The pattern recognition receptor (RAGE) is a counterreceptor for leukocyte integrins: a novel pathway for inflammatory cell recruitment

Abstract

The pattern recognition receptor, RAGE (receptor for advanced glycation endproducts), propagates cellular dysfunction in several inflammatory disorders and diabetes. Here we show that RAGE functions as an endothelial adhesion receptor promoting leukocyte recruitment. In an animal model of thioglycollate-induced acute peritonitis, leukocyte recruitment was significantly impaired in RAGE-deficient mice as opposed to wild-type mice. In diabetic wild-type mice we observed enhanced leukocyte recruitment to the inflamed peritoneum as compared with nondiabetic wild-type mice; this phenomenon was attributed to RAGE as it was abrogated in the presence of soluble RAGE and was absent in diabetic RAGE-deficient mice. In vitro, RAGE-dependent leukocyte adhesion to endothelial cells was mediated by a direct interaction of RAGE with the beta2-integrin Mac-1 and, to a lower extent, with p150,95 but not with LFA-1 or with beta1-integrins. The RAGE-Mac-1 interaction was augmented by the proinflammatory RAGE-ligand, S100-protein. These results were corroborated by analysis of cells transfected with different heterodimeric beta2-integrins, by using RAGE-transfected cells, and by using purified proteins. The RAGE-Mac-1 interaction defines a novel pathway of leukocyte recruitment relevant in inflammatory disorders associated with increased RAGE expression, such as in diabetes, and could provide the basis for the development of novel therapeutic applications.

Figure 1.

The contribution of RAGE to inflammatory reactions in vivo. After thioglycollate injection into the mouse peritoneum to induce acute inflammation, the number of neutrophils in the peritoneal lavage was analyzed after 4 h. (A) Prior to thioglycollate administration, nondiabetic or diabetic mice were treated by intraperitoneal injection with PBS (black bars), with a blocking mAb against ICAM-1 (gray bars), with soluble RAGE (white bars), or a combination of the blocking mAb against ICAM-1 and soluble RAGE (hatched bars). *, P < 0.0001 as compared with control (nondiabetic mice treated with PBS); +, P < 0.0001 as compared with control (diabetic mice treated with PBS); #, P < 0.001; ns, not significant (P = 0.1169). (B) The number of emigrated neutrophils into the peritoneum of nondiabetic (black bars) or diabetic (gray bars) wild-type or RAGE^−/− mice was compared 4 h after thioglycollate injection. *, P < 0.0001; #, P = 0.0059; ns, not significant (P = 0.2656). (C) The number of neutrophils infiltrated into the peritoneum of wild-type mice (black bars), RAGE^−/− mice (white bars), or tie2-RAGE×RAGE^−/− mice (gray bars) was compared 4 h after thioglycollate injection. *, P < 0.0001; ns, not significant (P = 0.6976). (D) After thioglycollate injection into the mouse peritoneum the number of macrophages in the peritoneal lavage of wild-type (wt, black bars) or RAGE ^−/− (white bars) mice was analyzed after 40 and 72 h. *, P < 0.0001. Data are mean ± SD (n = 5 mice per treatment) of typical experiments; similar results were obtained in three separate sets of experiments.

Figure 1.

The contribution of RAGE to inflammatory reactions in vivo. After thioglycollate injection into the mouse peritoneum to induce acute inflammation, the number of neutrophils in the peritoneal lavage was analyzed after 4 h. (A) Prior to thioglycollate administration, nondiabetic or diabetic mice were treated by intraperitoneal injection with PBS (black bars), with a blocking mAb against ICAM-1 (gray bars), with soluble RAGE (white bars), or a combination of the blocking mAb against ICAM-1 and soluble RAGE (hatched bars). *, P < 0.0001 as compared with control (nondiabetic mice treated with PBS); +, P < 0.0001 as compared with control (diabetic mice treated with PBS); #, P < 0.001; ns, not significant (P = 0.1169). (B) The number of emigrated neutrophils into the peritoneum of nondiabetic (black bars) or diabetic (gray bars) wild-type or RAGE^−/− mice was compared 4 h after thioglycollate injection. *, P < 0.0001; #, P = 0.0059; ns, not significant (P = 0.2656). (C) The number of neutrophils infiltrated into the peritoneum of wild-type mice (black bars), RAGE^−/− mice (white bars), or tie2-RAGE×RAGE^−/− mice (gray bars) was compared 4 h after thioglycollate injection. *, P < 0.0001; ns, not significant (P = 0.6976). (D) After thioglycollate injection into the mouse peritoneum the number of macrophages in the peritoneal lavage of wild-type (wt, black bars) or RAGE ^−/− (white bars) mice was analyzed after 40 and 72 h. *, P < 0.0001. Data are mean ± SD (n = 5 mice per treatment) of typical experiments; similar results were obtained in three separate sets of experiments.

Figure 2.

The adhesive properties of RAGE. (A) Adhesion of PMA-stimulated human neutrophils to immobilized RAGE is shown in the absence (-) or presence of EDTA (5 mM), soluble RAGE (10 μg/ml), mAb to β1-integrin, mAb to β2-integrin, mAb to Mac-1, mAb to LFA-1, or mAb to α4-integrin (each antibody at 20 μg/ml). (B) Adhesion of PMA-stimulated human neutrophils to CHO cells (gray bars) or RAGE-transfected CHO cells (black bars) is shown in the absence (-) or presence of mAb to β1-integrin, mAb to β2-integrin, mAb to Mac-1, or mAb to LFA-1 (each antibody at 20 μg/ml), or soluble RAGE (10 μg/ml). Cell adhesion is represented either as absorbance of labeled cells at 590 nm or as percentage of total added cells. All data are mean ± SD (n = 3) of a typical experiment, similar results were obtained in at least three separate experiments.

Figure 3.

RAGE is a counter-receptor for β2-integrins. (A) Adhesion of nontransfected K562 cells, LFA-1-transfected K562 cells, Mac-1-transfected K562 cells, or p150,95-transfected K562 to immobilized RAGE is shown in the absence (gray bars) or presence (black bars) of β2-integrin–stimulating antibody Kim185 (10 μg/ml). (B) Adhesion of nontransfected K562 cells, LFA-1–transfected K562 cells, Mac-1–transfected K562 cells, or p150,95-transfected K562 cells, which were preincubated with Kim185 (10 μg/ml), to nontransfected CHO cells (gray bars) or RAGE-transfected CHO cells (black bars) is shown. (C) Adhesion of Mac-1–transfected K562 cells, which were preincubated with Kim185 (10 μg/ml), to RAGE-transfected CHO cells is shown in the absence (-) or presence of mAb to Mac-1 (20 μg/ml) or soluble RAGE (10 μg/ml). Cell adhesion is represented either as absorbance at 590 nm or as percentage of total added cells. All data are mean ± SD (n = 3) of a typical experiment, similar results were observed in at least three separate experiments.

Figure 4.

Contribution of RAGE to neutrophil adhesion to cultured endothelial cells. PMA-stimulated adhesion of human neutrophils to endothelial cells is shown in the absence (-) or presence of mAb to β2-integrin, mAb to Mac-1, mAb to ICAM-1 (each antibody at 20 μg/ml), soluble RAGE (10 μg/ml), or a combination of soluble RAGE and mAb to ICAM-1. Cell adhesion is represented as percentage of total added cells. All data are mean ± SD (n = 3) of a typical experiment, similar results were observed in at least three separate experiments.

Figure 5.

The interaction between RAGE and Mac-1. (A) The binding of ICAM-1 or soluble RAGE (each 5 μg/ml) to immobilized LFA-1 (gray bars) or Mac-1 (black bars) is shown. (B) Dose-dependent specific binding of soluble RAGE to immobilized Mac-1 is shown. Specific binding is expressed as absorbance at 405 nm. Data are mean ± SD (n = 3) of a typical experiment; similar results were observed in at least three separate experiments.

Figure 6.

Influence of Mac-1 ligands on the interaction between RAGE and Mac-1. (A) The specific binding of soluble RAGE (100 nM) to immobilized Mac-1 is shown in the absence (-) or presence of FBG, HK, ICAM-1, or the isolated I-domain of Mac-1 (each 500 nM). (B) The specific binding of soluble RAGE (100 nM) to immobilized Mac-1 is shown in the absence or presence of increasing concentrations of FBG (black circles) or HK (white squares). (C) The specific binding of FBG (100 nM) to immobilized Mac-1 is shown in the absence or presence of increasing concentrations of soluble RAGE. Specific binding is expressed as absorbance at 405 nm. Data are mean ± SD (n = 3) of a typical experiment; similar results were observed in at least three separate experiments.

Figure 7.

Influence of RAGE ligands on the interaction between RAGE and Mac-1. (A) The specific binding of soluble RAGE (5 μg/ml) to immobilized Mac-1 is shown in the absence or presence of increasing concentrations of S100-B (black circles) or CML (white squares). Specific binding is expressed as absorbance at 405 nm. Data are mean ± SD (n = 3) of a typical experiment; similar results were observed in at least three separate experiments. (B) PMA-stimulated adhesion of U937 cells to immobilized RAGE is presented in the absence (-) or presence of mAb to Mac-1 (20 μg/ml), CML (800 nM), S100-B (800 nM), or the combination of S100-B and mAb to Mac-1. Cell adhesion is represented as percent of control. Data are mean ± SD (n = 3) of a typical experiment; similar results were observed in at least three separate experiments.