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B S Bochner

Publications and source records attributed to B S Bochner.

At least 37 records · Page 2Linked to original sources
PubMed2000

Road signs guiding leukocytes along the inflammation superhighway.

The term inflammation is used to describe the localized tissue changes, including leukocyte extravasation, that occur as part of the response to tissue damage, infection, or other immunologic responses. This carefully orchestrated series of events requires the existence of highly specific, regulated mechanisms for control of leukocyte recruitment and is dependent on both the inciting event and organ involved. This review summarizes recent developments in our understanding of how adhesion molecules and chemokines interact to facilitate tissue-specific and leukocyte subtype-specific influx during inflammation. Novel mechanisms believed to be responsible for capture and compartmentalization of B and T lymphocytes within lymph nodes are discussed, along with a description of adhesion molecule- and chemokine-mediated pathways that are believed to be involved in selective recruitment of lymphocytes and eosinophils to a variety of tissues, including the skin, gut, and lung. This growing knowledge and its potential importance provide enthusiasm for future anti-inflammatory therapies that target these recruitment pathways.

Animals↗
PubMed1999

The leukocyte integrin alpha D beta 2 binds VCAM-1: evidence for a binding interface between I domain and VCAM-1.

The trafficking of leukocytes through tissues is supported by an interaction between the beta 2 (CD18) integrins CD11a/CD18 (LFA-1) and CD11b/CD18 (Mac-1) and their ligand ICAM-1. The most recently identified and fourth member of the beta 2 integrins, alpha D beta 2, selectively binds ICAM-3 and does not appear to bind ICAM-1. We have reported recently that alpha D beta 2 can support eosinophil adhesion to VCAM-1. Here we demonstrate that expression of alpha D beta 2 in a lymphoid cell that does not express alpha 4 integrins confers efficient binding to VCAM-1. In addition, a soluble form of alpha D beta 2 binds VCAM-1 with greater efficiency relative to ICAM-3. The I domain of alpha D contains a binding site for VCAM-1 since recombinant alpha D I domain binds specifically to VCAM-1. In addition, alpha D mAb that block cellular binding to VCAM-1 bind the alpha D I domain. Using VCAM-1 mutants we have determined that the binding site on VCAM-1 for alpha D beta 2 overlaps with that of alpha 4++ integrins. Substitution of VCAM-1 aspartate at position 40, D40, within the conserved integrin binding site, diminishes binding to alpha D beta 2 and abrogates binding to the alpha D I domain. The corresponding integrin binding site residue in ICAM-3 is also essential to alpha D beta 2 binding. Finally, we demonstrate that alpha D beta 2 can support lymphoid cell adhesion to VCAM-1 under flow conditions at levels equivalent to those mediated by alpha 4 beta 1. These results indicate that VCAM-1 can bind to an I domain and that the binding of alpha D beta 2 to VCAM-1 may contribute to the trafficking of a subpopulation of leukocytes that express alpha D beta 2.

Animals↗
PubMed1999

Intracellular expression and release of Fc epsilon RI alpha by human eosinophils.

Although Fc epsilon R have been detected on human eosinophils, levels varied from moderate to extremely low or undetectable depending on the donor and methods used. We have attempted to resolve the conflicting data by measuring levels of IgE, Fc epsilon RI, and Fc epsilon RII in or on human eosinophils from a variety of donors (n = 26) and late-phase bronchoalveolar lavage fluids (n = 5). Our results demonstrated little or no cell surface IgE or IgE receptors as analyzed by immunofluorescence and flow cytometry. Culture of eosinophils for up to 11 days in the presence or absence of IgE and/or IL-4 (conditions that enhance Fc epsilon R on other cells) failed to induce any detectable surface Fc epsilon R. However, immunoprecipitation and Western blot analysis of eosinophil lysates using mAb specific for Fc epsilon RI alpha showed a distinct band of approximately 50 kDa, similar to that found in basophils. Western blotting also showed the presence of FcR gamma-chain, but no Fc epsilon RI beta. Surface biotinylation followed by immunoprecipitation again failed to detect surface Fc epsilon RI alpha, although surface FcR gamma was easily detected. Since we were able to detect intracellular Fc epsilon RI alpha, we examined its release from eosinophils. Immunoprecipitation and Western blotting demonstrated the release of Fc epsilon RI alpha into the supernatant of cultured eosinophils, peaking at approximately 48 h. We conclude that eosinophils possess a sizable intracellular pool of Fc epsilon RI alpha that is available for release, with undetectable surface levels in a variety of subjects, including those with eosinophilia and elevated serum IgE. The biological relevance of this soluble form of Fc epsilon RI alpha remains to be determined.

Adult↗
PubMed1999

Down-regulation of human basophil IgE and FC epsilon RI alpha surface densities and mediator release by anti-IgE-infusions is reversible in vitro and in vivo.

Previously, infusions of an anti-IgE mAb (rhumAb-E25) in subjects decreased serum IgE levels, basophil IgE and FcepsilonRIalpha surface density, and polyclonal anti-IgE and Ag-induced basophil histamine release responses. We hypothesized that these effects would be reversed in vivo by discontinuation of infusions and in vitro by exposing basophils to IgE. Subjects received rhumAb-E25 biweekly for 46 wk. Blood samples taken 0-52 wk after rhumAb-E25 were analyzed for serum IgE and basophil expression of IgE, FcepsilonRIalpha, and CD32. Basophil numbers were unaffected by infusions. Eight weeks after infusions, free IgE levels rose in vivo but did not reach baseline. Basophil IgE and FcepsilonRIalpha rose in parallel with free IgE while CD32 was stable. FcepsilonRI densities, measured by acid elution, returned to 80% of baseline, whereas histamine release responses returned to baseline. Basophils cultured with or without IgE or IgG were analyzed for expression of IgE, FcepsilonRIalpha, and CD32. By 7 days with IgE, expression of IgE and FcepsilonRIalpha rose significantly, whereas cultures without IgE declined. IgE culture did not effect CD32. IgG culture did not effect expression of any marker. The present results strongly suggest that free IgE levels regulate FcepsilonRIalpha expression on basophils.

Adult↗
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↗
PubMed1999

Functional expression of transmembrane 4 superfamily molecules on human eosinophils.

BACKGROUND: Transmembrane 4 superfamily (TM4SF) molecules are exclusively found on hematopoietic cells. Several members of the TM4SF are reported to be associated with other cell surface molecules, including integrins, and might participate in signal transduction, but little is known about their role on eosinophils. In the present study, we determined the expression and function of TM4SF molecules on human eosinophils. METHODS: Surface expression of TM4SF molecules on purified peripheral blood eosinophils was examined using indirect immunofluorescence and flow cytometry. Purified eosinophils were incubated with anti-TM4SF monoclonal antibodies (mAbs) for up to 24 h. Eosinophil activation was evaluated by measuring eosinophil homotypic aggregation as well as changes in surface expression of CD11b or CD62L by flow cytometry. RESULTS: Freshly isolated eosinophils expressed CD9, CD37, CD53, CD63 and CD81. Incubation with anti-CD9 mAb but not with anti-CD37, CD53, CD63 or CD81 mAb induced significant eosinophil homotypic aggregation. Incubation with any of the anti-TM4SF mAb for 30 min failed to alter the expression of either CD11b or CD62L on eosinophils. In contrast, the expression of CD11b was significantly enhanced after 24 h of incubation with anti-CD53 mAb, while the expression of CD62L was significantly reduced with anti-CD81 mAb. CONCLUSIONS: Cross-linking of some surface TM4SF molecules induced significant eosinophil homotypic aggregation, upregulation of CD11b expression, or CD62L shedding, consistent with activation of eosinophils. Our data suggest that several TM4SF molecules are functionally expressed on human eosinophils, and therefore might participate in allergic inflammation.

Antigens, CD↗
PubMed1999

Inhibition of VCAM-1 expression in human bronchial epithelial cells by glucocorticoids.

We have demonstrated previously that cytokines induce surface expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) on BEAS-2B bronchial epithelial cells in vitro. The present studies demonstrate glucocorticoid inhibition of cytokine-induced VCAM-1 expression as detected using flow cytometry and Northern blot analysis. Several commonly used inhaled glucocorticoids were tested for their ability to inhibit VCAM-1 and ICAM-1 expression. All glucocorticoids tested inhibited VCAM-1 expression in a dose-dependent manner. No inhibition of ICAM-1 expression was observed. The most potent of the glucocorticoids tested for inhibition of VCAM-1 expression were mometasone furoate and fluticasone propionate (FP), which had IC50 values (i.e., concentrations at which each glucocorticoid produced 50% inhibition) of under 10 pM. Budesonide, triamcinolone acetonide, and beclomethasone dipropionate (BDP) had intermediate potency, and hydrocortisone and the BDP metabolite beclomethasone-17-monopropionate were the least potent of the steroids tested. Kinetic analysis of the ability of FP to inhibit VCAM-1 expression revealed that preincubation with FP for 3 h completely inhibited VCAM-1 expression induced by tumor necrosis factor-alpha (TNF-alpha). FP inhibited VCAM-1 expression by 50% even when added as late as 6 h after stimulation with TNF-alpha. Using Northern blot analysis, we confirmed inhibition of VCAM-1 and ICAM-1 messenger RNA (mRNA) expression by FP. Pretreatment with FP (10(-11) M to about 10(-7) M, 24 h) inhibited TNF-alpha-induced VCAM-1 mRNA expression in BEAS-2B in a dose-dependent manner, but did not inhibit expression of ICAM-1 mRNA. Studies with actinomycin D indicate that FP treatment accelerated the degradation of TNF-alpha-induced VCAM-1 mRNA. FP (10(-7) M) also inhibited VCAM-1 mRNA expression induced by TNF-alpha in primary human bronchial epithelial cells as assessed by reverse transcription-polymerase chain reaction. These results suggest that suppression of epithelial VCAM-1 expression by glucocorticoids may contribute to their anti-inflammatory effects.

Androstadienes↗
PubMed1998

Lipopolysaccharide-induced leukocyte rolling and adhesion in the rat mesenteric microcirculation: regulation by glucocorticoids and role of cytokines.

A common side effect of high dose glucocorticoid therapy is increased susceptibility to bacterial infection, an effect that is in part mediated through inhibition of leukocyte recruitment to infected areas. However, the sites at which glucocorticoids act to prevent the multistep process of leukocyte recruitment have not been fully established. In this study, the effects of the glucocorticoid dexamethasone (DEX) on leukocyte-endothelial interactions, in response to bacterial LPS, were examined utilizing a model of rat mesenteric intravital microscopy. Pretreatment of rats with DEX (0.5 mg/kg) for 18 h or 30 min before stimulation with LPS significantly inhibited LPS-induced leukocyte rolling and adhesion in mesenteric postcapillary venules. Pretreatment with DEX also inhibited LPS-induced changes in expression of L-selectin and a shared epitope of CD11b/c on circulating neutrophils. These effects of DEX may be due to DEX inhibition of IL-1, TNF, and cytokine-induced neutrophil chemoattractant-1 (CINC-1) generation, since antagonists to these mediators were able to mimic DEX effects on leukocyte-endothelial interactions and circulating leukocyte phenotype. These data indicate that inhibition of cytokine- and chemokine-induced leukocyte-endothelial interactions may be a primary mechanism by which glucocorticoids inhibit leukocyte recruitment to bacterial agents and thus increase susceptibility to infection.

Animals↗
PubMed1998

alphadbeta2 integrin is expressed on human eosinophils and functions as an alternative ligand for vascular cell adhesion molecule 1 (VCAM-1).

The beta2 family of integrins, CD11a, CD11b, CD11c, and alphad, are expressed on most leukocytes. We show that the newest member of this family, alphad, is expressed on human eosinophils in peripheral blood, and surface expression can be upregulated within minutes by phorbol ester or calcium ionophore A23187. Culture of eosinophils with interleukin 5 (IL-5) leads to a two- to fourfold increase in alphad levels by 3-7 d without a change in alpha4 integrin expression. Eosinophils isolated from late phase bronchoalveolar lavage fluids express alphad at levels similar to that seen after 3 d of IL-5 culture. Regarding alphadbeta2 ligands, in both freshly isolated and IL-5-cultured eosinophils, as well as alphadbeta2-transfected Chinese hamster ovary cells, alphadbeta2 can function as a ligand for vascular cell adhesion molecule 1 (VCAM-1). This conclusion is based on the ability of monoclonal antibodies to alphad, beta2, or VCAM-1 to block cell attachment in static adhesion assays. In experiments with eosinophils, the relative contribution of alphadbeta2 integrin- mediated adhesion is enhanced after IL-5 culture. These experiments demonstrate that alphadbeta2 is an alternative ligand for VCAM-1, and this integrin may play a role in eosinophil adhesion to VCAM-1 in states of chronic inflammation.

Animals↗
PubMed1998

Association of the decreased expression of alpha3beta1 integrin with the altered cell: environmental interactions and enhanced soft agar cloning ability of c-myc-overexpressing small cell lung cancer cells.

Small cell lung cancer (SCLC) is a highly invasive and metastatic tumor, and the decreased expression of alpha3beta1 integrin may contribute to its virulence. Alpha3beta1 is a critical integrin for pulmonary development and epithelial integrity, and its reduced expression has been linked to the increased malignancy and invasion of other cancers. The amplification of the c-myc oncogene is seen frequently in relapsed SCLC tumors and is associated with a worsened prognosis. In the present study using a model of SCLC tumor progression, overexpression of c-myc in a classic SCLC cell line, NCI H209, enhanced in vitro features of tumorigenesis, altered the relationships between cell and environment, and markedly down-regulated the expression of the alpha3 integrin subunit at both the transcript and protein levels. This inverse relationship between the expression of the alpha3 integrin subunit and c-myc is mimicked by other c-myc-overexpressing SCLC cell lines. Restoring alpha3 expression in the myc-transfected 209 cells reversed the effects of c-myc: alpha3 transfection increased cell:cell adhesion and reduced soft agar cloning without affecting the in vitro doubling time. The diminished soft agar cloning produced by alpha3 transfection was reversed by an antibody that specifically engages alpha3beta1 integrins, P1B5. These results suggest first, that alpha3beta1 integrin mediates homotypic adhesion of SCLC cells, and second, that unengaged alpha3beta1 integrin suppresses the growth of disaggregated SCLC cells. Thus, the down-regulation of the alpha3 integrin subunit may contribute to the enhanced tumorigenicity of c-myc-overexpressing SCLCs by allowing the growth of tumor cells that have reduced contact with ligand-expressing substratum or cells, a condition that occurs during the growth of the primary tumor, tumor invasion, and metastasis.

Agar↗
PubMed1998

Rat neutrophils express alpha4 and beta1 integrins and bind to vascular cell adhesion molecule-1 (VCAM-1) and mucosal addressin cell adhesion molecule-1 (MAdCAM-1).

The alpha4 integrins, which are constitutively expressed on all human leukocyte subtypes except neutrophils, interact with vascular cell adhesion molecule-1 (VCAM-1) and mucosal addressin cell adhesion molecule (MAdCAM-1) on endothelium to mediate selective recruitment of leukocyte subpopulations, other than neutrophils, to sites of inflammation. However, here we report that a different paradigm of leukocyte recruitment may exist in the rat. Flow cytometric analysis of rat neutrophils using a panel of monoclonal antibodies which recognize rat alpha4 and beta1 integrins showed consistent, low levels of expression. Although alpha4 was expressed at lower levels on neutrophils than all other rat leukocytes, this level of expression was sufficient to mediate significant levels of alpha4- and beta1-dependent neutrophil adhesion to rat and human VCAM-1, and alpha4-dependent, but beta1-independent, adhesion to human MAdCAM-1. These data suggest that rat neutrophils, unlike other species, may use alpha4 integrins to traffic to sites of inflammation in vivo.

Animals↗
PubMed1998

In vitro regulation of FcepsilonRIalpha expression on human basophils by IgE antibody.

In vivo studies suggested the possibility of an IgE-dependent regulation of high-affinity (FcepsilonRI) IgE receptor expression on basophils. The current studies extend these observations to in vitro cultures of human basophils. Incubation of basophils for 3 to 4 weeks resulted in a slow dissociation of IgE antibody, during which time FcepsilonRI expression decreased, as measured by flow cytometry using the anti-FcepsilonRIalpha monoclonal antibody, 22E7, or by measuring FcRIalpha mass by Western blotting of whole-cell lysates. Culture of basophils with IgE resulted in upregulation of FcepsilonRIalpha expression by both flow cytometry and Western blotting of whole-cell lysates. Upregulation followed a linear time course during 2 weeks of culture. The relative increase in FcepsilonRIalpha density depended on the starting density; with starting densities of FcepsilonRIalpha of 10,000 to 170,000 per basophil, the upregulation varied 20- to 1.1-fold, respectively. Upregulation occurred in high-purity basophils, was not influenced by IgG at concentrations up to 1 mg/mL, and was inhibited by dimeric IgE. Heat-inactivated IgE was less effective and the monoclonal antibody CGP51901 that prevents IgE binding to FcepsilonRIalpha blocked the ability of IgE to induce upregulation. The dose-response curve for IgE-induced upregulation had an effective concentration50 of 230 ng/mL. Although the receptor through which IgE induces this upregulation is not yet known, several characteristics suggest that the upregulation is mediated by IgE interacting through FcepsilonRIalpha itself.

Antibodies, Monoclonal↗
PubMed1998

Beta 1 integrin-dependent binding of Jurkat cells to fibronectin is regulated by a serine-threonine phosphatase.

We investigated the effects of signaling molecule inhibitors on the expression and function of beta1 integrins in Jurkat cells. Jurkat cells expressed alpha4beta1 and alpha5beta1, with significant levels of constitutively activated beta1 integrins as assessed by labeling with mAb 15/7 that distinguishes between activation states. Adhesion to fibronectin (Fn) was mediated equally through alpha4 and alpha5 subunits, and was potentiated by the beta1 integrin activating mAb 8A2. Fn adhesion was decreased by okadaic acid through effects on both alpha4beta1, and alpha5beta1. Tyrphostin A23 also decreased adhesion but was less potent. Neither inhibitor had any effect on the surface expression of total or activated beta1 integrins. The effect of tyrphostin was completely reversed by 8A2; the effect of okadaic acid was only partially reversed. Using Calyculin A, we determined that Jurkat adhesion to Fn was regulated via protein phosphatase 1, independent of the levels of integrins or integrin activation epitopes. Activation of Jurkat cells with a CD3-stimulating mAb enhanced adhesion to Fn and was partially blocked by okadaic acid. These data demonstrate different regulatory pathways for constitutive versus activation-dependent adhesion via beta1 integrins, and implicate both tyrosine kinases and serine-threonine phosphatases in integrin function.

CD3 Complex↗
PubMed1998

Altered expression of CD11b and CD62L after cross-linking of CD45 isoforms on human eosinophils.

BACKGROUND: Phosphotyrosine phosphatase CD45 is exclusively found on nucleated hematopoietic cells. CD45 is an essential component of signaling of mast cel degranulation and of lymphocyte activation, but little is known about its role in eosinophils. In the present study, we have determined the expression and function of CD45 isoforms on human eosinophils. METHODS: Expression of CD45 isoforms on purified peripheral blood eosinophils was examined using indirect immunofluorescence and flow cytometry. Eosinophils were cultured with anti-CD45 isoform monoclonal antibodies (mAbs) for up to 24 h. Eosinophil activation was evaluated by measuring surface expression of CD11b or CD62L by flow cytometry. RESULTS: Fresh eosinophils express CD45, CD45RB, CD45RO epitopes but not CD45RA. Incubation with anti-CD45 isoform mAb for 30 min did not alter the expression of either CD11b or CD62L on eosinophils. In contrast, the expression of CD11b was significantly enhanced after 24 h of incubation with mAbs against CD45, CD45RB, or CD45RO. In addition, the expression of CD62L was also significantly reduced with anti-CD45RB and with anti-CD45RO mAbs. CONCLUSIONS: Cross-linking of surface CD45 isoforms for 24 h significantly induced upregulation of CD11b expression and CD62L shedding, consistent with activation of eosinophils. Our data suggest that CD45 isoforms are functionally expressed on human eosinophils, and are capable of modulating eosinophil function and might participate in allergic inflammation.

Antibodies, Monoclonal↗
PubMed1998

Intranasal beclomethasone reduces allergen-induced symptoms and superficial mucosal eosinophilia without affecting submucosal inflammation.

Previous investigations have suggested that nasal secretions, obtained by lavage or scraping, and the nasal submucosa, sampled by biopsy, are two distinct compartments. We investigated the effect of intranasal corticosteroids on antigen-induced eosinophil influx into both compartments. We performed a double-blind, placebo-controlled study in 15 patients with seasonal allergic rhinitis. Beclomethasone dipropionate, 84 microg twice a day, was delivered to one nostril while the other nostril received placebo for 1 wk. Subjects were then challenged with grass or ragweed extracts on each inferior turbinate. Nasal scrapings from both inferior turbinates were obtained before and 24 h after challenge, and bilateral inferior turbinate biopsies were obtained 24 h after challenge, with the subjects still receiving treatment. Intranasal steroids led to a significant reduction in sneezes and eosinophil influx in nasal secretions without affecting the number of eosinophils in the submucosa. Furthermore, intranasal steroids had no effect on the numbers of submucosal EG2+ (activated eosinophils) or CD25+ (IL-2-receptor-bearing) cells, nor did they decrease the endothelial expression of vascular cell adhesion molecule-1 (VCAM-1). These data show that pretreatment with intranasal steroids successfully inhibited the clinical response to allergen and reduced eosinophils in the superficial compartment of the nasal mucosa, but it had no effect on inflammation in the deeper compartment. This might be related to a different distribution of the active medication and antigen into the nasal mucosa or to a specific effect of the active medication on the epithelium resulting in inhibited migration of eosinophils across this layer.

Administration, Intranasal↗
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↗
PubMed1998

Antagonism of selectin-dependent adhesion of human eosinophils and neutrophils by glycomimetics and oligosaccharide compounds.

Early in inflammation, adhesion occurs between leukocytes and endothelium when selectins bind to sialyl Lewis X (sLex) and related oligosaccharides. We tested novel compounds that mimic sLex for their ability to inhibit selectin-mediated adhesion of human eosinophils and neutrophils in vitro. Neutrophils and eosinophils were isolated by density gradient centrifugation, and eosinophils were further purified by immunomagnetic negative selection. Adhesion to unstimulated or interleukin-1beta-stimulated (5 ng/ml, 4-6 h) umbilical vein endothelial monolayers was tested under static or rotating conditions, where adhesion is primarily E- or L-selectin dependent, respectively. P-selectin-dependent adhesion was tested on immobilized platelets treated with or without phorbol myristate acetate (10(-7) M, 10 min). Stimulus-induced adhesion was always at least 4-fold higher than without stimulus, and selectin dependence was confirmed with specific blocking monoclonal antibodies. E-selectin-dependent adhesion of eosinophils and neutrophils was inhibited by compound GM2296 (the concentration producing 50% inhibition of adhesion [IC50] approximately 0.5-1 mM). E-selectin-dependent adhesion of neutrophils, but not eosinophils, was also inhibited by another compound, sLex with a lipid tail (30 +/- 6% inhibition at 3 mM), whereas compound GM1292 slightly inhibited adhesion of both (23 +/- 5 and 20 +/- 6% inhibition, respectively, at 1 mM). L-selectin-dependent adhesion was more effectively inhibited by GM2296 (IC50 approximately 0.2-0.5 mM), although P-selectin-dependent adhesion was also inhibited (IC50 approximately 1 mM). Inhibition was reversible without affecting viability, and no effect was seen with these compounds in assays testing neutrophil adhesion to immobilized intercellular adhesion molecule-1. Thus, compound GM2296, a carbon-fucosylated derivative of glycyrrhetinic acid, inhibits E-, L-, and P-selectin-dependent eosinophil and neutrophil adhesion. The ability of these and perhaps other related glycomimetic compounds to interfere with the function of more than one type of selectin makes them desirable candidates as anti-inflammatory agents.

Antibodies, Monoclonal↗