αVβ3 integrin regulates macrophage inflammatory responses via PI3 kinase/Akt-dependent NF-κB activation

Abstract

Controlling macrophage responses to pathogenic stimuli is critical for prevention of and recovery from the inflammatory state associated with the pathogenesis of many diseases. The adhesion receptor αVβ3 integrin is thought to be an important receptor that regulates macrophage differentiation and macrophage responses to external signaling, but it has not been previously identified as a contributor to macrophage-related inflammation. Using an in vitro model of human blood monocytes (Mo) and monocyte-derived macrophages (MDMs) we demonstrate that αVβ3 ligation results in sustained increases of the transcription factor NF-κB DNA-binding activity, as compared with control isotype-matched IgG(1). Activation of NF-κB parallels the increase of NF-κB-dependent pro-inflammatory cytokine mRNA expression in MDMs isolated from individual donors, for example, TNF-α (8- to 28-fold), IL-1β (15- to 30-fold), IL-6 (2- to 4-fold), and IL-8 (5- to 15-fold) whereas there is more than a 10-fold decrease in IL-10 mRNA level occurs. Upon ligation of the αVβ3 receptor, treatment with TNF-α (10 ng/ml) or LPS (200 ng/ml, 1,000 EU) results in the enhanced and synergistic activation of NF-κB and LPS-induced TNF-α secretion. As additional controls, an inhibitor of αVβ3 integrin, cyclic RGD (10 µg/ml; IC(50) = 7.6 µM), attenuates the effects of αVβ3 ligation, and the natural ligand of αVβ3 integrin, vitronectin, reproduces the effects of αVβ3 activation by an immobilizing anti-αVβ3 integrin mAb. We hypothesize that αVβ3 activation can maintain chronic inflammatory processes in pathological conditions and that the loss of αVβ3 ligation will allow macrophages to escape from the inflammatory state.

Fig. 1

Ligation of αVβ3 integrin on MDMs results in NF-κB activation and increases in pro-inflammatory cytokines mRNA expression and enhanced TNF-α secretion. MDMs differentiated in vitro for 5–7 days in the presence of M-CSF were re-seeded on immobilized anti-αVβ3 integrin mAb, isotype-matched control IgG₁, or vitronectin (Vn) for the indicated time intervals. A: NF-κB DNA-binding activity was analyzed by EMSA in nuclear extracts as described in the Methods Section. Lane 2 shows basal NF-κB activity in MDMs prior to re-seeding; lanes 1 and 9 indicate free and excess unlabeled probes, respectively. B: In parallel with part A, TNF-α secretion by MDMs was measured by ELISA (n = 4; *P < 0.01 vs. IgG₁, t-test). C: MDMs from three donors were re-seeded on immobilized anti-αVβ3 mAb or IgG₁ for 24 h, mRNA amounts were analyzed by RT-PCR as described in the Methods Section. D: MDMs were re-seeded on immobilized anti-αVβ3 mAb, IgG₁, or vitronectin (Vn) in the presence or absence of cRGD (10 μg/ml) for 24 h and TNF-α secretion into medium was determined using the WEHI164 fibrosarcoma cell bioassay as described in the Methods Section (n=6; *P < 0.03, ^###P < 0.001 vs. IgG₁, ANOVA: Kruskal–Wallis test with Dunn’s post-test).

Fig. 2

Ligation of αVβ3 integrin on MDMs results in enhanced responsiveness to LPS and TNF-α stimulation. A: MDMs were re-seeded on the anti-αVβ3 integrin immobilized mAb or control IgG₁ for 24 h, and MDMs were exposed to LPS (200 ng/ml, 1,000 EU) or TNF-α (10 ng/ml) for 1–24 h. NF-κB-binding activity was analyzed using EMSA as described in the Methods Section. Lanes 2 and 3 show basal NF-κB activity prior to stimulation. Lanes 1 and 14 represent free and excess unlabeled probes, respectively. B: MDMs prepared as in A were stimulated with LPS (200 ng/ml, 1,000 EU) and TNF-α secretion into medium was measured by ELISA (n=4; *P < 0.0 vs. IgG₁, t-test).

Fig. 3

Ligation of αVβ3 integrin on MDMs results in a sustained hyperresponsiveness to LPS stimulation. A: MDMs were re-seeded on anti-αVβ3 integrin immobilized mAbs or IgG₁ and were cultured for an additional 4 days after re-seeding and then stimulated with LPS (200 ng/ml, 1,000 EU) for 5 h (lanes 3 and 4).Lanes 1 and 2 show basal NF-κB activity prior to stimulation. B:MDMs,re-seeded on the anti-αVβ3 integrin mAb or the control IgG₁, were stimulated with LPS (200 ng/ml) for 5 h on different days after re-seeding, and secretion of TNF-α was measured using ELISA (n=4, *P < 0.01 vs. IgG₁, t-test).

Fig. 4

NF-κB activation induced by αVβ3 integrin ligation and enhanced responsiveness to LPS stimulation is PI3 kinase/Akt-dependent and p38 MAPK-independent. A: MDMs were re-seeded on the immobilized anti-αVβ3 integrin mAb or control IgG₁, and equal amounts of whole cell extracts were analyzed by Western blot 24 h after re-seeding using phosphorylation-specific antibodies. B: MDMs were re-seeded on the immobilized anti-αVβ3 integrin mAb or control IgG₁ in the presence of the specific inhibitor of PI3 kinase, LY294002 (10 μM) (lanes 3 and 4), inhibitor of p38 MAPK, SB203580 (20 μM) (lanes 5 and 6), or vehicle (DMSO) (lanes 1 and 2). NF-κB-binding activity was examined 24 h after re-seeding using EMSA as described in the Methods Section. C: MDMs were re-seeded on the anti-αVβ3 integrin or the IgG₁ mAbs for 24 h in the presence of LY294002 (10 μM) (lanes 5 and 6), SB203580 (20 μM) (lanes 7 and 8), or vehicle (lanes 3 and 4), then stimulated with LPS (200 ng/ml, 1,000 EU)for 5 h NF-κB binding was measured using EMSA. Lanes 1 and 2 show basal NF-κB activity prior to LPS stimulation. Lane 9 shows an excess of unlabeled probe.

Fig. 5

αVβ3 integrin ligation on blood Mo results in their differentiation into sustained pro-inflammatory phenotype. A: Mo were seeded directly on the immobilized anti-αVβ3 integrin mAb or the control IgG₁ and were differentiated in vitro for 1 day (left part) or 5 days (right part). Basal (lanes 1 and 2) and LPS-induced (200 ng/ml, 1,000 EU for 5 h) NF-κB activities (lanes 3 and 4) were measured using EMSA. B: In parallel with part A experiments, Mo were seeded on immobilized mAbs or IgG₁; LPS-induced (200 ng/ml for 5 h) TNF-α secretion by Mo was assessed at different stages of their maturation by ELISA (n=4; *P < 0.01 vs. control IgG₁, t-test).

Fig. 6

Hypothetical model of the regulation of macrophage-related inflammation by αVβ3 integrin ligation. Macrophages as a part of the host defense system respond to external pro-inflammatory stimuli (e.g., TNF-α, LPS) by transient NF-κB activation which leads to physiologically relevant and transient inflammatory responses (blue arrows). The feedback mechanisms of NF-κB deactivation prevent progression of macrophage-related inflammation into a pathological state. We hypothesize that if αVβ3 integrin is ligated (red arrows), under the same conditions this ligation results in sustained activation of NF-κB and the transition of macrophages into a pro-inflammatory phenotype. Moreover, the increased release of pro-inflammatory mediators (dotted arrows) potentially may lead to formation of a pathogenic loop maintaining chronic inflammation in the lesions. We speculate that loss of αVβ3 ligation allows macrophages to escape from the sustained pro-inflammatory phenotype (green arrows). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com]