Spontaneous activation of circulating granulocytes in patients with acute myocardial and cerebral diseases.

Recent animal studies have suggested that there exists an activated subpopulation of circulating granulocytes which plays an important part in microvascular sequestration and tissue injury during shock and ischemia. In this respect, spontaneous granulocyte activation in form of pseudopod formation, a manifestation of actin polymerization, is a high risk for microvascular entrapment. The present investigation was carried out to determine if there is a significant difference in pseudopod formation in vitro between granulocytes obtained from healthy volunteers without symptoms and patients with acute cardiovascular illnesses. Blood samples from 25 healthy volunteers, 12 patients with acute myocardial infarction (AMI) and 12 patients with acute cerebral infarction (ACI) to determine spontaneous pseudopod formation in granulocytes with a high resolution light microscope over a period of several hours. The results revealed that the mean percentage of cells with pseudopod formation in the control group was below 10% in the first 3 hours, and increased to about 50% at 12 hours. In AMI patients, the level of activation within the first hour was not significantly different from the controls, but it rose rapidly to 90% in 4 to 5 hours. Patients with cerebral infarction, however, showed no significant difference from the control group. When the granulocytes of healthy subjects were incubated in plasma of AMI, the cells were activated similar to AMI granulocytes in their own plasma. When AMI plasma was serially diluted with Ringer's solution, the activation curve fell successively. These results indicate that AMI patients' blood contains plasma factor(s) which can activate granulocytes at a more rapid rate than controls.     

Leukocyte kinetics in the microcirculation

The transport of leukocytes in the microcirculation is specific for the type, size, and the rheological and adhesive properties, the microanatomy of the host organ, and the hemodynamics. The adhesion to the endothelium is determined largely by the degree of activation via chemotactic factors. Leukocyte motion differs from that of red cells or platelets in several respects. When granulocytes enter into capillaries, they are deformed just like red cells. Under normal flow conditions, the time to deform at the entry to capillaries is typically 1,000 times larger than for the red cell, leading to temporary obstruction of the capillaries. After entry, granulocytes move with lower velocity than red cells which causes a cell train formation inside the capillary. At the venular side, the granulocyte is displaced from the center stream toward the endothelium by faster moving red cells. This process leads to systematic attachment of the granulocytes to the endothelium. At a reduced perfusion pressure or in the presence of locally elevated levels of chemotactic factors, the granulocytes may not be able to pass through the capillary network, which leads to microvascular obstruction. Organs with a narrow capillary network may thereby become filters for circulating granulocytes. This event is accompanied in many situations with damage to the host organ.     

Composition and ultrastructure of elasmobranch granulocytes. II. Rays (Rajiformes)

The blood granulocyte composition of seven species of ray is given together with ultrastructural observations made on the epigonal organ and blood of Pavoraja spinifera and the spleen of a deepwater rajid skate. Under the light microscope three granulocyte types, eosinophils, eosinophilic granulocytes and neutrophilic granulocytes could be distinguished. At the EM level two granulocyte types were apparent, one with elongated granules containing longitudinal fibrils that consolidated to form an axial rod-like inclusion, and the other with large, spherical, uniformly electron-dense granules. Correlation of light and electron microscope observations indicated that the neutrophilic granulocytes with weakly basophilic, elongated granules become weakly eosinophilic, as eosinophilic granulocytes, and these in turn develop to eosinophils with granules containing axial rods. The other granulocyte type forms another population of eosinophils with spherical granules.
The inter-relationship of these granulocytes, the identification of eosinophilic granulocytes, or heterophils, as immature eosinophils, and the co-existence of two morphologically distinct eosinophil forms are discussed.     

Association of NKT cells and granulocytes with liver injury after reperfusion of the portal vein

Reperfusion of the liver was conducted by clamping the portal vein for 30 min in mice, followed by unclamping. Unique variation in the number of lymphocytes was induced and liver injury occurred thereafter. The major expander cells in the liver were estimated to be natural killer T cells (i.e., NKT cells), whereas conventional T cells and NK cells increased only slightly or somewhat decreased in number and proportion at that time. Reflecting the expansion of NKT cells in the liver, a Th0-type of cytokine profile was detected in sera, and cytotoxic activity was enhanced in liver lymphocytes. In NKT cell-deficient mice including CD1d (-/-) mice and athymic nude mice, the magnitude of liver injury decreased up to 50% of that of control mice. It was also suspected that accumulating granulocytes which produce superoxides might be associated with liver injury after reperfusion. This might be due to stress-associated production of catecholamines. It is known that granulocytes bear surface adrenergic receptors and that they are activated by sympathetic nerve stimulation after stress. The present results therefore suggest that liver injury after reperfusion may be mainly caused by the activation of NKT cells and granulocytes, possibly by their cytotoxicity and superoxide production, respectively.     

Consequences of activation and adenosine-mediated inhibition of granulocytes during myocardial ischemia

During acute myocardial ischemia, granulocytes accumulate and obstruct the microcirculation. Granulocytes remain plugged in individual myocardial capillaries on reperfusion and are the major cause of the no-reflow phenomenon. During 3 h of ischemia, the granulocyte content of myocardium measured by 111In labeling increases from 1.0 X 10(6) to 1.5 X 10(6) cells/g, and after 5 min of reperfusion increases to 2.4 X 10(6) cells/g. The effects of granulocytes during 1 h of acute ischemia were determined by comparing agranulocytic to whole blood perfusion. With whole blood collateral flow decreased, water content increased (edema), ventricular fibrillation was common, and 27% of capillaries had no-reflow, whereas in the absence of granulocytes, collateral flow increased, there was no edema, arrhythmias were rare, and the no-reflow phenomenon was completely prevented. It is unfortunate that the inflammatory signals triggered by ischemia remain active on acute reperfusion, limit tissue salvage, and perhaps cause reperfusion injury. Several activating stimuli for granulocytes are known, but what inhibits them? Adenosine is known to inhibit superoxide radical formation by granulocytes, and 5-amino-4-imidazole carboxamide-riboside (AICA-riboside) augments adenosine release from energy-deprived cells. In dogs subjected to 1 h of ischemia, AICA-riboside pretreatment augmented adenosine release by nearly 10-fold, which was accompanied by a significant increase in collateral blood flow and decreased arrhythmias. We propose a new hypothesis: adenosine acts as a natural antiinflammatory autacoid during transient injury linking the ability to catabolize ATP (an indicator of viability) to granulocyte inhibition, thus preventing premature activation of the inflammatory response to cell death. Granulocytes are active participants in acute myocardial ischemia and means to prevent their activation, remove them from the reperfusate, or inhibit them will be necessary for optimum reperfusion salvage.     

Structural alterations of marrow during inflammation

In response to infections and inflammations, bone marrow reacts to mobilize its granulocyte reserve. Three sets of factors are involved in this mobilization. The structure of the sinus wall is altered and adventitial cells retract to permit interaction of migrating cells with the endothelium. During the maturation process, granulocytes lose their binding potential to the supporting stroma, but their motility, chemotactic ability, and deformability increase. Consequently, they move toward the sinus endothelium with which they interact to enter the circulation. Soluble factors are also involved in granulocyte mobilization. The best characterized of these factors is C3e, an acidic fragment of the alpha chain of C3 with MW of 10-12 KD and ability to bind to granulocyte membrane. Other soluble factors may also be involved, but due to lack of adequate methodology, this area has been relatively underexplored.     

Capillary plugging by granulocytes and the no-reflow phenomenon in the microcirculation

Granulocytes are large, stiff viscoelastic cells that adhere naturally to the vascular endothelium. On their passage through the capillary network they have to be deformed, and recent evidence indicates that they may impose a significant hemodynamic resistance. The entry time of granulocytes into capillaries is about three orders of magnitude longer than that for red cells. Inside the capillary the granulocytes move with a lower velocity than red cells. Under conditions when the capillary perfusion pressure is reduced and/or elevated levels of inflammatory products are present that increase the adhesion stress to the endothelium, granulocytes may become stuck in the capillary. In such a situation, the granulocytes form a large contact area with the capillary endothelium, they obstruct the lumen, and they may initiate tissue injury. After the restoration of the perfusion pressure the granulocytes may not be removed from the capillary owing to the adhesion to the endothelium. Capillary plugging by granulocytes appears to be the mechanism responsible for the no-reflow phenomenon, and together with oxygen free radical formation and lysosomal enzyme activity may constitute the origin for ischemic injury as well as other microvascular occlusive diseases.     

Composition and ultrastructure of elasmobranch granulocytes. III. Sharks (Lamniformes)

The peripheral blood granulocyte composition of five species of shark is given together with ultrastructural observations made on the epigonal organ and blood of Mustelus lenticulatus and spleen of Apristurus sp. Three granulocyte lineages occurred in Mustelus . Ultrastructurally, eosinophilic granulocytes contained granules that were very variable in size and contained parallel fibrillar arrays that were unidirectional in small granules, but which were often orientated in several directions in larger granules. This dense core material sometimes formed angular crystalloids.
Eosinophils had ovoid or irregular granules that were usually uniformly electron-dense, but some had an eccentric ovoid area of greater electron-density. Some eosinophils enter the blood where they are phagocytic, but others, in blood and epigonal organ, showed an unusual but characteristic process of disintegration, leaving a ragged cell full of cellular debris.
The third granulocyte type had features of mast cells and basophils of higher vertebrates and was tentatively identified as belonging to that lineage. The inter-relationships of these cells, and of them with granulocytes of other elasmobranchs, are discussed.     

Calpain inhibitor I reduces the activation of nuclear factor-kappaB and organ injury/dysfunction in hemorrhagic shock.

There is limited evidence that inhibition of the activity of the cytosolic cysteine protease calpain reduces ischemia/reperfusion injury. The multiple organ injury associated with hemorrhagic shock is due at least in part to ischemia (during hemorrhage) and reperfusion (during resuscitation) of target organs. Here we investigate the effects of calpain inhibitor I on the organ injury (kidney, liver, pancreas, lung, intestine) and dysfunction (kidney) associated with hemorrhagic shock in the anesthetized rat. Hemorrhage and resuscitation with shed blood resulted in an increase in calpain activity (heart), activation of NF-kappaB (kidney), expression of iNOS and COX-2 (kidney), and the development of multiple organ injury and dysfunction, all of which were attenuated by calpain inhibitor I (10 mg/kg i.p.), administered 30 min prior to hemorrhage. Chymostatin, a serine protease inhibitor that does not prevent the activation of NF-kappaB, had no effect on the organ injury/failure caused by hemorrhagic shock. Pretreatment (for 1 h) of murine macrophages or rat aortic smooth muscle cells (activated with endotoxin) with calpain inhibitor I attenuated the binding of activated NF-kappaB to DNA and the degradation of IkappaBalpha, IkappaBbeta, and IkappaBvarepsilon. Selective inhibition of iNOS activity with L-NIL reduced the circulatory failure and liver injury, while selective inhibition of COX-2 activity with SC58635 reduced the renal dysfunction and liver injury caused by hemorrhagic shock. Thus, we provide evidence that the mechanisms by which calpain inhibitor I reduces the circulatory failure as well as the organ injury and dysfunction in hemorrhagic shock include 1) inhibition of calpain activity, 2) inhibition of the activation of NF-kappaB and thus prevention of the expression of NFkappaB-dependent genes, 3) prevention of the expression of iNOS, and 4) prevention of the expression of COX-2. Inhibition of calpain activity may represent a novel therapeutic approach for the therapy of hemorrhagic shock.     

Granulocyte interactions with GM-CSF and G-CSF secretion by endothelial cells and monocytes

We have, in previous studies, characterized the cytokine and cellular regulation of GM-CSF and G-CSF production by monocytes and endothelial cells. In this study, we investigated the regulatory role of granulocytes. The addition of granulocytes to endotoxin-stimulated monocytes dose-dependently decreased both GM-CSF and G-CSF concentrations, presumably by absorbing the cytokines. A similar dose-dependent decrease in GM-CSF concentration was found when granulocytes were added to IL-1-stimulated endothelial cells. In contrast, G-CSF secretion by endothelial cells responded to granulocytes in a biphasic fashion. At low granulocyte concentrations, endothelial cells responded with an increased G-CSF secretion, but at high concentrations of granulocytes G-CSF secretion was down modulated. Our results suggest that there exist two loops between granulocytes and endothelial cells for regulating G-CSF activity. Granulocytes can stimulate G-CSF secretion by activated endothelial cells but can also decrease the biological activity by absorbing the cytokine. These mechanisms might be involved in the regulation of the local and systemic levels of granulocytes.     

Effects of intravenous IL-8 administration in nonhuman primates.

IL-8, a cytokine known for its potent and specific neutrophil activation and chemoattractant properties, has been recently detected in the circulation during septic shock, endotoxemia, and after IL-1 alpha administration. Because of its observed in vitro actions, it has been hypothesized that IL-8 may contribute to the dynamics of circulating granulocytes and to the pathologic sequelae seen in sepsis. Here, human rIL-8 is administered to healthy nonhuman primates as a single i.v. injection or as a continuous 8-h i.v. infusion. We demonstrate that both methods of i.v. administration result in a rapid but transient, severe granulocytopenia, followed by a granulocytosis that persists as long as IL-8 levels are detectable in the circulation. There were no hemodynamic changes after IL-8 administration, and animals remained clinically stable during the 24-h observation period. No detectable circulating TNF-alpha, IL-1 beta, or IL-6 response was induced by either IL-8 administration regimen. Histopathologic examination revealed mild to moderate neutrophilic margination in lung, liver, and spleen, of greater severity in baboons receiving the 8-h infusion. There was no associated neutrophilic infiltration or tissue injury. Thus, IL-8 modulates circulating granulocyte dynamics and likely directs their actions, but when administered i.v. to healthy animals, either as a bolus dose or as a continuous infusion for up to 8 h, does not induce the hemodynamic and metabolic aberrations or the acute organ damage seen during sepsis.     

Expression of the GM-CSF gene after retroviral transfer in hematopoietic stem cell lines induces synchronous granulocyte-macrophage differentiation

Multipotent murine stem cell lines (FDC-Pmix) depend on IL-3 for self-renewal and proliferation and can be induced to differentiate into multiple hematopoietic lineages. Single FDC-Pmix cells infected with retroviral vectors expressing GM-CSF are induced to differentiate into granulocytes and macrophages. This results in a complete loss of clonogenic cells if IL-3 is not exogenously supplied; however, multipotent variants can be selected that do not terminally differentiate if cells are kept in the presence of IL-3. Unidirectional and synchronous granulocyte and macrophage differentiation accompanied with loss of self-renewal capacity is induced when IL-3 is removed. Our data indicate that activation of the GM-CSF receptor induces differentiation of stem cells by an instructive mechanism that can be blocked by the activated IL-3 receptor. A model of how receptors can induce proliferation and cell-specific differentiation by two separate pathways is discussed.     

Endotoxin-mediated pulmonary endothelial cell injury

Infusion of endotoxin into sheep results in physiological and structural damage to the pulmonary endothelium. It is uncertain whether complement activation and granulocyte sequestration in the pulmonary microcirculation and the ensuing granulocyte migration into the interstitium seen with endotoxemia contribute to the endothelial damage. We have shown that infusion of complement-activated plasma into sheep, although causing the same degree of granulocyte sequestration in the lungs, results in only modest and transient endothelial damage. In addition, migration or chemotaxis of granulocytes across the endothelial layer of intimal explants is not accompanied by either structural evidence of endothelial damage or a detectable increase in vascular permeability. Such studies indicate that neither complement/granulocyte activation nor granulocyte migration across a vessel wall is entirely responsible for the severe endothelial damage seen with endotoxin. In vitro studies of bovine pulmonary endothelial monolayers indicate that endotoxin can cause direct damage to the endothelium; the damage is dose-dependent and more severe in the presence of serum. Structural studies show endothelial cell retraction, pyknosis, and sloughing. Prostacyclin production and lactic dehydrogenase release are increased, as are permeability to small solutes and hydraulic conductance across the endothelium. It seems that endotoxin can cause a direct injury to pulmonary endothelium but complement and granulocyte activation may enhance the damage.     

Studies on the interaction of leucocytes and the myocardial vasculature

Granulytes play an important role in increasing the infarct size after ischemia and reperfusion by the release of oxygen-derived free radicals (ODFR) and lysosomal enzymes. It has been shown that the number of granulocytes adhering to the vascular endothelium increases after occlusion of the coronary artery, and that the area of myocardial damage can be reduced by preventing granulocyte adherence with monoclonal antibodies directed against adhesion receptors. The underlying mechanism of granulocyte activation under these conditions is not yet known. We have investigated whether granulocytes can be activated directly by reduced oxygen tensions. Granulocytes were suspended in a hypoxic buffer and incubated on fibronectin and gelatin coated microtitre plates at 1–3% ambient oxygen to study their ability to adhere to these matrices. The results showed that the adherence of granulocytes to fibronectin was dependent on the duration of hypoxia. After 30 min of incubation under hypoxia granulocyte adherence increased 1.3 to 1.8 fold compared to the normoxic control. The adherence to fibronectin could be inhibited partially by anti-CD18 antibody, a monoclonal antibody to the common beta chain of a class of extracellular matrix receptors. This direct activation of granulocytes due to hypoxic conditions may have implications for the interaction of these cells with the vascular endotheliumin vivo, (Mol Cell Biochem116: 197–202, 1992)     

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

Granulocytes play a key role in the body’s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte function, in general these are limited in terms of the information they can provide. For example, most existing assays merely provide a percentage of how many granulocytes are activated following a single, fixed length incubation. Complicating matters, most assays focus on only one aspect of function due to limitations in detection technology. This report demonstrates a technique for simultaneous measurement of granulocyte phagocytosis of bacteria and oxidative burst. By measuring both of these functions at the same time, three unique phenotypes of activated granulocytes were identified: 1) Low Activation (minimal phagocytosis, no oxidative burst), 2) Moderate Activation (moderate phagocytosis, some oxidative burst, but no co-localization of the two functional events), and 3) High Activation (high phagocytosis, high oxidative burst, co-localization of phagocytosis and oxidative burst). A fourth population that consisted of inactivated granulocytes was also identified. Using assay incubations of 10, 20, and 40-min the effect of assay incubation duration on the redistribution of activated granulocyte phenotypes was assessed. A fourth incubation was completed on ice as a control. By using serial time incubations, the assay may be able to able to detect how a treatment spatially affects granulocyte function. All samples were measured using an image-based flow cytometer equipped with a quantitative imaging (QI) option, autosampler, and multiple lasers (488, 642, and 785 nm).     

Identification of polymorphonuclear leukocytes in cytologic samples for flow cytometry.

Inflammatory cells are commonly present in cytologic specimens obtained for flow cytometry, and may interfere with the analysis of epithelial cells. We have found that detergent (Triton X-100) pretreatment in the two-step acridine orange staining procedure disrupts granulocyte cell membranes to yield bare nuclei; bladder epithelial and squamous cells on the other hand are quite resistant to the detergent treatment. Being deprived of their cytoplasmic RNA, the granulocytes lose red fluorescence. Moreover, the shearing forces in the cytometer extend the multisegmented granulocyte nuclei and align them in the direction of flow. Thus, they present as elongated objects in the measuring system, giving a large DNA fluorescence pulsewidth (nuclear size). These two phenomena make it possible to identify granulocytes in the recorded data, where they are discernible from the mononucleated leukocytes and from epithelial cells. By data selection the granulocytes can be excluded, rendering epithelial cell populations more amenable to analysis. This method may make it unnecessary to remove physically leukocytes from the specimen before flow cytometry; it may also provide a way to analyze the morphology of granulocyte nuclei and to assess methods to manipulate their membrane stability. Full protection from membrane disruption is accomplished by alcohol fixation, and partial protection by 20-30% serum.     

The analysis of cellular elements in coelomic fluid during early regeneration of of the starfish Asterias rubens L

Three main cell types were found in the coelomic fluid (CF) of intact starfishes: agranulocytes (55-80%) varying in size and form (spherical and ovoid) and with occasional pseudopodia, granulocytes (15-45%), and small cells (up to 2 %) with a high nuclear-cytoplasmic ratio. The starfish response to injury depends on the degree of coelomic fluid loss. After a slight wounding, when only insignificant portion of CF is lost, the cellular composition of circulating fluid changed only slightly. Unlike, a significant injury resulted in rising the share of small cells, regarded presumably as young cells. Besides, after injury the functional characteristics of SF also changed: the proportion of cells with decondensed chromatin and stained nucleoli increased, and coelomocytes acquired ability to form nets at adhesion. Moreover, some new cell types can be found (fusiform cells), with granulocyte proportion in nets increasing. We suppose that after slight wounding circulating coelomocytes may restore from the existing store of differentiated cells beyond the circulation, whereas after significant injury young undifferentiated coelomocytes are involved in the process of restoration.     

Natural killer cells in a lower invertebrate, Nereis diversicolor.

Globular, non-adherent coelomocytes, called "G3 granulocytes", of the polychaetous annelid Nereis diversicolor display spontaneous cytotoxicity. These cells were found capable of killing invertebrate as well as vertebrate target cells by a contact-dependent cytolytic process. Cytotoxic activity of G3 granulocytes against foreign cells develops in three steps. At first, the cells become motile and form lamellipodia. In a second step, short, pointed pseudopodia arise from the edge of the lamellipodia and are making contact with the stimulating foreign object. In a third step, the G3 granulocytes release dense granules by exocytosis onto the foreign substrate or cell which finally will undergo lysis. Within few minutes after activation, the G3 granulocyte will alter its polarity, realigning both Golgi apparatus and centrosome towards the target cell. A pore-forming protein may be involved in the cytotoxic activity of the G3 granulocytes. These cells were observed to burst after contact with and release of granules onto an abiotic solid substrate, indicating that under certain circumstances the G3 granulocytes may be sensitive to their own cytotoxic activity. These data support the postulate of Franceschi et al. (Eur. J. Immunol. 21, 489-493 (1991) that a primitive natural killer cell-like activity had been developed early in phylogenesis. A simple method for preparing invertebrate coelomocytes for scanning electron microscopy is described.     

Composition and ultrastructure of elasmobranch granulocytes. I. Dogfishes (Squaliformes)

The blood granulocyte composition of 10 species of dogfish is given, together with ultrastructural observations made on Etmopterus baxteri Leydig organ and blood, and on spleens of Oxynotus bruniensis, Deania calcea, Scymnodon plunketi and blood of Centroscymnus crepidator . Neutrophilic granulocytes, which were common, had spherical granules that developed a dense core, which then lost contents to become lucent. Eosinophilic granulocytes had ovoid or elongated granules with a fibrillar content that became aligned longitudinally, and rarely formed an axial rod. Eosinophils had large spherical granules that were electron-dense but in early stages had a disorganised fibrillar content. These cells correspond to the neutrophils, heterophils and eosinophils, respectively, of other elasmobranchs.
Dogfish granulocytes are compared with those of other elasmobranchs, and their lack of similarity to those of higher vertebrates is noted.     

Granulocyte kinetics with radioactive diisopropylfluorophosphate (DF32P) and radiochrome (51Cr)]

Determining granulocyte kinetics with DF32P allows various parameters to be gained during the in-vitro marking, such as the total blood granulocyte pool, circulating granulocyte pool, marginal granulocyte pool, daily granulocyte exchange rate and half decay period of granulocytes. The half decay period of granulocytes, bone-marrow reserve in myelocytes, metamyelocytes and band cells as well as polymorphonuclear neutrophils can be determined by in-vitro marking, with DF32P being intravenously injected. The combination of both procedures with DF32P will reveal the half decay period, pool sizes and exchange rates of the proliferating myelocyte compartiment in bone-marrow and mature blood granulocytes. If 51Cr is used for determining granulocyte kinetics the surface activities of various organs, such as heart, liver, spleen, and lungs, can mainly be determined in addition to the half-life of leucocytes, indicating the degradation or storage of cells in certain areas of the body. In addition to normal values those findings are principally presented which were obtained with in-vitro marking by DF32P and 51Cr in chronic myeloid leukaemia, osteomyelofibrosis or osteomyelosclerosis respectively and in hypersplenism.