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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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Immunohistochemical  analysis of c-kit receptor expression  on freshly prepared human peripheral blood buffy coats. Peripheral  blood buffy coats were incubated  with either negative control mAb  P3 (IgG matched) (a), or anti-human c-kit mAb SR-1 (b), and  cytocentrifugation slides were  prepared. After application of secondary antibody-conjugated gold  particles and a silver enhancement  procedure, the slides were counterstained with hematoxylin and eosin,  and analyzed with a Leica microscope. Arrows indicate the eosinophils. Other leukocytes, as shown  in the same field, were negative for  surface c-kit expression. Higher  magnification views of individual  eosinophils are shown in the upper  right corners.
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Figure 2: Immunohistochemical analysis of c-kit receptor expression on freshly prepared human peripheral blood buffy coats. Peripheral blood buffy coats were incubated with either negative control mAb P3 (IgG matched) (a), or anti-human c-kit mAb SR-1 (b), and cytocentrifugation slides were prepared. After application of secondary antibody-conjugated gold particles and a silver enhancement procedure, the slides were counterstained with hematoxylin and eosin, and analyzed with a Leica microscope. Arrows indicate the eosinophils. Other leukocytes, as shown in the same field, were negative for surface c-kit expression. Higher magnification views of individual eosinophils are shown in the upper right corners.

Mentions: To confirm c-kit expression by peripheral blood eosinophils and determine its potential expression by other circulating leukocytes, peripheral blood buffy coats from two separate donors (1 atopic and 1 nonatopic) were incubated with either SR-1 or control P3 antibody, and were subjected to immunohistochemical analysis using secondary antibody-conjugated gold particles and a silver enhancement procedure, followed by counterstaining with hematoxylin and eosin. As indicated by the counterstaining and shown for the nonatopic donor (Fig. 2), 100% of the eosinophils in the buffy coats were positive for c-kit, and eosinophils were the only cells displaying a signal for c-kit receptor. Identical results were obtained for the atopic donor (data not shown). A similar positive signal was also detected on freshly isolated human peripheral blood eosinophils after MACS column purification (data not shown).


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)

Immunohistochemical  analysis of c-kit receptor expression  on freshly prepared human peripheral blood buffy coats. Peripheral  blood buffy coats were incubated  with either negative control mAb  P3 (IgG matched) (a), or anti-human c-kit mAb SR-1 (b), and  cytocentrifugation slides were  prepared. After application of secondary antibody-conjugated gold  particles and a silver enhancement  procedure, the slides were counterstained with hematoxylin and eosin,  and analyzed with a Leica microscope. Arrows indicate the eosinophils. Other leukocytes, as shown  in the same field, were negative for  surface c-kit expression. Higher  magnification views of individual  eosinophils are shown in the upper  right corners.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198988&req=5

Figure 2: Immunohistochemical analysis of c-kit receptor expression on freshly prepared human peripheral blood buffy coats. Peripheral blood buffy coats were incubated with either negative control mAb P3 (IgG matched) (a), or anti-human c-kit mAb SR-1 (b), and cytocentrifugation slides were prepared. After application of secondary antibody-conjugated gold particles and a silver enhancement procedure, the slides were counterstained with hematoxylin and eosin, and analyzed with a Leica microscope. Arrows indicate the eosinophils. Other leukocytes, as shown in the same field, were negative for surface c-kit expression. Higher magnification views of individual eosinophils are shown in the upper right corners.
Mentions: To confirm c-kit expression by peripheral blood eosinophils and determine its potential expression by other circulating leukocytes, peripheral blood buffy coats from two separate donors (1 atopic and 1 nonatopic) were incubated with either SR-1 or control P3 antibody, and were subjected to immunohistochemical analysis using secondary antibody-conjugated gold particles and a silver enhancement procedure, followed by counterstaining with hematoxylin and eosin. As indicated by the counterstaining and shown for the nonatopic donor (Fig. 2), 100% of the eosinophils in the buffy coats were positive for c-kit, and eosinophils were the only cells displaying a signal for c-kit receptor. Identical results were obtained for the atopic donor (data not shown). A similar positive signal was also detected on freshly isolated human peripheral blood eosinophils after MACS column purification (data not shown).

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