Magnus Braide

Effects of E-selectin and P-selectin blockade on neutrophil sequestration in tissues and neutrophil oxidative burst in burned rats

OBJECTIVE Neutrophil deposition in tissues (leukosequestration) after shock may produce local tissue injury from proteases and high-energy oxygen species released from sequestered neutrophils. The initial step in the binding of neutrophils to capillary endothelium is the interaction of adhesion molecule (selectin) receptors between neutrophils and endothelial cells. We quantified leukosequestration in the tissues of burned rats using two methods of analysis: a) measurement of lung myeloperoxidase; and b) measurement of radiolabeled neutrophils and erythrocytes deposited in multiple tissues. We then determined the ability of a selectin receptor blocking agent to affect neutrophil deposition in tissues after burn injury. DESIGN Prospective, controlled, laboratory study. SETTING University research laboratory. SUBJECTS Male Wistar rats (200 to 300 g). INTERVENTIONS After tracheostomy and venous cannulation, rats received 17% total body surface area full-thickness contact burns and were resuscitated with saline (20 mL i.p.). Experimental animals received 2 mg/kg body weight i.v. administration of a P- and E-selectin blocking monoclonal antibody, CY-1747, immediately after burn. Lung tissue neutrophils were estimated by measuring myeloperoxidase in lung tissue. Neutrophil retention in lung, liver, spleen, gut, skin, muscle, kidney, and brain tissues was determined by removing (preburn) and differentially radiolabeling neutrophils (111In) and erythrocytes (51Cr), reinfusing cells 4.5 hrs after burn, and measuring tissue radioactivity 30 mins later. Edema was estimated by measuring extravasated 125 I-labeled albumin in the various tissues. Peripheral blood neutrophils were analyzed for intracellular hydrogen peroxide content, utilizing a fluorescent dye that reacts with hydrogen peroxide, coupled with analysis of cell fluorescence by flow cytometry. MEASUREMENTS AND MAIN RESULTS Myeloperoxidase concentration was increased in lungs 5 hrs after burn (p < .05), indicating neutrophil deposition. Radioisotope studies demonstrated significant (p < .05) leukosequestration into the lung, gut, kidney, skin, and brain tissues at 5 hrs after burn. Flow cytometry showed increased intracellular hydrogen peroxide content in peripheral blood neutrophils 5 hrs after burn. Tissue edema, manifested by radiolabeled albumin retention, was not seen in any tissues. Postburn neutrophil deposition in lungs and liver was blocked (p < .05) by administration of CY-1747 after burn, but maximal neutrophil hydrogen peroxide content was unaffected. CONCLUSION Burn injury in rats results in accumulation of neutrophils in multiple tissues. Neutrophil deposition in the lungs and liver is blocked by administration of the E/P-selectin blocking antibody, CY-1747. Since sequestration of metabolically active neutrophils may induce tissue injury, therapies that block postburn leukosequestration may improve clinical outcomes by limiting remote tissue injury.

Optimized density gradient separation of leukocyte fractions from whole blood by adjustment of osmolarity

Some of the compounds used for density gradient separation of blood cells have high osmolarities at the concentrations needed to create the required specific densities. Several mixed media use a combination of hyperosmolar shrinkage and red cell aggregation to improve cell separation. Due to the characteristics of Percoll density gradient medium the density and osmolarity of the gradient can be controlled separately. In the present study, Percoll gradients were used to determine the buoyant densities of different human blood cells at the osmolarities 300 mosM, 350 mosM and 400 mosM. Cell volumes were measured at the same osmolarities using a Coulter counter with channelyzer. As expected, the cell buoyant densities increased and the cell volumes decreased at the higher osmolarities used. There were, however, quantitative differences between the cells with respect to the effects of an increased osmolarity, making a 350 mosM density gradient the most effective in separating mononuclear leukocytes from polymorphonuclear leukocytes. A 400 mosM gradient offered the best possibilities to separate red blood cells from polymorphonuclear leukocytes. A one-step centrifugation procedure, based on these principles, is presented. This procedure makes possible the simultaneous purification of mononuclear leukocytes and polymorphonuclear leukocytes, suitable for functional assays.

Leukocyte margination during hemorrhagic shock correlates to preshock margination and is reduced by fucoidin.

Systemic and pulmonary circulation kinetics for 51Cr-erythrocytes and 111In-leukocytes were measured in rats during experimental hemorrhagic shock and normotension with or without pretreatment with the antirolling agent fucoidin. Leukocyte margination was expressed as transit factors (white blood cell transit time/red blood cell transit time) for polymorphonuclear and mononuclear cells. There was an increased pooling of leukocytes in the pulmonary and systemic vascular beds during shock with a maximum after 60 min when the transit factors had increased 2.90–3.72 times in the pulmonary vascular bed and 2.00–3.52 times in the systemic vascular bed for mononuclear and polymorphonuclear cells, respectively. High preshock pooling levels lead to a more pronounced increase in pooling during shock. Pretreatment with fucoidin significantly reduced the pooling increase in the systemic vascular bed. Granulocyte oxidative activity (nitro blue tetrazolium test) invariably increased during shock and was not affected by fucoidin.

Very High Daily Intraperitoneal Doses of Carbonyl Compounds Affect the Morphology, but Not the Exchange Characteristics, of Rat Peritoneum

Glucose degradation products (GDP) are carbonyl compounds, that are formed by heat sterilization of conventional peritoneal dialysis (PD) fluids. Carbonyl compounds are known to be toxic in vitro and potentially toxic also in vivo. The aim of this study was to evaluate the effects of daily, short-term exposure of the peritoneum to very high concentrations of GDP in vivo on peritoneal transport parameters and on peritoneal morphology in a well-established rat model of PD. Rats were exposed to three daily intraperitoneal (IP) injections (10 ml) for 9 days of a largely neutral (pH 7.2) PD fluid containing 1.5% glucose and sterilized by filtration, with (n = 8) or without (n = 8) the presence of different carbonyl compounds in concentrations 100 times higher than those reported in commercial PD fluids. Seven rats, not subjected to any exposure, served as controls. After the exposure, the rats were subjected to acute PD in 4-hour dwells. Twenty milliliters of 4% glucose dialysis fluid were instilled into the rat peritoneal cavity. Blood and dialysate samples were taken during the dwell for measurements of dialysate sodium, and for assessments of the mass transfer area coefficient (PS) for glucose and 51Cr-EDTA and of transperitoneal clearance (Cl) or radiolabelled albumin (RISA). At the end of the dwell, parts of the liver, diaphragm and peritoneum were removed for measurements of tissue cell density and thickness of the submesothelial peritoneal tissue. The exposure of the peritoneum to very high doses of carbonyl compounds did not affect the peritoneal transport of fluid and small solutes significantly, but seemed to slightly reduce lymph flow and albumin clearance out of the peritoneal cavity. Assessed after a hypertonic dwell, and compared to the situation in nontreated rats after the same kind of dwell, there was a significant thinning of the submesothelial tissue, but no difference in tissue cell density. It is concluded that short-term exposure of the peritoneum in vivo to very high doses of GDP resulted in almost no signs of acute toxicity.

Differences in lodgement of tumour cells in muscle and liver

Differences in the lodgement of circulating tumour cells in various organs are considered an important factor in metastatic organ selection. The present vital microscopic studies show that the pattern of intravascular arrest of tumour cells in muscle after intra-arterial injection is similar to that observed earlier, in the liver, after intraportal injection. However, parallel isotope studies on the lodgement process (at 5 min and 3 h after injection) showed that the tumour cells trapped in the muscle microvasculature were destroyed at a higher rate than in the liver. Tumour cells kept in test tubes, and thus not being subjected to the shearing forces of the circulation, had a higher survival rate than cells trapped in the muscle. The results indicate that stronger retardation forces acting on the tumour cells in muscle (arterial dissemination) than in the liver (venous dissemination) may be one mechanism behind the increased tumour cell destruction in muscle.

Effects of recombinant bactericidal/permeability-increasing protein (rBPI23) on neutrophil activity in burned rats

Bactericidal/permeability-increasing protein (BPI) is a neutrophil granule protein with potent bactericidal and lipopolysaccharide (LPS)-neutralizing activities. The purpose of this study was to determine if a human recombinant BPI product, rBPI23, would influence neutrophil (PMN) sequestration into various tissues in a rat burn injury model. Leukosequestration may produce local tissue injury from proteases and high-energy oxygen species released from PMNs. Rats received tracheostomy and venous cannulation, then received 17 to 20% total body surface area full-thickness contact burns and resuscitation with 20 ml, of intraperitoneal saline. Ten mg/kg body weight rBPI23 in saline was given by intravenous injection immediately after burn injury, followed by intravenous doses of 2 mg/kg at 2 and 4 hours. Control animals received intravenous saline only. PMN retention in lung, liver, spleen, gut, skin, muscle, kidney, and brain tissues was determined by removing (before burn injury) and differentially radiolabeling PMNs (111In) and erythrocytes (51Cr), reinfusing cells 4.5 hours after burn injury, and measuring tissue radioactivity 30 minutes later. Edema was estimated by measuring extravasated 125I-labeled albumin in the various tissues, 30 minutes after injection. Peripheral blood PMNS were analyzed for intracellular H2O2 content by flow cytometry using a fluorescent dye that reacts with H2O2. Radioisotope studies demonstrated significant (p < 0.05) leukosequestration into lung, liver, gut, kidney, and skin tissues at 5 hours after burn injury. Tissue edema, manifested by radiolabeled albumin retention, was not observed in any tissues. Postburn PMN deposition in lungs and skin was decreased (p < 0.05) by the immediate administration of rBPI23 after burn injury. Flow cytometry showed increased intracellular H2O2 content in peripheral blood PMNs 5 hours after burn injury (p < 0.05), which was unaffected by administration of rBPI23. Since sequestration of metabolically active PMNs may induce tissue injury, therapies that block leukosequestration after burn injury may improve clinical outcomes by limiting remote tissue injury.

Peritoneal dialysis fluid-induced angiogenesis in rat mesentery is increased by lactate in the presence or absence of glucose.

Angiogenesis may be an important mechanism behind the functional deterioration of the peritoneum leading to ultrafiltration failure in peritoneal dialysis. The present study was designed to compare the angiogenic properties of lactate-, bicarbonate-, and pyruvate-buffered fluids, evaluated separately with and without glucose. Five different fluids (lactate and bicarbonate with and without 2.5% glucose and pyruvate without glucose) were studied for 5 weeks of twice-daily injections in rats. The respective buffers (40 mmol/l) were adjusted to pH 7.2, and sodium, chloride, calcium, and magnesium were present at standard concentrations. The mesenteric window model, based on observation of the translucent peritoneal sections of the small intestine mesentery, was used for immunohistochemical imaging of microvessels (RECA-1 antigen) and macrophages (ED1 and ED2 antigens). All fluids induced angiogenesis as compared with untreated controls. The lactate-buffered fluids induced larger vascularized zones than did their bicarbonate- and pyruvate-buffered counterparts. Angiogenesis was accompanied by a local recruitment of ED1 macrophages from blood. Addition of glucose to the lactate- and bicarbonate-buffered fluids did not seem to alter their pro-angiogenic properties. In conclusion, intraperitoneal exposure to lactate buffer, compared with bicarbonate, stimulates angiogenesis in the presence or absence of glucose.

Spontaneous nitroblue-tetrazolium (NBT) reduction related to granulocyte priming and activation

The aim of the present study was to explore the relationship between the increasing level of spontaneous NBT-reduction and the tendency for PMNs to marginate during experimental hemorrhagic shock in rats. Rat PMNs, isolated on Percoll® density gradients or suspended in blood, were examined by chemiluminescence (CL), NBT-test and by their CD-18 expression and F-actin formation. The NBT-test generally produced higher numbers of activated PMNs when the cells were suspended in buffer than in whole blood, probably due to the scavenging properties of blood. The level of spontaneous NBT-reduction of PMNs in blood correlated with the magnitude of the NBT-response to f-MLP stimulation in blood and buffer. On the contrary, there were no significant correlations between spontaneous NBT reduction, CD18 expression and F-actin content. Thus, high levels of spontaneous NBT reduction in blood were associated with priming of the separated PMNs rather than increased rigidity (F-actin) or adhesiveness (CD18).

Migration of human granulocytes in filters: effects of gravity and movable gradients of f-MLP.

The Boyden chamber technique for chemotaxis uses a mesh filter that constitutes a matrix for cell locomotion and, at the same time, creates a local restriction for convective fluid movements that allows the establishment of a diffusive concentration gradient of chemotactic substance in the filter. In the present study, the Boyden chamber was modified by the introduction of a filter sandwich that allowed cell migration both upwards and downwards and by the use of a fluid density gradient controlling cell buoyancy and mechanically supporting a movable chemotactic gradient. This method was used to study chemotaxis and random migration of human granulocytes under the influence of gravitational forces and movable gradients of f-MLP. The results show that gravity affected cell motion significantly during random migration but not during chemotaxis. The rate of chemotactic migration was dependent on the steepness of the spatio-temporal f-MLP gradients. A stationary spatial gradient produced less migration than a gradient that was slowly moved through the filter sandwich in a direction opposite to that of the cell migration. The presence of f-MLP at constant concentration caused a minor, statistically insignificant, increase of the rate of random migration.

Citrate supplementation of PD fluid: effects on net ultrafiltration and clearance of small solutes in single dwells

BACKGROUND Inflammatory reactions affect the general performance as well as the technique survival of peritoneal dialysis (PD). Anti-inflammatory additives like heparin and sodium citrate have shown favourable results in these respects. The present study is the first to evaluate citrate-supplemented PD fluids (PDFs) in humans. METHODS Crossover design was used to evaluate sodium citrate and heparin-supplemented Gambrosol Trio (2.5% glucose) in 28 stable outpatients from the PD unit. Comparisons were made between single dwells of each fluid. Citrate supplementation at 5 mM/L was compared with standard PDF, and citrate supplementation at 10 mM/L was compared with low-molecular-weight heparin (4500 units of tinzaparin) supplementation and standard PDF. The initial osmolarity of the fluids was equalized by adding sodium chloride. RESULTS Citrate supplementation at 5 mM/L significantly increased net ultrafiltration, measured as drained volume gain, by 126 mL. Creatinine and phosphate clearance, but not glucose clearance, was significantly improved by supplementation with citrate or heparin. Heparin supplementation created an insignificant trend towards an increased ultrafiltration (P = 0.08). No negative side effects were reported for any of the treatments; however, citrate supplementation led to a small calcium loss by the drained PD fluid (0.4 mmol) and a transient fall in the plasma concentration (0.04 mM/L) of free calcium ions at 5 mM/L citrate. Effects on plasma bicarbonate concentration were insignificant. CONCLUSIONS Citrate supplementation of PD fluid improved ultrafiltration and clearance of small solutes with only minor effects on calcium turnover. The mechanism is unknown and, according to the results, not related to complement inhibition.