Expression of CD35 (CR1) and CD11b (CR3) on circulating neutrophils and eosinophils from allergic asthmatic children

Migration from blood to tissue modulates eosinophil function, possibly through interactions with endothelial cells.The effects of contact with and migration through endothelial cells on eosinophil expression of surface markers and release of leukotriene C 4 were evaluated.A small proportion (2.6%) of eosinophils spontaneously migrated through endothelial cell monolayers. Activation of endothelial cells by interleukin (IL)-4 or IL-1b slightly increased this migration (to 12.4%), which became much greater when a chemoattractant was placed in the lower chamber (84.3%). However, the chemotactic effect was downregulated by pretreating endothelial cells with interferon gamma (IFN-c; 63.1%). At baseline, 5% of eosinophils expressed CD69; this increased to 30.7% in culture on untreated endothelial cells and to 50.9% on IL-1b-pretreated endothelial cells. This effect was mediated through intercellular adhesion molecule-1/CD11b interaction. Eosinophil migration through endothelial cells further increased CD69 expression to 63.9% and also increased CD35 expression from 83.3 to 91.3%. Upon stimulation, eosinophils that had migrated through endothelial cells produced more leukotriene C 4 than control cells (872.4 and 103.9 pg?mL -1 , respectively). Endothelial cell pretreatment with IL-4 or IL-1b further increased leukotriene C 4 release (1,789.1 and 2,895.1 pg?mL -1 , respectively), whereas pretreatment with IFN-c decreased it (293.7 pg?mL -1 ). These data show that in vitro interactions with endothelial cells upregulate eosinophil membrane receptor expression and mediator release and that these effects are differently modulated by T-helper cell type 1 and 2 cytokines. These eosinophil modulations may play an important role in asthma pathogenesis. Eur Respir J 2003; 21: 918-924.

Section: Discussionmentioning

confidence: 99%

Endothelial cells modulate eosinophil surface markers and mediator release

Mj¹,

Ferland²,

Pagé³

et al. 2003

“…Fluorescence gain was standardized with Quick Cell 3 beads (Flow Cytometry Standards Co., San Juan, Puerto Rico). Neutrophils and eosinophils were discriminated by means of depolarized orthogonal light scattering, as described previously [28,31,33]. In samples with low eosinophil numbers, the threshold for depolarized light scatter was increased in order to exclude neutrophils from the 10,000 events that were collected.…”

Section: Flow Cytometrymentioning

confidence: 99%

“…Cells were stimulated and labelled in whole blood, according to a slightly modified procedure described previously [31]. Briefly, ethylenediamine tetra-acetic acid (EDTA)-anticoagulated blood was washed twice with excess PBS at room temperature.…”

Section: Cell Stimulationmentioning

confidence: 99%

Inhibition of PAF-induced expression of CD11b and shedding of L-selectin on human neutrophils and eosinophils by the type IV selective PDE inhibitor, rolipram

Berends¹,

Dijkhuizen²,

Monchy

et al. 1997

We quantitatively determined whether the selective phosphodiesterase (PDE) inhibitor, rolipram, inhibits changes in the adhesion molecules CD11b and L-selectin on platelet-activating factor (PAF)-stimulated human neutrophils and eosinophils in vitro.Incubations were performed in human whole blood obtained from healthy volunteers, to restrict activation by purification procedures and to simulate in vivo conditions, in which different cell types may interact, more closely. Receptor expression was measured after fixation of cells, using monoclonal antibodies and flow cytometry.Concentration-dependent inhibition of the PAF-induced CD11b expression and L-selectin shedding for neutrophils and eosinophils was observed with rolipram, dibutyryl cyclic adenosine monophosphate (cAMP), prostaglandin E 2 (PGE 2 ), and isoproterenol. However, these inhibitions did not exceed 50%. Preincubation with rolipram (10 -8 M) and subsequent incubation with isoproterenol (0.5×10 -8 M) or PGE 2 (10 -8 M) induced a cumulative, but not synergistic, effect. Using the combination of rolipram with isoproterenol or PGE 2 , inhibition of PAF-induced Lselectin shedding from eosinophils was as high as 71±28 and 67±21%, respectively. Other inhibitions were below 50%.In conclusion, rolipram inhibits CD11b expression and L-selectin shedding of platelet-activating factor-stimulated neutrophils and eosinophils in whole blood in a concentration-dependent fashion. Inhibitions did not exceed 50%, even at high concentrations. The inhibition of platelet-activating factor induced shedding of Lselectin from eosinophils with a combination of rolipram and prostaglandin E 2 or isoproterenol, however, was found to be approximately 70%. Inhibition of rolling adhesion of eosinophils may, therefore, be a mode of action of type IV phosphodiesterase inhibitors.

“…Percentages of hypodense neutrophils and eosinophils were calculated as described previously [10]. In addition, the effect of theophylline and/or isoproterenol on the PAF-induced modulation of CD11b and L-selectin expression on eosinophils and neutrophils in whole-blood samples was determined using flow cytometric analysis [23]. …”

Section: Methodsmentioning

confidence: 99%

Effect of theophylline on CD11b and L-selectin expression and density of eosinophils and neutrophils in vitro

Spoelstra¹,

Berends²,

Dijkhuizen³

et al. 1998

The nonspecific phosphodiesterase inhibitor theophylline, widely used in asthma therapy, may cause a decrease in inflammatory responses of airways. In asthma, eosinophils migrate to the airway wall and become activated. Activated eosinophils are characterized by low cell density, as well as increased expression of CD11b and reduced expression of L-selectin, two adhesion molecules involved in transendothelial migration. To study the anti-inflammatory effect of theophylline on granulocyte adhesion molecules in vitro, the platelet-activating factor (PAF)-induced density shift was determined by density centrifugation and the modulation of CD11b and L-selectin expression by flow cytometry on eosinophils and neutrophils in human whole blood. A relatively high concentration of theophylline (10(-3) M) inhibited the increase in the percentage of hypodense eosinophils and neutrophils in whole-blood samples after PAF stimulation in vitro. A more pharmacological concentration (10(-4) M) inhibited the CD11b upregulation and L-selectin shedding induced by PAF (10(-7) M) on both eosinophils and neutrophils. The effect of isoproterenol on the inhibitory effect of theophylline was mainly additive, but a small synergistic effect could not be excluded. In conclusion theophylline can attenuate eosinophil and neutrophil activation in vitro at the level of adhesion molecule expression and changes in cell density. This may have implications for transendothelial migration of these cells in asthma.