Function and expression of adhesion molecules on human basophils.
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Basophils↗
Biomedical subjects
B S Bochner
Publications and source records attributed to B S Bochner.
The effects of glucocorticoids on human eosinophils.
It is readily apparent that eosinophils are important targets of steroid effects. Disruption of the cytokine network by glucocorticoids can indirectly diminish eosinophil generation, survival, and function. In addition, glucocorticoids appear to have direct effects on the survival, and perhaps on the function, of these cells. Rapid advances in our knowledge of the biochemistry of signal transduction, as well as the interaction between cell surface receptors and RNA transcription machinery, will allow detailed analysis of the mechanisms by which the function and life cycle of eosinophils are influenced by glucocorticoids.
Animals↗
Immunological aspects of allergic asthma.
Asthma is a chronic obstructive disease of the lower airways. It is characterized by episodic exacerbations of at least partially reversible airflow limitation, along with bronchial hyperreactivity and airway inflammation. This latter characteristic, airway inflammation, is now a major focus of investigation, in large part because of a growing body of evidence to support its critical role in the pathogenesis of this disorder. Investigations of the histopathologic, cytologic, and chemical events accompanying fatal and nonfatal asthma, as well as experimental allergen-induced allergic reactions of the lower airways, have confirmed that a wide variety of immunologic phenomena occur, including local release of preformed mediators, newly synthesized metabolites of arachidonic acid, and soluble pro-inflammatory proteins including kinins and cytokines; alterations in vascular permeability and the development of airways edema; leukocyte activation and accumulation; and immune-mediated neurogenic responses that affect airway smooth muscle and other local responses. This information has led to new pharmacologic approaches designed to antagonize each of these elements of the asthmatic diathesis, with varying degrees of success. Nevertheless, the development of novel antagonists of mediators, pro-inflammatory proteins, cells, adhesion molecules, neuropeptides, and their respective receptors have begun to yield additional insight into their relative importance in the immunologic basis of asthma.
Asthma↗
Identification of IgE-bearing cells in the late-phase response to antigen in the lung as basophils.
We have carried out studies to ascertain whether the histamine-containing, IgE-bearing cells found in the bronchoalveolar lavage (BAL) fluid obtained during the late-phase response following subsegmental antigen challenge of human airways are predominantly basophils or mast cells. Four lines of evidence suggest that most are basophils: (1) The cells fulfill morphologic criteria for light microscopy. (2) Cell surface markers determined by immunofluorescence and flow cytometry revealed that the IgE-bearing cells express the leukocyte antigens Fc gamma RII and the beta 2 integrins, LFA-1 and Mac-1, but do not express the mast cell-associated c-kit receptor for stem cell factor. (3) The late-phase histamine-containing cells in late-phase BAL fluids have the functional characteristics of basophils in their secretory responses to anti-IgE, the f-met peptide, and phorbol ester TPA. (4) The cells have a functional histamine type 2 receptor, a characteristic of basophils, not mast cells. We conclude that basophils infiltrate the lower airways hours after antigen exposure. These cells may be responsible for the mediator release observed at that time.
Alcian Blue↗
Expression of a functional laminin receptor (alpha 6 beta 1, very late activation antigen-6) on human eosinophils.
The mechanisms responsible for the accumulation of eosinophils at sites of allergic and other inflammatory reactions are unknown, but recent studies have implicated both eosinophil and endothelial adhesion molecules in this process. However, less well studied have been the adhesive interactions between eosinophils and the subendothelial basement membrane and interstitial connective tissues. To test the hypothesis that eosinophils might interact with extracellular matrix proteins, we analyzed purified human eosinophils for the expression and function of various beta 1 integrins. Using indirect immunofluorescence and flow cytometry, purified eosinophils from mildly allergic donors were found to consistently express the integrin subunits beta 1 (CD29), alpha 4 (CD49d, very late activation antigen [VLA]-4 alpha), and alpha 6 (CD49f, VLA-6 alpha). No significant expression of the alpha 1, alpha 2, alpha 3, alpha 5, or beta 4 subunits was detected. Platelet contamination of the eosinophil preparations was excluded by light microscopy and by the inability to detect expression of platelet glycoproteins alpha v, CD41b, and CD42b. Immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of eosinophils confirmed the expression of cell-surface beta 1, alpha 4, and alpha 6. Furthermore, eosinophils purified from allergic donors were shown to adhere to plate-bound laminin, but not to type 1 or type 4 collagen. Adhesion to laminin was concentration-dependent, required divalent cations, and was completely and specifically inhibited by the anti-alpha 6 monoclonal antibody (MoAb) GoH3 and by the anti-beta 1 MoAb 33B6. Interestingly, the anti-beta 1 MoAb 18D3 (which like 33B6 blocks eosinophil binding to VCAM-1) did not inhibit eosinophil adhesion to laminin, suggesting that there are functionally distinct epitopes on the beta 1 subunit. Eosinophils purified from 4 healthy, nonallergic donors also showed alpha 6-dependent adhesion to laminin, although these cells adhered less well. These studies establish the expression of alpha 6 beta 1 on human eosinophils and document its function as a laminin receptor. Interaction of eosinophil alpha 6 beta 1 with laminin, eg, in basement membranes, may contribute to the localization of these cells at inflammatory sites in vivo.
Animals↗
GM-CSF regulates human eosinophil responses to F-Met peptide and platelet activating factor.
Evidence is accumulating that suggests that granulocyte macrophage CSF (GM-CSF) may have inflammatory actions in addition to its effects as a hematopoietic growth factor. In these studies, the effects of culture of purified human eosinophils with GM-CSF on cellular responses, including intracellular calcium responses, the expression of adherence molecules, and adhesion, were examined. In freshly isolated eosinophils expression of the adhesion molecule CD11b was only weakly increased by a 10-min incubation with platelet-activating factor (PAF) (118 +/- 3, 121 +/- 4, 117 +/- 2, n = 8) and FMLP (100 +/- 1, 106 +/- 6, and 105 +/- 4% of control for 10(-8), 10(-7), and 10(-6) M, respectively, n = 4). Lyso PAF never increased CD11b expression. Exposure of eosinophils to GM-CSF (0.5 ng/ml for 2 days) induced an increase in CD11b responses to PAF (132 +/- 3, 144 +/- 4, and 140 +/- 7) and FMLP (123 +/- 8, 145 +/- 10, and 137 +/- 4% of control for 10(-8), 10(-7), and 10(-6) M concentrations, respectively, n = 4). In 3-day culture studies, responses were greatest on days 2 and 3. The half-maximal GM-CSF concentration for the increase in the response to FMLP (10(-7) M, day 2) was approximately 100 pg/ml. GM-CSF (0.5 ng/ml) induced an increase in basal CD11b expression by day 1, which declined toward the initial baseline on days 2 and 3. Dexamethasone (10(-7) M) hastened the decline in baseline CD11b expression but did not inhibit the increase in the response to either PAF or FMLP. After 3 days of culture with GM-CSF (0.08 ng/ml), FMLP (10(-6) M)-induced elevations of intracellular calcium were increased (277 +/- 27% of day 0 control, n = 4, p < 0.01), whereas the response induced by PAF was not (70 +/- 36% of day 0 control, p = NS). A 30-min exposure to GM-CSF caused a potentiation of both PAF- and FMLP-induced adherence of eosinophils to gelatin-coated plastic. Longer incubation with GM-CSF (3 days) potentiated only FMLP-induced adherence. We conclude that expression of adherence molecules, transmembrane signaling, and adherence responses of eosinophils are enhanced by exposure to GM-CSF. These effects of GM-CSF could potentiate the adherence to endothelium and subsequent migration of eosinophils to inflammatory sites in vivo.
Calcium↗
The effects of the potent glucocorticoid budesonide on adhesion of eosinophils to human vascular endothelial cells and on endothelial expression of adhesion molecules.
An important early event in the formation of the allergic late-phase cellular infiltrate is the adhesion of eosinophils to vascular endothelium. Because glucocorticoids are potent inhibitors of this late-phase inflammatory reaction, we examined the effect of the glucocorticoid budesonide on in vitro adhesion of human eosinophils to cultured human umbilical vein endothelial cells (HUVEC). Pretreatment of eosinophils, HUVEC or both with budesonide (10(-7) M) did not inhibit either baseline adhesion or that stimulated by interleukin-1 beta (50 U/ml, 4 hr) or interleukin-4 (100 U/ml, 21-23 hr) or the leukocyte activators N-formyl-methionyl-leucyl-phenylalanine (10(-6) M, 10 min) or platelet-activating factor (10(-6) M, 10 min). Pretreatment of HUVEC with budesonide also failed to affect expression of the adhesion molecules E-selectin, intercellular adhesion molecule-1, or vascular cell adhesion molecule-1 on resting HUVEC or after stimulation with interleukin-1 beta (10 U/ml, 4 hr), tumor necrosis factor (6 U/ml, 4 hr) or interleukin-4 (50 U/ml, 24 hr). Because budesonide failed to inhibit stimulus-induced eosinophil adhesion responses or endothelial adhesion molecule expression, we speculate that glucocorticoids inhibit the formation of eosinophil-enriched tissue infiltrates by inhibiting the production rather than the activity of eosinophil- or endothelial-activating factors or by altering the survival of eosinophils in tissues.
Budesonide↗
Adhesion molecule antagonists: future therapies for allergic diseases?
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Animals↗
Eosinophil transendothelial migration induced by cytokines. I. Role of endothelial and eosinophil adhesion molecules in IL-1 beta-induced transendothelial migration.
IL-1 beta promotes adhesiveness in human umbilical vein endothelial cells (HuVEC) for eosinophils through expression of adhesion molecules including intercellular adhesion molecules-1 (ICAM-1), E-selectin, and vascular cell adhesion molecule-1 (VCAM-1). Using an in vitro endothelial monolayer system, we examined whether IL-1 beta or TNF-alpha can promote eosinophil transendothelial migration. We also evaluated the contributions of ICAM-1, E-selectin, VCAM-1, leukocyte adhesion complex (CD11/18), and very late Ag-4 (CD11b/18) (VLA-4) in this process using blocking mAb, and determined the changes in expression of CD11b and L-selectin on eosinophils that had undergone transmigration. IL-1 beta and TNF-alpha treatment of HuVEC (4 h, 5 ng/ml) induced significant transendothelial migration of eosinophils (a 4.1 +/- 0.4-fold (IL-1 beta) and 2.0 +/- 0.9-fold (TNF-alpha) increase from the spontaneous value of 3.2 +/- 0.3%). Increased CD11b expression and shedding of L-selectin were observed on eosinophils following IL-1 beta-induced eosinophil transendothelial migration. Studies with mAb revealed that blockade of either ICAM-1 or CD11/18 inhibited transmigration, while antibodies against VCAM-1 and VLA-4 had no inhibitory effect. Among antibodies which block beta 2 integrins, anti-CD18 mAb had the best inhibitory effect (88% inhibition). The combined inhibitory effect of anti-CD11a mAb and anti-CD11b mAb was roughly equal to that of anti-CD18, although anti-CD11a (31% inhibition) and anti-CD11b (52% inhibition) were less effective individually. Anti-ICAM-1 by itself inhibited IL-1 beta-induced eosinophil transendothelial migration (24% inhibition) whereas neither anti-E-selectin nor anti-VCAM-1 were effective inhibitors. Interestingly, the combination of anti-E-selectin and anti-VCAM-1 with anti-ICAM-1 inhibited IL-1 beta-induced eosinophil transendothelial migration significantly better (53% inhibition) than anti-ICAM-1 alone. These results suggest that although the initial attachment of eosinophils to IL-1 beta-activated endothelial cells involves VCAM-1, E-selectin, and ICAM-1, the subsequent transendothelial migration process relies heavily on ICAM-1 and CD11/18. Finally, the changes that eosinophils have been observed to undergo during infiltration in vivo, namely increased expression of CD11/18 and shedding of L-selectin, appear to take place as a direct result of the interaction between eosinophils and endothelial cells.
Antibodies, Monoclonal↗
The human recombinant c-kit receptor ligand, rhSCF, induces mediator release from human cutaneous mast cells and enhances IgE-dependent mediator release from both skin mast cells and peripheral blood basophils.
The gene product of the steel locus of the mouse represents a growth factor for murine mast cells and a ligand for the c-kit proto-oncogene receptor, a member of the tyrosine kinase receptor class of oncogenes (for review, see O. N. Witte. 1990. Cell 63:5). We have studied the effect of the human recombinant c-kit receptor ligand stem cell factor (rhSCF) on the release of inflammatory mediators from human skin mast cells and peripheral blood basophils and compared its activity to that of rhIL-3, rhSCF (1 ng/ml to 1 microgram/ml) activated the release of histamine and PGD2 from mast cells isolated from human skin. Analysis by digital video microscopy indicated that purified human skin mast cells (84 +/- 5% pure) responded to rhSCF (0.1 to 1 microgram/ml) challenge with a rapid, sustained rise in intracellular Ca2+ levels that was accompanied by secretion of histamine. A brief preincubation (10 min) of mast cells with rhSCF (0.1 pg/ml to 1 ng/ml) significantly enhanced (100 +/- 35%) the release of histamine induced by anti-IgE (3 micrograms/ml), but was much less effective on IgE-mediated release of PGD2. In contrast, a short term incubation with rhSCF did not potentiate the secretion of histamine activated by substance P (5 microM). A 24-h incubation of mast cells with rhSCF did not affect the release of mediators induced by anti-IgE (3 micrograms/ml), probably due to receptor desensitization, rhSCF (1 ng/ml to 3 micrograms/ml) neither caused release of histamine or leukotriene C4 (LTC4) release from leukocytes of 14 donors, nor induced a rise in intracellular Ca2+ levels in purified (greater than 70%) basophils. Brief preincubation (10 min) of leukocytes with rhSCF (1 ng/ml to 3 micrograms/ml) caused an enhancement (69 +/- 11%) of anti-IgE-induced release of histamine that was significant at concentrations as low as 3 ng/ml (p less than 0.05), whereas it appeared less effective in potentiating IgE-mediated LTC4 release. In contrast, a prolonged incubation (24 h) with rhSCF (0.1 pg/ml to 100 ng/ml) did not enhance the release of histamine or LTC4 induced by anti-IgE (0.1 microgram/ml), whereas rhIL-3 (3 ng/ml) significantly potentiated the release of both mediators.(ABSTRACT TRUNCATED AT 400 WORDS)
Antibodies, Monoclonal↗
Histamine-containing cells obtained from the nose hours after antigen challenge have functional and phenotypic characteristics of basophils.
The pattern of mediators and appearance of cells that stain with alcian blue during human experimental early and late phase allergic reactions suggest that basophils accumulate in nasal secretions within hours of local Ag stimulation. To further explore whether the histamine containing cells that enter the nose after Ag challenge are mast cells or basophils, we studied their functional and phenotypic characteristics. Approximately 24 h after intranasal Ag provocation of subjects with allergic rhinitis, nasal lavage was performed, and the cells were isolated for degranulation studies, analysis of surface Ag, and viability. The average histamine content per alcian blue staining cell was 0.78 +/- 0.2 pg (n = 7), similar to that reported for peripheral blood basophils. Nasal cells were challenged in vitro with anti-IgE, ragweed Amb a I, and FMLP and their responses were compared to those of peripheral blood basophils isolated simultaneously from the same donors. Nasal leukocytes released histamine maximally at 0.1 micrograms/ml of anti-IgE (35.8 +/- 7.8%, n = 7) and responded to FMLP (25.4 +/- 9.9%, n = 7). The response of the cells to ragweed Amb a I and anti-IgE was attenuated compared to peripheral blood basophils. Anti-IgE-induced histamine release was calcium and temperature dependent. Dual color immunofluorescence and flow cytometric analysis of the recovered nasal cells coexpressed CD18, a leukocyte marker not expressed by mast cells. The nasal cells consistently had high levels of spontaneous histamine release (19.5 +/- 2.0%, n = 22). The viability of all cells, assessed by erythrosin B dye exclusion, was 70 +/- 2% (n = 15). However, the viability of IgE-bearing cells was only 28.3 +/- 5.7% (n = 4). The characteristics of histamine release and the nature of the cellular surface markers provide functional proof that the histamine-containing cells accumulating after nasal Ag challenge are basophils and not mast cells.
Adult↗
IL-4 induces adherence of human eosinophils and basophils but not neutrophils to endothelium. Association with expression of VCAM-1.
The present studies were performed to explore potentially selective mechanisms of leukocyte adhesion in an attempt to understand how preferential recruitment of eosinophils and basophils might occur during allergic and other inflammatory reactions. Stimulation of human vascular endothelial cells for 24 h with IL-4 (30 to 1,000 U/ml) induced adhesion for eosinophils (up to approximately four-fold of control) and basophils (up to approximately twofold of control) but not neutrophils (less than 125% of control). Analysis of endothelial expression of adhesion molecules by flow cytometry revealed that IL-4 treatment induced vascular cell adhesion molecule-1 (VCAM-1) expression without significantly affecting the expression of other adhesion molecules, namely endothelial-leukocyte adhesion molecule-1 (ELAM-1) or intercellular adhesion molecule-1 (ICAM-1). The concentration-response curve for IL-4-induced VCAM-1 expression paralleled that for adhesion. Endothelial cells stimulated with IL-4 expressed adhesive properties for eosinophils by 3 h; the response increased steadily during a 24-h time course study. Eosinophils and basophils adhered to plates coated with a recombinant form of VCAM-1. This adhesion was blocked with antibodies to VCAM-1 but not ELAM-1. mAb directed against either VCAM-1 or VLA-4 inhibited (by approximately 75%) the binding of eosinophils and basophils to IL-4-stimulated endothelial cells. Because VLA-4 and VCAM-1 have been demonstrated to bind to each other in other adhesion systems, these results suggest that IL-4 stimulates eosinophil and basophil adhesion by inducing endothelial cell expression of VCAM-1 which binds to eosinophil and basophil VLA-4. The lack of expression of VLA-4 on neutrophils and the failure of IL-4 to stimulate neutrophil adherence support this conclusion. It is proposed that local release of IL-4 in vivo in allergic diseases or after experimental allergen challenge may partly explain the enrichment of eosinophils and basophils (vs neutrophils) observed in these situations.
Antibodies, Monoclonal↗
Immunophenotyping and functional analysis of purified human uterine mast cells.
Human mast cells have been purified from uterine tissues, and their surface marker profile and function have been evaluated as part of ongoing studies of mast cell heterogeneity. Using a panel of antibodies, purified uterine mast cells (UMC; 81% +/- 7% purity, n = 10) were analyzed by immunofluorescence and flow cytometry for surface expression of various antigens. Consistent with previous analyses of mast cells from other tissues, UMC expressed HLA class I, IgE, c-kit receptor, CD9, CD33, CD43, CD45, and CD54, while CD11a, CD11b, CD14, CD16, CD23, and CD64 were not detected. Unlike other mast cells, UMC expressed CD11c/CD18 (p150,95) and CD32 (Fc gamma RII). Additional antigens not previously studied on mast cells included the selectin LECAM-1 (Leu-8) and several beta 1 and beta 3 integrins; expression of very late activation antigen-4 (VLA-4) (CD49d/CD29), VLA-5 (CD49e/CD29), and the vitronectin receptor (CD51/CD61) was seen. Functional studies showed that treatment of human umbilical vein endothelial cells with interleukin-1 (5 ng/mL for 4 hours) resulted in a twofold to threefold increase in adhesiveness for UMC. Purification procedures did not alter histamine release responses to anti-IgE or the calcium ionophore A23187, and treatment of UMC with an anti-CD32 monoclonal antibody (IV.3) did not induce histamine release or alter anti-IgE-induced release. These data suggest that UMC may possess unique phenotypic characteristics, and support the concept of mast cell heterogeneity.
Antibodies, Monoclonal↗
Mechanisms of basophil recruitment in allergic diseases.
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Animals↗
Altered adhesion molecule expression and endothelial cell activation accompany the recruitment of human granulocytes to the lung after segmental antigen challenge.
Mounting evidence suggests that inflammatory cells recruited to the lung can contribute to the pathogenesis of asthma. The factors governing the activation and recruitment of circulating cells to the lung remain unknown, but an early step in this process is the interaction of adhesion molecules on circulating cells with those on endothelial cells. We used a segmental antigen challenge model followed 18 h later by bronchoalveolar lavage (BAL) to study granulocyte recruitment to the lung in 14 allergic subjects. Using immunofluorescence and flow cytometry, we determined the expression of the adhesion molecules CD11b, L-selectin (LECAM-1), and VLA-4 on BAL and peripheral blood granulocytes. Total cell count and percentages of recovered eosinophils and basophils were significantly increased in BAL fluids from antigen-challenged segments. Compared with their peripheral blood counterparts, CD11b expression was increased 2- to 3-fold on BAL eosinophils, basophils, and neutrophils (n = 9, P less than 0.05). In contrast, L-selectin expression was significantly decreased on BAL cells (n = 3 to 4, P less than 0.05). Similar phenotypic changes were observed on all three cell types, and on neutrophils recovered from saline-challenged control lung segments. In two subjects, VLA-4 alpha (CD49d) expression on BAL eosinophils was 78 +/- 5% of that seen on peripheral blood eosinophils. Because ELAM-1 (endothelial leukocyte adhesion molecule-1, E-selectin) expression occurs during allergic inflammation and is shed after endothelial activation, we used a sensitive enzyme-linked immunosorbent assay to analyze BAL supernatants for a soluble form of this molecule (sELAM-1).(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
Endothelial leukocyte adhesion molecule-1 and intercellular adhesion molecule-1 mediate the adhesion of eosinophils to endothelial cells in vitro and are expressed by endothelium in allergic cutaneous inflammation in vivo.
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Cell Adhesion↗
Adhesion of human basophils, eosinophils, and neutrophils to interleukin 1-activated human vascular endothelial cells: contributions of endothelial cell adhesion molecules.
Cytokines such as interleukin 1 (IL-1) promote adhesiveness in human umbilical vein endothelial cells for leukocytes including basophils, eosinophils, and neutrophils, and induce expression of adherence molecules including ICAM-1 (intercellular adhesion molecule-1), ELAM-1 (endothelial-leukocyte adhesion molecule-1), and VCAM-1 (vascular cell adhesion molecule-1). In the present study, blocking monoclonal antibodies (mAb) recognizing ICAM-1, ELAM-1, and VCAM-1 have been used to compare their roles in IL-1-induced adhesion of human basophils, eosinophils, and neutrophils. IL-1 treatment of endothelial cell monolayers for 4 hours induced a four- to eight-fold increase in adhesion for each cell type. Treatment of endothelial cells with either anti-ICAM-1 or anti-ELAM-1 mAb inhibited IL-1-induced adherence of each cell type. In contrast, treatment with anti-VCAM-1 mAb inhibited basophil and eosinophil (but not neutrophil) adhesion, and was especially effective in blocking eosinophil adhesion. The effects of these mAb were at least additive. Indirect immunofluorescence and flow cytometry demonstrated expression of VLA-4 alpha (very late activation antigen-4 alpha, a counter-receptor for VCAM-1) on eosinophils and basophils but not on neutrophils. These data document distinct roles for ICAM-1, ELAM-1, and VCAM-1 during basophil, eosinophil, and neutrophil adhesion in vitro, and suggest a novel mechanism for the recruitment of eosinophils and basophils to sites of inflammation in vivo.
Antibodies, Monoclonal↗