Interaction between integrin alpha9beta1 and vascular cell adhesion molecule-1 (VCAM-1) inhibits neutrophil apoptosis - PubMed (original) (raw)

Interaction between integrin alpha9beta1 and vascular cell adhesion molecule-1 (VCAM-1) inhibits neutrophil apoptosis

Ewan A Ross et al. Blood. 2006.

Abstract

According to the prevailing paradigm, neutrophils are short-lived cells that undergo spontaneous apoptosis within 24 hours of their release from the bone marrow. However, neutrophil survival can be significantly prolonged within inflamed tissue by cytokines, inflammatory mediators, and hypoxia. During screening experiments aimed at identifying the effect of the adhesive microenvironment on neutrophil survival, we found that VCAM-1 (CD106) was able to delay both spontaneous and Fas-induced apoptosis. VCAM-1-mediated survival was as efficient as that induced by the cytokine IFN-beta and provided an additive, increased delay in apoptosis when given in combination with IFN-beta. VCAM-1 delivered its antiapoptotic effect through binding the integrin alpha9beta1. The alpha9beta1 signaling pathway shares significant features with the IFN-beta survival signaling pathway, requiring PI3 kinase, NF-kappaB activation, as well as de novo protein synthesis, but the kinetics of NF-kappaB activation by VCAM-1 were slower and more sustained compared with IFN-beta. This study demonstrates a novel functional role for alpha9beta1 in neutrophil biology and suggests that adhesive signaling pathways provide an important extrinsic checkpoint for the resolution of inflammatory responses in tissues.

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Figures

Figure 1

Figure 1. Soluble VCAM-1 delays the functional senescence of neutrophils

(A) Neutrophils were cultured in the presence or absence of soluble adhesion molecules for 18 hours at the concentrations stated (micrograms per milliliter). Apoptosis was measured by DiOC6 staining. Results are expressed as the percentage of apoptotic neutrophils compared with neutrophils cultured in medium alone; mean ± SD of 3 separate experiments. (B) Neutrophils were cultured for 18 hours in the presence or absence of soluble adhesion molecules (VCAM-1, ICAM-1, PECAM-1 all at 10 μg/mL) or IFN-β (1000 U/mL). Apoptosis is evident in medium and ICAM-1– and PECAM-1–treated cells by the presence of small shrunken cells with condensed nuclei. (C) Neutrophils were cultured in the presence or absence of 10 μg/mL sVCAM-1, 1000 U/mL IFN-β, or both together and apoptosis measured by DiOC6 staining. Each point is a single experiment. Bar is the mean; ***P < .001. (D) Neutrophils were incubated with VCAM-1 in the presence or absence of the anti-VCAM antibody 1G11 (10 μg/mL). Cells were cultured alone (□) or in the presence of VCAM-1 (■). An isotype-matched control antibody is shown. Apoptosis was measured by DiOC6 staining. Data are the mean ± SD of 3 separate experiments. (E) VCAM-1 delays the functional senescence of neutrophils. Superoxide generation in response to PMA was determined by measuring oxidation of cytochrome c at the time of neutrophil isolation (t = 0) or following 18 hours of incubation with medium alone, 1000 U/mL IFN-β, 10 μg/mL sVCAM-1, or both together. Data are the mean ± SD of 3 separate experiments.

Figure 2

Figure 2. VCAM-1 inhibits FAS-induced neutrophil apoptosis

(A) Neutrophils were incubated with 10 μg/mL sVCAM-1, 1000 U/mL IFN-β, or both together in the absence (□) or presence (■) of 50 ng/mL CD95 antagonist antibody CH-11 for 6 hours and apoptosis measured by DiOC6 staining. Data are the mean ± SD of 3 separate experiments. (B) Caspase 3 activation was measured in freshly isolated neutrophils (T = 0) or neutrophils incubated for 6 hours in medium alone (□) or presence of 1000 U/mL IFN-β (■), 10 μg/mL soluble VCAM-1 (■), or both together (■) with or without 50 ng/mL of the CD95 activating antibody CH-11. Data are the mean ± SD of 3 separate experiments.

Figure 3

Figure 3. VCAM-1 inhibits neutrophil but not lymphocyte apoptosis

(A) CD31, ICAM-1, and VCAM-1 were immobilized at the concentrations shown (micrograms per milliliter) and neutrophils added for 18 hours before apoptosis was measured by DiOC6 staining. Results are expressed as the percentage of apoptotic neutrophils compared with neutrophils cultured in medium alone; mean ± SD of 3 separate experiments. (B) VCAM-1 was immobilized in a 96-well plate, and neutrophils were added in the absence (□) or presence (■) of 1000 U/mL IFN-β. After 18 hours, apoptosis was measured by DiOC6 staining. (C) Cells from a CD4 T-cell line were incubated in the presence or absence of the survival factors IL-2 (50 U/mL), IFN-β (1000 U/mL), or sVCAM-1 (10 μg/mL). Apoptosis was measured by DiOC6 staining. Data in panels B-C are from a single experiment that is representative of 3 performed.

Figure 4

Figure 4. The integrin α9β1 is the VCAM-1–binding receptor mediating neutrophil survival

Potential VCAM-1–binding integrins expressed on neutrophils (A) or on CD4 T cells (B) were quantified using flow cytometry. (C) VCAM-1–coated beads were cross-linked to the cell surface of neutrophils, and beads were washed and run on a reducing PAGE gel as described. Control human IgG-coated beads (for bead conjugation, see “Immunoblots and Western blotting” under “Materials and methods”) were used to show nonspecific binding of surface molecules to the beads after cross-linking. Proteins were detected by Western blot with anti-α9 and anti-β1–specific antibodies. The blots shown are representative of 3 separate experiments performed. Neutrophils were incubated for 18 hours (D) in the presence or absence of the anti-α9β1 antibody Y9A2 (10 μg/mL). Cells were cultured alone (□) or in the presence of VCAM-1 (■). An isotype-control antibody is shown. Apoptosis was measured by DiOC6 staining. Results are expressed as the percentage of apoptotic neutrophils compared with neutrophils cultured in medium alone with no VCAM-1; mean ± SD of 3 separate experiments.

Figure 5

Figure 5. VCAM-1–mediated rescue shares similar signaling pathways to IFN-β and activates NF-κB

Neutrophils were cultured for 18 hours in the absence (□) or presence of 20 μM PI3K inhibitor LY294002 (■) with or without 1000 U/mL IFN-β, 10 μg/mL sVCAM-1, or both together (A); in the absence (□) or presence of 100 μg/mL NF-κB inhibitor SN50 (■) with or without 1000 U/mL IFN-β, 10 μg/mL sVCAM-1, or both together (B); and in the absence (□) or presence of 1 μg/mL cycloheximide (■) with or without 1000 U/mL IFN-β, 10 μg/mL sVCAM-1, or both together (C). Apoptosis was measured by DiOC6 staining; mean ± SD of 3 separate experiments. (D) NF-κB activation was measured and (E) quantified (see “Materials and methods”) following the exposure of neutrophils to medium alone, 1000 U/mL IFN-β, 10 μg/mL VCAM-1, or both together after 60, 300, and 1800 seconds.

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