by leukocytes in blood incubated with human serum albumin (HSA). Furthermore, plasma from blood incubated with HbAo exhibited chemotactic activity and stimulated human umbilical vein endothelial cells to become adherent to neutrophils. These activities were 3.3 and 2.6 times those measured in plasma from blood incubated with HSA. Hydrocortisone (0.05 micromol/L to 50 micromol/L) inhibited cytokine release in a dose-dependent manner with ED50 values of 0.23 micromol/L, 0.19 micromol/L, and 0.10 micromol/L for IL-6, IL-8, and TNF-alpha, respectively. The release of proinflammatory cytokines in whole blood after exposure to hemoglobin solutions is consistent with the possibility that an inflammatory reaction could develop on infusion of hemoglobin, whereas inhibition of cytokine release by hydrocortisone suggests that the inclusion of anti-inflammatory compounds in hemoglobin solutions may prevent undesirable effects caused by inflammation after infusion.

Hemoglobin stimulates mononuclear leukocytes to release interleukin-8 and tumor necrosis factor alpha.

Incubation of human mononuclear leukocytes (MNL) with human stroma-free hemolysate (SFH), purified adult hemoglobin Ao (HbAo), and oxidized HbAo (METHb) caused MNL to release compounds into the supernate that mediated neutrophil (polymorphonuclear leukocytes, PMN) chemotaxis and PMN adherence to human umbilical vein endothelial cells (HUVEC). Chemotaxis and PMN adherence to HUVEC were reduced significantly when supernates were preincubated with neutralizing antibodies to interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-alpha), respectively, suggesting that IL-8 and TNF-alpha played significant roles in mediating these activities. Greatest chemotactic activity was observed in supernates of MNL treated with HbAo; while greatest PMN/endothelial cell (EC) adherence activity was observed in supernates of MNL treated with METHb. Furthermore, PMN/EC adherence activity was a function of METHb content in each hemoglobin solution. PMN chemotaxis, PMN adherence to HUVEC, and cytokine release increased as a function of increasing incubation time. Chemotactic activity was detected in HbAo-treated and METHb-treated MNL supernates after incubation for 6 hours and was maximal by 10 hours. IL-8 was detected in both HbAo and METHb-MNL supernates by 4 hours. PMN/EC adherence activity was detected in HbAo-MNL supernates at 10 hours and in METHb-MNL supernates at 4 hours. TNF-alpha was detected in METHb and HbAo-MNL supernates at 4 and 12 hours, respectively. These results suggest that hemoglobin solutions stimulate MNL to release IL-8 and TNF-alpha in quantities sufficient to induce PMN chemotaxis and PMN adherence to HUVEC. This is a US government work. There are no restrictions on its use.

Quantitation of polymorphonuclear leukocyte adherence to endothelial cells by electronic particle size discrimination.

A method for quantitating the number of polymorphonuclear leukocytes (PMNs) adhering to endothelial cells in vitro is presented. Confluent human umbilical vein endothelial cells cultured in 24-well multiplates and treated with tumor necrosis factor alpha (TNF alpha) were incubated subsequently with PMNs which adhere to the endothelial cell as a function of TNF alpha concentration. Adherent PMNs and endothelial cells were proteolytically dissociated from the multiwell and, using an electronic particle counter, the number of endothelial cells and PMNs were determined simultaneously on the basis of size. The average number of PMNs adhering per endothelial cell was then calculated. The method is rapid and precise and offers an alternative to both tedious microscopic counting and the hazardous radiolabeling of PMNs.

A method for isolating neutrophils from moderate volumes of human blood.

The Flow Laboratories procedure for isolating polymorphonuclear leukocytes (PMNs) from human blood whereby whole blood is centrifuged through Mono-Poly resolving medium (MPRM) has been modified. Using this modification, as much as 30 ml of whole blood could be processed in a single centrifuge tube, and erythrocyte contamination of the final PMN suspension was reduced significantly. PMN suspensions were composed of 96% neutrophils, and neutrophil recovery was 42-55% depending upon the volume of blood processed. Cell viability was 98% as determined by trypan blue exclusion. Greater than 99% of the neutrophils were spherical suggesting that the neutrophils were not primed during isolation.

The mechanism of peroxidase-mediated cytotoxicity. II. Role of the heme moiety.

Various peroxidases in the presence of hydrogen peroxide and a halide ion have been shown to exert a cytolytic activity against erythrocytes and other cells. However, few studies have been done to elucidate the active site on the enzymes that is responsible for the cytotoxic activity. In addressing this question we found that boiling of horseradish peroxidase only partially abolishes its cytotoxic activity, suggesting that an intact tertiary structure of the protein may not be essential for the cytotoxic activity. This conclusion was confirmed by demonstrating that microperoxidase, hemin, and hematoheme also exert cytotoxic activity in the presence of hydrogen peroxide and iodide, the kinetics of which were identical to those obtained with the peroxidases. Fluoride, bromide, and thiocyanate could not replace iodide in any of these systems. These results indicate that the active site for the cytotoxic activity of the peroxidases is located within the heme moiety, whereas the protein portions of the enzymes affect the cytotoxic activity of the enzymes only in an indirect manner. We also tested a variety of compounds for their ability to inhibit the cytolytic reaction toward erythrocytes. We found that compounds such as thiourea, thionicotinamide, and uric acid are much more potent inhibitors of the cytolytic reaction than tyrosine and histidine. These observations support the concept that oxidative reactions rather than halogenation reactions are the primary cause of the peroxidase-mediated lysis of erythrocytes.

Studies on the mechanism of carbon monoxide-induced vasodilation in the isolated perfused rat heart.

We investigated the effects of dissolved CO on isolated potassium-arrested (K+) perfused rat hearts. Hearts from male Sprague-Dawley rats were perfused via the aorta with oxygenated Krebs-Henseleit solution containing 20 mM K+. Coronary flow (Qt) averaged 48.8 +/- 1.6 (SE), 48.1 +/- 1.7, and 55.6 +/- 1.7 ml/min/g dry wt when the perfusate was equilibrated with 95% O2-5% CO2, 5% N2-90% O2-5% CO2, and 5% CO-90% O2-5% CO2, respectively. The change in Qt was statistically significant when CO was present in the perfusion medium, but was not significant when N2 was present. Furthermore, the effect was reversible because coronary flow returned to control levels when CO was removed. Myocardial oxygen consumption (MVO2) did not change significantly when hearts were perfused with either N2 or CO. The magnitude of CO-induced vasodilation was not affected significantly by the addition of either 5 microM propranolol, 2 microM phentolamine, 1 unit of adenosine deaminase, or 0.1 mM indomethacin to the perfusate. In addition, CO reversed the vasoconstrictive effects of the alpha-agonist methoxamine. These results indicate that CO exerts a vasodilatory effect on coronary vasculature that is not the result of decreased O2 content in the perfusate and is not mediated by adrenergic influences, adenosine, or prostaglandins.

The mechanism of peroxidase-mediated cytotoxicity. I. Comparison of horseradish peroxidase and lactoperoxidase.

The kinetics of the cytolytic activity expressed by lactoperoxidase and horseradish peroxidase toward erythrocytes in the presence of H2O2 and iodide have been investigated at physiological pH. The action of both enzymes was found to be very similar with respect to their kinetic mechanisms. Both enzymes showed saturation kinetics at higher enzyme concentrations under conditions where substrate concentrations were not limiting. Optimal concentrations of H2O2 and iodide were found to be 40 and 25 microM, respectively, for both enzymes. Higher concentrations of H2O2 inhibited the cytolytic activity. The pH dependence of the cytolytic reaction is also very similar for both enzymes, showing maximal activity at about pH 6.3. Moreover, the cytolytic activities of both enzymes were inhibited by tyrosine, tryptophan, cysteine, and to a lesser extent by histidine. We conclude from these data that the mechanisms of horseradish peroxidase and lactoperoxidase in promoting the lysis of erythrocytes are closely related if not identical.

Observations on the cytolytic activity of lactoperoxidase using a continuous assay.

A turbidometric assay that allows continuous monitoring of the cytolytic activity of toxic agents toward various target cells has been developed. This assay monitors the change in absorbance at 600 nm (due to light scattering) of a suspension of human red blood cells as a function of time. The rate of cell lysis, delta A600/delta t, can be expressed as the number of cells lysed per minute, which facilitates the determination of kinetic constants. Using this procedure we observed that the cytolytic activity exerted by various peroxidases in the presence of hydrogen peroxide and a halide ion proceeds in at least two stages. During the first stage no lysis occurs, but scanning electron microscopy showed that alterations in the target cell membrane take place. During the second stage the target cells lyse, resulting in a simultaneous release of metabolites and macromolecules. We conclude that the lytic action of peroxidases is directed toward the target cell membrane, which appears to acquire an increased rigidity and subsequently disintegrates.