Activation of human polymorphonuclear neutrophilic granulocytes by immuno-modulating cytokines: an ultrastructural study

Activated granulocytes play an important role in the propagation of inflammatory reactions and are capable of mediating tissue damage by the release of reactive oxygen species and lysosomal contents. Cytokines produced by immunocompetent and other cells were recently suggested to influence granulocyte functions. Therefore, in the present study, we investigated the effect of relevant immuno-modulating cytokines on isolated human granulocytes by ultrastructural criteria: scanning and transmission electron microscopy, ultrastructural detection of H2O2. The following recombinant human cytokines were tested: tumor necrosis factor (TNF), lymphotoxin (LT), GM-CSF, M-CSF, G-CSF, interleukin 1 (IL 1) alpha and beta, IL 2, IL 3, IL 4, IL 6, interferon (IFN)-alpha and gamma. Only TNF, LT, GM-CSF and IL 3 (at high concentrations) induced significant morphological changes (increased adherence to plastic layers, typical polarization, development of intracellular vesicles) and production of H2O2. None of the other cytokines tested induced any detectable effect on isolated granulocytes even at unphysiological concentrations. The results clearly demonstrate that only certain cytokines are capable of influencing granulocytes. Release of these mediators represents a specific signal for granulocyte activation in inflammatory disease states.     

Activation of neutrophilic granulocytes by products of human lymphocytes

Materials produced by stimulated primary cultures of human lymphocytes and by a continuous human lymphoid cell line, RPMI 1788, were tested for granulocyte phagocytosis promoting activity. Blood lymphocytes of healthy donors were stimulated with tuberculin (PPD) or concanavalin A (con A) and the culture fluids collected after 3 days of in vitro incubation. Fluids of the lymphoid cell line were collected from 2-day-old cultures. All three kinds of preparations were found to induce a significant increase in neutrophilic granulocyte phagocytic activity. Studies on the kinetics of granulocyte activation revealed that it is a relatively rapid process, being maximal within 60-120 min. This is in contrast to the considerably slower activation of monocytes in a comparable system. Intracutaneous inoculation of the active materials in human volunteers resulted in reactions that grossly and histologically resembled accelerated delayed type hypersensitivity reactions.     

The effect of adherence on the generation of reactive oxygen species by human neutrophilic granulocytes

Human neutrophilic granulocytes (PMN) suspended in protein containing salt solution or adherent on protein coated nylon fibers were tested for the production of H₂O₂ and O ₂ ^– in response to various PMN stimulants. Upon stimulation with the chemotactic factors formyl-methionyl-leucyl-phenylalanine, C5a and platelet activating factor, the non-chemotactic ionophore A23187, and the chemotaxis inhibitors tumor necrosis factor (TNF) and lymphotoxin (TNF ) adherent PMN produced considerably more reactive oxygen metabolites than suspended cells. The relative amounts of the two metabolites varied with the stimulus and its concentration, TNF and TNF favoring H₂O₂ production, C5a eliciting more O ₂ ^– than H₂O₂ and the other active stimulants being in between. Leukotriene B₄ and a novel monocytederived chemotaxin were inactive in releasing either oxygen derivative from adherent or suspended PMN. The data indicate that attachment of PMN to endothelial cells or to connective tissue substances can strongly enhance its ability to respond to a given stimulus with the production of reactive oxygen metabolites. The findings may in part explain the priming phenomenon since many PMN-priming mediators increase the cells' adherence.     

Activity of defensins from human neutrophilic granulocytes against Mycobacterium avium-Mycobacterium intracellulare.

We have examined the activity of defensins from human neutrophilic granulocytes against Mycobacterium avium-Mycobacterium intracellulare. M. avium-M. intracellulare at 2.5 x 10(6)/ml or 2.5 x 10(8)/ml was cultured in the presence of defensins at 37 degrees C from 4 to 48 h. After incubation, CFU were enumerated. Human neutrophil peptide 1 (HNP-1) at 5 micrograms/ml had the ability to kill M. avium-M. intracellulare. Treatment with HNP-1 resulted in significant (96.3 to 97.7%) killing of M. avium-M. intracellulare, even after taking clumping into consideration. This activity was not affected by the presence of calcium (0.5 and 1.0 mM), magnesium (0.5 and 1.0 mM), or sodium chloride (25, 50, and 100 mM). The optimal pH for bactericidal activity was higher than 5. We tested numerous M. avium-M. intracellulare strains, and HNP-1 was successful in killing every strain, although the degree of killing varied among them (34.2 to 87.2%). Additionally, this activity was independent of colonial morphology. We also examined the activity of HNP-2 and HNP-3 against M. avium-M. intracellulare and found that they were as effective in killing M. avium-M. intracellulare as HNP-1 was. These observations suggest that defensins may play an important role in the host defense against M. avium-M. intracellulare.     

Stimulation of particle-induced chemiluminescence in human granulocytes by various Escherichia coli strains

The patterns of neutrophil chemiluminescence stimulated by groups of E. coli strains opsonized with pooled normal human serum were compared. All strains of E. coli were obtained from patients with chronic pyelonephritis. 'O' serovars which lacked capsular (K) antigen produced significantly higher chemiluminescence than strains possessing a variety of K antigens. Chemiluminescence produced by a range 'O' serovars bearing the K1 antigen did not differ significantly from those containing other K antigens. The lowest chemiluminescence values were obtained with a group of O2:K1 strains. Rough strains containing K1 antigen were also poor stimulators of chemiluminescence.     

Cortisol inhibits apoptosis in carp neutrophilic granulocytes

The direct effect of cortisol treatment on carp neutrophil viability was examined in vitro. Cortisol treatment caused an inhibition of neutrophil apoptosis. The effect was blocked by glucocorticoid receptor blocker RU486, showing that rescue from apoptosis was receptor mediated. Using binding studies with radioactive cortisol, a single class of glucocorticoid receptors was detected with high affinity (Kd=2.6 nM) and low capacity (497 receptors/cell) for cortisol binding. Both in vitro and in vivo cortisol treatment did not affect neutrophil respiratory burst activity. These data indicate that cortisol can augment the supply of functional neutrophilic granulocytes in conditions of acute stress, which may be essential for survival, since phagocytes form the first line of defence against micro-organisms.     

Functions of human neutrophilic granulocytes after in vivo exposure to interferon alpha.

The ability of neutrophilic granulocytes to phagocytize yeast particles and to reduce Nitro Blue Tetrazolium at rest and on activation with bacterial stimuli was monitored in 32 patients receiving treatment with human interferon alpha. The ability of these cells to attach to and ingest yeast particles was not altered to any major extent during 1 year of interferon treatment. In most patients, the Nitro Blue Tetrazolium-reducing activity increased after the first injection of interferon. During prolonged treatment with interferon alpha, 1 week to 1 year, granulocytes activated with bacteria exhibited a reduced Nitro Blue Tetrazolium activity in most patients.     

Stimulation of particle-induced chemiluminescence in human granulocytes by various Escherichia coli strains.

The patterns of neutrophil chemiluminescence stimulated by groups of E. coli strains opsonized with pooled normal human serum were compared. All strains of E. coli were obtained from patients with chronic pyelonephritis. ''O'' serovars which lacked capsular (K) antigen produced significantly higher chemiluminescence than strains possessing a variety of K antigens. Chemiluminescence produced by a range ''O'' serovars bearing the K1 antigen did not differ significantly from those containing other K antigens. The lowest chemiluminescence values were obtained with a group of O2:K1 strains. Rough strains containing K1 antigen were also poor stimulators of chemiluminescence.     

Morphologic features of the phagocytosis of filamentous forms of bacteria by neutrophilic granulocytes

A model system has been developed (B. megaterium + granulocyte) imitating the mechanisms of phagocytosis of filamentous forms of bacteria by neutrophilic granulocytes. In the capture of multicellular microbial filaments by one or several granulocytes predominate the mechanisms of cell membrane invagination and formation of exocytic secretory system providing the inhibition of the activity of the phagocytized agent and release of microbicidal factors and enzymes into the extracellular environment. The importance and frequency of occurrence of the observed phenomenon in different pathological forms have not been elucidated.     

Clinico-morphological tests to assess the function of neutrophilic granulocytes

Clinico-morphological tests for the estimation of neutrophil granulocyte function were compared by their reproducibility, reliability and availability for standard clinical laboratories, and the results were analysed. Lysosomal-cationic test was offered for clinical practice, as it gives qualitative and quantitative assessment of the phagocytosis process, allows prediction of complications before their clinical onset, performance of rapid evaluation of the therapy used, reveals shifts in the level of non-specific resistance of the body. Methods of application of lysosomal-cationic test in the pathoanatomic practice have been developed for the study of intravital biopsy samples and surgical material.     

Functional activity of neutrophilic granulocytes in patients with relapsing herpes

Neutrophilic granulocytes exhibit different degrees of functional activity in patients with relapsing herpetic infection in various phases of the disease. A high level of myeloperoxidase is detected in the serum over the entire course of the disease and a three-fold increase of serum lactoferrin appears at the beginning of remission. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 120, No 9, pp. 329–331, September, 1995 Presented by B. I. Tkachenko, Member of the Russian Academy of Medical Sciences     

Morphological variants of neutrophilic granulocytes in blood of practically normal humans

Verification of presumed inertness of blood neutrophil granulocytes revealed their morphological heterogeneity in practically healthy donors. At light microscopic level it was expressed as a varying degree of cell cytoplasm granularity--58% of the cells were highly granular, 30% contained moderate amount of granules and 12% were completely devoid of granules. According to the ultrastructural analysis, neutrophils were subdivided into four groups: intact cells (60%), cells with slight (26%), moderate (12%) and severe (2%) changes. The criteria for this classification included changes in neutrophil shape and ultrastructure during its activation: formation of pseudo- and lobopodia, spatial redistribution of organelles, degranulation etc. Presence of neutrophils with the signs of activation in the circulation suggests that the neutrophil system normally is not inert, but is in a state of so-called working tone, thus providing high antibacterial resistance of the macroorganism exposed to natural bacterial environment.     

Superantigen-dependent accelerated death of bovine neutrophilic granulocytes in vitro is mediated by blood mononuclear cells

While classical interactions of bacterial superantigens (SAgs) with antigen presenting cells and T cells have been studied intensively, the potential interactions of SAgs with granulocytes (PMNs) have gained much less attention. We investigated if in the bovine system SAgs have any direct or indirect influence on the fate of granulocytes, which are among those cells primarily responsible for the elimination of superantigen-producing bacteria. The tested SAgs (SEA, SEB) had no apparent direct effect on PMN viability (neutrophils and eosinophils). However, in the presence of blood mononuclear cells (MNCs), SAgs led to an accelerated death of neutrophils but not of eosinophils. Compared to medium controls, in SAg-stimulated cultures only about 20-50% of the neutrophils survived after 24 hours in vitro. Accelerated death of neutrophils required the presence of at least 10% MNC and started between 2.5-24 h after initiation of the co-culture between MNC and PMN. Minimal effective SEA concentrations ranged between 10-100 pg/l (SEB 0.1-10 ng/l). The effect could be mimicked by culture supernatants of SAg-stimulated MNCs, suggesting that direct cell-cell interactions are not required for the killing. In the human system, where we tested the role of TNF-alpha, an antibody specific for this cytokine was not able to abolish the death of human neutrophils. Brefeldin A, an inhibitor of golgi transport and cytokine secretion, which blocked the SAg-induced activation of bovine MNC did not abolish the killing of neutrophils. Blocking of nitric oxide generation or PGE2 synthesis also could not alter the SAg-induced killing of bovine neutrophils. The observed indirect negative effects of SAgs on neutrophils may provide new insights in mechanisms by which superantigens modulate the hosts immune response.     

Stimulation of the respiratory burst and promotion of bacterial killing in human granulocytes by intravenous immunoglobulin preparations.

We have examined the effect of two i.v. immunoglobulin preparations on the metabolic and functional activities of neutrophil granulocytes from the peripheral blood. Production of superoxide anion (O2-) by granulocytes was measured through superoxide dismutase inhibitable reduction of ferricytochrome C after incubation of cells for various times together with immunoglobulin (concentration ranging from 0.25 to 5.0 mg/ml). The results showed dose-dependent response of O2- production independent of the incubation time. Granulocytes containing ingested Staphylococcus aureus released a significantly (P less than 0.001) larger amount of O2- and killed a higher number (P less than 0.001) of viable bacteria in the presence of 5 mg/ml immunoglobulin than did cells incubated in the absence of extracellular i.v. immunoglobulin. These data raise the possibility that immunoglobulin concentrates for i.v. use may enhance the anti-bacterial activities of phagocytic cells through direct stimulation of the respiratory burst. Inflammatory reactions observed during i.v. immunoglobulin infusion in hypo- or agammaglobulinaemic patients may also be related to phagocytic cell activation.     

Stimulation of naive and memory T cells by cytokines

Summary: On the basis of cell surface markers, mature T cells are considered to have either a naïve or a memory phenotype. These cells exhibit distinct types of kinetic behaviour in vivo. While naive-phenotype cells persist long term in a non-dividing state. memory phenotype T cells include cycling cells and exhibit a more rapid rate of turnover; this has also been shown to be true for cells that can be definitively identified as naive or memory T cells respectively. The number of memory-phenotype (CD44in) CD8⁺ T cells entering cell cycle is greatly increased after In vivo exposure to viruses, bacteria or components of bacteria. Accelerated turnover of memory T cells also occurs after the injection of a variety cytokines that are induced by infectious agents, including type I interferon (IFN-I), Although naive-phenotype T cells do not divide in response to these cytokines, they do exhibit signs of activation, including upregulation of CD69 after exposure to lFN-1, These findings suggest that the dissimilar in vivo kinetics of naive- and memory-phenotype T cells might reflect their divergent responses to cytokines. Furthermore, the ability of infection- induced cytokines to stimulate non-specific proliferation of memory phenotype T cells and partial activation of naive-phenotype T cells implies that they play a complex role during primary immune responses w infectious agents.     

Scanning probe microscopy for the study of morphological parameters of neutrophilic granulocytes

Morphological parameters of human blood neutrophilic granulocytes were studied by scanning probe microscopy. The basic morphological characteristics of the living (unfixed) and fixed cells were compared. It was found that using the method described, the dimensions of the cells changed substantially and significantly depending upon the mode of fixation applied. The methanol fixation allowed to demonstrate the internal structure of a cell, though it sharply deformed the object of study. After glutaraldehyde fixation cells possessed the morphological characteristics similar to those of the living cells, but the diameter and height of the fixed cells were again different from those of living cells. The resolution of a scanning probe microscope was higher when the fixed cells were studied, permitting the visualization of a detailed cell structure, including the nucleus and cytoplasmic granules (in particular, when methanol fixation was used). However, this method of study is unreliable for the determination of cell dimensions (diameter and height).