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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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Cytofluorographic analysis of c-kit receptor expression on  freshly isolated, human peripheral blood eosinophils. (A) Eosinophils from  donors 1 and 2 were analyzed with three different mouse anti–human c-kit  mAbs, SR-1 (——), YB5.B8 (----), and 95C3 (.....), as well as a control  mouse mAb, P3 (——), (IgG control). (B) Eosinophils from an additional  6 donors (3–8) were analyzed with SR-1 and P3 mAbs. The values expressed on the y axis are values of the MFI units of SR-1 staining divided  by the MFI units of P3 control mAb staining in each donor.
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Figure 1: Cytofluorographic analysis of c-kit receptor expression on freshly isolated, human peripheral blood eosinophils. (A) Eosinophils from donors 1 and 2 were analyzed with three different mouse anti–human c-kit mAbs, SR-1 (——), YB5.B8 (----), and 95C3 (.....), as well as a control mouse mAb, P3 (——), (IgG control). (B) Eosinophils from an additional 6 donors (3–8) were analyzed with SR-1 and P3 mAbs. The values expressed on the y axis are values of the MFI units of SR-1 staining divided by the MFI units of P3 control mAb staining in each donor.

Mentions: Cytofluorographic analyses of surface epitopes expressed by human peripheral blood eosinophils with or without rhSCF stimulation were performed by established procedures (52). Freshly isolated human peripheral blood eosinophils were resuspended in RPMI 1640 medium containing 10% FCS at a concentration of 106 cells/ml and were divided into two identical fractions. rhSCF was added to one fraction to a final concentration of 100 ng/ml (5 nM). Alternatively, rh eotaxin was used at a final concentration of 24 nM and both fractions were incubated for 15 min at 37°C. The cells were harvested and washed once with cold PBS containing 0.5% HSA and 0.02% sodium azide (FACS buffer). Samples of 5 × 105 cells were then incubated for 1 h on ice with primary antibodies (purified mAbs at a final concentration of 10 μg/ml or ascites at a final dilution of 1:500 or P3 culture supernatant at 1:4 dilution). The cells were washed once with FACS buffer and incubated in the dark for 1 h on ice with fluorescein isothiocyanate–conjugated goat anti–mouse IgG (GIBCO BRL) at a final dilution of 1:100. The cells were washed again with the FACS buffer, resuspended in 0.25 ml of PBS, and analyzed on a FACSort® machine (Becton Dickinson, Oxnard, CA). For c-kit expression (Fig. 1), the results are presented as overlaid histograms and the fold increase of mean fluorescence intensity (MFI). The fold increase of c-kit expression was calculated by dividing the MFI units of SR-1 staining by the MFI units of P3 control mAb staining in each donor.


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)

Cytofluorographic analysis of c-kit receptor expression on  freshly isolated, human peripheral blood eosinophils. (A) Eosinophils from  donors 1 and 2 were analyzed with three different mouse anti–human c-kit  mAbs, SR-1 (——), YB5.B8 (----), and 95C3 (.....), as well as a control  mouse mAb, P3 (——), (IgG control). (B) Eosinophils from an additional  6 donors (3–8) were analyzed with SR-1 and P3 mAbs. The values expressed on the y axis are values of the MFI units of SR-1 staining divided  by the MFI units of P3 control mAb staining in each donor.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2198988&req=5

Figure 1: Cytofluorographic analysis of c-kit receptor expression on freshly isolated, human peripheral blood eosinophils. (A) Eosinophils from donors 1 and 2 were analyzed with three different mouse anti–human c-kit mAbs, SR-1 (——), YB5.B8 (----), and 95C3 (.....), as well as a control mouse mAb, P3 (——), (IgG control). (B) Eosinophils from an additional 6 donors (3–8) were analyzed with SR-1 and P3 mAbs. The values expressed on the y axis are values of the MFI units of SR-1 staining divided by the MFI units of P3 control mAb staining in each donor.
Mentions: Cytofluorographic analyses of surface epitopes expressed by human peripheral blood eosinophils with or without rhSCF stimulation were performed by established procedures (52). Freshly isolated human peripheral blood eosinophils were resuspended in RPMI 1640 medium containing 10% FCS at a concentration of 106 cells/ml and were divided into two identical fractions. rhSCF was added to one fraction to a final concentration of 100 ng/ml (5 nM). Alternatively, rh eotaxin was used at a final concentration of 24 nM and both fractions were incubated for 15 min at 37°C. The cells were harvested and washed once with cold PBS containing 0.5% HSA and 0.02% sodium azide (FACS buffer). Samples of 5 × 105 cells were then incubated for 1 h on ice with primary antibodies (purified mAbs at a final concentration of 10 μg/ml or ascites at a final dilution of 1:500 or P3 culture supernatant at 1:4 dilution). The cells were washed once with FACS buffer and incubated in the dark for 1 h on ice with fluorescein isothiocyanate–conjugated goat anti–mouse IgG (GIBCO BRL) at a final dilution of 1:100. The cells were washed again with the FACS buffer, resuspended in 0.25 ml of PBS, and analyzed on a FACSort® machine (Becton Dickinson, Oxnard, CA). For c-kit expression (Fig. 1), the results are presented as overlaid histograms and the fold increase of mean fluorescence intensity (MFI). The fold increase of c-kit expression was calculated by dividing the MFI units of SR-1 staining by the MFI units of P3 control mAb staining in each donor.

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