Azurophil Granule Marker Research Articles

Sputum from People with Cystic Fibrosis Reduces the Killing of Methicillin-Resistant Staphylococcus aureus by Neutrophils and Diminishes Phagosomal Production of Reactive Oxygen Species.

Cystic fibrosis (CF) airway disease is characterized by chronic polymicrobial infections and an infiltration of neutrophils (PMNs). Staphylococcus aureus has been the most prevalent respiratory pathogen in CF. In particular, methicillin-resistant S. aureus (MRSA) represents a huge clinical burden in CF due to its association with lung disease and increased resistance to antibiotics. In CF, PMNs are unable to kill and clear MRSA. The reason for this remains largely unknown. Our study found that CF PMNs are as equally capable of killing MRSA as healthy PMNs. We show that the CF sputum, however, significantly impairs the ability of human PMNs to kill CF MRSA isolates. In the absence of CF sputum, PMNs kill MRSA via intracellular mechanisms mediated by phagocytosis, rather than extracellular mechanisms via NET formation. CF sputum does not affect the phagocytosis of MRSA via healthy or CF PMNs. Our results demonstrate that CF sputum exposure impairs phagosomal levels of reactive oxygen species (ROS) in MRSA-phagocytosing PMNs. While phagosomal co-localizations of MRSA with primary granule markers, myeloperoxidase and cathepsin D, were significantly reduced upon CF sputum exposure, that of a third azurophilic granule marker, neutrophil elastase, remained unaffected. This suggests that CF sputum does not compromise the fusion of primary granules with phagosomes but diminishes phagosomal ROS levels via another, likely more specific, mechanism. Overall, we identified the airway environment as an important factor that restricts neutrophils' oxidative microbicidal activities in CF against MRSA. These results deliver new details of the complex host-pathogen interactions present in the CF lung.

Lysophosphatidylcholine induces azurophil granule translocation via Rho/Rho kinase/F-actin polymerization in human neutrophils.

Translocation of azurophil granules is pivotal for bactericidal activity of neutrophils, the first-line defense cells against pathogens. Previously, we reported that lysophosphatidylcholine (LPC), an endogenous lipid, enhances bactericidal activity of human neutrophils via increasing translocation of azurophil granules. However, the precise mechanism of LPC-induced azurophil granule translocation was not fully understood. Treatment of neutrophil with LPC significantly increased CD63 (an azurophil granule marker) surface expression. Interestingly, cytochalasin B, an inhibitor of action polymerization, blocked LPC-induced CD63 surface expression. LPC increased F-actin polymerization. LPC-induced CD63 surface expression was inhibited by both a Rho specific inhibitor, Tat-C3 exoenzyme, and a Rho kinase (ROCK) inhibitor, Y27632 which also inhibited LPC-induced F-actin polymerization. LPC induced Rho-GTP activation. NSC23766, a Rac inhibitor, however, did not affect LPC-induced CD63 surface expression. Theses results suggest a novel regulatory mechanism for azurophil granule translocation where LPC induces translocation of azurophil granules via Rho/ROCK/F-actin polymerization pathway.

Regulation of neutrophil secretion by the lysosomal amino acid transporter, cystinosin

Neutrophil exocytosis is an important immune response, but secretion must be tightly regulated because exacerbated release of the neutrophil toxic mediators is injurious to the host and mediates inflammation. Here we show that neutrophil exocytosis of azurophilic granules is regulated by the cystine lysosomal transporter cystinosin. Cystinosin localized at azurophilic granules and lysosomes in neutrophils. Isolated bone‐marrow‐derived Ctns−/− neutrophils showed exacerbated azurophilic granule secretion under unstimulated conditions, suggesting dysregulation of a cell‐intrinsic mechanism. The defect was specific for azurophilic granules because secretion of gelatinase granules was not affected. Using phalloidin staining, we show that Ctns−/− neutrophils have decreased F‐actin, suggesting disruption of the azurophilic granule‐specific actin barrier. In addition, phalloidin‐based staining showed increased filopodial protrusion formation in Ctns−/− neutrophils, thus affecting cell morphology. Induction of either actin polymerization or depolymerization by jasplakinolide (JK) or cytochalasin, respectively, followed by stimulation with the bacteria‐derived peptide, fMLF, showed that secretion was still significantly increased in Ctns−/− neutrophils, suggesting mechanistic defects beyond impaired actin remodeling. Using Total Internal Reflection Fluorescence Microscope (TIRFM) and Super‐Resolution Radial Fluctuations (SRRF), we show increased numbers of docked granules at the exocytic actin zone in Ctns−/− neutrophils. Immunofluorescence analysis showed increased colocalization of the small GTPase Rab27a with its effector JFC1 in Ctns−/− neutrophils, suggesting that Rab27a drives the increased exocytosis observed in Ctns−/− neutrophils. This was confirmed by treatment with the Rab27a‐JFC1 binding‐specific inhibitor Nexinhib20 (NEI20), which decreased granule docking and impaired secretion of the azurophilic granule marker myeloperoxidase (MPO) in unstimulated and stimulated Ctns−/− neutrophils. Combined treatment with NEI20 and JK further decreased MPO secretion in Ctns−/− neutrophils suggesting the uncoupling of Rab27a activation and actin depolymerization in Ctns−/− neutrophils. This was confirmed by the observation that the number of docking granules decreases in response to the combined treatment. Blocking ERM (Ezrin/Radixin/Moesin) also decreased the number of docked azurophilic granules at the exocytic active zone and impaired MPO secretion in Ctns−/− neutrophils, suggesting the participation of ERM on azurophilic granule exocytosis. In vivo, we found elevated levels of azurophilic granule cargo in plasma from cystinosin‐deficient mice, and Ctns−/− mice showed increased neutrophilic tissue infiltration, denoting systemic activation. Our data suggest that cystinosin is a negative regulator of exocytosis and that neutrophil‐mediated inflammation may play a significant role in disease progression in cystinosis.Support or Funding InformationCRFNIH

Neutrophil Degranulation of Azurophil and Specific Granules.

Neutrophils play a pivotal role in innate immunity and in the inflammatory reactions. Upon activation, neutrophils release several toxic molecules directed against microbial pathogens into the phagosome. These molecules include reactive oxygen species (ROS), myeloperoxidase, glucosidases, proteases, and antibacterial peptides. In resting cells these proteins and the enzyme responsible for ROS production (NOX2) are stored inside or at the membranes of different granules called azurophil or primary, specific or secondary, gelatinase or tertiary, and the secretory vesicles. Each granule has a specific density, content, and markers. Myeloperoxidase (MPO) is the azurophil granule marker, and the neutrophil-gelatinase-associated lipocalin (NGAL) is the specific granule marker. After cell activation by different stimuli, granule contents are released into the phagosome or in the extracellular space through a process called degranulation. Also during this process, membrane granules fuse with the phagosome and plasma membrane allowing expression of new markers at the cell surface. The degranulation can be assessed by measuring either the release of different proteins by neutrophils or the expression of granule markers at the plasma membrane. In this chapter, we describe the techniques used to measure degranulation of azurophil and specific neutrophil granules using different approaches such as measurement of MPO enzymatic activity and detection of MPO and NGAL proteins by SDS-PAGE and Western blot.

Pro-oxidant versus anti-oxidant effects of seeds aglycone extracts of Lepidium sativum and Eruca vesicaria Linn., in vitro, and on neutrophil nitro-oxidative functions.

This study evaluated the anti-inflammatory and antioxidant properties of seeds aglycone extracts from Lepidium sativum (LS) and Eruca vesicaria (EV) Linn., on oxidative damages in vitro and on neutrophil nitro-oxidative functions. The results showed that LS and EV aglycone extracts attenuated liver microsomal lipids and proteins oxidation through a potent antioxidant effect as attested by the dose dependent quenching of DPPH radical scavenging activity. LS and EV aglycone extracts inhibited dose dependently the production of superoxide anion by BALB/c mice-derived peritoneal neutrophils, whereas they slightly enhanced exocytosis of myeloperoxidase (MPO), a marker of azurophilic granules. Interestingly, only LS replenished glutathione (GSH) and nitric oxide levels, indicating a fine differential effect. This study highlighted the subtle oxidative and antioxidant capacity of LS and EV seeds aglycone extracts. These health promoting compounds could be used to finely modulate critical events involved in microbial infection, inflammation and nitro-oxidative stress.

Peptoanaerobacter stomatis Primes Human Neutrophils and Induces Granule Exocytosis.

Peptoanaerobacter stomatis is a newly appreciated taxon associated with periodontal diseases; however, little is known about the organism's pathogenic potential or its interaction with the host immune response. Neutrophils are the most abundant innate immune cell present in the gingival tissue and function to constrain the oral microbial challenge. However, some periodontal pathogens have developed strategies to evade phagocytosis and killing by neutrophils. Therefore, to begin to understand the role of P. stomatis in periodontitis, we studied its interactions with human neutrophils. Our data showed that after 30 min of incubation, neutrophils failed to engulf P. stomatis efficiently; however, when P. stomatis was internalized, it was promptly eradicated. P. stomatis challenge induced a robust intracellular respiratory burst; however, this response did not contribute to bacterial killing. Minimal superoxide release was observed by direct bacterial challenge; however, P. stomatis significantly increased N-formyl-methionyl-leucyl phenylalanine (fMLF)-stimulated superoxide release to an extent similar to that of cells primed with tumor necrosis factor alpha (TNF-α). When neutrophils were challenged with P. stomatis, 52% of the bacterium-containing phagosomes were enriched for the specific granule marker lactoferrin and 82% with the azurophil granule marker elastase. P. stomatis challenge stimulated exocytosis of the four neutrophil granule subtypes. Moreover, P. stomatis susceptibility to extracellular killing could be attributed to the exocytosis of antimicrobial components present in neutrophil granules. Priming neutrophils for an enhanced respiratory burst together with promoting granule content release could contribute to the chronic inflammation and tissue destruction that characterize periodontal diseases.

Phosphoglycerate Kinase-A Novel Streptococcal Factor Involved in Neutrophil Activation and Degranulation.

Neutrophils have been proposed as important contributors to the hyperinflammatory responses that are associated with severe invasive Streptococcus pyogenes infections. In particular, streptococcal surface proteins have been implicated as potent neutrophil activators. Here we explore the impact of streptococcus-secreted factors on neutrophil activation and degranulation. Primary human neutrophils were exposed to supernatants prepared from cultures of invasive S. pyogenes strains of varying serotypes in the stationary growth phase. Neutrophil activation was assessed by measurement of secreted resistin, an azurophilic granule marker, and by determination of the secretome profile, using mass spectrometry. Marked variation in resistin release and the neutrophil secretome profile were observed following exposure to different strains. A high resistin response was triggered exclusively by SpeB-negative strains, suggesting that at least 1 stimulatory factor is susceptible to SpeB proteolytic degradation. Further analysis, including proteomics and stimulation analyses, identified phosphoglycerate kinase as a stimulatory factor for neutrophils. Taken together, results of this study reveal a novel secreted streptococcal factor that, in the absence of SpeB, can trigger neutrophil activation and degranulation. This finding is of interest in light of reports of hypervirulent SpeB-negative S. pyogenes variants present during invasive infections.

Targeting of Neutrophil Lewis X Blocks Transepithelial Migration and Increases Phagocytosis and Degranulation

Polymorphonuclear leukocytes (PMNs) are innate immune cells whose principal function is to migrate from the blood to sites of inflammation, where they exert crucial anti-infectious and immunomodulatory effects. However, dysregulated migration of PMNs into mucosal epithelial tissues is characteristic of chronic inflammatory disorders, including inflammatory bowel disease. Carbohydrate-mediated binding interactions between PMN Lewis glycans and endothelial glycan-binding proteins are critical for initial migration of PMN out of the vasculature. However, the role of Lewis glycans during transepithelial migration (TEM) has not been well characterized. Herein, we show that antibody blockade of Lewis X (Le(x)) displayed as terminal glycan residues on the PMN surface blocks chemotaxis and TEM while enhancing PMN-adhesive interactions with intestinal epithelia. Unexpectedly, targeting of subterminal Le(x) residues within glycan chains had no effect on PMN migration or adhesive interactions. There was increased surface expression of Le(x) on PMN after TEM, and blockade of terminal Le(x) regulated post-migratory PMN functions, increasing PMN phagocytosis and the surface mobilization of azurophilic (CD63, myeloperoxidase, and neutrophil elastase) and specific (CD66b and lactoferrin) granule markers. These findings suggest that terminal Le(x) represents a potential target for regulating PMN trafficking and function in inflamed mucosa. Furthermore, given its abundant expression on migrating PMN, Le(x) may be a rational target for modulating inflammation in diseases where dysregulated PMN influx is associated with host tissue damage.

Targeting of PMN Expressed Lewis‐X Blocks PMN Transepithelial Migration and Increases Phagocytosis and Surface Mobilization of Granule Contents

Dysregulated transepithelial migration (TEM) of neutrophils (PMN) is pathognomonic of numerous inflammatory disorders including inflammatory bowel disease (IBD), yet mechanisms controlling PMN TEM remain poorly defined. The glycan determinant Lewis X (Galβ1‐4 (Fucα1‐3) GlcNAc‐β‐R), expressed by PMN surface glycoproteins (including MAC‐1 and LFA‐1), has previously been implicated in adhesive interactions between PMN and endothelium. However, little is known about the wider role of Lex in PMN function following extravasation. Studies revealed that engagement of terminal Lex glycans (but not the related glycan sLex) increased PMN adhesive interactions, both with other PMNs and with intestinal epithelial cells, blocking PMN chemotaxis and TEM. Further, flow cytometry analyses revealed increased surface expression of Lex on human PMN following TEM. In addition to blocking PMN trafficking, targeting of Lex also altered post‐migratory PMN functions, increasing both PMN phagocytosis and the surface mobilization of azurophilic granule (CD63 and MPO) and specific granule (CD66b and lactoferrin) markers. Finally, immunohistochemistry analyses revealed robust expression of Lex by PMN in the crypt abscesses of individuals with active IBD. Therefore, Lex represents a potential target for regulating PMN trafficking into the intestinal lumen and PMN luminal function. Further, given its abundant expression on migrating PMN and during inflammation, Lex may be a rational target for modulating inflammation in IBD.

Stimulation of human neutrophils with sera containing HLA Class I alloantibody causes preferential degranulation of azurophilic granules and secretory vesicles

The activation of neutrophils by human leukocyte antigen (HLA) Class I alloantibody is thought to be involved in transfusion-related acute lung injury. Neutrophils contain various biological substances in four groups of granules, including secretory vesicles, azurophilic granules, specific granules and gelatinase granules. To characterize the activation of neutrophils by HLA Class I alloantibody, we investigated whether HLA Class I alloantibody could cause the degranulation of these groups of granules either coordinately or selectively. Sera containing HLA-A24 alloantibody were incubated with neutrophils in a washed whole blood system. CD11b expression (secretory vesicles) on neutrophils was analysed by flow cytometry, and the secretion of markers of each granule was determined by ELISA. The treatment of cross-matching-positive neutrophils with sera containing HLA-A24 alloantibody caused the significant expression of CD11b, and the significant secretion of neutrophil elastase and myeloperoxidase, azurophilic granule markers and heparin-binding protein (HBP), which is localized in secretory vesicles and azurophilic granules when compared with cross-matching-negative neutrophils. In contrast, no significant differences were observed in the secretion of lactoferrin, a marker of specific granules, and matrix methalloproteinase-9, a marker of gelatinase granules between cross-matching-positive and cross-matching-negative cells upon stimulation with sera. CD11b expression and secretion of HBP by serum was partially inhibited by p38 mitogen-activated protein (MAP)-kinase inhibitors. Neutrophils activated with sera containing HLA Class I alloantibody caused the preferential degranulation of azurophilic granules and secretory vesicles. This process was at least in part mediated by p38 MAP kinase-involved signal transduction.

PMN degranulation in relation to CD63 expression and genetic polymorphisms in healthy individuals and COPD patients

Polymorphonuclear neutrophils (PMNs) play an important role in chronic obstructive pulmonary disease (COPD) pathogenesis. The tetraspanin CD63 is a membrane marker of azurophilic granules and is actively involved in the process of PMN endocytosis and azurophilic granule exocytosis. In this study, we investigated genetic polymorphisms of the CD63 gene, quantified CD63 expression and PMN myeloperoxidase (MPO) release in healthy individuals and COPD patients. We evaluated the potential correlations between genetic polymorphisms and gene expression and MPO release. COPD patients had significantly lower CD63 expression and released less MPO upon chemokine stimulation compared with the healthy individuals. Eleven putative polymorphisms in the CD63 gene were investigated but only three were polymorphic in our study subjects. None of the polymorphisms was associated with CD63 expression in either the healthy subjects or the COPD patients. However, the 8041C/G polymorphism, which is located 3' to the CD63 gene, was associated with MPO release in the healthy subjects. The CC genotype was associated with greater MPO release than the GG genotype (P=0.007). These results suggest that COPD patients have different patterns of CD63 expression and PMN mediator release than healthy individuals. It is likely that genetic variants have limited effect on CD63 expression and MPO release in the context of COPD but their role in other diseases has yet to be determined.

In Vivo Treatment With Granulocyte Colony-Stimulating Factor Results in Divergent Effects on Neutrophil Functions Measured In Vitro

We have studied the effects of granulocyte colony-stimulating factor (G-CSF) administration to normal individuals on a variety of functional and biochemical neutrophil characteristics that relate to host defense. G-CSF adversely affected neutrophil (polymorphonuclear leukocyte [PMN]) chemotaxis. While this could be partially explained by reduced assembly of neutrophil F-actin, we also recognized an elevated cytosolic calcium mobilization and a normal upregulation of neutrophil CD11b. G-CSF resulted in reduced PMN killing of Staphylococcus aureus with a 10:1 (bacteria:neutrophil) ratio and normal killing with a 1:1 ratio. In association with this, we demonstrated divergent effects on the respiratory burst of intact cells and divergent effects on the content of marker proteins for neutrophil granules. While G-CSF may have resulted in increased content of cytochrome b558 in the cell membrane, it did not alter the amounts of cytosolic oxidase components. After therapy, there was normal content of the azurophilic granule marker, myeloperoxidase, decreased content of the specific granule marker, lactoferrin, and normal content of lysozyme (found in both granules classes). Finally, G-CSF therapy markedly reduced the apoptotic rate of the isolated neutrophil. Therefore, considering disparate functional and biochemical activities, the real benefit of G-CSF therapy may lie in enhanced number and survival of neutrophils.

In Vivo Treatment With Granulocyte Colony-Stimulating Factor Results in Divergent Effects on Neutrophil Functions Measured In Vitro

AbstractWe have studied the effects of granulocyte colony-stimulating factor (G-CSF) administration to normal individuals on a variety of functional and biochemical neutrophil characteristics that relate to host defense. G-CSF adversely affected neutrophil (polymorphonuclear leukocyte [PMN]) chemotaxis. While this could be partially explained by reduced assembly of neutrophil F-actin, we also recognized an elevated cytosolic calcium mobilization and a normal upregulation of neutrophil CD11b. G-CSF resulted in reduced PMN killing of Staphylococcus aureus with a 10:1 (bacteria:neutrophil) ratio and normal killing with a 1:1 ratio. In association with this, we demonstrated divergent effects on the respiratory burst of intact cells and divergent effects on the content of marker proteins for neutrophil granules. While G-CSF may have resulted in increased content of cytochrome b558 in the cell membrane, it did not alter the amounts of cytosolic oxidase components. After therapy, there was normal content of the azurophilic granule marker, myeloperoxidase, decreased content of the specific granule marker, lactoferrin, and normal content of lysozyme (found in both granules classes). Finally, G-CSF therapy markedly reduced the apoptotic rate of the isolated neutrophil. Therefore, considering disparate functional and biochemical activities, the real benefit of G-CSF therapy may lie in enhanced number and survival of neutrophils.

Biosynthesis of Granule Proteins in Normal Human Bone Marrow Cells. Gelatinase Is a Marker of Terminal Neutrophil Differentiation

Differentiation and maturation of myeloid cells is characterized by the sequential acquisition of two distinct cytoplasmic granule subsets, azurophil granules and specific granules. We recently showed the existence of a third granule subset, gelatinase granules. To investigate whether appearance of gelatinase granules marks a further step in maturation of myeloid cells beyond the appearance of specific granules, we sorted normal human bone marrow cells into one of three groups according to maturity by centrifugation on Percoll density gradients. The biosynthesis of myeloperoxidase (MPO) (an azurophil granule marker), lactoferrin and neutrophil gelatinase-associated lipocalin NGAL (specific granules markers) and gelatinase was then studied in each of these groups. We found that gelatinase was synthesized mainly in the group containing band cells and segmented cells. This contrasted with lactoferrin and NGAL, which were synthesized almost exclusively in the group containing myelocytes and metamyelocytes, and with MPO, which was mainly synthesized in the group containing myeloblasts and promyelocytes. Immunocytochemistry was in full agreement with the biosynthesis data, and showed that gelatinase appears in band cells, whereas NGAL and lactoferrin both appear in myelocytes. Thus, acquisition of gelatinase granules marks a step in neutrophil differentiation beyond the appearance of specific granules.

Modulation of Rat Peripheral Polymorphonuclear Leukocyte Response by Nitric Oxide and Arginine

The effect of nitric oxide (NO) on the luminol-dependent chemiluminescence (LCL) response of rat polymorphonuclear leukocytes (PMNLs) was analyzed by using sodium nitroprusside (SNP), a NO donor, and L-arginine (L-arg), a NO precursor. A significant reduction in the LCL intensity was observed in presence of SNP (100 mumol/L) or L-arg (5 or 10 mmol/L) in arachidonic acid (AA) phorbol ester (PMA) and formyl-methionyl-leucyl-phenylalanine stimulated PMNLs. However, opsonized zymosan-induced LCL was not attenuated significantly. Reduction in hydroxyl radical and superoxide generation was also observed in SNP- or L-arg-pretreated cells. D-Arg (10 mmol/L) pretreatment did not inhibit PMNLs' LCL response. Furthermore, methylene blue (5 mumol/L) and L-NG-mono methyl-L-arginine (100 or 300 mumol/L) significantly attenuated the LCL response, as induced by various agonists. Cyclic GMP did not alter the reactive oxygen species generation from rat PMNLs. In addition, AA-induced release of myeloperoxidase, a marker of azurophilic granules, was found to be enhanced in L-arg- (10 mmol/L) pretreated PMNLs. The results suggest that NO inhibits free radical generation from rat PMNLs.

Modulation of rat peripheral polymorphonuclear leukocyte response by nitric oxide and arginine

The effect of nitric oxide (NO) on the luminol-dependent chemiluminescence (LCL) response of rat polymorphonuclear leukocytes (PMNLs) was analyzed by using sodium nitroprusside (SNP), a NO donor, and L-arginine (L-arg), a NO precursor. A significant reduction in the LCL intensity was observed in presence of SNP (100 mumol/L) or L-arg (5 or 10 mmol/L) in arachidonic acid (AA) phorbol ester (PMA) and formyl- methionyl-leucyl-phenylalanine stimulated PMNLs. However, opsonized zymosan-induced LCL was not attenuated significantly. Reduction in hydroxyl radical and superoxide generation was also observed in SNP- or L-arg-pretreated cells. D-Arg (10 mmol/L) pretreatment did not inhibit PMNLs' LCL response. Furthermore, methylene blue (5 mumol/L) and L-NG- mono methyl-L-arginine (100 or 300 mumol/L) significantly attenuated the LCL response, as induced by various agonists. Cyclic GMP did not alter the reactive oxygen species generation from rat PMNLs. In addition, AA-induced release of myeloperoxidase, a marker of azurophilic granules, was found to be enhanced in L-arg- (10 mmol/L) pretreated PMNLs. The results suggest that NO inhibits free radical generation from rat PMNLs.

Differential up-regulation of specific and azurophilic granule membrane markers in electropermeabilized neutrophils

We have developed an alternative method to study the degranulation in electropermeabilized human neutrophils by measuring the up-regulation of the specific membrane markers CD63 (residing in the azurophilic granules of resting neutrophils) and CD67 (present in specific granules). The expression of these marker proteins was measured by the binding of specific antibodies to paraformaldehyde-fixed cells and subsequent flow cytometry. We first investigated whether the changes in CD63 and CD67 expression after stimulation of intact cells were comparable with earlier measurements of neutrophil degranulation, in which the release of soluble marker proteins was measured. These experiments indicated that this new method compare favourably with earlier studies, both with respect to kinetics and stimulus dependency. Subsequently, we applied this method (which does not include centrifugation of the cells)_to study degranulation in electropermeabilized neutrophils. In permeabilized neutrophils, a clear up-regulation of the specific granule marker CD67 was observed upon incubation with a free Ca 2+ concentration of 1 μM, a value of the cytosolic free Ca 2+ concentration occurring in formymethionyl-leucyl-phenylalanine (FMLP)-activated neutrophils. The azurophilic granule marker CD63 required GTP-γ-S besides 1 μM Ca 2+ for a significant up-regulation. Hence, our study indicates a different requirement for intracellular signals of the two main types of granules in human neutrophils.

RTNFα Facilitates Human Polymorphonuclear Leukocyte Adherence to Fibrinogen Matrices With Mobilization of Specific and Tertiary but Not Azurophilic Granule Markers

rTNF alpha facilitates highly reproducible adherence of polymorphonuclear leukocyte (PMN) to fibrinogen-coated surfaces in a concentration- and time-dependent manner. The adhesion was maximal with 1.0 nM rTNF alpha within 40-50 min at 37 degrees C. A monoclonal antibody (1B4) directed toward the beta 2-chain of the integrin receptor for fibrinogen (CD11b, CD18) completely inhibited the rTNF alpha induced adhesion. TNF alpha caused a time-dependent secretion of the granule markers gelatinase and lactoferrin but no liberation of myeloperoxidase and minimal production of A alpha(1-21), a specific cleavage product of fibrinogen generated by elastase, as markers for the azurophilic granule. PMN adhered to fibrinogen in the presence of rTNF alpha could be further stimulated with cytochalasin B and N-formyl-methionyl-leucyl-phenylalanine (FMLP) to release azurophilic granule markers as measured by increasing MPO activity and A alpha(1-21) production over time. Thus the rTNF alpha-facilitated adherence of PMN to a fibrinogen matrix provides a system for partial activation of PMN resulting in release of markers of specific and tertiary but not azurophilic granules. Moreover, these conditions should provide an opportunity to define more clearly the signal transduction processes involved in azurophilic granule release.

Inhibition of neutrophil response by curcumin

Blood neutrophils, when exposed to appropriate stimuli, aggregate, degranulate and generate superoxide anion. Curcumin, a non-steroidal antiinflammatory agent, modulated these functions, depending upon the kind of stimulus used. It inhibited monkey neutrophil aggregation induced by chemotactic peptide fmlp and zymosan activated plasma (ZAP) but did not affect that induced by serum treated zymosan (STZ) and arachidonic acid (AA). Generation of O2- radical was inhibited by curcumin, when cells were stimulated by AA, STZ and fmlp. Curcumin inhibited the release of myeloperoxidase, an azurophilic granule marker enzyme. Release of lysozyme was less susceptible to inhibition by curcumin. The results suggest that curcumin interferes with neutrophil responses to various physiological stimuli and a part of its antiinflammatory action is mediated via inhibition of neutrophil function. Inhibition of neutrophil function by curcumin appears to be mediated via calcium dependent mechanisms.

Natural inhibitor from Candida albicans blocks release of azurophil and specific granule contents by chemotactic peptide-stimulated human neutrophils

A product released by Candida albicans hyphae which was previously determined to block the neutrophil respiratory burst also inhibited the degranulation response elicited by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP). When neutrophils were incubated with 100 micrograms of this Candida hyphal inhibitory product (CHIP) per ml and stimulated with 1,000 nM FMLP, release of the azurophil granule marker beta-glucuronidase and the specific granule marker lactoferrin was decreased to 31.2 +/- 5.7 and 35.7 +/- 5.3% of control, respectively. Inhibition was concentration dependent, with a half-maximal reduction of beta-glucuronidase and lactoferrin release being induced by 4 and 1 micrograms of CHIP per ml, respectively. In contrast to these striking alterations in response to FMLP, CHIP had no significant effect on lactoferrin release stimulated by phorbol myristate acetate. Moreover, actin polymerization, which has been suggested to be involved in regulation of degranulation, was unaffected by CHIP whether the neutrophils were stimulated by FMLP or phorbol myristate acetate. The selectivity of inhibition of neutrophil degranulation thus provides additional confirmation that CHIP is a useful agent for exploring human neutrophil activation pathways.