Biomedical subjects

C A Bickel

Publications and source records attributed to C A Bickel.

17 recordsLinked to original sources

PubMed2001

Increased renal Na-K-ATPase, NCC, and beta-ENaC abundance in obese Zucker rats.

Renal sodium retention, as a result of increased abundance of sodium transporters, may play a role in the development and/or maintenance of the increased blood pressure in obesity. To address this hypothesis, we evaluated the relative abundances of renal sodium transporters in lean and obese Zucker rats at 2 and 4 mo of age by semiquantitative immunoblotting. Mean systolic blood pressure was higher in obese rats relative to lean at 3 mo, P < 0.02. Furthermore, circulating insulin levels were 6- or 13-fold higher in obese rats compared with lean at 2 or 4 mo of age, respectively. The abundances of the alpha(1)-subunit of Na-K-ATPase, the thiazide-sensitive Na-Cl cotransporter (NCC or TSC), and the beta-subunit of the epithelial sodium channel (ENaC) were all significantly increased in the obese rats' kidneys. There were no differences for the sodium hydrogen exchanger (NHE3), the bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2 or BSC1), the type II sodium-phosphate cotransporter (NaPi-2), or the alpha-subunit of ENaC. These selective increases could possibly increase sodium retention by the kidney and therefore could play a role in obesity-related hypertension.

Animals↗

PubMed2000

A study of contemporary levels and temporal trends in inbreeding in the Tangier Island, Virginia, population using pedigree data and isonymy.

In this study we describe inbreeding in a large pedigree from Tangier Island, Virginia, in which we compare two commonly used methods to estimate inbreeding in humans: pedigree and isonymy (identical surnames of spouses). Genealogical data on 3,512 individuals dating back to 1722 were used. Using the pedigree method, we determined an average inbreeding coefficient (F) of 0.00873 for the community as a whole, and 0.018 for inbred individuals. Analysis of temporal trends showed that inbreeding began around 1800 and peaked at 0.0109 in 1824-1849 and 1875-1899. Thereafter, inbreeding steadily declined to 0.00565 in 1975-1997. Analysis of pedigree structure complexity over time showed that close consanguinity contributes to inbreeding in the earlier cohorts, and remote consanguinity accounts for much of the inbreeding in the later cohorts. The number of common ancestors increases over time, as does the number of paths connecting inbred individuals to these common ancestors. Inbreeding estimates based on the isonymy approach yielded a 2.2-fold higher value of F (0.01945) compared to the pedigree method. Total isonymy estimates over 25-year cohorts overestimated inbreeding values from pedigree data between 1. 5-8-fold. We speculate that the overestimation is probably due to the inability of our data to satisfy the method's assumption of monophyletic origin of each surname. In conclusion, inbreeding in the Tangier Island population is consistent with the isolated nature of its population, and temporal trends reflect patterns in emigration and a breakdown in isolation over time.

Adolescent↗

PubMed2000

Endogenous glucocorticoids and antigen-induced acute and late phase pulmonary responses.

BACKGROUND: Several studies suggest that endogenous glucocorticoids can dampen the severity of experimental allergic reactions in animals. OBJECTIVE: To investigate the influence that endogenous glucocorticoids have on the course of IgE-mediated pulmonary early and late phase reactions. METHODS: Twenty-one allergic asthmatic and six healthy control subjects underwent inhaled antigen challenge with measurements of plasma cortisol and cortisone by gas chromatography-mass spectrometry. RESULTS: There were no differences between the asthmatic and control groups in the baseline levels of cortisol or cortisone. However, the asthmatic subjects had significantly higher cortisol levels (67.2 +/- 8.6 vs 35.1 +/- 4.5 ng/mL; P = 0.04) and had higher cortisol/cortisone ratios (4.8 +/- 0. 6 vs 3.0 +/- 0.2; P = 0.01) 8 h after challenge compared to the control subjects. Among the asthmatic subjects, those whose FEV1 recovered rapidly had higher baseline levels of cortisol and those who displayed a late phase reaction had lower levels of cortisol during the late phase period. CONCLUSION: The results suggest that endogenous glucocorticoids may play a significant role in the modulation of airway responses to antigen challenge, and that antigen challenge may induce cortisol production in allergic subjects.

Adult↗

PubMed1999

An in vitro comparison of commonly used topical glucocorticoid preparations.

BACKGROUND: Glucocorticoids (GC) are potent inhibitors of peripheral blood eosinophil, basophil, and airway epithelial cell function. OBJECTIVES: We compared in vitro the inhibitory activity of synthetic GC used for topical treatment in asthma and rhinitis on basophil histamine release (HR), eosinophil viability, and expression of vascular cell adhesion molecule-1 (VCAM-1) in the human bronchial epithelial cell line BEAS-2B. METHODS: Cells were treated for 24 hours with increasing concentrations (range 10(-13) to 10(-6) mol/L) of fluticasone propionate (FP), mometasone furoate (MF), budesonide (BUD), beclomethasone dipropionate (BDP), triamcinolone acetonide (TAA), hydrocortisone (HC), or dimethyl sulfoxide diluent before challenge. HR was measured by a fluorometric assay, viability of purified eosinophils was assessed by erythrosin B dye exclusion, and expression of VCAM-1 was measured by flow cytometry. RESULTS: GC induced a concentration-dependent inhibition of anti-IgE-induced HR. Maximum inhibition ranged from 59. 7% to 81%, with a rank order of GC potency of FP > MF > BUD > BDP congruent with TAA >> HC. Three-day treatment of eosinophils with GC concentration-dependently inhibited IL-5-induced eosinophil viability, with a rank of potency almost identical to that observed with basophil HR. The rank order of potency of GC for inhibition of the expression of VCAM-1 in BEAS-2B cells was MF congruent with FP >> BUD > TAA > HC congruent with BDP. Inhibitory concentration of 50% values revealed that epithelial cells were the most sensitive and eosinophils were the least sensitive. CONCLUSIONS: These data, combined with information on pharmacodynamics of these drugs in vivo, may be useful in estimating GC local anti-inflammatory effects.

Administration, Topical↗

PubMed1999

Macrophage-derived chemokine induces human eosinophil chemotaxis in a CC chemokine receptor 3- and CC chemokine receptor 4-independent manner.

BACKGROUND: Chemokines are believed to contribute to selective cell recruitment. Macrophage-derived chemokine (MDC) is a CC chemokine that causes chemotaxis of dendritic cells, monocytes, and activated natural killer cells. MDC binds to CC chemokine receptor 4 (CCR4) but not to CCR1, CCR2, CCR3, CCR5, CCR6, or CCR7. OBJECTIVE: Our aim was to determine the in vitro activity of MDC on human eosinophils by using chemotaxis and calcium flux assays. METHODS: Eosinophils were purified from peripheral blood of allergic donors, and chemotactic activity of MDC and other CC chemokines was compared in microchemotaxis chamber assays. The role of CCR3 in these assays was determined by using a CCR3-blocking antibody. Measurements of cytosolic Ca++ mobilization were performed by using fura-2AM labeling, with eosinophils and cell lines transfected with CCR3 or CCR4. Eosinophil expression of CCR3 and CCR4 mRNA was determined by using RT-PCR. RESULTS: MDC (0.1 to 100 nmol/L) caused dose-dependent chemotaxis of purified human eosinophils (maximum approximately 3-fold control). Compared with other CC chemokines, the potency and efficacy for eosinophil chemotaxis were similar for MDC and eotaxin but were less than that observed for RANTES, monocyte chemoattractant protein (MCP)-4, and eotaxin-2. Although MDC can act by means of CCR4, RT-PCR analysis failed to reveal CCR4 mRNA in eosinophils. Effects of MDC on eosinophils was also independent of CCR3, as a blocking mAb to CCR3 failed to inhibit MDC-induced chemotaxis. Furthermore, CCR3-transfected human embryonic kidney cells labeled with Fura-2AM exhibited a rapid rise in intracellular free calcium after stimulation with eotaxin, eotaxin-2, or MCP-4, but not with MDC. Eosinophils cultured for 72 hours in 10 ng/mL IL-5 also demonstrated increased intracellular free calcium after stimulation with eotaxin-2 or MCP-4, but not with up to 100 nmol/L MDC. CONCLUSION: MDC is a CCR3- and CCR4-independent activator of eosinophil chemotaxis, but it does not appear to elicit measurable cytosolic calcium elevations during these responses. MDC appears to act by means of another receptor in addition to CCR4 and may therefore contribute to eosinophil accumulation without working through CCR1 to CCR7.

Calcium Signaling↗

PubMed1998

CD44 and CD69 represent different types of cell-surface activation markers for human eosinophils.

Eosinophils (EOS) purified from peripheral blood or late-phase bronchoalveolar lavage (BAL) were analyzed with 473 monoclonal antibodies (mAbs) from the Fifth International Workshop on Human Leukocyte Antigens in an attempt to identify markers of EOS activation. Two strategies were used: (1) to look for surface markers absent on fresh EOS but present after in vivo activation (e. g., in late-phase BAL fluid [BALF]) or after in vitro culture for up to 72 h with cytokines (<= 10 ng/ml of interleukin-3 [IL-3], IL-5, or granulocyte-macrophage colony-stimulating factor [GM-CSF]); and (2) to look for markers constitutively expressed on fresh EOS that were increased after activation in vivo or after culture in vitro. With indirect immunofluorescence and flow cytometry, the first approach revealed that among approximately 350 mAbs tested, only those recognizing CD69 became bound to late-phase BALF EOS or cytokine-cultured EOS, but not to fresh EOS. Using the second approach, we observed statistically significant concentration- and time-dependent increases in CD44 expression in EOS cultured with IL-3, IL-5, or GM-CSF (approximately 2-fold increase in fluorescence intensity, P < 0.05), but not with interferon-gamma (IFN-gamma) (up to 100 ng/ml), whereas levels of 15 other constitutively expressed markers were unchanged. Despite increased expression, neither fresh nor cytokine-cultured EOS adhered to immobilized hyaluronate, a ligand for CD44. Additionally, simultaneous comparison of hypodense (specific gravity < 1.085 g/liter) and normodense (specific gravity > 1.085 g/liter) EOS from allergic donors consistently revealed higher levels of CD44 expression (approximately 3- to 8-fold) but not CD69 expression on hypodense EOS. We conclude that CD69 and CD44 represent different types of activation markers for human EOS. These findings may be useful in assessing the state of EOS activation in vitro and in vivo.

Antibodies, Monoclonal↗

PubMed1997

Cutaneous injection of RANTES causes eosinophil recruitment: comparison of nonallergic and allergic human subjects.

RANTES, a member of the C-C chemokine family, is a potent chemoattractant for T lymphocytes and eosinophils, but not neutrophils. To determine the effect of RANTES on cell recruitment in vivo, we injected up to 4 microg of RANTES intradermally into both allergic and nonallergic subjects and obtained biopsies 30 min, 6 h, and 24 h later. A dose- and time-dependent recruitment of eosinophils, CD45RO+ cells, and CD3+ cells was observed, with no effect seen on polymononuclear, cutaneous lymphocyte Ag+, CD68+, or tryptase+ cells. Eosinophil recruitment occurred more rapidly in allergic subjects than in nonallergic subjects. No eosinophil infiltrate was observed in nonallergic biopsies at 30 min and 6 h, whereas significant eosinophil recruitment was observed in allergic subjects by 30 min, reaching near-maximum levels by 6 h. The peak responses at 24 h were similar in both groups (nonallergic, 110 +/- 24 eosinophils/mm2; allergic, 113 +/- 38 eosinophils/mm2). The two groups had comparable numbers of circulating eosinophils. Major basic protein staining demonstrated eosinophil degranulation in both allergic and nonallergic groups. RANTES injection resulted in activation of endothelial E-selectin expression at 24 h. Incubation of cultured HUVECs with RANTES had no effect on adhesion molecule expression, suggesting that the in vivo effect may have been indirect. Our studies demonstrate that RANTES is a potent chemoattractant for eosinophils, CD3+ cells, and CD45RO+ cells in human skin. The accelerated eosinophil recruitment in allergic subjects provides support for the hypothesis that eosinophils from these subjects are primed in vivo.

Administration, Cutaneous↗

PubMed1997

Regulation of alpha 4 integrin-mediated adhesion of human eosinophils to fibronectin and vascular cell adhesion molecule-1.

BACKGROUND: Eosinophils selectively accumulate at sites of allergic inflammation. Their recruitment is dependent on both the expression and functional activity of cell adhesion molecules. How the functional activity of cell adhesion molecules on eosinophils is regulated is poorly understood. OBJECTIVE: Our objective was to examine the functional activity of alpha 4 integrins on human eosinophils and its regulation by various agents. METHODS: Function of alpha 4 integrins on human eosinophils was examined by testing adhesion to immobilized fibronection and vascular cell adhesion molecule-1 (VCAM-1) in the presence or absence of a monoclonal antibody (mAb) (8A2) that activates beta 1 integrin function. RESULTS: Spontaneous eosinophil adhesion to VCAM-1 was enhanced by 8A2, but adhesion to fibronectin could only be detected in the presence of 8A2. Concentrations of 8A2 that were approximately 100-fold less than saturating induced maximal eosinophil adhesion. Adhesion to VCAM-1 in the presence of 8A2 was effectively inhibited by alpha 4 and beta 1 integrin mAbs: beta 7 mAb had partial inhibitory activity. Connecting segment-1 peptide and alpha 4 mAb blocked 8A2-dependent fibronectin binding: beta 1, beta 2, and beta 7 integrin mAbs had partial inhibitory activity. Eosinophils obtained from bronchoalveolar lavage fluids and blood eosinophils stimulated with IL-5, platelet-activating factor, or RANTES displayed increased beta 2 integrin-dependent, not alpha 4 integrin-dependent, attachment. Spontaneous adhesion of eosinophils to VCAM-1 was significantly reduced by the tyrosine kinase inhibitor tyrphostin B46 (inhibitory concentration of 50% approximately equal to 20 mumol/L); this effect was reversed by 8A2. CONCLUSIONS: The functional activity of integrins on eosinophils can be positively and negatively regulated. Altered integrin avidity may influence eosinophil recruitment in vivo.

Antibodies, Monoclonal↗

PubMed1997

Phenotypic analysis of alveolar macrophages and monocytes in allergic airway inflammation. I. Evidence for activation of alveolar macrophages, but not peripheral blood monocytes, in subjects with allergic rhinitis and asthma.

Macrophages and monocytes play important proinflammatory roles in allergic inflammation. We hypothesized that these cells would express an activated phenotype in allergic disease of the airways. We therefore compared the expression of 17 activation markers on the surface of alveolar macrophages (AM) and peripheral blood monocytes (PBM) in 13 subjects with asymptomatic allergic asthma (AA), nine subjects with asymptomatic allergic rhinitis (AR), and 11 nonallergic (N). AM were obtained by BAL, and PBM were simultaneously obtained by phlebotomy; both were analyzed for expression of surface markers using a new two-color flow cytometry method that essentially eliminates background autofluorescence. The proportions of AM in BAL fluid from AA, AR, and N subjects were 84 +/- 2, 85 +/- 4, and 91 +/- 1%, respectively; viability always exceeded 92%. Expression of eight markers (CD16, CD18, CD32, CD44, CD71, HLA Class I, HLA DR, and HLA DQ) was significantly (p < 0.05) higher on AM of AA than on N; expression of six markers (CD11a, CD16, CD18, CD71, HLA Class I, and HLA DR) was higher on AM of AR than on N, with differences in CD44 levels approaching statistical significance (p = 0.07). Expression of one marker, CD44, was significantly higher on AM of AA than on those of AR, with differences in HLA Class I levels approaching statistical significance (p = 0.07). In contrast, no significant differences were found among the three groups in the expression in eight other markers (CD11b, CD14, CD23, CD29, CD33, CD35, CD63, and CD64). Finally, similar analysis of PBM from these same subjects failed to find any difference between the three groups in any of the 17 activation markers studied. These data suggest that AM are activated in allergic respiratory diseases, and that levels of HLA Class I and CD44 on AM are altered during allergic inflammation in the upper and lower airways.

Adult↗

PubMed1996

Detection of the chemokine RANTES and endothelial adhesion molecules in nasal polyps.

BACKGROUND: To better understand the mechanisms of eosinophil recruitment into the upper airways, we examined human nasal polyps for the expression of the chemotactic cytokine RANTES and the endothelial adhesion molecules E-selectin and vascular cell adhesion molecule-1 (VCAM-1). METHODS: Routine histologic examination and immunostaining with antibodies to RANTES, E-selectin, and VCAM-1 were performed on three types of tissues: nasal polyps, sinus mucosa, or turbinates from patients undergoing other elective procedures (S/T), and nasal biopsy specimens from nonallergic volunteers (NA). To further quantify the expression of endothelial adhesion molecules, some tissue samples were homogenized, and the resulting supernatants were assayed with sandwich ELISAs for VCAM-1 and E-selectin. RESULTS: Polyp eosinophil counts ranged from 19/mm2 to 1818/mm2 (763 +/- 120/mm2, mean +/- SEM) and were significantly higher than those found in the control tissues (5 +/- 2 in S/T samples and 20 +/- 9 in NA samples, p < 0.002). Immunochemical staining for RANTES was observed in 11 of 14 polyps; intense staining for RANTES (grade 3) was observed in six of 14 polyps. None of nine S/T samples or five NA samples demonstrated grade 3 staining. Staining with anti-RANTES was largely localized to airway and glandular epithelium. There was no significant correlation between counts of eosinophils or the combined total of eosinophils plus mononuclear cells and the intensity of epithelial RANTES staining in all nasal tissues. Staining for VCAM-1, as well as for E-selectin, was detected in 11 of 14 polyps and eight of 13 control tissues. VCAM-1 detected by ELISA in polyp tissues (6.8 +/- 1.3 micrograms/gm) was higher than that found in six S/T samples (1.2 +/- 0.3 micrograms/gm, p < 0.005) and in two NA samples (1.8 +/- 0.02 micrograms/gm, p = 0.08). E-selectin values in polyps (1.4 +/- 0.3 micrograms/gm) were not statistically different from those detected in six S/T samples (0.5 +/- 0.2 microgram/gm) or two NA samples (1.6 +/- 0.4 microgram/gm). Counts of eosinophils and eosinophils plus mononuclear cells displayed a strong correlation with VCAM-1 ELISA values (p < 0.005 and p < 0.004, respectively) but not with VCAM-1 staining. VCAM-1 staining correlated with EG2-positive eosinophils in nasal polyp tissues (p < 0.01). E-selectin staining did not correlate with either neutrophil or eosinophil counts. CONCLUSIONS: These studies demonstrate that the chemokine RANTES is produced in vivo predominantly to nasal epithelium. Endothelial activation, as indicated by adhesion molecule expression, occurs in human nasal polyp tissues and in control tissues, possibly reflecting the continued antigen exposure of the nasal mucosa. The correlations found in this study suggest that expression of VCAM-1 plays a role in the selective recruitment of eosinophils and mononuclear cells into nasal polyp tissues and that RANTES may be more important in localizing eosinophils to the epithelium.

Adult↗

PubMed1996

Glucocorticosteroid inhibition of cytokine production: relevance to antiallergic actions.

We believe that there are the following four classes of glucocorticoid-sensitive cytokines that are involved in cell recruitment: (1) those that activate endothelium nonspecifically; (2) those that activate endothelium specifically; (3) those that activate, prime, and prolong the survival of eosinophils; and (4) those that stimulate movement of cells up into the epithelium. Glucocorticoids inhibit the generation of these cytokines and thereby prevent several different aspects of inflammation, including the activation and recruitment of inflammatory cells (eosinophils, basophils, and lymphocytes) and the release of inflammatory mediators. We believe such pleiotropic actions account for the efficacy and widespread use of glucocorticoids in the treatment of asthma.

Anti-Allergic Agents↗

PubMed1995

Carboxyfluorescein diacetate labeling does not affect adhesion molecule expression or function in human neutrophils or eosinophils.

Fluorescently labeled leukocytes are commonly used in in vitro and in vivo experimental systems. However, the effects of fluorescent labeling on the expression and function of leukocyte adhesion molecules has not been examined in part because the extreme intensity of fluorescence tends to obscure signals from other fluorochromes used for dual color analysis. We have utilized a novel technique involving a 7-amino-4-methylcoumarin-3-acetic acid (AMCA) fluorophore-conjugated F(ab')2 fragment excitable in the ultraviolet wavelength range (350-450 nm) and dual-laser flow cytometry to determine if labeling of human neutrophils and eosinophils with the fluorescent dye 5-(6)-carboxyfluorescein diacetate (CFDA) alters surface expression of the primary leukocyte adhesion molecules involved in leukocyte-endothelial interactions. Simultaneously, adhesion molecule function was assessed by comparing the ability of CFDA-labeled vs. control cells to adhere to cultured human umbilical vein endothelial cells (HUVEC) and purified immobilized adhesion molecules. Isolated human eosinophils and neutrophils were fluorescently labeled by incubation with CFDA. Flow cytometric comparisons of labeled and unlabeled cells demonstrated that fluorescence labeling of neutrophils and eosinophils with CFDA did not alter basal surface expression of the beta 2 integrins (i.e., CD11a CD11b or, CD18). Stimulation of neutrophils with fMLP and eosinophils with PMA resulted in increased surface expression of CD11b and CD18 which was not altered by CFDA labeling. Likewise, CFDA labeling of neutrophils and eosinophils did not significantly alter their integrin-dependent adhesion to activated HUVEC under static or rotational conditions. Similarly, adhesion to immobilized recombinant E- and P-selectin was unaltered. These data demonstrate that fluorescent labeling of human neutrophils and eosinophils with CFDA does not alter surface expression or function of several adhesion molecules necessary for leukocyte-endothelial interactions. The use of CFDA-labeled cells in experiments employing intravital microscopy should therefore provide valid information on adhesion molecule function in vivo.

Cell Adhesion↗

PubMed1995

Comparison of human eosinophil and neutrophil ligands for P-selectin: ligands for P-selectin differ from those for E-selectin.

Eosinophils (EOS) and neutrophils (PMN) display different patterns of accumulation during various inflammatory reactions. We hypothesized that EOS and PMN may differ in their ligands for P-selectin, and that these ligands may differ from those previously identified for E-selectin. Recombinant human P-selectin was immobilized on plastic surfaces and adhesion of 51Cr-labeled human EOS or PMN was compared. EOS and PMN adhered in a concentration-dependent fashion, with similar maximal net adhesion. Preincubation with a blocking P-selectin antibody inhibited adhesion of both cell types, whereas a non-blocking antibody did not. To determine if the counterligands were sialylated proteins, cells were treated with various glycosidases and proteases before testing adhesion. Neuraminidase treatment markedly inhibited binding of both cell types, while endo-beta-galactosidase had no significant effect. Pretreatment with several proteases reduced adhesion of both cell types, although they consistently caused a greater inhibition of PMN binding than EOS binding. To determine whether the P-selectin ligands were surface structures whose expression or function may be altered by cell activation, leukocytes were pretreated with various stimuli; only platelet-activating factor (PAF) treatment reduced the capacity of leukocytes to adhere to P-selectin. Thus, the counterligands for P-selectin on EOS and PMN are similar sialylated, protease-sensitive, endo-beta-galactosidase-resistant structures, whose function and/or expression is reduced following treatment with PAF. These characteristics are clearly different than those reported for EOS and PMN ligands for E-selectin, and suggest disparate roles for P-selectin and E-selectin during EOS and PMN recruitment during inflammatory responses in vivo.

Cell Adhesion↗

PubMed1994

Comparison of human eosinophil and neutrophil adhesion to endothelial cells under nonstatic conditions. Role of L-selectin.

To simulate adhesion that occurs under conditions of flow, we investigated the attachment of eosinophils to endothelium under rotational conditions. Tissue-culture plates containing monolayers of HUVEC were placed on a horizontal rotator (80 revolutions per minute (rpm)), and equal numbers of purified human eosinophils or neutrophils were added to separate wells at 4 degrees C. Binding of eosinophils and neutrophils to unstimulated endothelial cells was 15 +/- 3 and 31 +/- 11 cells/four high power fields (HPF), respectively. After preincubation of HUVEC with IL-1 beta (1 ng/ml, 4 h, 37 degrees C), adhesion increased to 56 +/- 4 and 290 +/- 26 cells/four HPF, respectively (p < 0.0002 for both, n = 8-14). Eosinophils with reduced levels of L-selectin (blood eosinophils activated in vitro or eosinophils obtained from bronchoalveolar lavage (BAL) performed after segmental lung allergen challenge of allergic subjects) demonstrated reduced binding under rotating conditions. Several L-selectin Abs inhibited adhesion of eosinophils and neutrophils (e.g., LAM1-3: 43 +/- 14% vs 63 +/- 3% inhibition; LAM1-6: 73 +/- 5% vs 36 +/- 6% inhibition, respectively, n > or = 6). Interestingly, one additional L-selectin Ab, LAM1-11, inhibited eosinophil but not neutrophil adhesion (51 +/- 2% vs 1 +/- 7% inhibition, respectively, n > or = 5). We conclude that eosinophils, like neutrophils, use L-selectin to bind to activated endothelial cells under conditions of flow, although mAb LAM1-11 can selectively inhibit eosinophil attachment to stimulated endothelial cells in vitro, suggesting different functional epitopes on L-selectin among eosinophils and neutrophils.

Cell Adhesion↗

PubMed1994

Differences between human eosinophils and neutrophils in the function and expression of sialic acid-containing counterligands for E-selectin.

Both neutrophils and eosinophils have been shown to bind to the inducible endothelial cell adhesion molecule E-selectin. For neutrophils, one of the reported ligands for E-selectin is the sialylated Lewis X Ag (sLe(x)). To analyze the counterligands on eosinophils for E-selectin, adhesion assays were performed in which purified leukocytes were allowed to adhere to a soluble recombinant form of the molecule immobilized on plastic plates. Eosinophils, like neutrophils, bound to immobilized E-selectin, but significantly more neutrophils than eosinophils adhered in this assay. Consistent with the greater ability of neutrophils to bind E-selectin was the observation by flow cytometry that neutrophils expressed significant levels of sLe(x) and a sialylated dimeric form of the Le(x) Ag (sialyl-dimeric Le(x), or sialyl-stage-specific embryonic Ag-1, recognized by mAb FH6), whereas the expression of these epitopes on eosinophils was extremely low or undetectable. Expression was similar on eosinophils from allergic and nonallergic donors, and was not altered on eosinophils after induction of L-selectin shedding in vitro by treatment with platelet-activating factor. For both eosinophils and neutrophils, treatment with sialidase was associated with the complete elimination of sLe(x) and sialyl-dimeric Le(x) surface expression, and abolished leukocyte adhesion to E-selectin. Another glycosidase, endo-beta-galactosidase, which specifically cleaves the beta 1-4 galactose linkage to N-acetyl-glucosamine when it exists in an extended chain form such as that found in sialyl-dimeric Le(x), significantly inhibited eosinophil and neutrophil adhesion and expression of sialyl-dimeric Le(x). Such treatment also reduced sLe(x) expression on eosinophils, while having little effect on total neutrophil sLe(x) expression. For both eosinophils and neutrophils the sialylated ligand did not appear to be a glycoprotein because pretreatment of leukocytes with several proteases had no effect on adhesion to E-selectin or on expression of sLe(x) and sialyl-dimeric Le(x). These data suggest that eosinophils, like neutrophils, use sialylated, protease-resistant structures to bind to E-selectin, although the eosinophil expresses much lower levels of these structures on its surface. A major proportion of the sLe(x)-containing E-selectin ligand on the surface of eosinophils appears to be in the form of sialyl-dimeric Le(x), whereas this represents a minor proportion on the surface of neutrophils. Based on results using endo-beta-galactosidase, it appears that these cells may rely disproportionately upon the cell surface sialyl-dimeric Le(x) to bind to E-selectin.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies, Monoclonal↗

PubMed1993

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↗

PubMed1992

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↗