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Human peripheral blood eosinophils express a functional c-kit receptor for stem cell factor that stimulates very late antigen 4 (VLA-4)-mediated cell adhesion to fibronectin and vascular cell adhesion molecule 1 (VCAM-1).

Yuan Q, Austen KF, Friend DS, Heidtman M, Boyce JA - J. Exp. Med. (1997)

Bottom Line: We evaluated mature peripheral blood eosinophils for their expression of the surface tyrosine kinase, c-kit, the receptor for the stromal cell-derived cytokine, stem cell factor (SCF).The uniform and selective expression of c-kit by eosinophils was confirmed by immunohistochemical analysis of peripheral blood buffy coats.Cell adhesion to FN40 was completely inhibited with antibodies against the alpha4 and beta1 integrin subunits, revealing that the SCF/c-kit adhesion effect was mediated by a single integrin heterodimer, very late antigen 4 (VLA-4).

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
We evaluated mature peripheral blood eosinophils for their expression of the surface tyrosine kinase, c-kit, the receptor for the stromal cell-derived cytokine, stem cell factor (SCF). Cytofluorographic analysis revealed that c-kit was expressed on the purified peripheral blood eosinophils from 8 of 8 donors (4 nonatopic and 4 atopic) (mean channel fluorescence intensity 2.0- 3. 6-fold, average 2.8 +/- 0.6-fold, greater than the negative control). The uniform and selective expression of c-kit by eosinophils was confirmed by immunohistochemical analysis of peripheral blood buffy coats. The functional integrity of c-kit was demonstrated by the capacity of 100 ng/ml (5 nM) of recombinant human (rh) SCF to increase eosinophil adhesion to 3, 10, and 30 microg/ml of immobilized FN40, a 40-kD chymotryptic fragment of plasma fibronectin, in 15 min by 7.7 +/- 1.4-, 5.3 +/- 3.3-, and 5.4 +/- 0. 2-fold, respectively, and their adhesion to 0.1, 0.5, and 1.0 microg/ml vascular cell adhesion molecule-1 (VCAM-1), by 12.7 +/- 9. 2-, 3.8 +/- 2.5-, and 1.7 +/- 0.6-fold, respectively. The SCF-stimulated adhesion occurred without concomitant changes in surface integrin expression, thereby indicating an avidity-based mechanism. rhSCF (100 ng/ml, 5 nM) was comparable to rh eotaxin (200 ng/ml, 24 nM) in stimulating adhesion. Cell adhesion to FN40 was completely inhibited with antibodies against the alpha4 and beta1 integrin subunits, revealing that the SCF/c-kit adhesion effect was mediated by a single integrin heterodimer, very late antigen 4 (VLA-4). Thus, SCF represents a newly recognized stromal ligand for the activation of eosinophils for VLA-4-mediated adhesion, which could contribute to the exit of these cells from the blood, their tissue localization, and their prominence in inflammatory lesions.

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Effect of rh eotaxin on the adhesion of human eosinophils to  FN40 (30 μg/ml) in the presence of anti-integrin mAbs and control IgG  mAb. Isolated human eosinophils were incubated with mouse anti–human α4  (A4-PUJ1), anti–human α4β7 (Act-1), anti–human β1 (4B4) or IgG control mAb anti–human CD3 (HIT3a) for 10 min at 4°C before being assayed for adhesion with or without rh eotaxin (24 nM) in 96-well plates  coated with FN40 (30 μg/ml) for 15 min at 37°C. Data are presented as  mean ± SD of three independent experiments, each performed in triplicate.
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Figure 8: Effect of rh eotaxin on the adhesion of human eosinophils to FN40 (30 μg/ml) in the presence of anti-integrin mAbs and control IgG mAb. Isolated human eosinophils were incubated with mouse anti–human α4 (A4-PUJ1), anti–human α4β7 (Act-1), anti–human β1 (4B4) or IgG control mAb anti–human CD3 (HIT3a) for 10 min at 4°C before being assayed for adhesion with or without rh eotaxin (24 nM) in 96-well plates coated with FN40 (30 μg/ml) for 15 min at 37°C. Data are presented as mean ± SD of three independent experiments, each performed in triplicate.

Mentions: Eotaxin, a selective eosinophil chemoattractant (61, 62), belongs to the CC chemokine family. Other members of this family are known to be stimuli for eosinophil adhesion via integrin α4 (63). Preliminary dose-dependence experiments indicated that the effect of 24 nM rh eotaxin was similar to the effect of 5 nM rhSCF for augmenting eosinophil adhesion to 30 μg/ml FN40. Thus, the effects of these concentrations on eosinophil adhesion were compared over three concentrations of FN40 (Fig. 7). Eotaxin at 24 nM increased eosinophil binding to FN40 to the same extent as 100 ng/ml (5 nM) rhSCF at each static input of FN40 ligand. Eotaxin did not change the surface expression of c-kit, α4, α6, β1, and α4β7 by cytofluorographic analysis (data not shown), and the augmented adhesion of these cells to FN40 produced by eotaxin was completely blocked by mAbs against α4 and β1 (Fig. 8).


Human peripheral blood eosinophils express a functional c-kit receptor for stem cell factor that stimulates very late antigen 4 (VLA-4)-mediated cell adhesion to fibronectin and vascular cell adhesion molecule 1 (VCAM-1).

Yuan Q, Austen KF, Friend DS, Heidtman M, Boyce JA - J. Exp. Med. (1997)

Effect of rh eotaxin on the adhesion of human eosinophils to  FN40 (30 μg/ml) in the presence of anti-integrin mAbs and control IgG  mAb. Isolated human eosinophils were incubated with mouse anti–human α4  (A4-PUJ1), anti–human α4β7 (Act-1), anti–human β1 (4B4) or IgG control mAb anti–human CD3 (HIT3a) for 10 min at 4°C before being assayed for adhesion with or without rh eotaxin (24 nM) in 96-well plates  coated with FN40 (30 μg/ml) for 15 min at 37°C. Data are presented as  mean ± SD of three independent experiments, each performed in triplicate.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2198988&req=5

Figure 8: Effect of rh eotaxin on the adhesion of human eosinophils to FN40 (30 μg/ml) in the presence of anti-integrin mAbs and control IgG mAb. Isolated human eosinophils were incubated with mouse anti–human α4 (A4-PUJ1), anti–human α4β7 (Act-1), anti–human β1 (4B4) or IgG control mAb anti–human CD3 (HIT3a) for 10 min at 4°C before being assayed for adhesion with or without rh eotaxin (24 nM) in 96-well plates coated with FN40 (30 μg/ml) for 15 min at 37°C. Data are presented as mean ± SD of three independent experiments, each performed in triplicate.
Mentions: Eotaxin, a selective eosinophil chemoattractant (61, 62), belongs to the CC chemokine family. Other members of this family are known to be stimuli for eosinophil adhesion via integrin α4 (63). Preliminary dose-dependence experiments indicated that the effect of 24 nM rh eotaxin was similar to the effect of 5 nM rhSCF for augmenting eosinophil adhesion to 30 μg/ml FN40. Thus, the effects of these concentrations on eosinophil adhesion were compared over three concentrations of FN40 (Fig. 7). Eotaxin at 24 nM increased eosinophil binding to FN40 to the same extent as 100 ng/ml (5 nM) rhSCF at each static input of FN40 ligand. Eotaxin did not change the surface expression of c-kit, α4, α6, β1, and α4β7 by cytofluorographic analysis (data not shown), and the augmented adhesion of these cells to FN40 produced by eotaxin was completely blocked by mAbs against α4 and β1 (Fig. 8).

Bottom Line: We evaluated mature peripheral blood eosinophils for their expression of the surface tyrosine kinase, c-kit, the receptor for the stromal cell-derived cytokine, stem cell factor (SCF).The uniform and selective expression of c-kit by eosinophils was confirmed by immunohistochemical analysis of peripheral blood buffy coats.Cell adhesion to FN40 was completely inhibited with antibodies against the alpha4 and beta1 integrin subunits, revealing that the SCF/c-kit adhesion effect was mediated by a single integrin heterodimer, very late antigen 4 (VLA-4).

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
We evaluated mature peripheral blood eosinophils for their expression of the surface tyrosine kinase, c-kit, the receptor for the stromal cell-derived cytokine, stem cell factor (SCF). Cytofluorographic analysis revealed that c-kit was expressed on the purified peripheral blood eosinophils from 8 of 8 donors (4 nonatopic and 4 atopic) (mean channel fluorescence intensity 2.0- 3. 6-fold, average 2.8 +/- 0.6-fold, greater than the negative control). The uniform and selective expression of c-kit by eosinophils was confirmed by immunohistochemical analysis of peripheral blood buffy coats. The functional integrity of c-kit was demonstrated by the capacity of 100 ng/ml (5 nM) of recombinant human (rh) SCF to increase eosinophil adhesion to 3, 10, and 30 microg/ml of immobilized FN40, a 40-kD chymotryptic fragment of plasma fibronectin, in 15 min by 7.7 +/- 1.4-, 5.3 +/- 3.3-, and 5.4 +/- 0. 2-fold, respectively, and their adhesion to 0.1, 0.5, and 1.0 microg/ml vascular cell adhesion molecule-1 (VCAM-1), by 12.7 +/- 9. 2-, 3.8 +/- 2.5-, and 1.7 +/- 0.6-fold, respectively. The SCF-stimulated adhesion occurred without concomitant changes in surface integrin expression, thereby indicating an avidity-based mechanism. rhSCF (100 ng/ml, 5 nM) was comparable to rh eotaxin (200 ng/ml, 24 nM) in stimulating adhesion. Cell adhesion to FN40 was completely inhibited with antibodies against the alpha4 and beta1 integrin subunits, revealing that the SCF/c-kit adhesion effect was mediated by a single integrin heterodimer, very late antigen 4 (VLA-4). Thus, SCF represents a newly recognized stromal ligand for the activation of eosinophils for VLA-4-mediated adhesion, which could contribute to the exit of these cells from the blood, their tissue localization, and their prominence in inflammatory lesions.

Show MeSH
Related in: MedlinePlus