Failure to detect IL-3-binding sites on human mast cells

IL-3, a pleiotropic lymphokine, has been termed mast cell growth factor because it promotes growth and differentiation of murine mast cells. Murine mast cells, in turn, express cell surface receptors for IL-3. Human rIL-3 has been shown to induce proliferation and differentiation of human basophils and to activate basophils via high affinity binding sites. To investigate whether human mast cells express IL-3R, binding studies with 125I-radiolabeled human rIL-3 were performed on HMC-1, a novel human mast cell line, and on pure populations (i.e., 93 to 99% purity) of human tissue mast cells obtained with mAb and C from dispersed lung (n = 2). Unexpectedly, neither enriched human lung mast cells nor HMC-1 cells bound radiolabeled human rIL-3 specifically. Moreover, human rIL-3 failed to promote uptake of [3H]thymidine, synthesis of histamine, histamine releasability, or changes in expression of mast cell differentiation Ag (YB5B8, CD54/ICAM-1, CD9/p24, CD33/gp67) on either human lung mast cells or HMC-1 cells. It is hypothesized that the fundamental difference in the biologic response to IL-3 between human and murine mast cells is due to a loss during evolution of mast cell high affinity IL-3 binding sites.     

Basophil-derived mouse mast cell protease 11 induces microvascular leakage and tissue edema in a mast cell-independent manner

Mouse mast cell protease 11 (mMCP-11) is the most recently identified member of the mouse mast cell tryptase family. This tryptase is preferentially produced by basophils in contrast to other members that are expressed by mast cells but not basophils. Although blood-circulating basophils have long been considered as minor and redundant relatives of tissue-resident mast cells, recent studies illustrated that basophils and mast cells play distinct roles in vivo. To explore the in vivo role of basophil-derived mMCP-11, here we prepared recombinant mMCP-11 and its protease-dead mutant. Subcutaneous injection of the wild-type mMCP-11 but not the mutant induced edematous skin swelling with increased microvascular permeability in a dose-dependent manner. No apparent infiltration of proinflammatory cells including neutrophils and eosinophils was detected in the skin lesions. The cutaneous swelling was abolished by the pretreatment of mice with indomethacin, a cyclooxygenase inhibitor, suggesting the major contribution of prostaglandins to the microvascular leakage. Of note, the cutaneous swelling was elicited even in mast cell-deficient mice, indicating that mast cells are dispensable for the mMCP-11-induced cutaneous swelling. Thus, basophil-derived mMCP-11 can induce microvascular leakage via prostaglandins in a mast cell-independent manner, and may contribute to the development of basophil-mediated inflammatory responses.     

Peptides of major basic protein and eosinophil cationic protein activate human mast cells

The eosinophil granule proteins, major basic protein (MBP) and eosinophil cationic protein (ECP), activate mast cells during inflammation; however the mechanism responsible for this activity is poorly understood. We found that some theoretical tryptase-digested fragments of MBP and ECP induced degranulation of human cord blood-derived mast cells (HCMCs). The spectrum of activities of these peptides in HCMCs coincided with intracellular Ca²⁺ mobilization activities in Mas-related G-protein coupled receptor family member X2 (MRGPRX2)-expressing HEK293 cells. Two peptides corresponding to MBP residues 99–110 (MBP (99–110)) and ECP residues 29–45 (ECP (29–45)), respectively, induced degranulation of HCMCs and intracellular Ca²⁺ mobilization in MRGPRX2-expressing HEK293 cells in a concentration-dependent manner. Stimulation with MBP (99–110) or ECP (29–45) induced the production of prostaglandin D2 by HCMCs. The activities of MBP (99–110) and ECP (29–45) in both HCMCs and MRGPRX2-expressing HEK293 cells were inhibited by MRGPRX2-specific antagonists. In conclusion, these results indicated that MBP and ECP fragments activate HCMCs, and it may occur via MRGPRX2. Our findings suggest that tryptase-digested fragments of eosinophil cationic proteins acting via the MRGPRX2 pathway may further our understanding of mast cell/eosinophil communication.     

Glycomic analysis of human mast cells, eosinophils and basophils

In allergic diseases such as asthma, eosinophils, basophils and mast cells, through release of preformed and newly generated mediators, granule proteins and cytokines, are recognized as key effector cells. While their surface protein phenotypes, mediator release profiles, ontogeny, cell trafficking and genomes have been generally explored and compared, there has yet to be any thorough analysis and comparison of their glycomes. Such studies are critical to understand the contribution of carbohydrates to the induction and regulation of allergic inflammatory responses and are now possible using improved technologies for detecting and characterizing cell-derived glycans. We thus report here the application of high-sensitivity mass spectrometric-based glycomics methodologies to the analysis of N-linked glycans derived from isolated populations of human mast cells, eosinophils and basophils. The samples were subjected to matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) screening analyses and MALDI-TOF/TOF sequencing studies. Results reveal substantive quantities of terminal N-acetylglucosamine containing structures in both the eosinophil and the basophil samples, whereas mast cells display greater relative quantities of sialylated terminal epitopes. For the first time, we characterize the cell surface glycan structures of principal allergic effector cells, which by interaction with glycan-binding proteins (e.g. lectins) have the possibility to dictate cellular functions, and might thus have important implications for the pathogenesis of inflammatory and allergic diseases.     

Stimulation of basophil and rat mast cell histamine release by eosinophil granule-derived cationic proteins

Major basic protein (MBP), an arginine-rich basic polypeptide that constitutes the crystalloid core of the large specific eosinophil granule, has previously been shown to stimulate noncytolytic histamine release from human basophils and rat mast cells by an IgE-independent mechanism. Two additional basic polypeptides present in eosinophil granules, eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN), were examined for similar activity in the present study. Acid-solubilized eosinophil granules were fractionated by chromatography on a Sephadex G-50 column. Incubation of basophil-containing human mononuclear cells with the individual column fractions demonstrated that histamine release occurred only with the fractions that contained MBP. The selectivity of the basophil response for MBP was confirmed by using equimolar concentrations of purified MBP, ECP, and EDN. In contrast, both MBP and ECP, but not EDN, stimulated histamine release from purified rat peritoneal mast cells. Reduction and alkylation of the MBP molecule diminished the response of human basophils to MBP but enhanced the potency of the molecule with rat mast cells. The distinct potency of MBP as a stimulus for histamine secretion from human basophils suggests that eosinophil release of MBP may be a specific event in the augmentation of immediate hypersensitivity reactions and other disorders characterized by eosinophilia.     

Major basic protein homolog (MBP2): a specific human eosinophil marker

Human eosinophil granule major basic protein (MBP1) is an exceedingly basic (isoelectric point >11) 14-kDa protein, comprising the core of the secondary eosinophil granule. Recently, a less cationic homolog of MBP, termed MBPH or simply, MBP2, has been discovered. We prepared a panel of mAbs to MBP2 and used these Abs to localize and quantitate this molecule in leukocytes and biological fluids. Specific mAbs for MBP2 were selected using slot-blot analyses and used in a two-site immunoassay, Western blotting, and immunofluorescence microscopy. The sensitivity of the immunoassay was markedly improved by reduction and alkylation of MBP2. MBP1 is more abundant than MBP2 in lysates of eosinophils and their granules, as judged by immunoassay and Western blotting. By immunofluorescence, MBP1 is present in eosinophils, basophils, and a human mast cell line (HMC1), whereas MBP2 is only detected in eosinophils. Neither MBP1 nor MBP2 could be detected in any other peripheral blood leukocyte. MBP2 levels measured in plasma and serum were essentially identical. In contrast to past measurements for MBP1, MBP2 was not detected above normal levels in sera from pregnant donors. However, measurement of serum MBP2 discriminated patients with elevated eosinophils from normal subjects, and MBP2 was also detectable in other biological specimens, such as bronchoalveolar lavage, sputum, and stool. These results indicate that MBP2 is present only in eosinophils and that it may be a useful biomarker for eosinophil-associated diseases.     

Evaluation of human peripheral blood leukocytes for mast cell tryptase

Murine monoclonal and goat polyclonal antibodies against tryptase, the dominant neutral protease and protein component in secretory granules of human mast cells, were used to assess the presence of tryptase in peripheral leukocytes. Carnoy's fluid-fixed cytocentrifuge preparations of enriched populations of lymphocytes, monocytes, eosinophils, and neutrophils showed no reactivity with anti-tryptase antibodies by a sensitive indirect immunoperoxidase procedure. Dispersed human lung mast cells showed strong granular cytoplasmic staining with both antibodies, whereas only approximately 50% of the peripheral blood basophils detectable with Wright's stain were detected with anti-tryptase antibodies, and these showed a staining pattern that was faint, granular, and cytoplasmic at high concentrations of antibody. At lower antibody concentrations mast cell staining was still intense, whereas basophils were not stained. Extracts of neutrophils and lymphocytes of up to 90% purity had undetectable amounts of tryptase by an ELISA sandwich immunoassay, as well as undetectable enzymatic activity with tosyl-L-gly-pro-lys-p-nitroanilide (a sensitive substrate for tryptase) in the presence of soybean trypsin inhibitor. Extracts of basophil-enriched (6 to 50% purity) preparations contained 0.046 +/- 0.013 pg of tryptase per basophil by the immunoassay along with 2 X 10(-9) +/- 0.8 X 10(-9) U of tryptase-like enzyme activity per basophil, compared with corresponding values of 12 pg, 480 X 10(-9) U of tryptase per human lung mast cell. Thus very small amounts of tryptase are present in human basophils (approximately 0.4% of that found in mast cells), but not in other peripheral leukocytes.     

Tat protein is an HIV-1-encoded beta-chemokine homolog that promotes migration and up-regulates CCR3 expression on human Fc epsilon RI+ cells

Human basophils and mast cells express the chemokine receptor CCR3, which binds the chemokines eotaxin and RANTES. HIV-1 Tat protein is a potent chemoattractant for basophils and lung mast cells obtained from healthy individuals seronegative for Abs to HIV-1 and HIV-2. Tat protein induced a rapid and transient Ca(2+) influx in basophils and mast cells, analogous to beta-chemokines. Tat protein neither induced histamine release from human basophils and mast cells nor increased IL-3-stimulated histamine secretion from basophils. The chemotactic activity of Tat protein was blocked by preincubation of FcepsilonRI(+) cells with anti-CCR3 Ab. Preincubation of Tat with a mAb anti-Tat (aa 1-86) blocked the migration induced by Tat. In contrast, a mAb specific for the basic region (aa 46-60) did not inhibit the chemotactic effect of Tat protein. Tat protein or eotaxin desensitized basophils to a subsequent challenge with the autologous or the heterologous stimulus. Preincubation of basophils with Tat protein up-regulated the level of CCR3 mRNA and the surface expression of the CCR3 receptor. Tat protein is the first identified HIV-1-encoded beta-chemokine homologue that influences the directional migration of human FcepsilonRI(+) cells and the expression of surface receptor CCR3 on these cells.     

Characterization of the anti-inflammatory effect of FK-506 on human mast cells.

We have examined the effects of FK-506 and of the struturally related macrolide rapamycin, which bind with high affinity to a specific binding protein (FKBP), to evaluate the involvement of this protein in the release of preformed (histamine) and de novo synthesized inflammatory mediators (sulfidopeptide leukotriene C4 and prostaglandin D2) from mast cells isolated from human lung parenchyma. FK-506 (0.1 to 300 nM) concentration dependently inhibited histamine release from lung parenchymal mast cells activated by anti-IgE. FK-506 was more potent in lung mast cells than in basophils (IC50 = 1.13 +/- 0.46 nM vs 5.28 +/- 0.88 nM; p less than 0.001), whereas the maximal inhibitory effect was higher in basophils than in lung mast cells (88.4 +/- 2.5% vs 76.4 +/- 3.8%; p less than 0.01). FK-506 had little or no inhibitory effect on histamine release from lung mast cells challenged with compound A23187, whereas it completely suppressed A23187-induced histamine release from basophils. FK-506 also inhibited the de novo synthesis of 5-lipoxygenase (sulfidopeptide leukotriene C4) and cyclo-oxygenase (prostaglandin D2) metabolites of arachidonic acid from mast cells challenged with anti-IgE. Unlike in basophils, Il-3 (3 to 30 ng/ml) did not modify anti-IgE- or A23187-induced histamine release from lung mast cells nor did it reverse the inhibitory effect of FK-506. Rapamycin (3 to 300 nM) had little or no effect on the release of histamine from lung mast cells, but it was a competitive antagonist of the inhibitory effect of FK-506 on anti-IgE-induced histamine release from human mast cells with a dissociation constant of about 12 nM. These data indicate that FK-506 is a potent anti-inflammatory agent that acts on human lung mast cells presumably by binding to a receptor site (i.e., FKBP).     

Basic characteristics of human lung mast cell desensitization

Human lung parenchymal mast cells displayed both specific and nonspecific desensitization. The kinetics of both release and desensitization were approximately equal to 3 times faster than human basophils, but a similar relationship between release and desensitization suggests similar biochemistries in basophils and mast cells. Arachidonic acid metabolite (PGD2 and LTC4) release was slower to desensitize (t1/2 of 8 min) than histamine release (t1/2 of 3 min), the ratio of which is similar to the ratio observed in basophils. Ionophore A23187-induced release was unaffected by desensitization to anti-IgE antibody, and calcium-45 uptake was inhibited by desensitization, suggesting that desensitization inhibits the early post-cross-linking "influx" of calcium that is necessary for mediator release in mast cells. In contrast to the above similarities in basophil and mast cell desensitization, mast cell desensitization, unlike that of basophils was not inhibited by diisopropylfluorophosphate.     

Antigen inhalation induces mast cells and eosinophils infiltration in the guinea pig esophageal epithelium involving histamine-mediated pathway

Antigen challenge in sensitized guinea pig esophagus in vitro induces mast cell degranulation and histamine release. This study tests the hypothesis that antigen inhalation in vivo induces infiltration of the esophageal epithelium by mast cells and eosinophils via a histamine pathway. Actively sensitized guinea pigs were exposed to inhaled 0.1% ovalbumin. One or 24 h after inhalation exposure, the esophagus was processed for immunofluorescent staining of mast cell tryptase and eosinophil major basic protein (MBP). Additional animals were pretreated with thioperamide, a histamine H4/H3 receptor antagonist. Total tryptase- and MBP-labeled cells and percent of positive cells in the epithelial layer were counted. The total number of mast cells was unchanged after inhalation challenge, but the percentage in the epithelium increased 1 h after challenge. The total number of eosinophils increased 1 h after challenge, and the percentage migrating to the epithelium increased by 24 h after challenge. Mast cell migration into the mucosal epithelium preceded that of eosinophils. Thioperamide inhibited mast cell and eosinophil migration. In conclusion, antigen inhalation in sensitized animals induces mast cells and eosinophils to infiltrate in the esophageal epithelium via histamine-mediated mechanism.     

Identification of a chymotrypsin-like proteinase in human mast cells

An antiserum was produced against a chymotryptic proteinase purified from human skin. The antiserum did not cross-react with human leukocyte cathepsin G and elastase, rat mast cell proteinase I, and human skin tryptase. Indirect immunofluorescent staining of frozen skin sections to localize the proteinase showed cytoplasmic staining of cells scattered about the papillary dermis and around blood vessels and appendages. Restaining these sections with toluidine blue revealed that the fluorescently stained cells contained metachromatically staining granules, the major distinguishing feature of mast cells. A similar correlation was found in lung tissue. Ultrastructural studies employing the ferritin bridge technique to immunologically identify the proteinase additionally localized the proteinase to mast cell granules. Biochemical and immunochemical characterization of chymotryptic activity solubilized from isolated human lung mast cells identified a chymotryptic proteinase that may be identical to the skin chymotryptic proteinase. These studies establish that human skin mast cells contain a chymotrypsin-like proteinase that is a granule constituent and provide evidence that indicates a comparable proteinase is also present in lung mast cells.     

Histochemistry and morphology of porcine mast cells

Summary Mast cells have been described extensively in rodents and humans but not in pigs, and the objective of this study was to characterize porcine mast cells by histochemistry and electron microscopy. Carnoy's fluid proved to be a good fixative but fixation with neutral buffered formalin blocked staining of most mast cells. Alcian Blue stained more mast cells than did Toluidine Blue (pH 0.5), although Alcian Blue also stained goblet cells. In pigs, unlike rodents, the Alcian Blue method did not distinguish between mast cells in the intestinal mucosa and those in the connective tissue of the intestinal submucosa, tongue and skin. Mast cells were significantly larger in adult pigs than in piglets; in adult pigs and piglets, mast cells in the intestinal mucosa were significantly larger than those in submucosal connective tissue, and they were more varied in shape in piglets and adults. Granules in mast cells in the intestinal mucosa stained less intensely than those in mast cells in connective tissue of tongue, skin and intestinal submucosa. Mast cells in the connective tissue of the tongue, skin and intestinal submucosa fluoresced strongly when stained with berberine sulphate or with a mixture of berberine sulphate and Acridine Orange, but mast cells in the intestinal mucosa did not. All mast cells reacted positively in an enzyme-histochemical method previously used to detect human tryptase but not in a method previously used to detect human chymase. Mast cells in the medulla of thymus stained similarly to mast cells in the intestinal mucosa. Ultrastructural differences between mast cells were not detected.     

HIV-1 Nef promotes migration and chemokine synthesis of human basophils and mast cells through the interaction with CXCR4

Background

The Nef protein can be detected in plasma of HIV-1-infected patients and plays a role in the pathogenesis of HIV-1. Nef produced during the early stages of infection is fundamental in creating the ideal environment for viral replication, e.g. by reducing the ability of infected cells to induce an immune response.

Aim

Based on previous experience showing that both Tat and gp41 of HIV-1 are potent chemotactic factors for basophils and mast cells, and gp120 is a powerful stimulus for the release of histamine and cytokines (IL-4 and IL-13) from basophils, in this study we aimed to verify if the HIV Nef protein can exert some effects on basophils and mast cells purified from healthy volunteers through the interaction with the CXCL12 receptor, CXCR4.

Methods

Basophils purified from peripheral blood cells of 30 healthy volunteers and mast cells obtained from lung tissue of ten healthy volunteers were tested by flow cytometric analysis, chemotaxis and chemokine production by ELISA assays.

Results

Nef is a potent chemoattractant for basophils and lung mast cells obtained from healthy, HIV-1 and HIV-2 seronegative individuals. Incubation of basophils and mast cells with Nef induces the release of chemokines (CXCL8/IL-8 and CCL3/MIP-1α). The chemotactic activity of Nef on basophils and mast cells is mediated by the interaction with CXCR4 receptors, being blocked by preincubation of FcεRI⁺ cells with an anti-CXCR4 Ab. Stimulation with Nef or CXCL12/SDF-1α, a CXCR4 ligand, desensitizes basophils to a subsequent challenge with an autologous or heterologous stimulus.

Conclusions

These results indicate that Nef, a HIV-1-encoded α-chemokine homolog protein, plays a direct role in basophils and mast cell recruitment and activation at sites of HIV-1 replication, by promoting directional migration of human FcεRI⁺ cells and the release of chemokines from these cells. Together with our previous results, these data suggest that FcεRI⁺ cells contribute to the dysregulation of the immune system in HIV-1 infection.

     

Protein L. A bacterial Ig-binding protein that activates human basophils and mast cells.

Peptostreptococcus magnus strain 312 (10(6) to 10(8)/ml), which synthesizes a protein capable of binding to kappa L chains of human Ig (protein L), stimulated the release of histamine from human basophils in vitro. P. magnus strain 644, which does not synthesize protein L, did not induce histamine secretion. Soluble protein L (3 x 10(-2) to 3 micrograms/ml) induced histamine release from human basophils. The characteristics of the release reaction were similar to those of rabbit IgG anti-Fc fragment of human IgE (anti-IgE): it was Ca2(+)- and temperature-dependent, optimal release occurring at 37 degrees C in the presence of 1.0 mM extracellular Ca2+. There was an excellent correlation (r = 0.82; p less than 0.001) between the maximal percent histamine release induced by protein L and that induced by anti-IgE, as well as between protein L and protein A from Staphylococcus aureus (r = 0.52; p less than 0.01). Preincubation of basophils with either protein L or anti-IgE resulted in complete cross-desensitization to a subsequent challenge with the heterologous stimulus. IgE purified from myeloma patients PS and PP (lambda-chains) blocked anti-IgE-induced histamine release but failed to block the histamine releasing activity of protein L. In contrast, IgE purified from myeloma patient ADZ (kappa-chains) blocked both anti-IgE- and protein L-induced releases, whereas human polyclonal IgG selectively blocked protein L-induced secretion. Protein L acted as a complete secretagogue, i.e., it activated basophils to release sulfidopeptide leukotriene C4 as well as histamine. Protein L (10(-1) to 3 micrograms/ml) also induced the release of preformed (histamine) and de novo synthesized mediators (leukotriene C4 and/or PGD2) from mast cells isolated from lung parenchyma and skin tissues. Intradermal injections of protein L (0.01 to 10 micrograms/ml) in nonallergic subjects caused a dose-dependent wheal-and-flare reaction. Protein L activates human basophils and mast cells in vitro and in vivo presumably by interacting with kappa L chains of the IgE isotype.     

Ultrastructural studies of human basophils and mast cells.

Ultrastructural studies of human mast cells (HMCs) and basophils (HBs) are reviewed. Sources of HMCs include biopsies of tissue sites and in situ study of excised diseased organs; isolated, partially purified samples from excised organs; and growth-factor-stimulated mast cells that develop de novo in cultures of cord blood cells. Sources of HBs for study include partially purified peripheral blood basophils, basophils in tissue biopsies, and specific growth factor-stimulated basophils arising de novo from cord blood cells. The ultrastructural studies reviewed deal with identity, secretion, vesicles, recovery, and synthesis issues related to the biology of these similar cells.     

IL-33 induces Th17-mediated airway inflammation via mast cells in ovalbumin-challenged mice

Allergic asthma is characterized by infiltration of eosinophils, elevated Th2 cytokine levels, airway hyperresponsiveness, and IgE. In addition to eosinophils, mast cells, and basophils, a variety of cytokines are also involved in the development of allergic asthma. The pivotal role of eosinophils in the progression of the disease has been a subject of controversy. To determine the role of eosinophils in the progression of airway inflammation, we sensitized and challenged BALB/c wild-type (WT) mice and eosinophil-deficient ΔdblGATA mice with ovalbumin (OVA) and analyzed different aspects of inflammation. We observed increased eosinophil levels and a Th2-dominant response in OVA-challenged WT mice. In contrast, eosinophil-deficient ΔdblGATA mice displayed an increased proportion of mast cells and a Th17-biased response following OVA inhalation. Notably, the levels of IL-33, an important cytokine responsible for Th2 immune deviation, were not different between WT and eosinophil-deficient mice. We also demonstrated that mast cells induced Th17-differentiation via IL-33/ST2 stimulation in vitro. These results indicate that eosinophils are not essential for the development of allergic asthma and that mast cells can skew the immune reaction predominantly toward Th17 responses via IL-33 stimulation.     

Inflammatory cells of teleostean fish: a review focusing on mast cells/eosinophilic granule cells and rodlet cells

In contrast to the roles played by monocytes/macrophages, neutrophils and lymphocytes, the presence and functions of basophils, mast cells/eosinophilic granule cells, eosinophils and rodlet cells in teleosts are areas of controversy. The tissue distribution of mast cells/eosinophilic granule cells in species from a certain genus shows a characteristic pattern, and this pattern is usually also present at the family level. Functionally, the mast cells/eosinophilic granule cells of teleosts show close similarity to the mast cells of mammals. Acute tissue damage is causing mast cell/eosinophilic granule cell degranulation and release of mediators of inflammation, whereas an increase in the number of these cells is often found in chronically inflamed tissues. The mast cells/eosinophilic granule cells of teleosts show marked diversity in their staining properties, with both basophilic and acidophilic components in their granules. In some fish families, e.g. the labrids, the eosinophilic component is dominating, whereas in the pike the granules are strongly basophilic and show the metachromatic staining characteristics found in the granules of mast cells, but being more akin to the granules of the mucosal than to those of the connective tissue type of mast cells of mammals. With respect to rodlet cells, a cell type hitherto clearly demonstrated only in teleosts, a characteristic distribution pattern seems to be established in certain families. In other families rodlet cells are absent in some individuals and present in different tissues in others. However, there is a close relation between the presence of helminths or other noxious agents and the presence of rodlet cells. Massive aggregations of such cells can be seen in affected epithelia of gills or the intestinal tract, and in individuals of species from some fish families they also occur in association with mesothelial and endothelial tissues. The rodlet cell may represent a type of eosinophilic granulocyte that populates the tissues at its immature stage and mature in response to the appropriate stimuli, in a way similar to that of mast cell precursors. Present evidence points to a functional role for the rodlet cells of teleosts in host defence against parasites.     

Novel technique for the direct flow cytofluorometric analysis of human basophils in unseparated blood and bone marrow, and the characterization of phenotype and peroxidase of human basophils

BACKGROUND: No technique has been reported to analyze directly the antigen expression on basophil leukocytes when using a flow cytometer; therefore, the exact phenotype of human basophils and the character of the peroxidase in basophils are not well understood. METHODS: Human blood basophils were purified by using an antibody against high-affinity Fc epsilon receptor (hFcepsilonR) and a MACS magnetic cell sorting system and then cytochemically stained. The phenotype and peroxidase of the human basophils were flow cytofluorometrically analyzed directly in unseparated blood and bone marrow samples as hFcepsilonR+/MBP+ (major basic protein)/Hist+ (histamine) light-density cells distributed in the high sidescatter area of lymphocytes on light scattergrams. RESULTS: The peroxidase granules of human basophils were stained by an anti-eosinophil peroxidase (EPO) antibody. The human blood basophils had common granulocyte markers plus CD25, i.e., they were CD11a/ CD11b/CD11c/CD25/CD38/CD13/CD33/hFcepsi lonR/MBP/Hist/ EPO positive, CD71 dim positive, CD14/CD15 partially positive, and CD2/CD3/CD7/CD122/CD16/CD56/CD57/ CD10/CD19/CD20/CD22/HLA-DR/MPO (myeloperoxidase)/CD23 negative. Further examination was done to analyze the expression of colony-stimulating factor receptors on three lineages of granulocytes, i.e., basophils, eosinophils, and neutrophils. The neutrophils were CD114 (G-CSFR)/CD116 (GM-CSFR)/CD124 [interleukin (IL)-4R]/CD126 (IL-6R) positive and CD123 (IL-3R)/CD125 (IL-5R) negative. In contrast, the eosinophils and basophils were CD116/CD123/CD125/CD126 positive and CD114/CD124 negative. CONCLUSIONS: This novel technique for directly characterizing human basophil leukocytes with flow cytometry may be a convenient way to screen the expression of surface antigens and the cytoplasmic expression of CD antigens and other proteins in human blood basophils and to analyze alterations of the character of basophils by cytokines and other biological substances in vivo and in vitro.