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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 rhSCF on  the adhesion of human eosinophils to FN40, VCAM-1, and  laminin. Freshly isolated, human  peripheral blood eosinophils  were incubated for 15 min at  37°C in 96-well plates coated  with FN40, VCAM-1, or laminin (LN) in the presence (▪) or  absence of (□) rhSCF (100 ng/ ml, 5 nM) and assayed for adhesion. Data are presented as mean  ± SD of three independent experiments for FN40 and LN and  four independent experiments  for VCAM-1, each performed  in triplicate. Asterisks indicate  P <0.05.
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Figure 3: Effect of rhSCF on the adhesion of human eosinophils to FN40, VCAM-1, and laminin. Freshly isolated, human peripheral blood eosinophils were incubated for 15 min at 37°C in 96-well plates coated with FN40, VCAM-1, or laminin (LN) in the presence (▪) or absence of (□) rhSCF (100 ng/ ml, 5 nM) and assayed for adhesion. Data are presented as mean ± SD of three independent experiments for FN40 and LN and four independent experiments for VCAM-1, each performed in triplicate. Asterisks indicate P <0.05.

Mentions: To determine the functional integrity of the expressed c-kit receptor on eosinophils, the ability of rhSCF to augment their adhesion to ligands selective for integrin α4 and α6 was evaluated in a static adhesion assay. The 15-min time course for the adhesion assay was selected because a kinetic study showed that augmented adhesion peaked at 15 min, persisted for 30 min, and decreased to baseline by 45 min (data not shown). Adhesion to FN40 increased at all three inputs of 3, 10, and 30 μg/ml of immobilized FN40. In the absence of rhSCF, the adhesion of FN40 was limited (specific binding of 4.3 ± 3.5% at 30 μg/ml FN40). Concomitant stimulation with rhSCF (5 nM) augmented adhesion to 3, 10, and 30 μg/ml FN40 by 7.7 ± 1.4-fold, 5.3 ± 3.3-fold, and 5.4 ± 0.2-fold, respectively, compared with baseline (Fig. 3). Adhesion to rVCAM-1 also increased in relation to the input of ligand. In the absence of rhSCF, rVCAM-1 supported greater adhesion than FN40 (specific binding of 9.3 ± 9% and 22.5 ± 10% at 0.5 μg/ml and 1.0 μg/ml of rVCAM-1, respectively). Stimulation with rhSCF augmented adhesion to 0.1, 0.5, and 1.0 μg/ml rVCAM-1 by 12.7 ± 9.2-fold, 3.8 ± 2.5-fold, and 1.7 ± 0.6-fold, respectively, compared with baseline (Fig. 3). Higher baseline adhesion mediated by VCAM-1 compared with fibronectin has been previously reported (22). Cell adhesion to laminin also increased in a dose-dependent fashion with respect to ligand input but did not increase further with stimulation by rhSCF (Fig. 3). Treatment with rhSCF did not change the surface expression of integrin α4, α6, β1, α4β7, or c-kit receptor as evaluated by cytofluorographic analysis (Fig. 4), indicating that the increases in adhesion to the α4 ligands were not due to increased receptor expression.


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 rhSCF on  the adhesion of human eosinophils to FN40, VCAM-1, and  laminin. Freshly isolated, human  peripheral blood eosinophils  were incubated for 15 min at  37°C in 96-well plates coated  with FN40, VCAM-1, or laminin (LN) in the presence (▪) or  absence of (□) rhSCF (100 ng/ ml, 5 nM) and assayed for adhesion. Data are presented as mean  ± SD of three independent experiments for FN40 and LN and  four independent experiments  for VCAM-1, each performed  in triplicate. Asterisks indicate  P <0.05.
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Related In: Results  -  Collection

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Figure 3: Effect of rhSCF on the adhesion of human eosinophils to FN40, VCAM-1, and laminin. Freshly isolated, human peripheral blood eosinophils were incubated for 15 min at 37°C in 96-well plates coated with FN40, VCAM-1, or laminin (LN) in the presence (▪) or absence of (□) rhSCF (100 ng/ ml, 5 nM) and assayed for adhesion. Data are presented as mean ± SD of three independent experiments for FN40 and LN and four independent experiments for VCAM-1, each performed in triplicate. Asterisks indicate P <0.05.
Mentions: To determine the functional integrity of the expressed c-kit receptor on eosinophils, the ability of rhSCF to augment their adhesion to ligands selective for integrin α4 and α6 was evaluated in a static adhesion assay. The 15-min time course for the adhesion assay was selected because a kinetic study showed that augmented adhesion peaked at 15 min, persisted for 30 min, and decreased to baseline by 45 min (data not shown). Adhesion to FN40 increased at all three inputs of 3, 10, and 30 μg/ml of immobilized FN40. In the absence of rhSCF, the adhesion of FN40 was limited (specific binding of 4.3 ± 3.5% at 30 μg/ml FN40). Concomitant stimulation with rhSCF (5 nM) augmented adhesion to 3, 10, and 30 μg/ml FN40 by 7.7 ± 1.4-fold, 5.3 ± 3.3-fold, and 5.4 ± 0.2-fold, respectively, compared with baseline (Fig. 3). Adhesion to rVCAM-1 also increased in relation to the input of ligand. In the absence of rhSCF, rVCAM-1 supported greater adhesion than FN40 (specific binding of 9.3 ± 9% and 22.5 ± 10% at 0.5 μg/ml and 1.0 μg/ml of rVCAM-1, respectively). Stimulation with rhSCF augmented adhesion to 0.1, 0.5, and 1.0 μg/ml rVCAM-1 by 12.7 ± 9.2-fold, 3.8 ± 2.5-fold, and 1.7 ± 0.6-fold, respectively, compared with baseline (Fig. 3). Higher baseline adhesion mediated by VCAM-1 compared with fibronectin has been previously reported (22). Cell adhesion to laminin also increased in a dose-dependent fashion with respect to ligand input but did not increase further with stimulation by rhSCF (Fig. 3). Treatment with rhSCF did not change the surface expression of integrin α4, α6, β1, α4β7, or c-kit receptor as evaluated by cytofluorographic analysis (Fig. 4), indicating that the increases in adhesion to the α4 ligands were not due to increased receptor expression.

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