Metalloproteinases TACE and MMP-9 Differentially Regulate Death Factors on Adult and Neonatal Monocytes After Infection with Escherichia coli

Abstract

Background: Cleaving ligands and receptors of the tumor necrosis factor (TNF) superfamily can critically regulate the induction of apoptosis. Matrix metalloproteinases (MMPs) such as MMP-9 and tumor necrosis factor-α-converting enzyme (TACE) have been shown to cleave CD95-Ligand (CD95L) and TNF/(TNF receptor-1) TNFR1 which induce phagocytosis induced cell death (PICD) in adult monocytes. This process is reduced in neonatal monocytes. Methods: Here we tested in vitro, whether Escherichia coli infection mounts for activation of MMP-9 and TACE in monocytes and whether this process regulates PICD. Results: The surface expression of TACE was most prominent on infected adult monocytes. In contrast, surface presentation of MMP-9 was highest on infected neonatal monocytes. Selective blocking of MMP-9 decreased CD95L secretion, while inhibition of TACE left CD95L secretion unaltered. Blocking of MMP-9 increased surface CD95L (memCD95L) expression on infected neonatal monocytes to levels comparable to infected adult monocytes. Moreover, MMP-9 inhibition raised PICD of infected neonatal monocytes to levels observed for infected adult monocytes. In contrast, TACE inhibition decreased PICD in infected monocytes. Addition of extracellular TNF effectively induced memCD95L presentation and PICD of adult monocytes and less of neonatal monocytes. Conclusion: MMP-9 activity is crucial for downregulating cell-contact dependent PICD in E. coli infected neonatal monocytes. By this mechanism, MMP-9 could contribute to reducing sustained inflammation in neonates.

Keywords: apoptosis; inflammation; matrix-metalloproteinase; monocytes; phagocytosis.

Figure 1

TACE and MMP-9 expression is induced four hours p.i. by infection with E. coli. TACE expressing monocytes were assessed before and after infection with E. coli. (A) Histogram plots to the right compare isotype controls (filled) and anti-TACE stained PBMO which were non-infected or E. coli infected. Density plots (below) detail the distribution of E. coli infected PBMO regarding TACE surface expression (compare to the non-infected PBMO to the left). E. coli MMP-9 expressing monocytes were assessed before and after infection with E. coli. (B) (n = 5, *** p < 0.001, forked bars represent Student’s t-test, blunt-ended bars represent ANOVA). Blocking of TACE activity increases its substrate CD62L. (C) Monocytes were infected with GFP-E. coli for 4 h with or without indicated inhibitors and surface expression of CD62L was determined. (A–C); (n = 5, ** p < 0.01, *** p < 0.001, forked bars represent Student’s t-test, blunt-ended bars represent ANOVA).

Figure 2

Blockage of metalloproteinases modulated expression of memCD95L and memTNF. PBMO and CBMO were infected and/or treated with the indicated metalloproteinase inhibitors. Expression of memCD95L (A) and memTNF (B) were assessed on CD14-positive gated monocytes. The same groups were analyzed for secreted (s) CD95 (C) and TNF (D) 24 h p.i. via ELISA (n = 5, * p < 0.05, ** p < 0.01, *** p < 0.001, forked bars represent Student’s t-test, blunt-ended bars represent ANOVA).

Figure 3

Blockage of MMP-9 increased PICD of CBMO. PBMO and CBMO were infected and/or treated with the indicated metalloproteinase inhibitors. Apoptosis was determined by Nicoletti assay 24 h p.i. The right panel gives the ratio of the values highlighted in dark-grey and grey, respectively (n = 5, * p < 0.05, ** p < 0.01, *** p < 0.001, forked bars represent Student’s t-test, blunt-ended bars represent ANOVA).

Figure 4

TNF triggers expression of CD95L. PBMO and CBMO were stimulated with TNF and/or infected with E. coli for four hours and analyzed for CD95L expression. Some groups were pre-treated with etanercept. The number of PBMO and CBMO expressing memCD95L were determined as the percentage (A) and the mean concentration given as the mean intensity (MFI), (B); for A and B, n = 5, * p < 0.05, ** p < 0.01, *** p < 0.001, forked bars represent Student’s t-test, blunt-ended bars represent ANOVA.

Figure 5

Monocyte PICD is enhanced by cell–cell contact. Monocytes were infected with GFP-E. coli for one hour, washed and re-incubated for another 24 h in transwell chambers (experimental setup sketch); non-infected cells served as controls. Monocytes from the same donor were co-cultivated in compartments, separated by teflon membranes. Apoptosis was detected by hypodiploid DNA-content (A, n = 6 for PBMO and n = 4 for CBMO; * p < 0.05). PBMO and CBMO were sub-gated (B) in phagocyting/binding (GFP⁺) and non-phagocyting (GFP⁻) monocytes and analyzed for memCD95L expression. Subgated PBMO and CBMO were further analyzed for TACE (C, left panel) and MMP9 (C, right panel) expression. (D) The expression of memCD95L and memTNF of monocytes in the lower (“trans“) chamber was assessed. The percentage of surface expressing monocytes (panels to the left) and the mean values (MFI, panels to the right) were determined (n = 5, * p < 0.05, ** p < 0.01, *** p < 0.001, forked bars represent Student’s t-test, blunt-ended bars represent ANOVA).