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PubMed2001

IL-4 production by human basophils found in the lung following segmental allergen challenge.

BACKGROUND: Human blood basophils secrete high levels of IL-4 following activation with specific allergen, yet their role as cytokine-producing cells in allergic lesions has not been described. OBJECTIVE: Our objective was to investigate whether and under what conditions basophils infiltrating allergic lesions in the lung secrete IL-4 in vitro. METHODS: Bronchoalveolar lavage (BAL) cells were recovered 20 hours after segmental allergen challenge. Basophils were enriched with Percoll using a protocol commonly used for blood basophils. IL-4 and histamine were measured in culture supernatants following activation with a variety of stimuli. Two-color flow cytometry was performed to detect intracellular IL-4. RESULTS: IL-4 protein was detected in all basophil culture supernatants following a 4- to 5-hour incubation in medium alone; the levels obtained did not significantly increase with the addition of anti-IgE. BAL basophils failed to release histamine in response to specific allergen but showed nearly 60% histamine release with N-formyl-methionyl-leucyl-phenylalanine, suggesting that they were desensitized to IgE-mediated stimuli as a result of their activation in vivo. Using these same conditions, IL-4 was not detected in BAL cell fractions enriched for lymphocytes and eosinophils. Ionomycin induced IL-4 secretion by BAL basophils, and this response was reduced with the addition of phorbol myristate acetate. In contrast, phorbol myristate acetate promoted the secretion of IL-4 by BAL cells enriched for lymphocytes; both findings are identical to those reported for basophils and lymphocytes purified from blood. Flow cytometry confirmed the secretion of IL-4 by BAL basophils. CONCLUSIONS: These data suggest that basophils migrating to the lung following allergen challenge represent a major source of IL-4.

Allergens↗
PubMed2001

Basophil recruitment and IL-4 production during human allergen-induced late asthma.

BACKGROUND: Basophils represent an important source of inflammatory mediators and cytokines after IgE-dependent activation in human beings. OBJECTIVE: To assess the role of basophils in allergic asthma, we measured the number of basophils in the bronchial mucosa and their capacity to express IL-4 mRNA and protein during allergen-induced late asthmatic responses. METHODS: Fiberoptic bronchoscopic bronchial biopsies were obtained at 24 hours from sites of segmental bronchial allergen challenge and control sites in 19 patients with atopic asthma and 6 nonatopic healthy volunteers. Basophil numbers were assessed by immunohistochemistry through use of mAb 2D7. IL-4 mRNA--positive cells were detected through use of in situ hybridization and colocalized to basophils through use of sequential immunohistochemistry/in situ hybridization. IL-4 protein was detected and colocalized to basophils through use of dual immunohistochemistry. RESULTS: After allergen challenge, there was an increase in the median number of 2D7-positive basophils per square millimeter in the bronchial mucosa in patients with asthma (0.9 cells/mm(2) at baseline to 8.8 cells/mm(2) after challenge; P =.002), which also was significantly higher than what was seen in nonasthmatic controls (P =.01). Similarly, IL-4 mRNA--positive cells were increased at 24 hours in patients with asthma (1.4 to 14) in comparison with controls (0 to 0; P =.02). Colocalization studies revealed that 15% and 41% of the basophil population in patients with asthma after allergen-challenge expressed, respectively, IL-4 mRNA and protein. Conversely, 19% of IL-4 mRNA-positive cells and 72% of IL-4 protein--positive cells were accounted for by basophils. CONCLUSION: After allergen provocation in sensitive patients with atopic asthma, basophils are recruited to the bronchial mucosa and express IL-4 mRNA and protein, which might contribute to local IgE synthesis and/or tissue eosinophilia or other aspects of allergic inflammation during late responses and ongoing asthma.

Adult↗
PubMed2001

Src homology 2 domain-containing inositol 5' phosphatase is negatively associated with histamine release to human recombinant histamine-releasing factor in human basophils.

BACKGROUND: The human recombinant histamine-releasing factor (HrHRF) acts as a complete stimulus for histamine release and IL-4 secretion from a subpopulation of highly allergic donor basophils, termed IgE(+) basophils. Additionally, IgE(+) basophils release histamine to other secretogues, IL-3, and deuterium oxide. We hypothesized that IgE(+) basophils were hyperreleasable. OBJECTIVE: Deficiencies in early signal transduction events associated with Fc(epsilon)RI lead to a nonreleasable phenotype, whereas the Src homology 2 domain--containing inositol 5' phosphatase (SHIP) knockout mice have hyperreleasable mast cells. The purpose of this study was to ascertain whether a difference in intracellular signaling molecules could explain the hyperreleasable phenotype of human IgE(+) basophils. METHODS: Basophils were purified by means of double Percoll gradients and negative selection with magnetic beads. Cell lysates were Western blotted for the tyrosine kinases Lyn and Syk and the phosphatase SHIP. Additionally, histamine release to HrHRF was performed in addition to real-time RT-PCR to investigate mRNA for SHIP. RESULTS: We show a striking negative correlation between the amount of SHIP protein per cell equivalent, but not Lyn or Syk, and maximum histamine release to HrHRF. This deficiency of SHIP was observed in basophils, but not lymphocytes or monocytes, of these IgE(+) donors. Additionally, levels of mRNA for SHIP did not differ between IgE(+) and IgE(-) donor basophils, which is consistent with a posttranscriptional mechanism of protein regulation. SHIP and phosphatidylinositol 3-kinase reciprocally regulate phosphatidylinositol (3,4,5) triphosphate levels. We also demonstrated that Ly294002, the phosphatidylinositol 3 kinase inhibitor, prevented HrHRF-induced histamine release in IgE(+) donor basophils. CONCLUSION: Taken together, these data suggest that the hyperreleasability of IgE(+) donors is associated with low levels of SHIP and implicate SHIP as an additional regulator of secretion in human basophils.

Basophils↗
PubMed1982

Suppression of T cell-mediated cutaneous basophil hypersensitivity by serum from guinea pigs immunized with mycobacterial adjuvant.

Guinea pigs immunized with protein antigens emulsified with complete Freund's adjuvant (CFA) and skin tested at 3-4 wk have classical tuberculin-type delayed hypersensitivity (DH) reactions with few basophils present. However, recipients of T cells from these animals have delayed responses containing large basophil infiltrates and thus resemble basophil-rich cutaneous basophil hypersensitivity (CBH) responses that are elicited in animals immunized without CFA. This suggests that animals immunized with CFA have T cells with basophil-recruiting capacity but that this activity is suppressed. Using a transfer system, we found that immune serum from donors immunized with CFA had the ability to suppress the basophil-recruiting capacity of immune T cells. When immune serum and peritoneal exudate cells from guinea pigs immunized with CFA were co-transferred intravenously to normal recipients, the cell-mediated transfer of basophil-rich responses was suppressed. The responsible serum factor was antigen nonspecific, had an approximately 70,000 mol wt, and acted preferentially on cells from donors that express basophil-poor DH responses. Thus, tuberculin-type delayed hypersensitivity and CBH might be mediated by a common T cell, but the resulting basophil component of the delayed response depends on the modulation of T cell recruitment of basophils by factors in CFA-immune serum.

Animals↗
PubMed2003

Sensitive flow cytometric method to test basophil activation influenced by homeopathic histamine dilutions.

OBJECTIVE: In an experimental setting, human basophil degranulation was triggered by anti-IgE to measure the effects from homeopathic solutions in an in-vitro cell system. A 3-color flow cytometric method with enhanced accuracy was established. As an example we looked at the influence of histamine on anti-IgE activation of basophils. METHODS: Basophils were identified in the flow cytometer by their physical properties in the forward and side scatter light depiction and by gating on CD2(-), CD14(-), CD16(-), CD19(-), HLA-DR(-) negative and CD123-positive cells. CD63 expression on the cell surface of the anti-IgE-activated basophils served as an activation marker. RESULTS: With this method we were able to study basophil function of the 0.6-3.9% basophils out of the mononuclear blood cell fraction and to document their activation status upon anti-IgE activation. Optimal activation occurs at 0.6 microg/ml final anti-IgE concentration; not less than 10,000 basophils have to be counted per batch to reduce the variation of the measurement. The fixation method was able to stabilize activation for two days. After investigation and reduction of the source of measurement variability, an unequivocally inhibited basophil activation was documented in a partly optimized system with homeopathic dilutions of histamine (10(-22)M, 10(-23)M, 10(-24)M, and 10(-25)M histamine). Dilutions greater than 10(-20)M histamine (Avogadro's number 6.02 x 10(23)) account for less than 1.36 molecules of histamine in the test sample, indicating a true homeopathic effect. CONCLUSIONS: This test system is adequate for studying the effects of highly diluted mediators on basophil activation by anti-IgE. The systematic application of this experimental arrangement is recommended to study the effects of homeopathic dilutions on basophils.

Antibodies, Anti-Idiotypic↗
PubMed1988

Adherence of human basophils to cultured umbilical vein endothelial cells.

The mechanism by which circulating human basophils adhere to vascular endothelium and migrate to sites of allergic reactions is unknown. Agents have been identified which stimulate the adherence of purified basophils to cultured human umbilical vein vascular endothelial cells (HuVEC). Treatment of HuVEC with interleukin 1, tumor necrosis factor (TNF), bacterial endotoxin, and 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in time and dose-dependent increases of adhesiveness for basophils. Coincubation of basophils and HuVEC for 10 min with C5a, formyl-methionyl-leucyl-phenylalanine, the calcium ionophore A23187, platelet-activating factor, TNF, and TPA also resulted in significant dose-dependent increases in basophil adherence; this effect resulted from activation of the basophil. Adherence of basophils to HuVEC was time and temperature dependent, required divalent cations, and was unaffected by glucocorticoids. Monoclonal antibody 60.3, directed against the beta-subunit of the leukocyte adherence complex CD18, inhibited the binding of basophils to HuVEC. Adherence of basophils to vascular endothelium may be important in initiating basophil infiltrates in vivo.

Basophils↗
PubMed2004

Parasite antigen-driven basophils are a major source of IL-4 in human filarial infections.

Basophil contribution to the IL-4 pool in filarial infections was assessed using PBMC from 20 patients with active filarial infections and from 9 uninfected subjects. Patient basophils released histamine in response to Brugia malayi Ag (BmAg). They also released IL-4 within 2 h after exposure to BmAg, as assessed by intracellular cytokine flow cytometry. This IL-4 induction was Ag specific, as IL-4 was not detected in BmAg-exposed basophils obtained from uninfected subjects. Although there were, on average, 64 times more CD4(+) T cells than basophils in the peripheral circulation of filaria-infected patients, the absolute numbers of basophils and CD4(+) T cells producing IL-4 per 100000 PBMC were equivalent (geometric mean: 16 IL-4-producing basophils/100000 PBMC vs 22 IL-4-producing CD4(+) T cells/100000 PBMC). Basophils also released IL-4 in response to both low and high concentrations of BmAg, whereas CD4(+) T cells released IL-4 only after incubation with a high concentration of BmAg, raising the possibility that basophils, due to their lower threshold for activation, may actually release IL-4 more frequently than CD4(+) T cells in vivo. Furthermore, IL-4 production in vitro by Ag-stimulated purified basophils or CD4(+) T cells provided evidence that basophils release greater quantities of IL-4 per cell than CD4(+) T cells in response to BmAg. These results suggest that, when Ag-specific IgE is present in a filaria-infected individual, basophils function to amplify the ongoing Th2 response by releasing IL-4 in greater amounts and possibly more frequently than CD4(+) T cells in response to filarial Ag.

Animals↗
PubMed2004

Identification of selective basophil chemoattractants in human nasal polyps as insulin-like growth factor-1 and insulin-like growth factor-2.

In a search for novel leukocyte chemoattractants at sites of allergic inflammation, we found basophil-selective chemoattractant activity in extracts of human nasal polyps. The extracts were fractionated by reverse phase HPLC, and the resulting fractions were tested for leukocyte-stimulating activity using sensitive shape change assays. The basophil-selective activity detected was not depleted by a poxvirus CC-chemokine-binding protein affinity column. This activity was further purified by HPLC, and proteins in the bioactive fractions were analyzed by tandem electrospray mass spectrometry. Insulin-like growth factor-2 (IGF-2) was identified in these HPLC fractions, and the basophil-stimulating activity was inhibited by an anti-IGF-2-neutralizing Ab. Recombinant IGF-2 induced a substantial shape change response in basophils, but not eosinophils, neutrophils, or monocytes. IGF-2 stimulated chemokinesis of basophils, but not eosinophils or neutrophils, and synergized with eotaxin-1/CCL11 in basophil chemotaxis. IGF-2 also caused up-regulation of basophil CD11b expression and inhibited apoptosis, but did not stimulate degranulation or Ca(2+) flux. Recombinant IGF-1 exhibited similar basophil-selective effects as IGF-2, and both growth factors were detected in nasal polyp extracts by ELISA. This is the first demonstration of chemokinetic factors that increase the motility of basophils, but do not act on other granulocytes or monocytes. IGF-1 and IGF-2 could play a role in the selective recruitment of basophils in vivo.

Amino Acid Sequence↗
PubMed2004

Transendothelial migration of human basophils.

During allergic reactions, basophils migrate from the blood compartment to inflammatory sites, where they act as effector cells in concert with eosinophils. Because transendothelial migration (TEM) represents an essential step for extravasation of cells, for the first time we have studied basophil TEM using HUVEC. Treatment of HUVEC with IL-1beta significantly enhanced basophil TEM, which was further potentiated by the presence of a CCR3-specific ligand, eotaxin/CCL11. In addition to CCR3 ligands, MCP-1/CCL2 was also active on basophil TEM. Although stromal cell-derived factor-1/CXCL12, a CXCR4 ligand, failed to induce TEM in freshly isolated basophils, it caused strong TEM in 24-h cultured cells. IL-3 enhanced basophil TEM by increasing the chemokinetic response. Spontaneous TEM across activated HUVEC was inhibited by treatment of cells with anti-CD18 mAb, but not with anti-CD29 mAb, and also by treatment of HUVEC with anti-ICAM-1 mAb. Anti-VCAM-1 mAb alone failed to inhibit TEM, but showed an additive inhibitory effect in combination with anti-ICAM-1 mAb. In contrast, eotaxin- and IL-3-mediated TEM was significantly inhibited by anti-CD29 mAb as well as anti-CD18 mAb. These results indicate that beta2 integrins play the primary role in basophil TEM, but beta1 integrins are also involved, especially in TEM of cytokine/chemokine-stimulated basophils. In conclusion, the regulatory profile of basophil TEM is very similar to that reported for eosinophils. Our results thus support the previous argument for a close relationship between basophils and eosinophils and suggest that the in vivo kinetics of these two cell types are similar.

Animals↗
PubMed2004

Urokinase induces basophil chemotaxis through a urokinase receptor epitope that is an endogenous ligand for formyl peptide receptor-like 1 and -like 2.

Basophils circulate in the blood and are able to migrate into tissues at sites of inflammation. Urokinase plasminogen activator (uPA) binds a specific high affinity surface receptor (uPAR). The uPA-uPAR system is crucial for cell adhesion and migration, and tissue repair. We have investigated the presence and function of the uPA-uPAR system in human basophils. The expression of uPAR was found at both mRNA and protein levels. The receptor was expressed on the cell surface of basophils, in the intact and cleaved forms. Basophils did not express uPA at either the protein or mRNA level. uPA (10(-12)-10(-9) M) and its uPAR-binding N-terminal fragment (ATF) were potent chemoattractants for basophils, but did not induce histamine or cytokine release. Inactivation of uPA enzymatic activity by di-isopropyl fluorophosphate did not affect its chemotactic activity. A polyclonal Ab against uPAR inhibited uPA-dependent basophil chemotaxis. The uPAR-derived peptide 84-95 (uPAR84-95) induced basophil chemotaxis. Basophils expressed mRNA for the formyl peptide receptors formyl peptide receptor (FPR), FPR-like 1 (FPRL1), and FPRL2. The FPR antagonist cyclosporin H prevented chemotaxis induced by FMLP, but not that induced by uPA and uPAR84-95. Incubation of basophils with low and high concentrations of FMLP, which desensitize FPR and FPRL1, respectively, but not FPRL2, slightly reduced the chemotactic response to uPA and uPAR84-95. In contrast, desensitization with WKYMVm, which also binds FPRL2, markedly inhibited the response to both molecules. Thus, uPA is a potent chemoattractant for basophils that seems to act through exposure of the chemotactic uPAR epitope uPAR84-95, which is an endogenous ligand for FPRL2 and FPRL1.

Adult↗
PubMed1992

RANTES, a monocyte and T lymphocyte chemotactic cytokine releases histamine from human basophils.

Chemotaxis of different populations of cells and release of proinflammatory mediators in response to antigenic stimulation are important processes in allergic diseases. These lead to the late phase response, a hallmark of chronic allergic diseases. Recombinant RANTES, a member of the "intercrine/chemokine" family of cytokines, has been previously shown to be chemotactic for monocytes and T cells of memory/helper phenotype. In this manuscript, we show that it is capable of inducing histamine release from human basophils at concentrations as low as 10(-10) M and compare its activity with that of monocyte chemotactic and activating factor/monocyte chemoattractant protein-1 (MCAF/MCP-1), another intercrine/chemokine. RANTES (10(-7) M) caused histamine release from the leukocytes of 26 of 33 donors tested (mean 21.8 +/- 3.1%). In the same group of donors, MCAF/MCP-1, goat anti-human IgE (anti-IgE; 1 microgram/ml), and FMLP (10(-5) M) released 41.1 +/- 2.9%, 40.5 +/- 4.6%, and 44 +/- 3.1% histamine, respectively. The percent histamine release by RANTES in atopic vs nonatopics was 30.3 +/- 6.7 and 16.5 +/- 2.4, respectively (p less than 0.05), and histamine release by RANTES correlated significantly with histamine release by MCAF (r = 0.69; p less than 0.001) but not with histamine release by anti-IgE (r = 0.29; p greater than 0.05). Histamine release by RANTES and MCAF/MCP-1 was extremely rapid, reaching a maximum within 1 min. RANTES was also shown to activate highly purified basophils (80% pure), and its activity was inhibited by a polyclonal anti-RANTES antibody. At a suboptimal concentration (6 x 10(-9) M), RANTES did not prime basophils to enhance histamine release by secretagogues such as anti-IgE, C5a, or FMLP. On the other hand, preincubation of basophils with RANTES or MCAF/MCP-1 desensitized basophils to either factor but not to anti-IgE, C5a, or FMLP. Preincubation of basophils with pertussis toxin markedly diminished the basophil response to either RANTES or MCAF/MCP-1. These results suggest that RANTES and MCAF/MCP-1: 1) are potent activators of basophils; 2) may function via the same, or a closely related, receptor system in basophils; and 3) may represent a link between activation of monocytes, lymphocytes, and basophils in inflammatory disorders such as the late phase allergic reaction.

Basophils↗
PubMed2003

Characterization and function of the multifaceted peripheral blood basophil.

Basophils are derived from metachromatic hematopoietic precursor cells of myeloid origin. The basophilic granulocyte differentiates and matures in the bone marrow, circulates in the peripheral blood, and upon proper stimulation, migrates into the tissues. Peripheral blood basophils act as chief effector cells of the allergic response and as purveyors of various allergy-associated mediators. Under appropriate conditions, basophils can be induced to release their mediators into the extracellular space of tissues or blood of the host organism. The plasma membrane of basophils contains receptors for immunoglobulin E (IgE) homocytotropic antibody which exhibits high affinity for these granulocytes and their Fc epsilon receptors. IgE cytophilic antibody binds antigen at its Fab portion. When bound to the basophil plasma membrane, the antigen-antibody complex undergoes multivalent interactions, which create crosslinking of the Fc epsilon receptors on the basophil plasma membrane. This receptor cross-linking results in basophil degranulation and subsequent release of its pharmacologically active substances. The basophil exhibits considerable heterogeneity and is characterized as Type I, II, III, IV, V and VI based upon granule content and time of antigen stimulation. Evidence is presented showing the role of the basophil in hyperplasia, hypersensitivity, parasitic infections and other diseases.

Basophils↗
PubMed1990

Bimodal IgG4-mediated human basophil activation. Role of eosinophils.

The role of IgG4 antibodies in allergic disorders is suspected. Yet, their presence on human basophil membrane has not been demonstrated and the mechanism of the degranulation induced by anti-IgG4 antibodies remains unclear. As previously reported, we observed that monoclonal anti-IgG4 (10 to 100 micrograms/ml) induced histamine release in the presence of D2O from leukocytes of normal and atopic subjects. The release was accompanied by a decrease of the number of toluidine blue-positive basophils (TB+). Histamine release and TB+ decrease were also observed with lower concentrations of anti-IgG4 (1 to 100 pg/ml). Since basophil activation assessed by TB+ decrease was more sensitive than histamine release, we thus used the former method to further study the mechanisms of the anti-IgG4- vs anti-IgE-induced basophil activation. Basophil activation by anti-IgG4 at 1 to 100 pg/ml, but not by anti-IgG4 at 10 to 100 micrograms/ml or anti-IgE, required the presence of polymorphonuclear cells. Furthermore, anti-IgG4-stimulated purified eosinophils, but not neutrophils, released basophil-activating factors identified as cationic proteins from eosinophils. Thus, the human basophil can be activated by anti-IgG4 via two different mechanisms according to the antibody concentration. At high concentrations (10 to 100 micrograms/ml) basophil activation does not require the presence of polymorphonuclear cells whereas at lower concentrations (1 to 100 pg/ml) the presence of eosinophils is necessary. We propose that in the latter concentration range, basophil activation is a two-step process: 1) release by anti-IgG4 of eosinophil cationic proteins that 2) will, in turn, activate human basophils. This study lends support to the role of IgG4 and eosinophils in anaphylactic reactions.

Antibodies, Anti-Idiotypic↗
PubMed1990

IL-3 augments adhesiveness for endothelium and CD11b expression in human basophils but not neutrophils.

A number of natural and recombinant human cytokines have been tested for their ability to activate basophil and neutrophil adhesiveness for human umbilical vein endothelial cells in vitro. Coincubation of basophils and endothelial cell monolayers for 10 min with biologically relevant concentrations of rIL-1, natural IL-2, rIL-4, rIL-5, rIL-6, rIL-8, rGM-CSF, and rIFN-gamma had no effect on basophil adhesiveness. In contrast, rIL-3 induced basophil adhesiveness for endothelial cells (optimal at 1 ng/ml: 144 +/- 18% of control adherence (mean +/- SEM); control basophil binding, 13 +/- 3%, n = 9, p less than or equal to 0.05). This increase in adhesiveness was similar in magnitude to that induced by an optimal concentration of a known potent inducer of basophil adhesiveness (1 microM FMLP, 164 +/- 15% of control adherence, n = 9). Under these experimental conditions, the effects of rIL-3 occurred at concentrations of 0.1 to 30 ng/ml, were partially dependent on calcium, and were not accompanied by histamine release. Fixation experiments demonstrated that the effect of rIL-3 was directed against the basophil rather than the endothelial cell. Neither rIL-3 nor the other cytokines tested had any effect on the adherence of 51Cr-labeled neutrophils, even when tested simultaneously on cells from the same donors. Under experimental conditions that permitted histamine release, no correlation was seen between the ability of rIL-3 (0.3 to 300 ng/ml) to induce histamine release or enhance adhesiveness (n = 8). mAb blocking experiments demonstrated a role for both CD11 and CD18 adherence glycoproteins in basophil adherence induced by rIL-3, and indirect immunofluorescence and flow cytometric analysis revealed that rIL-3 treatment led to rapid and sustained increases in cell surface expression of CD11b antigens on basophils but not neutrophils (e.g., after 10 min: 217 +/- 29 vs 91 +/- 11% of control mean fluorescence intensity, p less than 0.05). However, no correlation was seen between the magnitude of changes in CD11b expression and changes in adhesion when tested simultaneously. These results suggest that local production of IL-3 during allergic reactions in vivo may selectively promote basophil activation, adhesion to endothelium, and recruitment to extravascular sites of inflammation.

Antigens, Differentiation↗
PubMed1986

Effect of disodium cromoglycate on cutaneous basophil anaphylaxis.

Cutaneous basophil anaphylaxis (CBA) was elicited by intradermal rechallenge of cutaneous basophil hypersensitivity (CBH) sites in guinea pigs sensitized 7 days previously with keyhole limpet hemocyanin (KLH). The antiallergy agent disodium cromoglycate (DSCG), administered i.v. immediately before rechallenge, inhibited the increased vasopermeability (measured by tissue dye uptake) and basophil degranulation (measured by light microscopic counts of intact basophils) characteristic of the CBA reaction. The antihistamine mepyramine, administered orally, inhibited vasopermeability but not basophil degranulation. The component contributed by DSCG inhibition of mast cell degranulation to the overall inhibition of the reaction was found to be minimal, since intact mast cells were found to be depleted at CBH sites and totally absent at CBA sites from animals treated with DSCG. Electron microscopic examination of basophils at CBA sites from DSCG-treated animals revealed the presence of ruffled perigranular membranes and enlarged perigranular spaces, but both the formation of degranulation sacs and the subsequent fusion of granule sac membranes with the plasma membrane were inhibited. DSCG also inhibited the vasopermeability and basophil degranulation of the CBA reaction elicited by KLH at day 14 and by C5a at day 7. When a basophil-enriched leucocyte preparation from KLH-sensitized guinea pigs was studied in vitro, DSCG inhibited both antigen-induced and C5a-induced basophil degranulation at 10(-5) and 10(-4) M. DSCG failed to inhibit the vasopermeability and the mast cell degranulation produced by either intradermal C5a or intradermal compound 48/80. These results indicate that anaphylactic degranulation of basophils, but not mast cells, is inhibited by DSCG in the guinea pig. This inhibition appears to take place independent of stimulus at an early stage of granule membrane fusion.

Anaphylaxis↗
PubMed1989

IgE-dependent regulation of human basophil adherence to vascular endothelium.

Mechanisms favoring the recruitment of circulating human basophils to extravascular sites of allergic inflammation are unknown. The basophil secretagogues anti-IgE, and pollen allergens rye grass I and ragweed Ag E (Lol p I and Amb a I) were tested for their ability to promote basophil adherence to umbilical vein endothelial cells. Co-incubation of endothelial cells and basophils with anti-IgE resulted in time and dose-dependent increases in basophil adhesion. These effects were due to activation of the basophil, required both magnesium and calcium, occurred before or in the absence of histamine release, and were seen at concentrations of stimulus below the usual range of secretagogue activity. In contrast, anti-IgE or Ag stimulation of neutrophils, or basophils from donors non-responsive to anti-IgE or Ag with respect to histamine release, had no effect on cell adherence. mAb 60.3, recognizing the CD18 leukocyte adhesion molecule, inhibited anti-IgE-induced enhancement of basophil-endothelial cell binding. Exposure of basophils to low concentrations of Ag in vivo may selectively initiate basophil infiltration into tissue sites of allergic inflammation by enhancing their adherence to endothelium.

Adjuvants, Immunologic↗
PubMed1989

Ultrastructural localization of the Charcot-Leyden crystal protein (lysophospholipase) to granules and intragranular crystals in mature human basophils.

Human blood basophils have been shown to form Charcot-Leyden crystals (CLC) and to contain quantities of CLC protein comparable to the eosinophil (Ackerman SJ, Weil GJ, Gleich GJ, J Exp Med 155:1597, 1982). We examined the subcellular localization of CLC protein in human basophils from peripheral blood using an ultrastructural postembedding immunogold method and affinity chromatography-purified polyclonal primary antibody to purified human eosinophil CLC protein. We found CLC protein to be uniquely associated with the main, large, particle-filled granule population of human basophils and particularly within intragranular Charcot-Leyden crystals in these granules. Rarely, CLC protein was localized within small, smooth perigranular vesicles. Neutrophils, lymphocytes, and monocytes present in preparations stained for the CLC protein were negative as were controls for the specificity of the immunogold staining. Mature eosinophils contained small numbers of positive crystalloid-free primary granules, as previously reported (Dvorak AM, Letourneau L, Login GR, Weler PF, Ackerman SJ, Blood 72:150, 1988). The presence of CLC protein in hexagonal and pyramidal crystals regularly present in activated types II and III basophils suggests two possibilities currently being investigated. Like eosinophils, basophils could synthesize CLC protein, or eosinophil-derived CLC protein could be internalized and stored in basophil granules. Regardless of the mechanism for acquisition of CLC protein (lysophospholipase) by basophils, the identification and subcellular localization of this enzyme in basophils requires that it be considered in basophil cell biology as well as in the pathobiology of basophil-rich reactions.

Basophils↗
PubMed1986

Stimulation of maturation of large immature histamine-containing basophilic cells from human peripheral blood, cord blood and bone marrow.

The development of FcR epsilon-bearing histamine-containing basophilic cells was studied in cultures of peripheral blood leucocytes and bone marrow leucocytes from normal individuals. The determination of basophilic cells was performed blindly. Before cultivation there were fairly similar numbers of basophilic cells in the samples from the three different sources (1.6 +/- 1.6 n = 13; 1.4 +/- 2.0 n = 21; 4.3 +/- 4.7 n = 8, respectively). During cultivation spontaneously appearing large blast-like basophilic cells were seen in good correspondence with a formation of histamine in the cultures (Spearman rank correlation coefficient = 0.9135, n = 13, P less than 0.001). This was more accentuated with bone marrow cells than with peripheral blood and cord blood cells. Conditioned medium (CM) was prepared from cells isolated from various tissues and stimulated by different means, e.g. peripheral blood from atopic individuals stimulated with allergen and unstimulated tonsil cells. Addition of the CM resulted in increased development of histamine-containing basophilic cells. Optimum stimulation was achieved with 10% CM. The basophilic stimulation by CM, as assessed as indices vs. unstimulated cultures, was more accentuated in cultures of peripheral blood cells than of bone marrow and cord blood cells (2.8 +/- 1.2; 1.8 +/- 0.5; 2.0 +/- 0.4, respectively). In contrast, the histamine formation was particularly evident in stimulated cultures of bone marrow cells, where more than four-fold increases of histamine were found. In bone marrow cells the histamine levels per basophilic cell also increased, whereas this was not the case in cord blood cells. A pronounced development of basophilic cells was achieved when using leukoagglutinin, provided the mitogen in the CM was eliminated. The formation of basophilic cells was prevented with mitomycin c and cycloheximide. In conclusion, the system described may provide important information on the development of histamine-containing basophilic cells at various maturation stages from different compartments, and mechanisms in a developing atopic disease.

Basophils↗