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To study the effect of polyclonal intravenous immunoglobulin G (IVIG) on neutrophils in vitro, adsorption of immunoglobulin G (IgG) to solid surfaces has to be prevented, because IgG bound to a solid surface can activate neutrophils through activating FcγRs. In this study we demonstrate that poloxamer 407, a non ionic surfactant, at low concentration (0.05%) prevented the adsorption of high concentrations of IgG (5 mg/ml) better than other blocking agents without interfering with the interaction of IgG with the neutrophils. Poloxamer 407 is therefore a suitable blocking agent to prevent the interaction of immunoglobulin with solid surfaces in cell-based in vitro experiments., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Neutrophil function relies largely on the ability of the cell to mobilize its different granules and vesicles to the cell surface and thereby expose and/or release effector molecules to the surrounding tissue. To properly identify these subcellular compartments is thus a prerequisite for studies of neutrophil physiology. A range of specific markers for the classical granules is available, but finding optimal markers for the secretory vesicles and plasma membrane has historically been more challenging. Latent and non-latent alkaline phosphatase activities are often used to distinguish these two light membrane structures, but the outcome using this technique depends on the level of cellular activation. Therefore, HLA-I was introduced some years ago as a specific, stimulation-independent marker for the plasma membrane. In this study we however report that detailed fractionation studies of neutrophil cytoplasts, lacking secretory vesicles, granules and other dense organelles, reveal that the HLA-I antigen is not only co-localizing with the plasma membrane marker ALP, but is also present in other, more dense organelles. Further, we found the mixed enzyme-linked immunosorbent assay (MELISA), detecting the beta(2)-microglobulin/HLA-I complex, to be negatively influenced by uncomplexed beta(2)-microglobulin present in the specific granules and secretory vesicles, making it difficult to use HLA-I as a plasma membrane marker during maturation of for example phagolysosomes.

Specific granule protein of 28 kDa (SGP28), also termed cysteine-rich secretory protein 3 (CRISP-3), is a glycoprotein that belongs to a family of cysteine-rich secretory proteins (CRISPs). SGP28 was originally discovered in human neutrophils, but transcripts are widely distributed in exocrine glands (salivary glands, pancreas, and prostate) and also found at lower levels in epididymis, ovary, thymus, and colon. The function of SGP28/CRISP-3 is not yet known. Similarities to pathogenesis-related proteins in plants and the expression in neutrophils and exocrine glands suggest that SGP28/CRISP-3 may play a role in innate host defense. We describe here the production of a recombinant, C-terminally truncated form of CRISP-3 (rCRISP-3Delta) and the generation of polyclonal antibodies against rCRISP-3Delta that are useful in immunoblotting and immunocytochemistry. We present a specific, accurate, and reproducible enzyme-linked immunosorbant assay (ELISA) for the measurement of CRISP-3 with a detection limit of 2 ng/ml. We further demonstrate the presence of CRISP-3 protein in human plasma (6.3 microg/ml), saliva (21.8 microg/ml), seminal plasma (11.2 microg/ml), and sweat (0.15 microg/ml), and describe the coexistence of two different molecular weight forms of CRISP-3, representing an N-glycosylated and a non-glycosylated form of the mature protein.

In this paper, we illustrate the fine structural localization of distinct marker proteins in the organelles of human neutrophils and outline our preferred methods for processing ultrathin cryosections for use with immunoelectron microscopy. Previous work has determined the subcellular localization of certain marker proteins within intact polymorphonuclear neutrophilic leukocytes (PMN) and PMN fractions. These are as follows: myeloperoxidase (MPO) for azurophilic granules, lactoferrin for specific/secondary granules, gelatinase for gelatinase/tertiary granules, albumin for the secretory vesicles, and HLA class I and L-selectin for the plasma membrane. In addition to analyzing the heterogeneity of the PMN granule populations, new information on the lysosomal system of this cell is reviewed and extended by the localization of the lysosome-associated membrane proteins (LAMPs) and the cation-independent mannose 6-phosphate receptor (CI-M6PR). LAMPs were absent in all identified granule populations, but were found in the membranes of vesicles, multivesicular bodies (MVB), and multilaminar compartments (MLC). We show here that MVB contain CI-M6PR whereas MLC do not. Furthermore, since MLC contain LAMPs but not the receptor, they probably correspond to the late endosome. By current criteria, the true lysosomes of the resting PMN are MVB and MLC. Finally, although azurophil granules contain acid hydrolases their membranes do not contain LAMPs and they cannot be classified as lysosomes, but rather are more similar to regulated secretory granules.

Human neutrophils are endowed with secretory vesicles, an intracellular reservoir of integral membrane proteins. Secretory vesicles fuse readily with the plasma membrane upon stimulation of the neutrophil, resulting in prompt transportation of various receptors and adhesion proteins to the neutrophil surface. This upregulation of membrane proteins has been shown to be crucial during the sequential steps preceding neutrophil extravasation. However, the lack of separation of secretory vesicles from the plasma membrane when the postnuclear supernatant from cavitated neutrophils is centrifuged on a Percoll density gradient has been an obstacle for the investigation of secretory vesicles. By use of Free Flow Electrophoresis (FFE) we were able to obtain a separation of secretory vesicles from the plasma membrane vesicles, and this procedure has been a valuable tool in the investigation of secretory vesicles. Methodological considerations and results obtained by FFE of light membranes from human neutrophils are presented in this paper.

Neutrophils contain a variety of proteins that endow the cell with its capacity to migrate towards and eliminate microbial pathogens. Many of these proteins are largely or exclusively localized to neutrophils. It is therefore of interest to quantitate these proteins in a variety of clinical settings as well as in basic research. The aim of this survey is to give an introduction to some of the more commonly used methods for quantitation of neutrophil proteins and to discuss advantages and problems of the different methods and the relevance of quantitating neutrophil proteins in different biological settings.

The neutrophilic granulocyte is the most numerous leukocyte in peripheral blood. The development from a multipotent progenitor cell to a mature neutrophil takes place in the bone marrow over a period of 10-14 days. In order to understand the cellular mechanisms behind this process, it is necessary to investigate cells from different stages of neutrophil differentiation. As no human cell line has the ability to faithfully reproduce the entire differentiation process from promyelocyte to segmented neutrophil the analysis of many maturation-dependent processes has to be done on neutrophil precursors from human bone marrow. For this purpose, a technique whereby neutrophil precursors can be isolated from the bone marrow and separated according to their maturity is required. Two different methods have been shown to be useful for isolation of immature neutrophils: density centrifugation on a Percoll gradient, where the increasing density of the cells with maturity forms the basis of the separation, and multidimensional flow cytometry, where a combination of size, granulation, and surface markers are used for the discrimination of different neutrophil precursors. This paper will review these two methods for separation of neutrophil precursors with special emphasis on Percoll density centrifugation and the use of cells isolated by this technique for the analysis of neutrophil-specific mRNAs and the biosynthesis of neutrophil granule proteins.

Ca(2+) signalling in neutrophils is important for triggering and coordinating the behaviour of neutrophils. Fluorescent probes for cytosolic free Ca(2+) concentration, e.g., fura2 and fluo3, have been widely used in neutrophils. These probes can be used to monitor Ca(2+) in the cytosol, the nucleus, near the plasma membrane and theoretically within Ca(2+) storage organelles. The longer wavelength indicators, e.g., fluo3 and calcium green, can be used confocally to monitor subcellular Ca(2+) changes in the cytosol of neutrophils and in the nucleus. Confocal techniques also permit "impossible views" imaging of Ca(2+) and newer scanning techniques promise very fast temporal resolution. Techniques using chlortetracycline (CTC) and DiOC(6)(3) are also described for monitoring the position of Ca(2+) storage sites in neutrophils and for manipulating their activity. Thus, in this review, a spectrum of new (and older) optical techniques are presented which are useful for measuring, monitoring and manipulating cytosolic free Ca(2+) concentration and Ca(2+) storage in neutrophils. With these techniques, it is hoped that more insight will be gained into both the mechanism of and the consequences of Ca(2+) signalling in neutrophils.

Methods for studying human neutrophils at the ultrastructural level by enzyme cytochemistry and immunocytochemistry are presented. The focus of these methods is on the analysis of the alkaline phosphatase-positive secretory organelle of these cells. These methods provide unique information which, when coupled with biochemical studies, provide for the most complete analysis of neutrophil structure and function.

In recent years, several investigators have addressed the question of whether mature polymorphonuclear neutrophils (PMN) are able to secrete cytokines. Their studies have brought forward new and exciting discoveries, by establishing that the release of inflammatory cytokines constitutes a novel and important aspect of the neutrophil biology, thereby emphasizing that PMN should no longer be regarded as cells that only release preformed mediators. Although it is still premature to assess the true biological significance of cytokine production by neutrophils, this new aspect of neutrophil biology opens novel perspectives as to the potential role of these cells in the inflammatory and immune responses. In this context, a correct methodological analysis and a detailed molecular investigation of the mechanisms regulating cytokine production by neutrophils in vitro is a critical and fundamental step to better understand how the release of cytokines by PMN may influence pathophysiological processes in vivo. We now describe and discuss the approach that we typically used throughout most of the last decade to characterize cytokine production by human neutrophils, as illustrated herein for a protein that is expressed and released by PMN, namely, vascular endothelial growth factor (VEGF).

Subcellular fractionation has been an important tool in the investigation of neutrophil structural organization including granule heterogeneity, composition and mobilization. The resolution of organelles obtained by subcellular fractionation was improved considerably after the introduction of nitrogen cavitation as an efficient but gentle means of disrupting neutrophils and with Percoll as a density medium. This paper describes in detail the methodology of subcellular fractionation of nitrogen cavitated neutrophils on one-, two-, and three-layer Percoll density gradients. Appropriate marker proteins are presented for neutrophil organelles including azurophil, specific and gelatinase granules, in addition to secretory vesicles and plasma membranes. The dynamics of granule and secretory vesicle exocytosis is demonstrated by subcellular fractionation of resting and activated human neutrophils. Finally, the paper describes the applications of subcellular fractionation in the investigation of the localization of neutrophil constituents, in protein purification schemes and in the study of translocation of cytosolic proteins to isolated neutrophil organelles.

hCAP-18 is a newly described protein of human neutrophilic granulocytes which belongs to the cathelicidin family of antimicrobial proteins. Members of this protein family share a common N-terminal sequence followed by a highly diverse antimicrobial, cationic C-terminus. The present work describes the production of recombinant hCAP-18, the generation of antibodies to the protein and the development of an accurate, sensitive and specific ELISA for the detection of hCAP-18 in cells, plasma and urine with a detection limit of 0.084 ng/ml. The amount of hCAP-18 in neutrophils is 0.627 microgram protein per 10(6) cells. The plasma level is 1.18 micrograms/ml which is several fold higher than for other neutrophil specific granule proteins. hCAP-18 is present in plasma as high molecular weight complexes. In accordance with this, hCAP-18 is barely excreted in the urine. The bone marrow appears to be the major source of plasma hCAP-18. The high level of hCAP-18 in plasma may provide an important defense against microorganisms and endotoxins.

NGAL is a newly described member of the lipocalin protein family, secreted from specific granules of human neutrophils upon activation of the cells. Its ability to bind the bacterial chemotactic formylpeptide FMLP indicates, that NGAL may have modulatory effects on the immune response. We here describe monoclonal and polyclonal antibodies against NGAL, which can be used for Western blotting and immunohistochemistry, and furthermore describe two ELISAs using either exclusively the polyclonal anti-NGAL antibodies or the polyclonal and monoclonal antibodies in combination. The assays are equally specific, reproducible, accurate, and sensitive, with a detection limit of 32 ng/l. The antibodies and assays will be valuable tools in the future investigation of NGAL expression in inflammatory and malignant disorders and in the elucidation of the function of NGAL as a modulator of the inflammatory response.

Indirect immunofluorescence (IIF) techniques have shown that anti-neutrophil cytoplasm autoantibodies (ANCA) are useful serological markers for certain small vessel vasculitides and the non-vasculitic inflammatory disorders. ELISA procedures, using purified molecules as solid phase ligands, helped to identify proteinase 3 (PR3) and myeloperoxidase (MPO) as two major ANCA antigens; and recently we characterised bactericidal/permeability-increasing protein (BPI) as another important ANCA antigen. ANCA against these three antigens are associated with different clinical disorders. Therefore purified antigens are needed to determine these different autoantibody specificities in order to help diagnosis and guide treatment. Here we describe a method using Orange-A dye ligand chromatography and cation exchange chromatography for the sequential purification of PR3, MPO and BPI, from the same starting material, an acid extract of normal human neutrophil granules. After separation the three antigens were free of contamination by each other and no traces were found of other known minor ANCA antigens.

We show that phagocyte production of reactive oxygen species can be measured using a microtitre plate based chemiluminescence blotting technique. The production of reactive oxygen species is determined by their ability to catalyze the oxidation of luminol or isoluminol, resulting in light emission which is recorded on a photographic film. The method permits the determination of NADPH oxidase activity from as few as 9000 cells. It could be used to detect NADPH oxidase defects in neutrophils (e.g. from patients suffering from chronic granulomatous disease), and to screen pharmaceuticals with scavenging activity for reactive oxygen species.

Intracellular oxidation of dihydrorhodamine 123 (DHR) to the fluorescent compound rhodamine 123 (Rho123) was used to detect the production of oxygen metabolites in activated neutrophils. Total leukocyte preparations can be used in this assay, which is a great advantage when priming of the respiratory burst is studied. We have defined the conditions that should be taken into account when priming is studied with this assay. We found that neither the extent nor the kinetics of DHR oxidation match those of NADPH oxidase activity. In addition, DHR oxidation is influenced by the absolute and relative number of neutrophils in the leukocyte suspension, by the DHR concentration and by myeloperoxidase availability. The results presented in this study emphasize the need for carefully designed experiments when DHR is used to study the respiratory burst in neutrophils.

A simple and reproducible radioreceptor assay (RRA) has been developed using membranes from CHO cells which can stably express human platelet-activating factor (PAF) receptor. The CHO cells expressing the PAF receptor, termed CHO.1F8, showed a significant intracellular Ca2+ response to PAF, and the same binding properties to [3H]WEB 2086, a PAF antagonist, as reported (Kd, 13.6 +/- 1.9 nM; Bmax, 2.5 +/- 0.4 pmol/mg protein (n = 6)). A competitive binding assay was done using the CHO.1F8 cell membranes and [3H]WEB 2086. The minimum detectable dose of PAF was 0.3 nM (approximately 30 pg per well) and the assay was highly specific for PAF. This method makes it possible to handle large numbers of samples rapidly and simultaneously, since the receptor membrane is prepared in advance and the binding assay can be completed within 3 h. Using this method, we have determined the production and cell association of PAF in human neutrophils.

Grancalcin is a newly discovered cytosolic calcium-binding protein, belonging to the group of EF-hand proteins. Grancalcin is specifically associated with cells originating in the bone marrow. Grancalcin binds reversibly to secretory vesicles and plasma membranes in human neutrophils and might therefore play a role in the regulation of vesicle/granule exocytosis. We describe here the production of recombinant grancalcin, the generation of antibodies to the protein and the development of a specific, accurate and sensitive ELISA for the detection of human leukocytic grancalcin. This ELISA may be useful for monitoring leukocyte infiltration into tissues.

Cyclophilins A and B (CyPA and CyPB) are known to be the main binding proteins for cyclosporin A (CsA), a potent immunosuppressive drug. Due to the high homology between the two proteins, antibodies to CyPB were found to cross-react with CyPA. In order to avoid this phenomenon, we raised specific antibodies against peptides copying the most divergent parts of the two sequences. These antibodies allowed us to develop an ELISA capture assay selective for either isotype. Thus, we showed that leukocyte CyPB concentration was almost ten times lower than that of CyPA, and that in contrast to the results described in the literature, only CyPB was released in plasma. Moreover, CyPB levels in leukocytes and plasma were found to correlate for the same donor, but no relationship was found with CyPA level.

Human neutrophil lipocalin (HNL) is a newly discovered protein from human neutrophil secretory granules. A double-antibody radioimmunoassay (RIA) was developed for the measurement of HNL in various body fluids and its high specificity was confirmed by the absence of cross-reaction with other granulocyte granule proteins. The RIA measures HNL within the range of 4-256 micrograms/l. The intra- and interassay coefficients of variation were less than 6% and 10%, respectively. When HNL was added to serum samples full recovery was obtained. Sera and plasma from 100 apparently healthy individuals revealed a mean level of 78.40 micrograms/l (range 37.95-190.87 micrograms/l) in serum and a mean level of 50.65 micrograms/l (range 30.51-105.8 micrograms/l) in EDTA-plasma. The distribution of HNL after gel filtration indicated that HNL exists mainly in two major forms, dimer and monomer. This, in addition to the excellent recovery, suggests that these major forms of HNL do not bind to compounds in serum or plasma that would interfere with the assay. The high specificity, sensitivity, reproducibility and accuracy of the present assay should facilitate the measurement of HNL in blood and other body fluids.

A sensitive sandwich ELISA has been developed to measure levels of native bactericidal/permeability-increasing protein (BPI) as well as two recombinant forms of BPI (rBPI and rBPI23) in human body fluids. The linear range for the rBPI and rBPI23 standard curves were 100-6000 pg/ml and 25-800 pg/ml respectively. Recovery of different concentrations of rBPI spiked into pooled human plasma samples averaged 83% and ranged from 65% at 300 ng/ml to 97% at 3 ng/ml. Recovery of rBPI23 averaged 56% and ranged from 30% at 0.5 ng/ml to 90% at 50,000 ng/ml. Because LBP is present in normal human plasma and shares sequence homology with BPI, the effects of rLBP on the BPI ELISA were also evaluated. Under standard assay conditions, rLBP caused minimal interference with BPI detection. At 100 micrograms/ml, rLBP generated a signal equivalent to 3 ng/ml of rBPI and 0.6 ng/ml of rBPI23. Matched serum and plasma samples were collected from 20 healthy adults to measure endogenous levels of BPI. The range of BPI concentrations was < 0.2-2.1 ng/ml in plasma and 4.9-72.1 ng/ml in serum. Western blot analysis indicated that the BPI ELISA immunoreactivity in plasma and serum correlated with the presence of a protein doublet (M(r) approximately 60,000), which comigrated with native BPI extracted from human neutrophils. These data demonstrate that low levels of holo-BPI are present in plasma, and suggest that additional quantities of BPI were released from neutrophils during the process of coagulation.

Superoxide dismutase (SOD)-inhibitable reduction of cytochrome c is the basis for a widely used assay to measure superoxide production. We report novel modifications leading to a dual wavelength, high throughput simultaneous kinetic and endpoint microplate assay with high reproducibility. Neutrophils were isolated using a modified elutriation procedure to minimize priming and adherence during isolation. Cytochrome c reduction was measured in a microplate reader using 96-well polystyrene plates with a modified (Plastek A*) surface to prevent the adherence and consequent activation of PMNs. Comparison of Plastek A* treated and untreated plates revealed a statistically significant difference in basal as well as stimulated levels of superoxide production. Absorption measurements were made at both 550 nm, the absorption maximum of reduced cytochrome c, and 557 nm, an isosbestic point. A significant increase in both well-to-well reproducibility and sensitivity (detection limit) was realized by using the normalized 550-557 nm difference values compared to the 550 nm absorbance values alone. These modifications represent an improved method for handling and assessing the function of superoxide production, providing greater experimental reproducibility and lessening the perturbations caused by the microplate.

We developed and optimized an enzyme immunoassay for human neutrophil defensins, cationic cysteine-rich peptides that participate in host defense and inflammation. The assay utilizes a sandwich design with a monoclonal capture antibody and a biotinylated monoclonal detecting antibody. Cetrimonium bromide is employed to obviate non-specific binding of defensins to surfaces. The assay has a sensitivity of 0.04-0.05 ng/ml and a working range of 0.05-10 ng/ml.