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)

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.

Figures

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 (–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.

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.

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.

Figure 4

Cytofluorographic analysis of the effect of rhSCF (100 ng/ml) on c-kit and integrin expression by eosinophils after incubation for 15 min at 37°C. Values on the x axis are the MFI units of each mAb staining. Data are presented as mean ± SD of MFI units from three independent donors. Medium alone (□); medium plus rhSCF (▪).

Figure 5

Effect of rhSCF 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 rhSCF (100 ng/ml, 5 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.

Figure 6

Dose effect of rhSCF on eosinophil adhesion to FN40. Human eosinophils were assayed for adhesion to FN40 (30 μg/ml)–coated 96-well plates in the presence of increasing concentrations of rhSCF. Data are presented as mean ± SD of three independent experiments each performed in triplicate. Single asterisk indicates significant stimulation relative to the unstimulated eosinophils and double asterisk indicates significant stimulation relative to a fourfold lesser concentration.

Figure 7

Comparison of the effects of rhSCF and eotaxin on eosinophil adhesion to FN40. Human eosinophils were assayed for adhesion to FN40-coated 96-well plates for 15 min at 37°C in medium alone (□) or in the presence of rhSCF (100 ng/ml, 5 nM) (▪) or rh eotaxin (24 nM) (▨ ). Data are presented as mean ± SD of three independent experiments each performed in triplicate.

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.

Figure 9

Effects of the combination of rhSCF and rh eotaxin on the adhesion of human eosinophils to FN40. Human eosinophils were assayed for adhesion to FN40 (30 μg/ml)–coated 96-well plates in the presence of increasing concentrations of rh eotaxin, either alone (□) or with a constant dose of rhSCF (25 ng/ml, 1.25 nM) (▪). Data are presented as mean ± SD of three independent experiments, each performed in triplicate.