Biomechanical regulation of human monocyte/macrophage molecular function

Abstract

When the monocyte infiltrates a tissue, adhesion to the extracellular matrix provides structural anchors, and the cell may be deformed through these attachments. To test the hypothesis that human monocytes/macrophages are mechanically responsive, we studied the effects of small cyclic mechanical deformations on cultured human monocytes/macrophages. When monocytes/macrophages were subjected to 4% strain at 1 Hz for 24 hours, neither matrix metalloproteinase (MMP)-1 nor MMP-3 was induced; however, in the presence of phorbol myristate acetate, strain augmented MMP-1 expression by 5.1 +/- 0.7-fold (P < 0.05) and MMP-3 expression by 1. 6 +/- 0.1-fold (P < 0.05). In contrast, MMP-9 expression was not changed by mechanical strain in the presence or absence of phorbol myristate acetate. Deformation rapidly induced the immediate early response genes c-fos and c-jun. In addition, mechanical deformation induced the transcription factor PU.1, an ets family member that is essential in monocyte differentiation, as well as mRNA for the M-CSF receptor. These studies demonstrate that human monocytes/macrophages respond to mechanical deformation with selective augmentation of MMPs, induction of immediate early genes, and induction of the M-CSF receptor. In addition to enhancing the proteolytic activity of macrophages within repairing tissues, cellular deformation within tissues may play a role in monocyte differentiation.

Figure 1.

Strain regulates MMP-1 expression by human monocytes/macrophages. Monocytes/macrophages were cultured on fibronectin-coated membranes for 4 to 5 days, fresh medium was exchanged, and cells were subjected to 4% mechanical strain at 1 Hz for 24 hours. Total RNA was isolated and analyzed by Northern blotting for MMP-1, MMP-3, and TIMP-1.

Figure 2.

A: Amplitude dependence of monocyte/macrophage augmentation of PMA-induced MMP-1. Human monocytes/macrophages cultured on fibronectin-coated membranes were subjected to 0%, 1%, 4%, and 9% strain for 24 hours. Deformation of 4% and above augmented MMP-1 expression. B: Time course of monocyte/macrophage augmentation of PMA-induced MMP-1 mRNA. All cells were treated with PMA in this experiment. Cyclohexamide blocked induction of MMP-1 mRNA.

Figure 3.

Strain promotes MMP-1 expression. Human monocytes/macrophages were cultured on fibronectin-coated membranes. After culturing 4 to 5 days, medium was exchanged and cells were subjected to 4% mechanical strain at 1 Hz for 24 hours. Media were analyzed with anti-human MMP-1 polyclonal antibody. Strain augments MMP-1 secretion by monocytes/macrophages.

Figure 4.

Strain regulates immediate early gene expression. Human monocytes/macrophages were cultured on fibronectin (2 μg/ml)-coated membranes in RPMI-medium containing 10% human serum for 4 to 5 days. Deformation was applied with 4% strain at 1 Hz. Total RNA was isolated at each time point and analyzed with Northern blotting for c-fos (A) and PU.1 and c-jun (B).

Figure 5.

A: Immediate early gene expression in the presence of PMA. Human monocytes/macrophages were cultured on fibronectin (2 μg/ml)-coated membranes in RPMI medium containing 10% human serum for 4 to 5 days. Deformation was applied with 4% strain at 1 Hz; PMA was added at the time strain was initiated or 2 hours before strain. Total RNA was isolated at each time point and analyzed with Northern blotting for c-fos (left) and c-jun (right). B: Electrophoretic mobility shift assay using oligonucleotides that contain the human MMP-1 AP-1 site; conditions were C (control), P (PMA), S (strain), and PS (strain + PMA). A cold oligonucleotide excess of 5× was used to compete for labeled oligonucleotide in conditions designated by *, and supershift assays with antibodies to AP-1 confirmed specificity of the assay (not shown). Strain itself did not activate the AP-1 site, although it modestly augmented the effects of PMA.

Figure 6.

Strain regulates c-fms expression by human monocytes/macrophages. Monocytes/macrophages were cultured on fibronectin-coated membranes for 4 to 5 days, fresh medium was exchanged, and cells were subjected to 4% mechanical strain at 1 Hz. Total RNA was isolated and analyzed by Northern blotting for c-fms.

Figure 7.

Effect of reactive oxygen species on MMP-1 regulation. Human monocytes/macrophages were cultured on fibronectin-coated membranes for 4 to 5 days. Before applying strain, cells were pretreated with 10 mmol/L of N-acetyl cysteine N-acetyl-l-cysteine for 30 minutes or 100 μmol of hydrogen peroxide (H₂O₂) and subjected to 4% mechanical strain at 1 Hz for 24 hours. Total RNA was isolated and analyzed with Northern blotting. N-acetyl-l-cysteine did not inhibit strain-augmentation of MMP-1.

Figure 8.

Effect of strain on MMP-1 regulation cultured on laminin. Human monocytes/macrophages were cultured on laminin (1 μg/ml)-coated membranes. After 4 to 5 days, medium was exchanged with fresh medium and cells were subjected to 4% mechanical strain at 1 Hz for 24 hours. Total RNA was isolated and analyzed by Northern blotting with a cDNA probe for human MMP-1.