Effects of recombinant human granulocyte colony-stimulating factor administration on neutrophil phenotype and functions

BACKGROUND AND OBJECTIVE: Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is currently used for treatment of various types of neutropenia, treatment of aplastic anemia, mobilization of peripheral blood progenitor cells. However, rhG-CSF is not only a growth factor for the myeloid lineage, but it also acts as a modulator of neutrophil behavior. The aim of the present review article is to examine the following aspects of rhG-CSF therapy: 1) does rhG-CSF influence neutrophil functions, and in particular their microbicidal properties? 2) does rhG-CSF modify neutrophil phenotype? 3) If so, what are the mechanisms potentially involved? EVIDENCE AND INFORMATION SOURCES: The author of the present article has been working in the field of rhG-CSF effects on neutrophil function, contributing original papers in peer-reviewed journals. In addition, the present review critically examines articles and abstracts published in journals covered by the Science Citation Index and Medline. STATE OF ART AND PERSPECTIVES: Treatment with rhG-CSF causes enhancement of functions such as phagocytosis, superoxide anion generation, chemiluminescence, bacterial killing, and ADCC. Neutrophil phenotype changes after rhG-CSF administration: immediate effects cause direct activation of circulating neutrophils, but delayed effects are characterized by increased surface expression of important effector molecules directly involved in neutrophil functions, such as CD14, CD32, CD64. These effects may have useful clinical consequence in patients who show an increased risk of infections, such as cancer patients, subjects with hematologic diseases (myelodysplasia, aplastic anemia), congenital diseases characterized by neutropenia, and patients with AIDS. Other changes which characterize neutrophils after rhG-CSF administration are represented by significant impairment of CD16 expression, chemotaxis, and reduced in vivo migration of neutrophils to inflammatory sites. These effects may be explained by bone marrow modification due to rhG-CSF therapy. In fact, treatment with rhG-CSF causes a significant acceleration of transit time of cells belonging to the myeloid lineage, along with amplification of the mitotic pool and a relative decrease of elements of the post-mitotic pool. It is possible that, because of the accelerated bone marrow transit time of myeloid cells, rhG-CSF causes a relative immaturity of circulating neutrophils. It is known that both CD16 expression and chemotaxis properties are acquired by neutrophils in the late stages of maturation, but the time necessary to acquire full functional maturity seems to be shortened by rhG-CSF administration, and this kinetic aspect may play a non-negligible role in the modification of neutrophil behavior.     

Up-regulation of neutral endopeptidase (CALLA) in human neutrophils by granulocyte-macrophage colony-stimulating factor

Neutral endopeptidase 24.11 (NEP/CALLA/CD10), an enzyme expressed on early lymphoid progenitors, neutrophils, and various other cell types, inactivates many biologically active peptides, including the bacterial chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLP). Inhibition of CD10/NEP on the surface of human neutrophils (PMNs) in vitro inhibits migration toward this chemotaxin, suggesting that enzymatic inactivation by NEP regulates the neutrophil response to fMLP. Because PMNs in inflammatory sites are exposed to various cytokines, we evaluated the effects of selected cytokines on CD10/NEP activity in vitro. Of five cytokines tested--interleukin-1 (IL-1), IL-6, and IL-8, granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor (GM-CSF)--GM-CSF provided the most consistent increase in surface NEP activity. Low concentrations (10(-9)-10(-7) M) of GM-CSF increased NEP activity in a time- and concentration-dependent manner to more than 225% that of control (phosphate-buffered saline-treated) cells. Cytofluorometry of cells stained with a fluorescent antibody to CD10 indicated that GM-CSF increased expression of surface CD10/NEP antigen in a similar manner. The effect of GM-CSF on NEP activity was enhanced still further by simultaneous exposure to IL-1, suggesting that combinations of cytokines may direct and regulate the neutrophil response within an inflammatory site. Rapid upregulation of CD10/NEP underscores the importance of this enzyme for control of peptide mediators of inflammation.     

Oleic acid increases cell surface expression and activity of CD11b on human neutrophils

Traumatic bone injury frequently results in the release of marrow-derived fatty material into the circulation. This may lead to the syndrome of fat embolism, associated with the generation of free fatty acids, the sequestration of neutrophils in the lungs, and the subsequent development of acute respiratory distress. Neutrophil accumulation in tissues requires their adherence to vascular endothelial cells and involves the beta2 integrin, CD11b/CD18 (Mac-1). We now report that the exposure of isolated human neutrophils to oleic acid causes a rapid increase in the cell surface expression and affinity state of CD11b, particularly under acidic conditions that are typical of inflammatory sites. Oleic acid also triggers neutrophil aggregation and neutrophil adherence to both fibrinogen-coated surfaces and confluent cultures of HUVEC. These processes are blocked by CD11b-specific inhibitors, including neutrophil-inhibitory factor and mAbs to CD11b. These observations may help explain the etiology of so-called fat embolism wherein trauma-induced release of fatty material causes pulmonary neutrophil accumulation and the development of acute respiratory distress.     

Granulocyte dysplasia and dysfunction, and CD11/CD18 defects in myelodysplastic syndromes

In myelodysplastic syndromes (MDS), dysplastic changes in neutrophils are a common feature reflecting the total degree of bone marrow dysplasia. Furthermore, granulocyte function is abnormal, so that a high risk of life-threatening infections has been documented. In this review we shall focus on the defects of both granulocytes and their CD11b/CD18 glycoprotein complex, which regulate granulocyte adherence, locomotion, diapedesis and migration into inflammatory sites, in patients suffering from primary MDS. The defective surface membrane glycoprotein expression of myelodysplastic phagocytes is not only a useful diagnostic tool, but also a powerful prognostic one, since MDS patients with such defects present both an increased susceptibility to infections and a decreased survival. Moreover, the administration of colony-stimulating factors is known to be able to elicit long-lasting improvement in neutrophil count, CD11b/CD18 expression and function, marrow myeloid maturation, and possibly to decrease bacterial infections in MDS patients.     

Effects of stem cell factor (SCF) on human marrow neutrophil, neutrophil/macrophage mixed, macrophage and eosinophil progenitor cell growth

The effects of stem cell factor (SCF) on the subpopulations of granulocyte/macrophage colony-forming units (CFU-GM) were examined. Hematopoietic progenitor cells were enriched from normal adult bone marrow specimens by immunomagnetic beads using an anti-CD34 antibody and lineage marker antibodies for positive selection and negative selection, respectively. SCF enabled neutrophil and neutrophil/macrophage mixed progenitors to respond to granulocyte/macrophage colony-stimulating factor (GM-CSF) or interleukin 3 (IL-3) and to develop the colony and further cluster formation. The neutrophil colonies stimulated by GM-CSF or IL-3 consisted mainly of immature cells, while the colonies stimulated by granulocyte colony-stimulating factor (G-CSF) consisted of mature neutrophils irrespective of the addition of SCF. In macrophage and eosinophil lineages, SCF augmented the colony formation in the presence of GM-CSF or IL-3, whereas the enhancement of total progenitor cell growth (colonies plus clusters) was not so marked as compared with the neutrophil lineage. Time-course observation revealed that SCF could stimulate macrophage and eosinophil progenitors to form colonies rapidly. These findings indicate that SCF acts on late myeloid progenitor cells in manners different from the lineages of commitment.     

Activation of human monocyte functions by tumor necrosis factor: rapid priming for enhanced release of superoxide and erythrophagocytosis, but no direct triggering of superoxide release

Tumor necrosis factor (TNF), like granulocyte-macrophage colony-stimul ating factor (GM-CSF), rapidly primed human monocytes for enhanced release of superoxide (O-2) stimulated by receptor-mediated agonists, N-formyl-methionyl-leucyl-phenylalanine (FMLP) and concanavalin A (Con A), but not by phorbol myristate acetate (PMA), which bypasses the receptors to stimulate the cells. The optimal priming was obtained by pretreatment of suspended monocytes with 10 U/mL TNF for 10 minutes at 37 degrees C. The potency of the maximal priming effect was TNF> GM-CSF, and the combined effect of TNF and GM-CSF was greater than that of each cytokine alone. GM-CSF induced an increase in cytoplasmic pH but TNF did not. These findings suggest that TNF and GM-CSF activate monocytes through different mechanisms. TNF and GM-CSF by themselves never triggered O-2 release in suspended monocytes or monocytes adherent to endothelial cells, although both cytokines triggered massive release of O-2 in human neutrophils. In additions, TNF and GM-CSF induced tyrosine phosphorylation of a 42-kD protein in neutrophils but not in monocytes. These findings suggest that the TNF-receptor- or GM-CSF-receptor-mediated signaling pathways for triggering O-(2) release is active in neutrophils but inactive or defective in monocytes. TNF also enhanced phagocytosis of sialidase-treated autologous erythrocytes by monocytes, and this effect was further potentiated in the presence of autologous fresh serum. The significant enhancement of erythrophagocytosis was obtained at 1 U/mL TNF. At this concentration of TNF, the expression of C3bi-receptor (CD11b/CD18) was upregulated. These findings show that TNF rapidly primes human monocytes for enhanced release of O-(2) and erythrophagocytosis and suggest that TNF activates monocytes through autocrine or paracrine mechanisms at the inflammatory sites inasmuch as TNF is primarily produced by activated monocytes/macrophages.     

Neutrophil apoptosis is associated with a reduction in CD16 (Fc gamma RIII) expression

Resolution of inflammation involves removal of recruited neutrophils from inflamed sites via a noninflammatory mechanism, possibly involving neutrophil apoptosis and engulfment/phagocytosis by macrophages. In this study, we describe the reduction in surface expression (> 90%) of the neutrophil molecule Fc gamma RIII (CD16) during in vitro culture at 37 degrees C, which was found to be temporally associated with the appearance of neutrophils with apoptotic morphology during in vitro culture and inhibitable by granulocyte-macrophage colony-stimulating factor (GM-CSF), which postpones apoptosis in the neutrophil. By using dual fluorescence analysis, CD16 "low" expressing neutrophils showed reduced staining with the DNA-binding dye propidium iodide, suggesting that CD16 low expressing neutrophils were apoptotic. Separation of CD16 "high" and CD16 "low" expressing neutrophils by fluorescence-activated cell sorting revealed that morphologically apoptotic cells exhibited the CD16 low phenotype. We did not observe similar marked changes in expression of other neutrophil surface molecules (including other phosphatidylinositol (PI)-linked molecules), indicating that generalized loss of surface molecules does not occur during apoptosis. We believe this to be the first reported cell type-specific membrane alteration in a surface glycoprotein associated with apoptosis, suggesting that the program of cell death in the neutrophil, in addition to morphologic and nuclear changes, includes alterations in expression of surface receptors.     

Raised neutrophil phospholipase A2 activity and defective priming of NADPH oxidase and phospholipase A2 in sickle cell disease

Intermittent painful crises due to vasoocclusion are the major clinical manifestation of sickle cell disease (SCD), but subclinical episodes may also occur. There is sparse evidence for the involvement of neutrophils in the pathophysiology of SCD, but production of cytokines by the damaged endothelium might influence neutrophil function and modulate responses to subsequent cytokine exposure. In addition, the activation of neutrophils in the microcirculation could itself exacerbate vasoocclusion. To test whether neutrophil inflammatory responses were altered in SCD, neutrophil phospholipase A2 and NADPH oxidase activity in response to in vitro priming by granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF-alpha) were measured both during and between painful crises. Resting levels of neutrophil phospholipase A2 activity in steady-state SCD (4.0% +/- 0. 5% of total cell radioactivity) were raised relative to control values (2.0% +/- 0.2%, n = 10, P = .008). There was no defect of agonist-stimulated phospholipase A2 or NADPH oxidase activity in steady-state SCD; however, the ability of phospholipase A2 to respond to priming with GM-CSF was attenuated to 63% +/- 17% of control values (n = 10, P = .04). Similarly, neutrophil NADPH oxidase activity after priming with GM-CSF and TNF-alpha was, respectively, 65% +/- 11% (n = 7, P = .03) and 57% +/- 7% of control (n = 10, P = .007) in steady-state disease, and was further reduced during painful vasoocclusive crises to 34% +/- 9% and 25% +/- 3% of control for GM-CSF and TNF-alpha, respectively. These data were not explained by poor splenic function or any racial factor, as normal cytokine responses were seen in splenectomized patients in remission from Hodgkin's disease and in healthy Afro-Caribbean subjects. Abnormal neutrophil cytokine priming responses were not observed in either patients with rheumatoid arthritis or iron-deficiency anemia. Our findings are indicative of an ongoing inflammatory state in SCD between painful crises involving neutrophil activation and an abnormality of cytokine-regulated neutrophil function, which may compromise the host defenses against certain microorganisms.     

Evidence for the activation of myeloperoxidase by f-Meth-Leu-Phe prior to its release from neutrophil granulocytes

Activity and release of myeloperoxidase (MPO) was measured in heparinized whole blood samples after activation of neutrophil granulocytes by the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP) using two different methods: (i) by determination of the amount of MPO released into the blood plasma using a MPO enzyme-immunoassay, and (ii) simultaneously, by measuring the remaining activity within the neutrophils by flow cytometry using the Bayer Technicon H3. Although a part of MPO was released immediately after addition of fMLP, remaining MPO activity within the neutrophils surprisingly increased during the first minutes after incubation. Subsequently, MPO activity dropped due to a continuous release of MPO. In addition to fMLP, granulocyte-macrophage colony stimulating factor (GM-CSF) enhanced MPO activity in neutrophils. These results indicate that MPO is present in resting granulocytes in an inactive or only partially active form and is activated by fMLP and GM-CSF.     

Isolation of cDNA encoding guinea pig neutrophil cationic antibacterial polypeptide of 11 kDa (CAP11) and evaluation of CAP11 mRNA expression during neutrophil maturation

Neutrophils contain various antibacterial polypeptides and proteins in the granules that contribute to the killing of microorganisms. Recently, we have purified a cationic antibacterial polypeptide of 11 kDa (CAP11) from guinea pig neutrophil granules. CAP11 is a homodimer of G1LRKKFRKTRKRIQKLGRKIGKTGRKVWKAWREYGQIPYPCRI43 joined with one disulfide bond. In this study, to understand the regulation of CAP11 expression, we isolated and analyzed cDNA encoding CAP11. Furthermore, we investigated the expression of CAP11 mRNA during neutrophil maturation and localization of CAP11 among neutrophil granule subsets. Sequence analysis of CAP11 cDNA isolated from guinea pig bone marrow cells using rapid amplification of cDNA ends technique indicated that CAP11 is synthesized as a precursor comprising 178 amino acid residues, which is composed of a signal peptide (N-terminal 29 residues), a propeptide (106 residues), and a C-terminal mature peptide (43 residues). Interestingly, the predicted CAP11 precursor displayed the characteristic features of cathelicidins, a novel protein family of antibacterial polypeptides with a conserved cathelin-like pro-region and a variable C-terminal antibacterial domain. Northern blot and Western blot analyses using neutrophils, macrophages, eosinophils, mononuclear cells, and bone marrow cells revealed that only neutrophils and bone marrow cells expressed CAP11 mRNA and contained CAP11, suggesting that expression of CAP11 is neutrophil lineage-specific. Furthermore, Northern blot analysis using bone marrow cells separated according to their maturation stages showed that CAP11 mRNA was predominantly expressed in the cells at later stages of neutrophil maturation. Consistent with this, in situ hybridization using CAP11-specific cRNA probe demonstrated that CAP11 mRNA was primarily expressed at metamyelocyte stage. In addition, extracellular release assay revealed that CAP11 was readily released from neutrophils accompanied with gelatinase by low concentrations of N-formyl-Met-Leu-Phe without release of specific and azurophil granule components, and CAP11 was found to be exclusively present in the fraction containing gelatinase granules, prepared by Percoll density gradient centrifugation. Together these observations indicate that CAP11 is a member of cathelicidin family and its mRNA is preferentially expressed at the later stage of neutrophil maturation (i.e. metamyelocyte stage). Furthermore, CAP11 may be stored in the granule subset, possibly the gelatinase granule.     

In vivo suppression of immune complex-induced alveolitis by secretory leukoproteinase inhibitor and tissue inhibitor of metalloproteinases 2

The pulmonary tree is exposed to neutrophil-derived serine proteinases and matrix metalloproteinases in inflammatory lung diseases, but the degree to which these enzymes participate in tissue injury remains undefined, as does the therapeutic utility of antiproteinase-based interventions. To address these issues, an in vivo rat model was examined in which the intrapulmonary deposition of immune complexes initiates a neutrophil-mediated acute alveolitis. In vitro studies demonstrated that rat neutrophils can release neutrophil elastase and cathepsin G as well as a neutrophil progelatinase, which was subsequently activated by either chlorinated oxidants or serine proteinases. Based on structural homologies that exist between rat and human neutrophil proteinases, rat neutrophil elastase and cathepsin G activities could be specifically regulated in vitro by recombinant human secretory leukoproteinase inhibitor, and rat neutrophil gelatinase activity proved sensitive to inhibition by recombinant human tissue inhibitor of metalloproteinases 2. When either of the recombinant antiproteinases were instilled intratracheally, in vivo lung damage as assessed by increased permeability or hemorrhage was significantly reduced. Furthermore, the coadministration of the serine and matrix metalloproteinase inhibitors almost completely prevented pulmonary damage while effecting only a modest decrease in neutrophil influx. These data support a critical role for neutrophil-derived proteinases in acute lung damage in vivo and identify recombinant human secretory leukoproteinase and recombinant human tissue inhibitor of metalloproteinases 2 as potentially efficacious interventions in inflammatory disease states.     

CD50 monoclonal antibodies inhibit neutrophil activation

The constitutive high expression of CD50 (ICAM-3) on resting leukocytes, coupled with the observation that CD50 is the primary LFA-1 ligand on resting T cells, suggests that CD50 may be an important LFA-1 ligand in the initiation of the immune/inflammatory response. CD50 mAbs have been reported to increase homotypic adhesion of lymphocytes, and lymphocyte adhesion to HUVEC and extracellular matrix proteins. In this study, the effects of CD50 mAbs on neutrophil activation were examined. CD50 mAbs were found to inhibit neutrophil adhesion induced by FMLP and 12-O-tetradecanoyl-phorbol-13-acetate to resting and TNF-activated HUVEC. CD50 mAbs also inhibited neutrophil adhesion stimulated by CD66a, CD66b, CD66c, and CD66d mAbs to HUVEC. CD50 mAbs inhibited the up-regulation of CD11b/CD18 to the neutrophil surface, and the down-regulation of surface CD62L expression. The potential contribution of src family kinases to the previously described tyrosine kinase activity associated with CD50 in neutrophils was also examined. hck and lyn were found to account for much of the tyrosine kinase activity associated with CD50 in neutrophils. The data indicate that CD50 in neutrophils functions not only as a potential ligand for LFA-1, but also regulates the surface expression and activity of CD11b/CD18 and CD62L. In contrast to the effects in lymphocytes, CD50 appears to function as a negative regulator of neutrophil activation.     

Rat blood neutrophils express very late antigen 4 and it mediates migration to arthritic joint and dermal inflammation

Blood neutrophils contribute to joint injury in human and experimental models of arthritis. Neutrophil migration out of the blood in joint inflammation involves both the CD18 (beta2) integrins and a CD18 integrin-independent pathway. To investigate this migration, radiolabeled rat blood neutrophils were used to measure neutrophil accumulation in the inflamed joints of rats with adjuvant arthritis and the role of leukocyte integrins in migration to these joints and to dermal inflammation was determined. Neutrophils migrated rapidly (<2 h) to the inflamed joints 14-18 d after immunization with adjuvant. Blocking monoclonal antibodies (mAbs) to both LFA-1 and Mac-1 together, as well as a mAb to CD18, inhibited neutrophil accumulation in the inflamed joints by 50-75%. However, migration to dermal inflammation induced by C5a(des Arg)' tumor necrosis factor alpha, lipopolysaccharide, and poly-inosine:cytosine was inhibited by approximately 90%. Flow cytometry revealed the expression of low levels of very late antigen 4 (VLA-4) on nearly all rat blood neutrophils. Treatment with anti-VLA-4 plus anti-LFA-1 but neither mAb alone, strongly (60-75%) inhibited neutrophil accumulation in arthritic joints. This mAb combination also inhibited neutrophil migration to dermal inflammatory reactions by 30-70%. Blocking VLA-4 together with the CD18 integrins inhibited neutrophil accumulation by 95-99%, virtually abolishing neutrophil accumulation in cutaneous inflammation. A similar blockade of VLA-4 and CD18 decreased neutrophil accumulation in the inflamed joints by 70-83%, but a significant portion of the neutrophil accumulation to these joints still remained. In conclusion, rat blood neutrophils express functional VLA-4 that can mediate neutrophil migration to both inflamed joints and dermal inflammatory sites. VLA-4 appears to be able to substitute for LFA-1 in this migration and is particularly important for accumulation in inflamed joints. However, there exists an additional CD18- and VLA-4-independent pathway of neutrophil migration to arthritic joints that is not involved in acute dermal inflammation.     

Age-dependent alterations of human recombinant GM-CSF effects on human granulocytes

The granulocyte-macrophage colony stimulating factor (GM-CSF) is an important in vivo regulator of granulopoiesis and neutrophil functions. It is well-known that the immune response and the transmembrane signalling in immune cells change with aging. We wished to elucidate the effects of GM-CSF in itself and in priming the activities of other inflammatory agents on neutrophils of elderly persons. Neutrophils of 20 healthy elderly (aged 60-90 years) and 20 healthy young (aged 20-25 years) subjects were studied for superoxide anion production, intracellular free calcium mobilization, antibody dependent cellular cytotoxicity (ADCC) and intracellular killing activities. It was found that GM-CSF is unable to prime neutrophils of elderly subjects to the action of FMLP, metenkephalin or opsonized zymosan. By the use of Pertussis toxin and H7 it was demonstrated that a different signal transduction pathway in neutrophils of elderly subjects is activated by GM-CSF or FMLP if compared to that of young subjects. These results suggest that the lack of priming could contribute to the greater susceptibility of the elderly to infections and that the change of the signal transduction mechanism in neutrophils of elderly subjects might partly explain this phenomenon.     

Epstein-Barr virus induces GM-CSF synthesis by monocytes: effect on EBV-induced IL-1 and IL-1 receptor antagonist production in neutrophils

Neutrophils play an important role in the control of viral infections by releasing a variety of potent agents. We previously demonstrated that Epstein-Barr virus (EBV) binds to human neutrophils and stimulates cytokine synthesis including interleukin-1 (IL-1) and IL-1 receptor antagonist (IL-1Ra). Since neutrophil functions are known to be modulated by the priming effect of granulocyte-macrophage colony-stimulating factor (GM-CSF), we therefore investigated the cellular source of GM-CSF synthesis following treatment of leukocytes with EBV and the effect of GM-CSF on the production of IL-1, IL-1Ra, and superoxide by EBV-treated neutrophils. In enriched-cell populations, only monocytes were found to produce GM-CSF in response to EBV, which was maximal after 12 h of incubation. The results obtained with UV-irradiated particles or EBV neutralized with monoclonal antibody 72A1 suggest that contact between the cell and the gp350 of the viral envelope is sufficient to induce the release of GM-CSF. On the other hand, GM-CSF differentially upregulated EBV-induced IL-1 and IL-1Ra production by neutrophils. Pretreatment of neutrophils with GM-CSF prior to EBV activation synergistically enhanced the production of IL-1 alpha and IL-1 beta, but only marginally affected IL-1Ra synthesis. In addition, GM-CSF was also found to synergistically enhance the superoxide production by neutrophils in response to EBV. Molecular analysis showed that GM-CSF did not alter the IL-1 beta and IL-1Ra mRNA synthesis induced by EBV, suggesting that GM-CSF could act at a posttranslational level. Local production of GM-CSF by monocytes in tissues invaded by EBV could serve to potentiate the host defense mechanisms directed toward the destruction of the infectious virus.     

Local anesthetic lidocaine inhibits the effect of granulocyte colony-stimulating factor on human neutrophil functions

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) enhanced superoxide (O2-) release in human neutrophils stimulated by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) and inversely regulated the surface expression of cellular adhesion molecules, leukocyte adhesion molecule-1 (LAM-1) and CD11b/CD18 leukocyte integrin, on human neutrophils; that is, rhG-CSF downregulated the expression of LAM-1 and upregulated the expression of CD11b on neutrophils. The cationic local anesthetic lidocaine inhibited not only FMLP-induced O2- release in neutrophils but also FMLP-induced CD11b upregulation and LAM-1 downregulation on neutrophils in a dose-dependent manner. Lidocaine also abolished the priming effect of rhG-CSF for enhanced release of O2- in neutrophils and inhibited rhG-CSF-induced CD11b upregulation and LAM-1 downregulation on neutrophils in a dose-dependent manner. These findings suggest that lidocaine inhibits human neutrophil functions, such as adherence to endothelial cells, by interfering with the expression of cellular adhesion molecules on neutrophils, and that lidocaine might have anti-inflammatory properties by inhibiting the effect of inflammatory cytokines.     

Superoxide dismutase mimetics inhibit neutrophil-mediated human aortic endothelial cell injury in vitro

In this study, we evaluated the ability of low molecular weight manganese-based superoxide dismutase mimetics to attenuate neutrophil-mediated oxygen radical damage to human aortic endothelial cells in vitro. Human neutrophils, when exposed to tumor necrosis factor-alpha and the complement compound C5a, induced endothelial damage assessed by the release of 51Cr into the medium. This damage correlated with the amount of superoxide generated by neutrophils. Three superoxide dismutase mimetics, with catalytic rate constants for superoxide dismutation ranging from 4 to 9 x 10(7) M-1 S-1, inhibited neutrophil- or xanthine oxidase-mediated endothelial cell injury in a concentration-dependent manner. A similar manganese-based compound with no detectable superoxide dismutase activity was ineffective in inhibiting injury. Fluorescent studies of the neutrophil respiratory burst showed that the superoxide dismutase mimetics were protective without interfering with the generation of superoxide by activated neutrophils. Catalase, elastase inhibitors, and desferrioxamine mesylate (an iron chelator and hydroxyl radical scavenger) were not protective against cell injury. This investigation demonstrates that neutrophil-mediated human aortic endothelial cell injury in vitro is mediated by the superoxide anion and that low molecular weight manganese-based superoxide dismutase mimetics are effective in abrogating this damage.     

Pseudomonas pyocyanin increases interleukin-8 expression by human airway epithelial cells

Pseudomonas aeruginosa, an opportunistic human pathogen, causes acute pneumonia in patients with hospital-acquired infections and is commonly associated with chronic lung disease in individuals with cystic fibrosis (CF). Evidence suggests that the pathophysiological effects of P. aeruginosa are mediated in part by virulence factors secreted by the bacterium. Among these factors is pyocyanin, a redox active compound that increases intracellular oxidant stress. We find that pyocyanin increases release of interleukin-8 (IL-8) by both normal and CF airway epithelial cell lines and by primary airway epithelial cells. Moreover, pyocyanin synergizes with the inflammatory cytokines tumor necrosis factor alpha and IL-1alpha. RNase protection assays indicate that increased IL-8 release is accompanied by increased levels of IL-8 mRNA. The antioxidant n-acetyl cysteine, general inhibitors of protein tyrosine kinases, and specific inhibitors of mitogen-activated protein kinases diminish pyocyanin-dependent increases in IL-8 release. Conversely, inhibitors of protein kinases C (PKC) and PKA have no effect. In contrast to its effects on IL-8 expression, pyocyanin inhibits cytokine-dependent expression of the monocyte/macrophage/T-cell chemokine RANTES. Increased release of IL-8, a potent neutrophil chemoattractant, in response to pyocyanin could contribute to the marked infiltration of neutrophils and subsequent neutrophil-mediated tissue damage that are observed in Pseudomonas-associated lung disease.     

Cytokine release by human bone marrow cells: analysis at the single cell level

Regulation of haemopoiesis is closely mediated by a number of growth factors in the marrow microenvironment. The identification of the cell type secreting these regulatory polypeptides is difficult due to the heterogeneity of bone marrow cells. To analyse the release of haemopoietic growth factors by normal human bone marrow cells at the single cell level, we employed the reverse haemolytic plaque assay (RHPA). Freshly isolated human marrow cells were examined for the release of interleukin-1 alpha (IL-1 alpha), IL-3, IL-6 and granulocyte-monocyte colony stimulating factor (GM-CSF). In order to identify various cytokine-secreting cell types, the RHPA was combined with immunocytochemical or enzymatic staining. The total of secreting marrow cells as well as the amount of several secretory haemopoietic subpopulations could be determined with this technique under various conditions. Following incubation with pure serum-free medium without addition of any mediator, only few cells secreting either IL-1 alpha, IL-3, IL-6 or GM-CSF could be observed. After 2 h incubation with recombinant human-IL-1 alpha (rhIL-1 alpha) (10.0 ng/ml) or rhGM-CSF (10.0 pg/ml) the number of cytokine-secreting cells significantly increased for all secretory products tested. Using cytochemical staining reactions, we were able to identify 55% of all cells secreting a specific cytokine. Glycophorin C-positive erythropoietic cells turned out to be the largest fraction (up to 89%) of cytokine-releasing haemopoietic cells, followed by neutrophil granulocytes (between 6 and 48%), and monocytes/macrophages (between 4 and 23%). Only few CD 61-positive cytokine-secreting megakaryocytes could be detected. Dose- and time-dependent kinetics after stimulation with rhGM-CSF revealed that the bulk of secretory activity originates from haemopoietic or rather from erythropoietic cells following low level stimulation and after short stimulation time. Thus, our data are in keeping with the assumption, that especially erythropoietic cells are producing a repertoire of cytokines that is thought to exhibit regulatory functions within marrow microenvironment. In the present study the RHPA is presented as an appropriate tool for measuring cytokine release not only of cells of the haematopoietic system but also of other tissues, for example solid tumours or malignant lymphomas.