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PubMed1978

Plasminogen activator of guinea pig basophilic leukocytes: probable localization to the plasma membrane.

The plasminogen activator (PA) activity of guinea pig basophil-enriched leukocyte preparations was localized to basophils, and not to contaminating lymphocytes and eosinophils, by correlating PA activity with basophil frequency and, more directly, by means of an improved cytochemical method here described. PA activity was fully expressed in living cells in the absence of immunologic stimuli and was suppressed/lost to a variable extent by different techniques of cell disruption. Conversely, killed, but not living, basophils expressed significant plasminogen-independent fibrinolytic activity, presumably reflecting access of cytoplasmic proteases of broken basophils to fibrin substrate. The PA activity of intact cells was destroyed by gentle trypsinization under conditions that did not impair cell viability. When disrupted cells were ultracentrifuged on a sucrose density gradient, PA activity was absent from purified granules and was confined to fractions containing cell membranes. The simplest explanation of these data is that guinea pig basophils have PA activity associated with their plasma membranes. This conclusion has several important implications for basophil functions in cell-mediated and other immunologic reactions in vivo.

Animals↗
PubMed1980

Basophils and eosinophils in three strains of rats and in athymic (nude) rats following infection with the nematodes Nippostrongylus brasiliensis or Trichinella spiralis.

A previous report showed that infection with the nematode Nippostrongylus brasiliensis stimulates a basophilia as well as an eosinophilia in the blood of August rats. The present study shows that blood levels of basophils and eosinophils were increased in two other rat strains, one inbred and one outbred, after infection with N. brasiliensis, and infection of two inbred rat strains with Trichinella spiralis also stimulated a basophilia as well as an eosinophilia. No increase occurred in basophils or eosinophils in athymic (nude) rats infected with N. brasiliensis, although both these cell types were found in the blood of control, specific pathogen free, nude rats in numbers comparable to those in specific pathogen free, heterozygote controls of the same strain. Rat basophils usually have few granules and in blood smears often appear as if they were partly degranulated. Basophils from uninfected nude rats contained more negative than positive staining granules compared with basophils from parasitized heterozygotes. The possession of small numbers of granules which vary in their reaction to stains of the Romanowski type is a normal feature of rat basophils in blood smears. Consequently rat basophils differ in these respects from those of other species.

Animals↗
PubMed1995

IL-3, IL-5, and granulocyte-macrophage colony-stimulating factor potentiate basophil mediator release stimulated by eosinophil granule major basic protein.

We have examined the potential of IL-3, IL-5, and granulocyte-macrophage (GM)-CSF to enhance basophil activation by eosinophil granule major basic protein (MBP). Preincubating basophil-containing mononuclear cells with 0.01 to 10 ng/ml IL-3 or IL-5 for 15 min at 37 degrees C caused a concentration-dependent enhancement of histamine release stimulated by 1.5 microM MBP. Statistically significant enhancement was evident at 1 ng/ml and was maximal at 10 ng/ml. Preincubation with GM-CSF similarly enhanced MBP-induced histamine release. A 10- to 15-min preincubation with IL-5 maximally increased the level of MBP-stimulated histamine release. Preincubation of cells with 10 ng/ml IL-3 or IL-5 reduced the MBP concentrations required for histamine release by three- to fourfold and enhanced the rate of MBP-induced histamine release. MBP-stimulated histamine release before or after cytokine priming was independent of cytotoxicity as measured by 51Cr release. Consistent with a direct action of the cytokines on basophils, flow cytometric analysis demonstrated the presence of IL-3 and GM-CSF receptors on basophils. MBP also stimulated low levels of leukotriene C4 (LTC4) release from basophils of 84 to 99% purity, and experiments using enriched (18-63%) basophil preparations demonstrated that preincubation with IL-3, IL-5, and GM-CSF also potentiated MBP-stimulated leukotriene C4 release up to threefold in parallel with histamine release. These results indicate that IL-3, IL-5, and GM-CSF may contribute to the pathogenesis of allergic and other disorders characterized by eosinophilia in part through potentiation of basophil mediator release stimulated by MBP.

Basophils↗
PubMed1993

Stimulation of human basophils results in homotypic aggregation. A response independent of degranulation.

For a better insight into the mechanisms determining the recruitment of human basophilic granulocytes from the circulation to sites of allergic reactions, we studied the homotypic aggregation of these cells. The aggregation was studied with > 95% pure basophil suspensions obtained from peripheral blood in a double-color flow cytometric analysis. Homotypic aggregation was induced by treatment of the basophils with anti-IgE, house dust mite allergen, the chemoattractant FMLP, PMA, or IL-3. The aggregation by anti-IgE was, in part, mediated by interactions with Fc gamma R-II as indicated by 43 +/- 15% inhibition after pretreatment with CD32 antibodies. The aggregation was mediated by beta 2-integrins, as was shown by inhibition of the response by CD18 antibodies. The aggregation induced by anti-IgE, allergen, and PMA displayed comparable kinetics (t1/2 max, 3 to 4 min), in contrast to the degranulation of basophils. FMLP induced the most rapid response (t1/2max, 1.6 min). Inhibition of protein kinase C by staurosporine resulted in a strong (> 90%) inhibition of the PMA-induced aggregation, whereas the FMLP-induced aggregation was more than doubled (from 11.7 +/- 1.9 to 24.4 +/- 1.9%). Staurosporine did not affect the extent of the anti-IgE-induced aggregation, but it induced a retardation of congruent to 10 min. In most experiments, no clear correlation was found between degranulation and aggregation of human basophils. Most strikingly, IL-3 did not induce degranulation but did induce aggregation. Thus, the homotypic aggregation response of human basophils is induced by intracellular signals not necessarily leading to degranulation. This might be important in the physiologic appearance of basophils at sites of allergic late-phase responses or inflammation.

Alkaloids↗
PubMed1993

In vivo characterization of the anti-inflammatory effect of cyclosporin A on human basophils.

We investigated whether cyclosporin A (CsA) in vivo exerts anti-inflammatory effects by inhibiting IgE- and non-IgE-dependent mediator release from human basophils. Six healthy volunteers were given oral CsA (5 mg/kg twice daily) or placebo for 5 days. Plasma CsA and basophil releasability in response to anti-IgE, FMLP, and A23187 were monitored 1 day before treatment, on alternate days during the treatment course, and 1 and 8 days after cessation of treatment. A constant plasma level of approximately 250 ng/ml CsA was obtained during CsA treatment. Basophil releasability in response to anti-IgE, FMLP, and A23187 was diminished by 20 to 60% throughout the course of CsA treatment. Placebo had no effect on basophil releasability. There was a significant correlation between plasma CsA and the decrease of histamine release induced by anti-IgE (rs = -0.66; p < 0.0005), FMLP (rs = -0.59; p < 0.001) and A23187 (rs = -0.68; p < 0.0001). In a second study, eight normal volunteers were given a single oral dose of CsA (7 mg/kg) or placebo, and plasma CsA and basophil releasability were monitored at different times thereafter. A rapid and significant reduction of histamine release induced by anti-IgE, FMLP, and A23187 paralleled a sharp increase of CsA plasma levels, which peaked at 5 h and lasted approximately 13 h. This study indicates that oral administration of CsA in normal subjects causes a rapid and significant inhibition of histamine release from basophils. This is the first evidence that in vivo administration of CsA can modulate the release of proinflammatory mediators from basophils obtained ex vivo.

Adolescent↗
PubMed1995

Quantitation of tryptase, chymase, Fc epsilon RI alpha, and Fc epsilon RI gamma mRNAs in human mast cells and basophils by competitive reverse transcription-polymerase chain reaction.

Competitive reverse transcription-PCR assays developed for human tryptase, chymase, Fc epsilon RI alpha, and Fc epsilon RI gamma mRNA molecules were applied to the HMC-1 leukemic mast cell line, the KU812 leukemic basophil cell line, mast cells dispersed from lung and skin, and peripheral blood basophils. Relative amounts of alpha-tryptase and beta-tryptase mRNA were determined by analysis of BseAI digests of PCR products. Tryptase expression was highest in tissue-derived mast cells, lowest in basophils and KU812 cells, and intermediate in HMC-1 cells. beta-Tryptase mRNA predominated in HMC-1 and KU812 cells; mixtures of alpha- and beta-tryptase were found in tissue mast cells; and alpha-tryptase predominated in basophils. Chymase mRNA was more abundant in skin-derived (nearly all of the MCTC type) than lung-derived (variable amounts of MCTC and MCT cells) mast cells. Small amounts of chymase mRNA were detected in HMC-1 cells; none was found in basophils, in KU812 cells, or in the one preparation of 100% MCT cells derived from lung. Comparable amounts of Fc epsilon RI alpha and Fc epsilon RI gamma mRNA molecules were measured in basophils and tissue-derived mast cells, lesser amounts were detected in KU812 cells, and almost none was detected in HMC-1 cells. Thus, steady state levels of the granule and membrane resident molecules examined in our study are transcriptionally regulated in mast cells and basophils.

Base Sequence↗
PubMedDate not supplied

Purification of rabbit blood basophils by isopycnic banding on Percoll.

This study defines the basophil densities in rabbits and also describes a relatively simple method of purifying basophils from peripheral blood through a two-step procedure. To evaluate the recovery of basophils, EDTA-anticoagulated blood in layered over Percoll of various densities, ranging from 1.070 to 1.080 g/ml. The cells retained in Percoll were collected separately after centrifugation at 600 g(av) for 30 min (Step I). Distribution of basophils indicated that basophils had densities of between 1.070 and 1.078 g/ml, with a peak at 1.074 g/ml. To enhance the basophil purity, a second centrifugation of the cells obtained from Step I over appropriate Percoll gradient led to further enhancement of basophil purity (30.4 +/- 0.7%) which was (20.6 +/- 0.2%) in Step I. The cells obtained by this method appeared morphologically normal and viable.

Animals↗
PubMed1996

Vesicular transport of histamine in stimulated human basophils.

Human basophils participating in experimentally produced contact allergy display progressive secretion of electron-dense secretory granule contents and retention of cytoplasmic granule containers in the absence of entire granule extrusion, a process termed piecemeal degranulation (PMD) and postulated to be effected by vesicular transport (Dvorak HF, Dvorak AM: Clin Hematol 4:651, 1975). Proof of this hypothesis was sought using models of human basophil-stimulated secretion, partially purified human peripheral blood basophils, and a morphometric analysis of the fraction of total cellular cytoplasmic vesicles loaded with histamine, a major proinflammatory mediator present in basophil secretory granules. The subcellular localization of histamine was accomplished using a new ultrastructural enzyme-affinity-gold method based on the affinity of diamine oxidase for its substrate, histamine (Dvorak et al: J Histochem Cytochem 41:787, 1993). Two models were selected for a kinetic analysis of stimulated vesicle transport of histamine based on known biochemical and ultrastructural characteristics (MacGlashan et al: J Immunol 136:2231, 1986; Warner et al: J Leukoc Biol 45:558, 1989; Dvorak et al: Am J Pathol 141:1309, 1992; Dvorak et al: Lab Invest 64:234, 1991). These models were selected to include the rapid release reaction stimulated by the bacterial peptide, FMLP, and the slow release reaction stimulated by the phorbol diester tumor promoter, TPA. The results of this study showed that the fraction of histamine-loaded cytoplasmic vesicles (%VG/TV/micron2) in TPA-stimulated basophils significantly exceeded the fraction in unstimulated cells, a process that persisted for 45 minutes after TPA stimulation and was associated with extensive PMD and no morphologic evidence of recovery. Similarly, the fraction of histamine-loaded cytoplasmic vesicles after FMLP stimulation significantly exceeded the fraction in unstimulated cells, a process that persisted for 10 minutes after FMLP stimulation and was associated with the morphologic continuum of PMD-->anaphylactic degranulation (characterized by extrusion of granules)-->recovery, a process largely complete in the 10-minute samples. These studies establish for the first time that an important proinflammatory mediator, histamine, traffic from secretory granules to the extracellular milieu in small cytoplasmic vesicles in stimulated human basophils. The association of this process with the ultrastructural release reaction defined as PMD produced primarily by TPA and in part by FMLP establishes vesicular transport as the mechanism for effecting this type of regulated secretion. Vesicular transport of histamine was also significant in the more complex stimulated secretory and recovery model produced by exposure of human basophils to the bacterial peptide FMLP.

Basophils↗
PubMed1996

Basophil priming by neurotrophic factors. Activation through the trk receptor.

There is increasing evidence that nerve growth factor (NGF) acts on cells of the immune system, apart from its neurotrophic effects. In human basophils, NGF potentiates mediator release and primes the cells to produce leukotriene C4 in response to C5a. It is, however, unknown whether other homologous neurotrophins also act outside the nervous system, and whether activation of basophils by NGF requires interaction with trk tyrosine kinase receptors, the low affinity NGF receptor (LNGFR), or both. A triple mutant NGF designed to interrupt binding to the LNGFR was found to activate basophils with equal efficacy as wild-type NGF, demonstrating that the LNGFR is not necessary. Despite a 10 times lower potency of mutant NGF, no LNGFR expression was detected by FACS analysis. Brain-derived neurotrophic factor, which interacts with trkB, was inactive at concentrations up to 1000 ng/ml (> 30,000-fold lower potency than NGF), while neurotrophin-3, which is thought to interact with trkC, trkB, and more weakly with trk, induced a threshold effect at 300 ng/ml (approximately 10,000-fold lower potency), demonstrating that 1) the LNGFR cannot deliver a direct signal; and 2) basophils do not express functional trkB and trkC receptors. In agreement with the functional data, basophils (in contrast to other granulocyte types) expressed mRNA for trk, but not trkB or trkC, and no or minimal mRNA for LNGFR. These data demonstrate that human blood basophils express functional trk receptors that do not require the participation of LNGFR, and that, among the neurotrophin family, NGF is unique in priming basophils.

Basophils↗
PubMed1989

Basophilic bodies of skeletal muscle in hypothyroidism: enzyme histochemical and ultrastructural studies.

Basophilic bodies of skeletal muscles from two patients with hypothyroidism were examined by enzyme histochemistry and ultrastructural study of ultrathin sections stained with periodic-acid-thiocarbohydrazide-silver proteinate for polysaccharides. Some additional characterizations of basophilic bodies were observed: basophilic bodies were found exclusively in type 1 fiber; basophilic bodies were devoid of myofibrillary adenosine triphosphatase, oxidative enzymes, and phosphorylase; and both fibrillary and granular components of basophilic bodies stained strongly for polysaccharides. The polysaccharide nature of basophilic bodies is in keeping with the previous suggestion that the formation of basophilic bodies in hypothyroid patients is related to an impairment of carbohydrate metabolism. Their selective involvement of type 1 fiber and preferential occurrence at the myotendinous junction remain obscure.

Adenosine Triphosphatases↗
PubMed2005

The basophil activation test by flow cytometry: recent developments in clinical studies, standardization and emerging perspectives.

The diagnosis of immediate allergy is mainly based upon an evocative clinical history, positive skin tests (gold standard) and, if available, detection of specific IgE. In some complicated cases, functional in vitro tests are necessary. The general concept of those tests is to mimic in vitro the contact between allergens and circulating basophils. The first approach to basophil functional responses was the histamine release test but this has remained controversial due to insufficient sensitivity and specificity. During recent years an increasing number of studies have demonstrated that flow cytometry is a reliable tool for monitoring basophil activation upon allergen challenge by detecting surface expression of degranulation/activation markers (CD63 or CD203c). This article reviews the recent improvements to the basophil activation test made possible by flow cytometry, focusing on the use of anti-CRTH2/DP2 antibodies for basophil recognition. On the basis of a new triple staining protocol, the basophil activation test has become a standardized tool for in vitro diagnosis of immediate allergy. It is also suitable for pharmacological studies on non-purified human basophils. Multicenter studies are now required for its clinical assessment in large patient populations and to define the cut-off values for clinical decision-making.

Journal Article↗
PubMed1978

Cytological changes of the pituitary basophils in rats slowly infused with LRH and with LRH and THR in combination.

Young male rats were iv infused with synthetic LRH (L) and with L and TRH (T) in combination for 1, 3, 24, 48 and 72 hrs at each dose of 1 microgram/hr. All the basophils of the controls infused with saline are divided into the continuous cyclic types, i.e., II-, II/III, III-, III/IV- and III/IV/II-types. The II-, III- and III/IV-type cells correspond, in fine structure, with those of the classical thyrotrophs, LH- and FSH-gonadotrophs, respectively. A 1-hr infusion of L does not induce any serious changes in all the basophils. After a 3-hr infusion of L, the II-type cells disperse from the gland, while the III/IV-type cells diminished the number of their small secretory granules, and the lumina of endoplasmic reticulum are closed. After a 24-hr infusion, the irregularly shaped III/IV/II-type cells which may revert from the III/IV- to the II-type cells are accumulated, whose secretory granules are remarkably reduced in diameter (50--100 nm). There is evidence to show the diffusion mechanism of the secretory granules into the ground matrix. After a 48-hr infusion of L, most basophils take an appearance of III/IV/II-type cells; after a 72-hr infusion, all the basophils show the homologous fine structure. Thus, the morphological changes of the basophils following the L infusion resemble intrinsically those following the T infusion (Soji, 1976b). From these results, it is tentatively concluded that L does not act only on the III-type cells analogous to the "LH-cells", but universally upon a series of basophils. The transformations of the III/IV-type cells into the III/IV/II-type ones and those of the II-type ones into the III- or III/IV-type ones due to a slow infusion of L are inhibited to some extent by the infusion of T + L. The granular releases from the III/IV- and II-type cells due to a slow infusion of L are also inhibited to some extent by the infusion of T + L. For this reason, both T and L may act, not synergistically but antogonistically, upon the transformation of a series of basophils and their granular release.

Animals↗
PubMed1997

Preliminary characterization of diacylglycerol generation in human basophils: temporal relationship to histamine release and resolution of degranulation.

Purified human basophils were examined for changes in diacylglycerol levels to determine whether the transient nature of a N-formyl-methionyl-leucyl-phenylalanine (fMLP) -stimulated elevation in membrane protein kinase C (PKC) activity could be explained by the transient production of diacylglycerol (DAG). In preliminary experiments total DAG levels were measured by the DAG kinase assay. Although elevations followed stimulation with 1 microM fMLP (basal levels of 15 pmol/10(6) basophils vs. 45 pmol/10(6) basophils at the 3-min time point), there were no detectable changes in the first 60 s of the reaction. Histamine release is typically complete by 30-45 s. Measurement of inositol trisphosphate indicated a rapid increase by 5 s of 2.5 pmol/10(6) basophils. If DAG were produced at similar levels, the DAG kinase assay would not have detected the elevation. Consequently, fMLP-stimulated basophils were examined for changes in 1-stearoyl, 2-arachidonoyl, 3-sn-glycerol (SA-DAG) and 1-oleoyl, 2-arachidonoyl, 3-sn-glycerol by GC-NICIMS (negative ion chemical ionization mass spectroscopy). A 5-s elevation in these two species averaged 2 pmol/10(6) basophils, consistent with the inositol trisphosphate levels and occurring during the period of histamine release. However, a much more pronounced second phase to the SA-DAG response also occurred, mirroring the total DAG levels. This second phase of the DAG response, either total or SA-DAG, was transient on a time scale temporally coincident with the appearance and resolution of degranulation sacs as measured by fluorescence microscopy. These data suggest that there is selective generation of DAG species in the early reaction and the later appearance of DAG may be related to the formation and resolution of granule structures that follow the secretion of histamine.

Basophil Degranulation Test↗
PubMed1993

Responsiveness of basophil granulocytes of horses suffering from chronic obstructive pulmonary disease to various allergens.

As basophils are the major effector cells of allergic reactions, confirmation of the allergic etiology of chronic obstructive pulmonary disease (COPD) was sought by the demonstration of a specific in vitro response of equine basophilic blood cells to some potential allergens (Aspergillus, Cladosporidium, Mucor, Penicillium, extracts of dust particles of hay and straw). The allergen induced degranulation of basophils and the histamine and protease release from basophils during incubation with the allergens were tested. By evaluating the results obtained from 14 COPD horses and eight controls it could be shown that the sensitivity of the basophils of affected horses was increased, particularly against the allergen extract of Mucor mucedo and Mucor spinosus. Further a greater percentage of COPD horses reacted positively with the Mucor allergen extract. The mitogenic stimulation of lymphocytes by PHA and by the allergen extracts used gave comparable results in affected and control horses. Thus the in vitro stimulation of basophils may be an easily to perform testing device for the identification of potential allergens involved in the pathogenesis of equine COPD.

Allergens↗
PubMed1995

Flow cytometric monitoring of allergen induced basophil activation.

Human basophils express many clustered differentiation antigens (CD), including CD45; however, none are specific for these cells. In a previous study, we described a two-color immunofluorescence procedure, employing antibodies to CD45 and IgE for the cytometric evaluation of basophils. In the present work, we show that when sensitized basophils are stimulated by allergenic preparations, they demonstrate an upregulation of CD45 as well as a decrease in anti-IgE binding. Since CD45 antigen modulation was observed with all aeroallergens tested and the decrease in IgE expression varied with allergenic preparations, the measurement of CD45 upregulation was used to evaluate basophil activation. Using this approach, reproducible results were observed when atopic patients were tested at different time intervals. In addition, we show that the upregulation of CD45 on allergen stimulated basophils is a very rapid phenomenon that is observed after a few minutes and that this rapid flow cytometric method can also be used for investigations on nonallergenic mediators of basophil stimulation such as calcium ionophores.

Adult↗
PubMed1994

Immunolocalization of intracellular interleukin-4 in normal human peripheral blood basophils.

The question as to whether other cell types apart from helper T lymphocytes are capable of producing interleukin-4 (IL-4) has gained much interest over the last years. Recent studies indicate that human basophils also produce IL-4, although direct proof is missing so far. In this study we demonstrate the presence of IL-4 in the cytoplasm of in vitro activated human peripheral blood basophils derived from normal donors. Cytokine-producing cells were revealed at the single-cell level by intracellular immunofluorescence staining using IL-4-specific monoclonal antibodies. Basophils showed a characteristic, apparently granular staining pattern easily discerned from the eccentric dot-shaped staining pattern in activated T cells used in control experiments. Cell counts following priming with IL-3 and stimulation with polyclonal sheep anti-IgE antibody or the anaphylatoxin C5a revealed a significant increase in IL-4-positive basophils to about 19% as compared with unprimed, unstimulated control cells (6%). The amount of IL-4 in the supernatant of these cell preparations paralleled these observations with an at least five- to sevenfold increase following stimulation as compared with control cells (< 5 ng/ml). Using confocal scanning laser microscopy, the intracellular presence of IL-4 was confirmed, and the cells were identified as being basophils on terms of their characteristic multilobed nucleus. This observation was supported by double labeling studies using antibodies to IL-4 and to the high-affinity IgE receptor (Fc epsilon R1). Interestingly, stimulation of cells led to a decrease in the number of Fc epsilon R1-positive cells. The above results show direct evidence that IL-4 is produced by activated human basophils.

Basophils↗
PubMed1989

Differential release of mediators from human basophils: differences in arachidonic acid metabolism following activation by unrelated stimuli.

We have examined the release of histamine and LTC4 from purified human basophils challenged with several different stimuli, both physiological and nonphysiological. Basophils (n = 16) challenged with 0.1 micrograms/ml anti-IgE released 38 +/- 4% of their available histamine and 39 +/- 12 ng LTC4/10(6) basophils within 15-30 min. F-Met peptide (n = 8) caused the release of 54 +/- 8% histamine and 42 +/- 25 ng LTC4/10(6) basophils within a period of 2-5 min. C5a caused the release of 22 +/- 3% histamine from selected donors but failed to initiate any LTC4 release unless combined with D2O or 5 mM extracellular calcium. The two nonphysiological stimuli A23187 and TPA caused extensive histamine release, 67 +/- 8 and 82 +/- 11%, respectively, and while A23187 initiated a large and rapid release of leukotriene, TPA failed to release any LTC4 even when combined with D2O or 2-5 mM extracellular calcium. Increased concentrations of extracellular calcium enhanced anti-IgE and f-Met peptide induced release of LTC4 but inhibited the A23187 induced release of leukotriene. A single peak of immunoreactive leukotriene C4 that comigrated with the authentic standard was identified using HPLC followed by radioimmunoassay. No LTD4 or LTE4 could be detected. Purified human basophils incubated with 0.2 microM [3H]AA incorporated 290 pmol/10(6) cells, or 32 +/- 5% of the available label within 60 min. The [3H]AA was taken principally into the phospholipids (73 +/- 5%), with 20 +/- 3% as neutral lipid, and only 5 +/- 2% remaining as the free acid. Three phospholipid subclasses, phosphatidylcholine, PC (24 +/- 2%), phosphatidylinositol, PI (22 +/- 1%), and phosphatidylethanolamine, PE (15 +/- 3%), accounted for the majority of the incorporated [3H]AA while the remainder of the phospholipids accounted for less than 5% of the total cpm. HPLC analysis of the lipid mediators released during stimulation with 0.1 micrograms/ml anti-IgE revealed [3H]LTC4 (2.4 +/- 1.0%), [3H]5HETE (1.0 +/- 0.1%), unmetabolized [3H]AA (91 +/- 2%), and an unidentified peak (3.4 +/- 1.4%). The unknown metabolite eluted with the prostaglandins, was inhibited by indomethacin, and appeared to have a relatively high specific activity. It may thus represent an artifact of the labeling procedure rather than a novel basophil-derived prostaglandin.

Arachidonic Acids↗
PubMed1994

Graded changes in the response of individual human basophils to stimulation: distributional behavior of early activation events.

These studies examine the distribution of single-cell responses in basophil preparations in the context of four events that may be associated with early activation by anti-immunoglobulin E (IgE) antibody and the bacterial peptide fMet-Leu-Phe (fMLP). In general, we measured the single-cell response distributions after challenge with a concentration of stimulus that resulted in an optimal response and compared this with the distribution that occurred after challenge with suboptimal concentrations of the same stimulus. The elevation in cytosolic calcium, as detected in Fura-2-labeled basophils, after challenge with anti-IgE or fMLP showed graded characteristics in that the distributions were unimodal under conditions of optimal or suboptimal challenge with little skewing from a normal distribution. Similarly, the up-regulation of the cell surface adhesion molecule CD11b, as determined by flow cytometry, showed graded unimodal increases after challenge with anti-IgE antibody at optimal and suboptimal concentrations. In addition, stimulation of basophils led to increased F-actin polymerization. After challenge with an optimal concentration of anti-IgE antibody, the F-actin content of basophils increased to a maximum between 10 and 15 min and returned to near prechallenge levels by 60 min. There was a close correlation between the maximum increase in F-actin content and histamine release regardless of the stimulus; anti-IgE antibody, fMLP, and phorbol ester (PMA) responses lay on the same regression line. The single-cell F-actin polymerization distributions were also unimodal and graded according to the magnitude of the histamine release response. During measurements of the calcium response under the microscope we noted that basophils underwent significant changes in morphology after challenge with any stimulus. These changes were related to both degranulation and nondegranulation events and could be quantitated by a series of image-processing algorithms, which are presented. The kinetics of the morphological change, measured as a change in cell perimeter, paralleled degranulation. Single-cell distributions of the morphologic changes were also unimodal under conditions of both optimal and suboptimal stimulation. Therefore, no evidence of all-or-nothing responses could be observed in the context of these four early activation events. In general, the response distributions resembled normal distributions at both optimal and suboptimal levels of stimulation, which indicated that single basophils responded in a graded manner.

Basophils↗