Methods for Analysis of Matrix Metalloproteinase Regulation of Neutrophil-Endothelial Cell Adhesion

Abstract

Recent evidence indicates novel role for matrix metalloproteinases (MMPs), in particular gelatinase A (MMP-2), in the regulation of vascular biology that are unrelated to their well-known proteolytic breakdown of matrix proteins. We have previously reported that MMP-2 can modulate vascular reactivity by cleavage of the Gly32-Leu33 bound in big endothelin-1 (ET-1) yielding a novel vasoactive peptide ET-1[1-32]. These studies were conducted to investigate whether gelatinolytic MMPs could affect neutrophil-endothelial cell attachment. ET-1[1-32] produced by MMP-2 up-regulated CD11b/CD18 expression on human neutrophils, thereby promoted their adhesion to cultured endothelial cells. ET-1[1-32] evoked release of gelatinase B (MMP-9), which in turn cleaved big ET-1 to yield ET-1[1-32], thus revealing a self-amplifying loop for ET-1[1-32] generation. ET-1[1-32] was rather resistant to cleavage by neutrophil proteases and further metabolism of ET-1[1-32] was not a prerequisite for its biological actions on neutrophils. The neutrophil responses to ET-1[1-32] were mediated via activation of ET(A)receptors through activation of the Ras/Raf-1/MEK/ERK signaling pathway. These results suggest a novel role for gelatinase A and B in the regulation of neutrophil functions and their interactions with endothelial cells. Here we describe the methods in detail as they relate to our previously published work.

Fig. 1

HPLC/MALDI-TOF analysis shows that big ET-1 (ET-1[1-38]) is cleaved to yield many smaller peptides by proteases in the cytosol and cell membrane of neutrophils. Based on their masses, these peptides were assigned the ET-sequences indicated in brackets. (A). Bar: peaks in the region of the HPLC chromatogram corresponding to neutrophil proteins, not ET peptides. Mass analysis on a MALDI TOF instrument reveals that MMP-9 (gelatinase B), cleaves big ET-1 to yield ET-1[1-32] (B).

Fig. 2

ET-1[1-32] induces gelatinase release from human neutrophils. Isolated neutrophils were challenged with ET-1[1-32] for 30 min at 37°C. Gelatinase released into the culture medium was measured as described in Methods and is expressed as percentage of total cellular enzyme activity. N=4, P<0.05 vs unstimulated.

Fig. 3

Effects of ET-1[1-32] on surface expression of adhesion molecules on human neutrophils. Neutrophils were challenged with 100 nM ET-1[1-32] for 30 min at 37°C, then stained with fluorescein-labeled anti-L-selectin and anti-CD11b monoclonal antibodies. C, negative control of immunostaining with irrelevant antibodies. Results are representative of 6 experiments.

Fig. 4

ET-1[1-32] activates the Ras/Raf-1 kinase/MEK/ERK 1/2 signaling pathway. A, Ras was detected by immunoblotting following affinity precipitation of GTP-bound active Ras from lysates of neutrophils. B, Raf-1 kinase activity was determined using a MAPK cascade system following immunoprecipitation of Raf-1. C, MEK and ERK 1/2 phosphorylation was detected by immunoblotting. Neutrophils were challenged with ET-1[1-32] for 2 min (A,B) or 5 min (C). Results are representative of 3 independent experiments.

Fig. 5

ET-1[1-32] enhances adhesion of neutrophils (PMN) to monolayers of human coronary artery endothelial cells (HCAEC). HCAEC were incubated with LPS (1 μg/ml) for 4 h, washed and PMN without or together with ET-1[1-32] (30 nM) were then added for 30 min at 37°C. Values are means ± SEM (n=5).