Granulocyte-macrophage colony-stimulating factor increases the synthesis of leukotriene B4 by human neutrophils in response to platelet-activating factor. Enhancement of both arachidonic acid availability and 5-lipoxygenase activation.

Granulocyte-macrophage CSF (GM-CSF) primes human neutrophils for increased functional responsiveness to a variety of inflammatory agonists. In the present report, we have investigated the effect of human GM-CSF on the ability of platelet-activating factor (PAF) to induce the synthesis of 5-lipoxygenase products in human neutrophils. Human neutrophils stimulated with PAF in the range of 10(-5) to 10(-7) M for 15 min released small quantities of leukotriene B4 and its omega-oxidation products, 20-OH- and 20-COOH-leukotriene B4 in amounts that were detectable by enzyme immunoassay. Preincubation of normal peripheral blood neutrophils with human rGM-CSF enhanced the synthesis of the 5-lipoxygenase products in a time- and dose-dependent manner. Treatment with GM-CSF enabled their detection in response to lower concentrations of PAF (greater than or equal to 10(-9) M). The PAF receptor antagonist BN52021 inhibited the synthesis of 5-lipoxygenase products by GM-CSF-treated neutrophils in response to PAF. In addition to its effect on PAF-induced leukotriene synthesis, GM-CSF also augmented intracellular calcium mobilization by PAF. This observation prompted us to examine the effect of GM-CSF on two calcium-dependent events that are essential for leukotriene synthesis, arachidonic acid liberation, and 5-lipoxygenase activation. GM-CSF by itself, did not directly activate either of these two processes, however, it consistently and markedly enhanced the ability of PAF to do so. These results indicate that preincubation of peripheral blood neutrophils with GM-CSF enhances the ability of PAF to stimulate leukotriene synthesis by increasing both arachidonic acid availability and 5-lipoxygenase activation in response to PAF. These observations provide additional evidence of an important role for GM-CSF in the modulation of inflammatory responses to endogenous agonists through enhancement of the production of potent cellular inflammatory mediators such as leukotrienes.     

Effects of Granulocyte-Macrophage Colony-Stimulating (GM-CSF) Factor on Corneal Epithelial Cells in Corneal Wound Healing Model

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that activates granulocyte and macrophage cell lineages. It is also known to have an important function in wound healing. This study investigated the effect of GM-CSF in wound healing of human corneal epithelial cells (HCECs). We used human GM-CSF derived from rice cells (rice cell-derived recombinant human GM-CSF; rhGM-CSF). An in vitro migration assay was performed to investigate the migration rate of HCECs treated with various concentrations of rhGM-CSF (0.1, 1.0, and 10.0 μg/ml). MTT assay and flow cytometric analysis were used to evaluate the proliferative effect of rhGM-CSF. The protein level of p38MAPK was analyzed by western blotting. For in vivo analysis, 100 golden Syrian hamsters were divided into four groups, and their corneas were de-epithelialized with alcohol and a blade. The experimental groups were treated with 10, 20, or 50 μg/ml rhGM-CSF four times daily, and the control group was treated with phosphate-buffered saline. The corneal wound-healing rate was evaluated by fluorescein staining at the initial wounding and 12, 24, 36, and 48 hours after epithelial debridement. rhGM-CSF accelerated corneal epithelial wound healing both in vitro and in vivo. MTT assay and flow cytometric analysis revealed that rhGM-CSF treatment had no effects on HCEC proliferation. Western blot analysis demonstrated that the expression level of phosphorylated p38MAPK increased with rhGM-CSF treatment. These findings indicate that rhGM-CSF enhances corneal wound healing by accelerating cell migration.     

Recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) induces human macrophage production of transforming growth factor-alpha

Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) is known as an inducer of proliferation and functional activation of myeloid cells. This study was carried out to characterize the effect of purified recombinant human GM-CSF (rhGM-CSF) on induction of TGF-alpha in macrophages. Using Northern blot analysis and immunoassays, we show here that rhGM-CSF markedly stimulates production of TGF-alpha messenger RNA and protein in normal tonsil macrophages. The findings are consistent with macrophages being a normal inducible source of TGF-alpha which may be an important mediator of various activities of GM-CSF both in hematopoietic and non-hematopoietic cells.     

Priming of the human neutrophil respiratory burst by granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-alpha involves regulation at a post-cell surface receptor level. Enhancement of the effect of agents which directly activate G proteins

Over the last few years, several studies showing that production of superoxide by neutrophils in response to chemotactic factors such as FMLP is enhanced after preincubation of the cells with granulocyte-macrophage (GM)-CSF or TNF-alpha have been published. Subsequent reports have indicated that this effect of the cytokines may be mediated by modulation of the number and/or affinity of surface receptors for FMLP. In the present study we have investigated the effect of preincubation with GM-CSF and TNF-alpha on the oxidative burst induced by sodium fluoride and guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)-agents which directly activate guanine-nucleotide binding proteins in neutrophils. Pretreatment of neutrophils with either GM-CSF or TNF-alpha dose-dependently enhanced the production of superoxide induced by NaF, as determined by the superoxide dismutase-inhibitable reduction of ferricytochrome c. Furthermore, preincubation of neutrophils with these cytokines enhanced the production of hydrogen peroxide induced by GTP gamma S in electroporated neutrophils. Because both NaF and GTP gamma S directly activate G proteins independently of external receptor-G protein interaction, these results imply that both GM-CSF and TNF-alpha alter the neutrophil signal transduction pathway in response to subsequent agonists independently of a modulation in the expression of the cell surface receptors for such agonists.     

Characterization of the priming effects of human granulocyte-macrophage colony-stimulating factor on human neutrophil leukotriene synthesis

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) is an in vitro and in vivo stimulator of human bone marrow myelomonocytic precursor cells and mature granulocyte and macrophage effector cells. We have compared the effect of GM-CSF on the synthesis of 5-lipoxygenase products induced by the chemotactic peptide fMet-Leu-Phe and the calcium ionophore A23187 in human neutrophils. Although GM-CSF alone did not stimulate detectable synthesis of products of the 5-lipoxygenase pathway, pre-incubation of neutrophils with 200 pM GM-CSF for 1 hour at 23 degrees C enhanced synthesis of leukotriene B4, its all-trans isomers and omega-oxidation products, and 5-hydroxyeicosatetraenoic acid in response to both the calcium ionophore A23187 (1.5 microM), and the chemotactic peptide fMet-Leu-Phe (0.1 microM). This priming effect of GM-CSF was maximal after a 60 min incubation at 23 degrees C, or after a 30 min preincubation at 37 degrees C. The effect of GM-CSF was maximal using a concentration of 1 nM. Enhancement of the leukotriene synthesis stimulated by A23187 was only observed when the cells were stimulated by the ionophore for periods of 3 minutes or less. In contrast, the enhancing effect of GM-CSF was still apparent when cells were exposed to fMet-Leu-Phe for as long as 15 minutes. Furthermore, the enhancing effect of GM-CSF was ablated when neutrophils were stimulated with A23187 and exogenous arachidonic acid. However, co-addition of exogenous arachidonic acid with fMet-Leu-Phe did not entirely mask the effect of GM-CSF. Possible mechanisms of action of GM-CSF are discussed.     

Glycosaminoglycans bind granulocyte-macrophage colony-stimulating factor and modulate its mitogenic activity and signaling in human osteoblastic cells

We recently demonstrated that granulocyte-macrophage colony-stimulating factor (GM-CSF) is an autocrine growth factor for human osteoblastic (hOB) cells. Since GM-CSF is a member of the heparin-binding factor family, we examined the interactions between GM-CSF and glycosaminoglycans (GAGs) present in the osteoblast microenvironment. Using a bioassay in which the mitogenic activity of recombinant human (rh) GM-CSF was measured after incubation in the presence of an hOB cell layer or extracellular matrix (ECM) produced by these cells, we showed that rhGM-CSF binds to GAG components present in the ECM and that the bound rhGM-CSF retains its ability to stimulate hOB cell proliferation. Heparan sulfate compounds on the hOB cell surface were also found to sequester GM-CSF. Moreover, treatment with sodium chlorate, an inhibitor of GAG sulfation, suppressed the mitogenic activity of rhGM-CSF on hOB cells. This inhibitory effect was rescued by a low dose of heparin. Heparin was also found to promote the effect of rhGM-CSF on hOB cell proliferation, allowing nonmitogenic high doses of rhGM-CSF to stimulate hOB cell growth. Western blot analysis showed that undersulfation of cellular GAGs by chlorate inhibited the increased tyrosine phosphorylation of proteins involved in GM-CSF signaling in cloned immortalized hOB cells. The data demonstrate that GM-CSF binds to proteoglycans on the hOB cell surface and in ECM produced by these cells and that the bound GM-CSF is biologically active. Furthermore, this study shows that cellular proteoglycans play an essential role in GM-CSF signaling and biological activity in hOBs. J. Cell. Physiol. 177:187–195, 1998. © 1998 Wiley-Liss, Inc.     

Evidence that de novo protein synthesis participates in a time-dependent augmentation of the chemotactic peptide-induced respiratory burst in neutrophils: Effects of recombinant human colony stimulating factors and dihydrocytochalasin B

We examined the effects of the recombinant human colony stimulating factors GM-CSF and G-CSF, cycloheximide (a protein synthesis inhibitor) and dihydrocytochalasin B (a microfilament disrupting agent) upon FMLP (N-formyl-methionyl-leucylphenylalanine)-stimulated O₂ ⁻ production by neutrophils. We confirmed a time dependent augmentation of O₂ ⁻ production following preincubation of neutrophils either alone or with colony stimulating factors. Furthermore, we found that GM-CSF, but not G-CSF, increased O₂ ⁻ production at some concentrations of the stimulus. Preincubation of neutrophils with cycloheximide in the absence of CSF caused a marked fall in O₂⁻-production that was first evident at 2 hours. The fall in O₂⁻-forming capacity caused by cycloheximide was much less pronounced if dihydrocytochalasin B was also included in the preincubation buffer. These findings suggest a previously unrecognized role for de novo protein synthesis in maintaining the ability of neutrophils to manufacture O₂⁻, and support earlier studies indicating that the cycling of FMLP receptors between the cell membrane and an intracellular compartment is important in determining the magnitude of the respiratory burst in FMLP-stimulated neutrophils.     

Enhanced Expression of Fc Receptors on Neutrophils from Calves with Leukocyte Adhesion Deficiency

The expression of Fc receptors for immunoglobulin G(IgG) and concanavalin A (con A)-binding receptors, luminol-dependent chemiluminescent (LDCL) responses, and the effect of anti-bovine IgG on LDCL responses were evaluated in neutrophils from Holstein calves with leukocyte adhesion deficiency (BLAD). Neutrophils from affected calves showed a 2.1- to 2.5-fold increase in Fc receptor expression compared with those of control calves by flow cytometric analysis. Con A-binding activities of neutrophils from affected calves were similar to those of control calves. Neutrophils from a calf with BLAD, when stimulated with zymosan opsonized with bovine serum (OPZ), heat-aggregated bovine IgG (Agg-bovine IgG), sheep red blood cells (SRBC) sensitized with anti-SRBC antibody (SRBC-anti-SRBC Ab), or con A had LDCL responses of 36 (P<0.05), 77, 126 and 119% of peak LDCL values of controls, respectively. The NBT-reducing value of neutrophils from a calf with BLAD when stimulated with Agg-bovine IgG after pretreatment with anti-bovine IgG was 116.5% of the values of neutrophils from control calves, but the difference was not significant. The LDCL responses of neutrophils from a control calf and a calf with BLAD stimulated with OPZ were inhibited markedly by pre-incubation with anti-bovine IgG antiserum at concentrations ranging from 1.25 to 20 or 40 μg/ml. Although an increase in Fc receptor expression on neutrophils from calves with BLAD was observed, the LDCL responses stimulated with SRBC-anti-SRBC Ab and NBT-reducing activity stimulated with Agg-bovine IgG after pretreatment with anti-bovine IgG did not correlate significantly with the increased Fc receptor expression. These results support that neutrophil functions mediated by the Fc receptors are associated synergistically with the presence of the complement receptor type 3 (CR3)(CD11b/CD18).     

Granulocyte-macrophage colony-stimulating factor as an autocrine survival-growth factor in human gliomas

We studied the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptors (GM-CSF.R) in 20 human brain gliomas with different tumor gradings and demonstrated constitutive high levels of both mRNA gene expression and protein production exclusively in the highest-grade tumors (WHO, III-IV grade). Five astrocytic cell lines were isolated in vitro from glioma cells, which had selectively adhered to plates pre-coated with rhGM-CSF. These cells were tumorigenic when xenografted to athymic mice, and produced GM-CSF constitutively in culture. Two lines, particularly lines AS1 and PG1, each from a patient with glioblastoma multiforme, constitutively over-expressed both GM-CSF and GM-CSF.R genes and secreted into their culture media biologically active GM-CSF. Different clones of the AS1 line, isolated after subsequent passages in vitro and then transplanted to athymic mice, demonstrated higher tumorigenic capacity with increasing passages in vivo. Cell proliferation was stimulated by rhGM-CSF in late-stage malignant clones, whereas apoptosis occurred at high frequency in the presence of blocking anti-GM-CSF antibodies. In contrast, rhGM-CSF did not induce any apparent effect in early-stage clones expressing neither GM-CSF nor GM-CSF.R. The addition of rhGM-CSF or rhIL-1β, to cultures induced the overproduction of both GM-CSF and its receptors and increased gene activation for several functional proteins (e.g. NGF, VEGF, VEGF.R1, G-CSF, MHC-II), indicating that these cells may undergo dynamic changes in response to environmental stimuli. These findings thus revealed: (1) that the co-expression of both autocrine GM-CSF and GM-CSF.R correlates with the advanced tumor stage; (2) that an important contribution of GM-CSF in malignant glioma cells is the prevention of apoptosis. These results imply that GM-CSF has an effective role in the evolution and pathogenesis of gliomas.     

Construction of a recombinant human GM-CSF/MCAF fusion protein and study on its in vitro and in vivo antitumor effects

A novel cytokine fusion protein was constructed by fusing granulocyte macrophage colony stimulat-ing factor (GM-CSF) with monocyte chemotactic activating factor (MCAF), which acts as a factor directing effector cells (monocytes) to a target site. The recombinant human GM-CSF/MCAF fusion protein could sustain the growth of GM-CSF-dependent cell line TF1 and was chemotactic for monocytes. The in vitro antitumor effect showed that rhGM-CSF/MCAF could activate monocytes to inhibit the growth of several human tumor cell lines, including a promyelocyte leukemia cell line HL-60, a lung adenocarcinoma cell line A549, a hepatoma cell line SMMC-7721 and a melanoma cell line Bowes. Furthermore, the cytotoxicity of monocytes activated by rhGM-CSF/MCAF against HL-60 and A549 was greater than that activated by GM-CSF or MCAF alone, even greater than that activated by a combina-tion of GM-CSF and MCAF, suggesting that the fusion protein has synergistic or enhanced effects. The in vivo anti-tumor effect indicated that     

Construction of a recombinant human GM-CSF/MCAF fusion protein and study on its in vitro and in vivo antitumor effects

A novel cytokine fusion protein was constructed by fusing granulocyte macrophage colony stimulating factor (GM-CSF) with monocyte chemotactic activating factor (MCAF), which acts as a factor directing effector cells (monocytes) to a target site. The recombinant human GM-CSF/MCAF fusion protein could sustain the growth of GMCSF-dependent cell line TF1 and was chemotactic for monocytes. Thein vitro antitumor effect showed that rhGM-CSF/MCAF could activate monocytes to inhibit the growth of several human tumor cell lines, including a promyelocyte leukemia cell line HL-60, a lung adenocarcinoma cell line A549, a hepatoma cell line SMMC-7721 and a melanoma cell line Bowes. Furthermore, the cytotoxicity of monocytes activated by rhGM-CSF/MCAF against HL-60 and A549 was greater than that activated by GM-CSF or MCAF alone, even greater than that activated by a combination of GM-CSF and MCAF, suggesting that the fusion protein has synergistic or enhanced effects. Thein vivo antitumor effect indicated that rhGM-CSF/MCAF had marked antitumor effect against A549 tumor in nude mice and even completely suppressed tumor formation. rhGM-CSF/MCAF was significantly more effective in inhibiting tumor growth than rhGM-CSF. Histological analysis showed that tumor site injected with rhGM-CSF/MCAF was infiltrated by a large number of monocytes while a sparse infiltration of monocytes was observed at the tumor site injected with rhGM-CSF or normal saline, suggesting that the antitumor effect of rhGM-CSF/MCAF was mediated by the recruitment of a large number of monocytes to the tumor site.     

Effect of opioid peptides on oxygen-dependent microbicidal activity of peripheral blood neutrophils

The effect of opioid peptides on the production of reactive oxygen by neutrophils in a nonfractionated leukocyte suspension and in a purified fraction of peripheral blood neutrophils was studied. Selective ??- and ??-agonists of peptide origin were shown to stimulate the spontaneous and inhibit zymosan-induced (15 ??g/mL) LDCL (luminol-dependent chemiluminescence) reactions of neutrophils in leukocyte suspension. ??-Endorphin caused a weaker inhibitory effect on zymosan-induced (15 ??g/mL) LDCL, and the ??2-agonost deltorphin II stimulated zymosan-induced (15 ??g/mL) LDCL only for 25 min of the experiment. ??-Endorphin and selective ??- and ??-agonists did not affect spontaneous or inhibited zymosan-induced (15 and 150 ??g/mL) LDCL in a purified fraction of peripheral blood neutrophils. Therefore, opioid peptides play an essential role in direct and indirect regulation of the oxygen-dependent system of neutrophil granulocyte bactericidal activity.     

Temporal adaptation of neutrophil oxidative responsiveness to n-formyl-methionyl-leucyl-phenylalanine. Acceleration by granulocyte-macrophage colony stimulating factor

This investigation was undertaken to clarify the mechanism by which purified recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) potentiates neutrophil oxidative responses triggered by the chemotactic peptide, FMLP. Previous studies have shown that GM-CSF priming of neutrophil responses to FMLP is induced relatively slowly, requiring 90 to 120 min of incubation in vitro, is not associated with increased levels of cytoplasmic free Ca2+, but is associated with up-regulation of cell-surface FMLP receptors. We have confirmed these findings and further characterized the process of GM-CSF priming. We found that the effect of GM-CSF on neutrophil oxidative responsiveness was induced in a temperature-dependent manner and was not reversed when the cells were washed extensively to remove the growth factor before stimulation with FMLP. Extracellular Ca2+ was not required for functional enhancement by GM-CSF and GM-CSF alone effected no detectable alteration in the 32P-labeled phospholipid content of neutrophils during incubation in vitro. Our data indicate that GM-CSF exerts its influence on neutrophils by accelerating a process that occurs spontaneously and results in up-regulation of both cell-surface FMLP receptors and oxidative responsiveness to FMLP. Thus, the results demonstrate that, with respect to oxidative activation, circulating endstage polymorphonuclear leukocytes are nonresponsive or hyporesponsive to FMLP; functional responsiveness increases dramatically as surface FMLP receptors are gradually deployed after the cells leave the circulation. Thus, as neutrophils mature, their responsiveness to FMLP changes in a manner which may be crucial for efficient host defense. At 37 degrees C, this process is markedly potentiated by GM-CSF. We conclude that endogenous GM-CSF, released systemically or at sites of infection and inflammation, potentially plays an important role in host defense by accelerating functional maturation of responding polymorphonuclear leukocytes.     

Construction of a recombinant human GM-CSF/MCAF fusion protein and study on itsin vitro andin vivo antitumor effects

A novel cytokine fusion protein was constructed by fusing granulocyte macrophage colony stimulating factor (GM-CSF) with monocyte chemotactic activating factor (MCAF), which acts as a factor directing effector cells (monocytes) to a target site. The recombinant human GM-CSF/MCAF fusion protein could sustain the growth of GMCSF-dependent cell line TF1 and was chemotactic for monocytes. Thein vitro antitumor effect showed that rhGM-CSF/MCAF could activate monocytes to inhibit the growth of several human tumor cell lines, including a promyelocyte leukemia cell line HL-60, a lung adenocarcinoma cell line A549, a hepatoma cell line SMMC-7721 and a melanoma cell line Bowes. Furthermore, the cytotoxicity of monocytes activated by rhGM-CSF/MCAF against HL-60 and A549 was greater than that activated by GM-CSF or MCAF alone, even greater than that activated by a combination of GM-CSF and MCAF, suggesting that the fusion protein has synergistic or enhanced effects. Thein vivo antitumor effect indicated that rhGM-CSF/MCAF had marked antitumor effect against A549 tumor in nude mice and even completely suppressed tumor formation. rhGM-CSF/MCAF was significantly more effective in inhibiting tumor growth than rhGM-CSF. Histological analysis showed that tumor site injected with rhGM-CSF/MCAF was infiltrated by a large number of monocytes while a sparse infiltration of monocytes was observed at the tumor site injected with rhGM-CSF or normal saline, suggesting that the antitumor effect of rhGM-CSF/MCAF was mediated by the recruitment of a large number of monocytes to the tumor site.     

The effect of cytokines on bactericidal activity of murine neutrophils

Abstract A range of recombinant cytokines have now been shown to modify aspects of the phenotype and function of human and murine neutrophils. However, few reports describe modification of the bactericidal activity of neutrophils. We therefore examined the recombinant murine cytokines tumor necrosis factor-α (TNF-α, 10–1000 ng ml⁻¹) and granulocyte macrophage-colony stimulating factor (GM-CSF, 10–1000 U ml⁻¹) for their ability to increase the bacterial killing capacity of murine neutrophils. Neutrophils from either bone marrow (fresh or cultured), or peritoneal exudates, or abscesses, were pre-incubated with either cytokine for 30–60 min and the killing of Proteus mirabilis, Escherichia coli , or Bacteriodes fragilis was examined in the presence or absence of serum over a 90 min period. Only for one combination was a small but significantly enhanced level of bacterial killing observed, the phagocytic killing of P. mirabilis by peritoneal exudate neutrophils in the presence of GM-CSF and serum. With this exception there was no enhancement of bacterial killing for the range of combinations of neutrophils and bacterial species tested. In contrast, at the concentrations tested for effect on bactericidal activity, TNF-α and GM-CSF were able to significantly upregulate CR3(but not FcγRII) expression on mouse neutrophils. There results indicate that upregulation of CR3 as an index of neutrophil activation does not necessarily correlate with increased bactericidal activity.     

Granulocyte/macrophage colony-stimulating factor primes human neutrophils for increased diacylglycerol generation in response to chemoattractant

Pretreatment of human neutrophils with granulocyte macrophage-colony stimulating factor (GM-CSF) augments several biological responses to chemoattractants (e.g. the respiratory burst, degranulation, and chemotaxis). However, little is known regarding the intracellular effects of priming with GM-CSF. In the present study, we have investigated the effects of GM-CSF on the generation of diacylglycerol (DAG), a proposed mediator of neutrophil responses. GM-CSF alone produced only a small increase in cellular DAG mass, which was most apparent after 30 min. GM-CSF pretreatment (60 min), however, caused a striking augmentation in DAG generation in response to the chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP), compared with neutrophils preincubated without GM-CSF. The augmentation in DAG generation correlated with an enhancement by GM-CSF of superoxide generation in response to fMLP. The data suggest that GM-CSF may exert some of its biological effects by enhancing DAG generation in response to a second agonist.     

The effects of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) on human osteoblast-like cells

The activity of human osteoblast-like cells cultured in vitro is regulated by a number of factors, which include systemic hormones as well as agents that can be produced locally within bone. Several cytokines and growth factors have been demonstrated to be produced by osteoblasts themselves, and this includes granulocyte-macrophage colony-stimulating factor (GM-CSF). In this report we show that recombinant human GM-CSF (rhGM-CSF) modulates the activities of osteoblast-like cells derived from human trabecular bone in vitro. rhGM-CSF stimulated the proliferation of the cultured human osteoblast-like cells, but antagonised the induction by 1,25(OH)2D3 of osteocalcin synthesis and alkaline phosphatase activity, two characteristic products of osteoblasts. rhGM-CSF however, had no appreciable effect on the production of prostaglandin E2, or on the plasminogen activator activity associated with human osteoblast-like cells. These results are the first report of which we are aware of an apparently direct action of GM-CSF on cells of the osteoblast phenotype. These studies indicate that GM-CSF represents another haematological factor that can potentially exert regulatory actions on human osteoblast-like cells. GM-CSF may therefore be a potential paracrine/autocrine regulator of osteoblast activity.     

Effect of opioid peptides on oxygen-dependent microbicidity of peripheral blood neutrophils

Investigation ofopioid peptide effect on the production of reactive oxygen species by neutrophils in non-fractionated leukocyte suspension and in purified fraction of peripheral blood neutrophils is disclosed in this work. It was determined that selective delta- and micro-agonists of peptide origin stimulated the spontaneous and suppressed 15 mkg/ml zymosan-induced LDCL (luminol-dependent chemiluminescence) reaction of neutrophils in leukocyte suspension. beta-endorphin was found to render less marked suppressive action on 15 mkg/ml zymosan-induced LDCL, and delta2-agonist deltorphin 2 promoted 15 mkg/ml zymosan-induced LDCL only toward the 25 minutes of the experiment. beta-endorphin and selective d- and m- agonists did not affect the spontaneous and suppressed 15 mkg/ml and 150 mkg/ml zymosan-induced neutrophil LDCL. Therefore, opioid peptides play essential role in the process of direct and indirect regulation of oxygen-dependent system of neutrophil granulocyte bactericidal activity.     

Redox state and O2*- production in neutrophils of Crohn's disease patients

The aim of this in vitro study was to evaluate the intracellular redox state and respiratory burst (RB) in neutrophils of patients with Crohn's disease (CD). The intracellular redox state and RB in neutrophils was assessed by the superoxide anion (O2*-) production induced in these cells after stimulation by various factors related to the molecular mechanisms that, if altered, may be responsible for an abnormal immune response. This can, in part, cause the onset of inflammation and tissue damage seen in CD. This study demonstrated a decreased glutathione/glutathione disulfide (GSH/GSSG) ratio index of an increased oxidative state in CD patient neutrophils. Moreover, our findings showed a decrease in tumor necrosis factor (TNF-alpha)- or phorbol 12-myristate 13-acetate (PMA)-induced O2*- production in CD patient neutrophils adherent to fibronectin as compared with controls. A decreased adhesion was also demonstrated. For this reason, the involvement of altered mechanisms of protein kinase C (PKC) and beta-integrin activation in CD patient neutrophils is suggested. These data also showed that the harmful effects of TNF-alpha cannot be caused by excessive reactive oxygen species (ROS) production induced by neutrophils. Decreased cell viability after a prolonged time of adhesion (20 hrs) was also measured in CD patient neutrophils. The findings of this study demonstrate, for the first time, that granulocyte-macrophage colony-stimulating factor (GM-CSF), a compound recently used in CD therapy, is able to activate the RB for a prolonged time both in control and CD patient neutrophils. Increased viability of CD patient neutrophils caused by GM-CSF stimulation was also observed. In conclusion, our results indicate that decreased O2*- production and adhesion, caused, in part, by an anomalous response to TNF-alpha, together with low GSH level and low cell viability, may be responsible for the defective neutrophil function found in CD patients. This can contribute to the chronic inflammation and relapses that characterize this pathology. A possible role of GM-CSF in inducing O2*- production and in restoring the defensive role of neutrophils in CD patients is suggested.     

Influence of neopterin on the generation of reactive oxygen species in human neutrophils

Neopterin is synthesized by human monocyte-derived macrophages primarily upon stimulation with the cytokine interferon-gamma. We studied the influence of neopterin on the generation of reactive oxygen species (ROS) in human peripheral blood neutrophils. Radical formation was measured using a biochemiluminometer. Neutrophils were isolated from peripheral blood of healthy donors. The generation of ROS by neutrophils suspended in Earl's solution (pH=7.4) at 37 degrees C was investigated by monitoring of chemiluminescence using luminol and lucigenin as light emitters. Neopterin induced chemiluminescence in suspensions of neutrophils in the presence of luminol, but not of lucigenin. Neopterin affected only adhesive cells. Addition of neopterin into the suspension of the cells involving D-mannitol, L-histidine and diazabicyclo[2.2.2]octane (DABCO) decreased luminol-dependent chemiluminescence (LDCL) of the neutrophils. The action of superoxide dismutase (SOD) and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) reduced neopterin-induced LDCL of neutrophils. Data suggest that neutrophils respond on exposure to neopterin with additional generation of singlet oxygen, hydroxyl radical and nitric oxide by nicotinamide adenine dinucleotide phosphate (NADPH)-independent pathways.