Neutrophil Primary Granules Research Articles

Cytokine signals through STAT3 promote expression of granulocyte secondary granule proteins in 32D cells

In a previous study, we showed that activation of a transfected human erythropoietin receptor (EPOR) in the murine myeloid cell line 32D resulted in the development of morphologic features of granulocytic differentiation and expression of the neutrophil primary granule protein myeloperoxidase. We now studied if EPOR signaling could also mediate secondary granule protein gene expression and investigated the signal transduction requirements for induction of secondary granule gene expression in 32D cells. Wild-type and variant 32D cells expressing normal or chimeric EPORs or receptors for granulocyte colony-stimulating factor (G-CSFRs) were stimulated with EPO or G-CSF and the expression of granulocyte-specific genes was analyzed by Northern blot analysis. To determine the signaling mechanisms required for secondary granule protein gene induction, the activation of STAT pathways following growth factor stimulation was studied by Western blot analysis. We found that EPO treatment of 32D cells engineered to express EPOR did not result in induction of the secondary granule protein genes encoding lactoferrin and 24p3 lipocalin, the mouse homolog of human N-Gal, or the myeloid transcription factor C/EBPepsilon. Replacement of the intracellular domain of EPOR with the intracellular domain of G-CSFR in a chimeric receptor was associated with EPO-mediated induction of lactoferrin, 24p3 lipocalin, and C/EBPepsilon genes. We found that STAT3 phosphorylation was mediated by the intracellular domain of G-CSFR, but not EPOR. Replacement of one or two of the STAT5 binding sites in the intracytoplasmic domain of the EPOR with STAT3 binding sites resulted in EPO-mediated STAT3 activation and a marked increase in the expression of the 24p3 lipocalin gene. Knockdown of STAT3 protein levels with siRNA caused significant decrease in 24p3 lipocalin gene induction. These results indicate that EPOR signaling cannot substitute for G-CSFR signaling to stimulate secondary granule protein gene expression in 32D cells. In addition, STAT3 is a critical mediator of 24p3 lipocalin gene expression in these cells.

Rac2 induces tissue destruction in acute lung injury by regulating neutrophil primary granule release

RATIONALE: Neutrophils are important in the tissue destruction observed in immune complex-mediated acute lung injury (ALI). Tissue injury in ALI is mediated by oxidative damage and degranulation. Degranulation leads to the release of neutrophil granule products, including neutrophil elastase and myeloperoxidase (MPO), which are stored in the primary granules. Rac2 GTPase is known to regulate primary granule release, superoxide production and chemotaxis in neutrophils. Hypothesis: Rac2-mediated primary granule release induces tissue destruction in ALI.

Neutrophil primary granule release and maximal superoxide generation depend on Rac2 in a common signalling pathway

Neutrophils play an integral role in innate immunity by undergoing degranulation and respiratory burst in response to inflammatory stimuli. Rac2, a monomeric GTP-binding protein, has been shown to be involved in several neutrophil functions, including primary granule release and superoxide (O(2)(-.) generation. We hypothesized that Rac2 is a common signalling molecule required for primary granule translocation and maximal O(2)(-.) production. Using bone marrow neutrophils from Rac2 knockout (KO) mice and wild type C57Bl/6 mice, we found that primary granule elastase and myeloperoxi dase release were absent in Rac2 KO neutrophils upon chemoattractant stimulation. Rac2 KO neutrophils also failed to produce maximal levels of extracellular O(2(-.) generation in response to phorbol myristate acetate (PMA). Although PMA was ineffective at eliciting primary granule mediator release, it induced secondary granule exocytosis in both WT and Rac2 KO neutrophils. Thus, the signalling pathway leading to primary granule release utilized Rac2, which was also necessary for full activation of O(2)(-.) generation in stimulated neutrophils. These findings indicate that O(2)(-.) release and secondary granule secretion may use protein kinase C (PKC) - dependent pathways, whereas primary granule exocytosis appears to rely on PKC-independent signalling events. These findings shed light on possible signalling mechanisms involved in granule secretion from activated neutrophils responding to different stimuli.

Pathogenesis of Pulmonary Vasculitis

Vasculitis is inflammation of blood vessels and can affect any type of vessel in any organ. Pulmonary vasculitis usually is a component of a systemic small vessel vasculitis. Three major forms of small vessel vasculitis that often affect the lungs are Wegener's granulomatosis, microscopic polyangiitis, and Churg-Strauss syndrome. These forms of vasculitis are strongly associated with antineutrophil cytoplasmic autoantibodies (ANCA) directed against enzymes contained in the primary granules of neutrophils and peroxidase-positive lysosomes of monocytes. This review discusses the evidence for a pathogenic role of ANCA. In vitro, ANCAs can activate cytokine-primed neutrophils and monocytes resulting in oxygen radical formation and release of lysosomal enzymes. In vivo, antimyeloperoxidase ANCA has been shown to induce crescentic glomerulonephritis and systemic vasculitis. Overall, the available data suggest that ANCA are indeed a pathogenic factor in the development of small-vessel vasculitis. Antiglomerular basement membrane (anti-GBM) disease also causes pulmonary vasculitis through immune attack on alveolar capillaries and glomerulonephritis through antibody mediated injury to glomerular capillaries. Thus, there is evidence that antibodies are important pathogenic factors in both ANCA disease and anti-GBM disease, however, there are also indications that T cells may play important pathogenic roles in both categories of disease as well.

Rac2 is critical for neutrophil primary granule exocytosis

AbstractNeutrophil degranulation is important in many inflammatory disorders, although the intracellular mechanisms underlying this process remain poorly understood. The Rho GTPase, Rac2, has been implicated in control of degranulation in earlier studies. We hypothesized that Rac2 selectively regulates neutrophil primary granule release. Using bone marrow and peritoneal exudate neutrophils from rac2-/- mice in comparison with similar cells from wild-type C57Bl/6 mice, we found that primary granule myeloperoxidase and elastase release was absent in Rac2-/- neutrophils in response to chemoattractant stimulation, cytochalasin B/f-Met-Leu-Phe (CB/fMLP), and CB/leukotriene B4. Rac2-/- neutrophils also failed to exhibit mobilization of the primary granule marker CD63+ during CB/fMLP stimulation as determined by confocal microscopy. Priming of Rac2-/- neutrophils with tumor necrosis factor (TNF) or by peritoneal elicitation did not rescue the defect in primary granule release. However, phosphorylation of p38 mitogen-activated protein (MAP) kinase in Rac2-/- neutrophils was evident in response to CB/fMLP and/or TNF. Primary granule density and morphology were normal in Rac2-/- neutrophils. Secondary specific and tertiary granule release, measured by lactoferrin immunoassay and zymography, was normal in response to CB/fMLP and adhesion to fibronectin. These findings suggest an obligatory role for Rac2 in regulation of primary granule release by neutrophils.

Proteolytic cleavage of granulocyte colony-stimulating factor and its receptor by neutrophil elastase induces growth inhibition and decreased cell surface expression of the granulocyte colony-stimulating factor receptor.

Neutrophil elastase (NE) is a serine protease stored in the primary granules of neutrophils that proteolytically cleaves multiple cytokines and cell surface proteins on release from activated neutrophils. Recent reports of mutations in the gene encoding this enzyme in some patients with neutropenic syndromes prompted us to investigate whether granulocyte colony-stimulating factor (G-CSF) and its receptor (G-CSFR) are also substrates for NE. To further address this, we examined the effect of NE on G-CSF and the G-CSFR both in solution and on intact cells. Incubation of recombinant G-CSF or a G-CSFR form corresponding to its extracellular domain with purified NE resulted in rapid proteolytic cleavage of both proteins. Addition of NE to tissue culture medium or pretreatment of G-CSF with NE before its addition to media suppressed the growth of G-CSF-responsive cells. NE also cleaved the G-CSFR on the surface of intact cells resulting in a time-dependent reduction in cell surface expression of the G-CSFR. Notably, decreased G-CSFR surface expression resulting from treatment of cells with NE was also associated with a reduction in cell viability and proliferation in response to G-CSF. These results are the first to demonstrate that G-CSF and G-CSFR are proteolytically cleaved by NE and that NE-induced degradation of these proteins correlates with a reduction in the biologic activity of the cytokine and a decrease in the signaling function of the receptor because of decreased G-CSFR surface expression. These findings provide additional insights into mechanisms by which G-CSF/G-CSFR interactions may be modulated.

Proteolysis in the lung.

Pulmonary emphysema is a major component of the morbidity and mortality of chronic obstructive pulmonary disease (COPD), a condition that has become the fourth leading cause of death in the USA, and is becoming epidemic worldwide. Emphysema is defined as enlargement of peripheral airspaces of the lung including respiratory bronchioles, alveolar ducts and alveoli, accompanied by destruction of the walls of these structures. The pathogenesis of emphysema can be dissected into three interrelated events: 1) chronic exposure to cigarette smoke may lead to inflammatory cell recruitment within the terminal airspaces of the lung; 2) these inflammatory cells release elastolytic proteinases that damage the extracellular matrix of the lung; and 3) ineffective repair of elastin and perhaps other extracellular matrix components result in pulmonary emphysema. Inherited deficiency of α1‐antitrypsin (α1‐AT), the primary inhibitor of neutrophil elastase (NE), predisposes individuals to early onset emphysema, and intrapulmonary instillation of elastolytic enzymes in experimental animals causes emphysema. Together, these findings led to the elastase/antielastase hypothesis for the pathogenesis of emphysema, which was proposed ∼40 yrs ago and remains the prevailing concept today. While the capacity of NE to initiate emphysema in patients deficient in α1‐AT is clear, the proteinases involved in the pathogenesis of the common form of emphysema associated with cigarette smoking is more complicated. In addition to NE, neutrophil primary granules contain other elastolytic serine proteinases including, cathepsin G and proteinase 3, and secondary granules possess matrix metalloproteinases; MMP‐8, a collagenase and MMP‐9, a 92 kDa gelatinase. While, macrophages are prominent inflammatory cells in smokers' lungs, the capacity of macrophages to degrade elastin was controversial until cysteine proteinases including cathepsins S and L were identified (K the most potent elastase has not been identified in lung macrophages). The author's group identified several MMPs produced by alveolar and interstitial …

Leptospires are killed in vitro by both oxygen-dependent and -independent reactions.

This study reports for the first time that leptospires are killed by H(2)O(2) and by low-molecular-weight primary granule components, which are agents normally released by neutrophils upon stimulation. Although both pathogenic and nonpathogenic strains were sensitive to H(2)O(2)-mediated killing, nonpathogenic organisms were found to be more susceptible. In addition, the killing of leptospires by H(2)O(2) was found to be independent of the presence of the neutrophil primary granule component myeloperoxidase and therefore not a consequence of halogenation reactions. We have also determined that leptospires are significantly sensitive only to primary granule components and, among those, to proteins and/or peptides of less than 30 kDa.

Discriminating between the Activities of Human Neutrophil Elastase and Proteinase 3 Using Serpin-derived Fluorogenic Substrates

Human neutrophil elastase (HNE) has long been linked to the pathology of a variety of inflammatory diseases and therefore is a potential target for therapeutic intervention. At least two other serine proteases, proteinase 3 (Pr3) and cathepsin G, are stored within the same neutrophil primary granules as HNE and are released from the cell at the same time at inflammatory sites. HNE and Pr3 are structurally and functionally very similar, and no substrate is currently available that is preferentially cleaved by Pr3 rather than HNE. Discrimination between these two proteases is the first step in elucidating their relative contributions to the development and spread of inflammatory diseases. Therefore, we have prepared new fluorescent peptidyl substrates derived from natural target proteins of the serpin family. This was done because serpins are rapidly cleaved within their reactive site loop whether they act as protease substrates or inhibitors. The hydrolysis of peptide substrates reflects the specificity of the parent serpin including those from alpha-1-protease inhibitor and monocyte neutrophil elastase inhibitor, two potent inhibitors of elastase and Pr3. More specific substrates for these proteases were derived from the reactive site loop of plasminogen activator inhibitor 1, proteinase inhibitors 6 and 9, and from the related viral cytokine response modifier A (CrmA). This improved specificity was obtained by using a cysteinyl residue at P1 for Pr3 and an Ile residue for HNE and because of occupation of protease S' subsites. These substrates enabled us to quantify nanomolar concentrations of HNE and Pr3 that were free in solution or bound at the neutrophil surface. As membrane-bound proteases resist inhibition by endogenous inhibitors, measuring their activity at the surface of neutrophils may be a great help in understanding their role during inflammation.

Characterization of a novel chemotactic factor for neutrophils in the bronchial secretions of patients with cystic fibrosis.

Chronic airway inflammation is a hallmark of cystic fibrosis (CF). Biological products with chemotactic activity are essential for neutrophil recruitment to sites of inflammation. The presence of a factor with chemotactic activity higher than that of interleukin (IL)-8 in the bronchial secretions of patients with CF has recently been reported. This article reports that the chemotactic activity of this factor remained unaffected by a variety of physical treatments and could be distinguished from those of IL-8, formylmethionylleucylphenylalanine, leukotreine B4, and platelet-activating factor. The factor induced chemotaxis and chemokinesis locomotion of neutrophils, and its chemotactic activity was sensitive to pertussis toxin and thapsigargin. Semipurified preparation of the chemotactic factor increased transiently intracellular Ca(2+) concentration but failed to stimulate the release of neutrophil primary granules and the production of superoxide, suggesting that the semipurified chemotactic factor is a Ca(2+)-dependent chemoattractant of neutrophils, acting via pertussin toxin-sensitive G protein-coupled surface receptors, that directs neutrophil movement toward the airway epithelium.

Anti-neutrophil cytoplasmic antibodies (ANCA) from patients with systemic vasculitis recognize restricted epitopes of proteinase 3 involving the catalytic site.

ANCA with specificity for proteinase 3 (PR3), a neutrophil primary granule enzyme, are of diagnostic value in Wegener's granulomatosis (WG) and certain other forms of systemic vasculitis. There is evidence to suggest that they play a pathogenic role in disease, and that the interaction of ANCA with PR3 is likely to be important. We showed, using a resonant mirror biosensor, that C-ANCA from different patients recognized the same or closely related epitopes on PR3. Studies using linear peptides in the SPOT system confirmed the highly restricted nature of this interaction and identified five linear epitopes. Fluid-phase inhibition studies, using a different set of peptides, validated the sequences involved. Using a computer-generated model of the structure of PR3, four of five epitopes were shown to be intimately linked with the catalytic site. The restricted number of epitopes, and their location at the catalytic site, has important implications for the role of C-ANCA in the pathogenesis of vasculitis.

Regulation of P-selectin binding to the neutrophil P-selectin counter-receptor P-selectin glycoprotein ligand-1 by neutrophil elastase and cathepsin G

In the inflammatory response, leukocyte rolling before adhesion and transmigration through the blood vessel wall is mediated by specific cell surface adhesion receptors. Neutrophil rolling involves the interaction of P-selectin expressed on activated endothelium and its counter-receptor on neutrophils, P-selectin glycoprotein ligand-1 (PSGL-1). Here, it is reported that P-selectin binding to neutrophils is lost under conditions that cause the release of proteinases from neutrophil primary granules. Treatment of neutrophils with the purified neutrophil granule proteinases, cathepsin G and elastase, rapidly abolished their capacity to bind P-selectin. This inactivation corresponded to loss of the N-terminal domain of PSGL-1, as assessed by Western blot analysis. A loss of intact PSGL-1 protein from the surfaces of neutrophils after the induction of degranulation was also detected by Western blot analysis. Cathepsin G initially cleaved near the PSGL-1 N-terminus, whereas neutrophil elastase predominantly cleaved at a more C-terminal site within the protein mucin core. Consistent with this, cathepsin G cleaved a synthetic peptide based on the PSGL-1 N-terminus between Tyr-7/Leu-8. Under conditions producing neutrophil degranulation in incubations containing mixtures of platelets and neutrophils, the loss of PSGL-1, but not P-selectin, from platelet-neutrophil lysates was detected. Cathepsin G- or neutrophil elastase-mediated PSGL-1 proteolysis may constitute a potential autocrine mechanism for down-regulation of neutrophil adhesion to P-selectin.

Absence of Cross-Reactivity to Myeloperoxidase of Anti-Thyroid Microsomal Antibodies in Patients with Autoimmune Thyroid Diseases

Thyroperoxidase is the major antigen of the thyroid microsomal antibodies (TMA) detected in autoimmune thyroid diseases. Its amino acid sequence has 44% homology with myeloperoxidase (MPO), an enzyme present in the primary granules of neutrophils and one of the major antineutrophil cytoplasmic antibodies (ANCA) antigens. The objective of the present study was to investigate the presence of cross-reactivity to MPO of TMA. We studied sera from 51 patients with autoimmune thyroid diseases, all of them TMA-positive. The presence of ANCA was investigated by indirect immunofluorescence and by capture enzyme-linked immunosorbent assay. ANCA were positive in 3.9% of the TMA-positive sera and none of them reacted with MPO. In contrast, the ANCA-positive sera revealed antielastase activity. None of the ANCA-positive cases presented clinical signs of vasculitis. However, these 2 patients had been on prolonged treatment with propylthiouracil. We conclude that there is no cross-reactivity to MPO of TMA in patients with autoimmune thyroid diseases, possibly because of difference in the spatial configuration of the immunodominant region. The presence of ANCA in patients with autoimmune thyroid diseases without evidence of vasculitis might result from propyIthiouracil-induced polyclonal activation.

Three-diaengional radiation therapy treatment planning and delivery

Anti-neutrophil cytoplasmic antibodies (ANCA) are antibodies directed against enzymes that are found mainly within the azurophil or primary granules of neutrophils. There are 3 types of ANCA that can be distinguished by the patterns they produce by indirect immunofluorescence when tested on normal ethanol-fixed neutrophils. Diffuse fine granular cytoplasmic fluorescence (cANCA) is typically found in Wegener’s granufomatosis, in some cases of microscopic polyarteritis and Churg Strauss syndrome, and in some cases of crescentic and segmental necrotising glomerulonephritis, but it is rare in other conditions. The target antigen is usually proteinase 3. Perinuclear fluorescence (pANCA) is found in many cases of microscopic polyarteritis and in other cases of crescentic and segmental necrotising glomerulonephritis. These antibodies are often directed against myeloperoxidase but other targets include elastase, cathepsin G, lactoferrin, lysozyme and beta-glucuronidase. The third group designated “atypical” ANCA includes neutrophil nuclear fluorescence and some unusual cytoplasmic patterns, and while a few of the target antigens are shared with pANCA, the others have not been identified. Sera that produce a pANCA or atypical ANCA pattern on alcohol-fixed neutrophils result in cytoplasmic fluorescence when formalin acetone fixation is used. pANCA or atypical ANCA occur in about 2/3 of all individuals with ulcerative colitis or primary sclerosing cholangitis, and they are found in a third of patients with Crohn’s disease. The reported incidence of ANCA in rheumatoid arthritis and SLE varies considerably but the patterns are predominantly pANCA and atypical ANCA. Where ANCA are associated with a small vessel vasculitis, such as Wegener’s granulomatosis and microscopic polyarteritis, antibody titres may parallel disease activity and have been used to monitor the response to treatment. In other non-vasculitic conditions ANCA levels do not necessarily reflect disease activity or the presence of a vasculitis. ANCA are also common in HIV infections where all fluorescence patterns are found, but the presence of these antibodies has no clinical significance. There have been many reports of ANCA occurring occasionally in other diseases.ANCA have not yet been demonstrated to be pathogenetic in the small vessel vasculitides. However there is in vitro evidence for 2 mechanisms, one of which focuses on neutrophil activation as the primary event and the other on antibody binding to *An update from the recent International and Australian Workshops. This paper is based on contemporary published data and observations presented at the 4th International Workshop on ANCA (Lubeck, May 1992) and 1st Australian Workshop on ANCA (Melbourne, October, 1992). vascular endothelium. Both mechanisms assume increased levels of circulating tumor necrosis factor or another cytokine that might result from the infections that often precede the onset of Wegener’s granulomatosis and microscopic polyarteritis. Successful new regimens in the treatment of Wegener’s granulomatosis and microscopic polyarteritis include the use of pooled immunoglobulin, and cyclosporin in combination with low dose prednisolone, but one report of 3-weekly pulse cyclophosphamide as a single agent indicated that it was effective in only 42% of patients.

A rapid, direct assay to measure degranulation of bovine neutrophil primary granules

A direct, rapid, quantitative colorimetric assay to determine neutrophil primary granule degranulation was adapted for use with bovine neutrophils. The assay measures the exocytosis of myeloperoxidase (MPO) using 3,3′,5,5′-tetramethylbenzidine as a substrate. The assay was validated by evaluating the effects of various stimulants and inhibitors of degranulation, the kinetics of primary granule exocytosis, and comparing the total myeloperoxidase content of neutrophils obtained from calves and adults. The results demonstrate that the assay is capable of detecting important differences that may occur in degranulation of bovine neutrophil primary granules and in total neutrophil myeloperoxidase content.

Proteinase-free myeloperoxidase increases airway epithelial permeability in a whole trachea model.

In cystic fibrosis the bronchiectatic conducting airways have large numbers of neutrophils in their walls and in their luminal contents. The neutrophil's primary granule enzyme activities of elastase and peroxidase are increased in the sputum of these patients. It has been postulated that these enzymes--together or individually--act to damage the airway epithelium. However, only peroxidase activity has consistently correlated with the degree of structural and functional airway disease in these patients with leakage of plasma protein into the airway lumen (Regelmann et al., Pediatr Pulmonol, 1995; 19:1-9). The present study was designed to test whether human neutrophil-derived myeloperoxidase can independently produce bronchial epithelial damage without the presence of proteases, as measured by increased permeability of the airway epithelium. Human peripheral blood neutrophils were purified, their primary granules isolated, and their peroxidase purified using affinity and ion exchange column chromatography. Activity of the proteinase-free peroxidase was measured using a chromogenic substrate. The effect of this peroxidase on the permeability of excised rat tracheas was measured using radioactive and fluorescent-labeled non-ionic molecules of varying molecular weight. Rat tracheas exposed to 15 minute treatments with either 130 U of peroxidase or hydrogen peroxide (10(-5) M) did not show a significant increase in the permeability of the epithelium to [3H]inulin, [14C]sucrose, and fluorescein isothiocyanate dextran 20 compared with control tracheas. However, those tracheas exposed to 130 U peroxidase followed by 10(-5) M hydrogen peroxide showed an increased permeability to each of the three test solutes. We conclude that proteinase-free myeloperoxidase, in the presence of non-toxic concentrations of its substrates, hydrogen peroxide and halide, produced increases in permeability to non-ionic molecules in the rat trachea within 15 minutes.

Proteinase‐free myeloperoxidase increases airway epithelial permeability in a whole trachea model

In cystic fibrosis the bronchiectatic conducting airways have large numbers of neutrophils in their walls and in their luminal contents. The neutrophil's primary granule enzyme activities of elastase and peroxidase are increased in the sputum of these patients. It has been postulated that these enzymes—together or individually—act to damage the airway epithelium. However, only peroxidase activity has consistently correlated with the degree of structural and functional airway disease in these patients with leakage of plasma protein into the airway lumen (Regelmann et al., Pediatr Pulmonol. 1995; 19: 1–9). The present study was designed to test whether human neutrophil-derived myeloperoxidase can independently produce bronchial epithelial damage without the presence of proteases, as measured by increased permeability of the airway epithelium. Human peripheral blood neutrophils were purified, their primary granules isolated, and their peroxidase purified using affinity and ion exchange column chromatography. Activity of the proteinase-free peroxidase was measured using a chromogenic substrate. The effect of this peroxidase on the permeability of excised rat tracheas was measured using radioactive and fluorescent-labeled non-ionic molecules of varying molecular weight. Rat tracheas exposed to 15 minute treatments with either 130 U of peroxidase or hydrogen peroxide (10−5 M) did not show a significant increase in the permeability of the epithelium to [3H]inulin, [14C]sucrose, and fluorescein isothiocyanate dextran 20 compared with control tracheas. However, those tracheas exposed to 130 U peroxidase followed by 10−5 M hydrogen peroxide showed an increased permeability to each of the three test solutes. We conclude that proteinase-free myeloperoxidase, in the presence of non-toxic concentrations of its substrates, hydrogen peroxide and halide, produced increases in permeability to non-ionic molecules in the rat trachea within 15 minutes. Pediatr. Pulmonol. 1997;24:29–34. © 1997 Wiley-Liss, Inc.

Activated neutrophils secrete stored alpha 1-antitrypsin.

Neutrophil elastase (NE), a potent serine protease, is stored in primary granules of neutrophils and released following neutrophil activation. Alpha-1-antitrypsin (alpha 1-AT), the major inhibitor of NE, is synthesized by mature neutrophils. In the context of the maintenance of tissue homeostasis, we hypothesized that neutrophils may be able to store alpha 1-AT, thus having it available for release concordantly with NE. Immunofluorescence and quantitative flow-cytometric studies of neutrophils and monocytes labeled with fluorescein-conjugated alpha 1-AT-antibody demonstrated larger amounts of cytoplasmic alpha 1-AT in neutrophils than in monocytes. [35S]methionine-labeling and anti-alpha 1-AT immunoprecipitation analysis showed that although both neutrophils and monocytes synthesize alpha 1-AT, the proportion of newly synthesized intracellular alpha 1-AT was much higher in neutrophils than in monocytes. Flow-cytometric analysis showed that in the presence of surface stimulation with cytochalasin B followed by formyl-methionyleucylphenylalanine (fMLP), mean intracellular alpha 1-AT was decreased in stimulated neutrophils compared with that in resting cells, suggesting that the stored alpha 1-AT was rapidly released following surface triggering. Evaluation of surface-stimulated neutrophils by [35S]methionine labeling and anti-alpha 1-AT immunoprecipitation demonstrated increased secretion of alpha 1-AT compared with that of resting neutrophils, with some of the secreted alpha 1-AT capable of forming complexes with NE. Thus, neutrophils respond to surface stimulation not only by secreting NE but also by secreting its inhibitor, alpha 1-AT, suggesting that these cells have an inherent mechanism for damping the local effects of NE, their most powerful proteolytic enzyme.

Cytokine-treated human neutrophils contain inducible nitric oxide synthase that produces nitration of ingested bacteria.

Although the production of NO within rodent phagocytes is well-characterized, its production and function within human phagocytes are less clear. We show here that neutrophils within human buffy coat preparations stimulated with a mixture of interleukin 1, tumor necrosis factor alpha, and interferon gamma contain inducible NO synthase mRNA and protein, one of the enzymes responsible for NO production. The protein colocalizes with myeloperoxidase within neutrophil primary granules. Using an inhibitor of NO synthase, L-N-monomethyl arginine, we show that activity of this enzyme is required for the formation of nitrotyrosine around phagocytosed bacteria, most likely through the intermediate production of peroxynitrite, a reaction product of NO and superoxide anions.

Distinct Temporal Patterns of Defensin mRNA Regulation During Drug-Induced Differentiation of Human Myeloid Leukemia Cells

Defensins are microbicidal peptides and the principal constituents of neutrophil primary granules. They are presumed to play a prominent role in innate host defenses. We examined defensin mRNA levels during drug-induced differentiation of the promyelocytic leukemia cell line, HL-60. Transcription was restricted to promyelocyte, myelocyte, and very early metamyelocyte stages of the granulocytic pathway. Complete downregulation occurred during late granulocytic maturation or early during phorbol ester-promoted differentiation along the monocyte/macrophage lineage. Retinoic acid (RA) was the strongest inducer of defensin mRNA accumulation, even at doses too low to effect morphologic changes; the initial (first 48 hours), gradual increase resulted from transcriptional activation and was enhanced by granulocyte colony-stimulating factor. In contrast, addition of hybrid polar compounds led to a transient, drug-specific downregulation within the same time period, apparently by means of selectively induced, biphasic degradation of transcripts. Subsequent increase in transcript levels was faster and more pronounced with hexamethylene bisacetamide, relative to dimethyl sulfoxide (DMSO). DMSO-promoted effects were strikingly different in serum-free medium or in the presence of the tyrosine kinase inhibitor, genistein. Under these conditions, and although differentiation was unaffected, early defensin mRNA downregulation was final. The effect did not occur with RA and expression of other myeloid-specific genes was also unchanged. Addition of selected cytokines caused a similar "dip," only at earlier times and uncoupled from differentiation. Tumor necrosis factor-alpha markedly induced defensin levels after 2 days in previously untreated HL-60 cells, but inhibited expression in RA-differentiated cells. These results begin to detail a complex regulation of defensin mRNA synthesis with both spatial and temporal control elements, and a unique modulation by chemical agents, cytokines, and serum-factors.