Matrix metalloproteinase-7-dependent release of tumor necrosis factor-alpha in a model of herniated disc resorption - PubMed (original) (raw)

Matrix metalloproteinase-7-dependent release of tumor necrosis factor-alpha in a model of herniated disc resorption

H Haro et al. J Clin Invest. 2000 Jan.

Abstract

Herniated disc (HD), one of the major causes of low back pain, is often resolved spontaneously without surgical intervention. Resorption is associated with a marked increase in infiltrating macrophages, and the matrix metalloproteinases (MMP) MMP-3 and MMP-7 have been implicated in this phenomenon. We developed a murine organ culture model in which intact intervertebral discs were cocultured with peritoneal macrophages to investigate the role of MMPs in HD resorption. Using macrophages isolated from MMP-null mice, we report that macrophage-produced MMP-7 was required for proteoglycan degradation, loss of wet weight, and macrophage infiltration of cocultured discs. The inability of MMP-7-deficient macrophages to infiltrate discs could not be attributed to a defect in macrophage migration. MMP-7 was required for the release of the cytokine TNF-alpha from peritoneal macrophages. The generation of soluble TNF-alpha was essential for the induction of MMP-3 in disc cocultures, which in turn is required for the generation of a macrophage chemoattractant and subsequent macrophage infiltration. TNF-alpha release from macrophages was necessary but insufficient for disc resorption, which required macrophage infiltration. We conclude that there is extensive communication between macrophages and chondrocytes in HD resorption and that an essential component of this communication is the requirement for MMPs to release soluble bioactive factors.

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Figures

Figure 1

Figure 1

Role of macrophage MMPs in in vitro disc resorption. Discs from wild-type (/wt) mice were cocultured with macrophages from wild-type (wt/), MMP-3–null (3–/), and MMP-7–null (7–/) mice. Wet weights were determined at day 0 and day 3, and the percent of starting wet weight was calculated for each individual disc. Values presented represent the mean and SD of 3–5 discs/group. *Significant difference from starting wet weight (P < 0.05).

Figure 2

Figure 2

Quantitative analysis of macrophage infiltration in MMP-null organ cultures. Macrophages from wild-type or MMP-null mice were cocultured with wild-type disc tissue. The genotype of the mice donating macrophages/disc tissue is indicated. wt = wild-type; 3– = MMP-3–null, 7– = MMP-7–null tissue. In a representative experiment, whole disc tissue was divided into 3 regions: nucleus pulposus (NP; inner gelatinous region), annulus fibrosus (AF; outer layer), and transition zone (TZ; intermediate layer), and the number of PKH26-labeled red fluorescent macrophages in 3 high-power fields in each region was determined. Data were analyzed with the Kruskal-Wallis test and were presented as mean ± SD. *Statistically significant difference (P < 0.05) compared with wild-type.

Figure 3

Figure 3

Quantitative analysis of macrophage migratory activity. Wild-type (wt), MMP-3–null (3–), and MMP-7–null (7–) macrophages were placed in the top well of a modified Boyden chamber, and the number of macrophages migrating through the filter was counted in 6 random ×400 high-power fields for each well. (a) BSA (200 μg/mL) in DMEM or FBS (10%) in DMEM was placed in the lower chamber. (b) Conditioned medium from intervertebral discs from wild-type mice (disc) or discs cocultured with wild-type macrophages (mφ/disc) was placed in the lower chamber. Data from 2 experiments performed in triplicate were analyzed with the Mann-Whitney test and presented as mean ± SD. *Statistically significant differences (P < 0.05) compared with control medium.

Figure 4

Figure 4

Role of MMP-7 in TNF-α induction of MMP-3. MMP-3 in the conditioned medium from 2-day cocultures of discs from wild-type mice (wt disc) and macrophages from wt (wt mφ) or MMP-7–null (7-mφ) mice was analyzed by Western blotting. (a) Anti-murine TNF-α antibody (α TNF-α, 1 ng/mL), anti-murine IL-1β (α IL-1β, 2 μg/mL) or control Ig G (1 ng/mL) was added to the cultures as indicated. (b) Recombinant murine TNF-α (rTNFα, 1 ng/mL), anti-murine TNF-α antibody (αTNF-α, 1 ng/mL), or recombinant MMP-7 (rMMP-7, 1 ng/mL) was added to the medium. Arrow indicates the migration of recombinant rat MMP-3.

Figure 5

Figure 5

Analysis of soluble and cell-surface TNF-α levels. (a) Forty-eight–hour conditioned medium from cocultures of wild-type discs (/wt) and wild-type (wt/) or MMP-7-null (7–/) macrophages was analyzed for soluble TNF-α levels by ELISA. The 7–/wt cocultures were incubated in the absence or presence of recombinant MMP-7 (rMMP-7, 100 ng/mL) for the duration of the coculture period. (b) Two-hour conditioned medium from wild-type (wt) or MMP-7–null (7–) macrophages either in the absence (to measure soluble TNF-α) or presence (to measure cell-surface TNF-α) of recombinant MMP-7(100 ng/mL). Where indicated, 10 μg/mL cycloheximide (CHX) was added for the duration of the conditioning. Results are mean and SD of duplicate samples from 2 separate experiments.

Figure 6

Figure 6

Requirement for TNF-α and macrophage infiltration for disc resorption. Discs from wild-type (/wt) mice were cocultured with macrophages from wild-type (wt/) and MMP-7–null (7–/) mice. Wet weights were determined at day 0 and day 3, and the percent of starting wet weight was calculated for each individual disc. Where indicated, anti-TNF-α (1 ng/mL), a control immunoglobulin (IgG, 1 ng/mL), or recombinant TNF-α (rTNFα, 1 ng/mL) was added to the cocultures. Values presented represent the mean and SD of 3–5 discs/group. *Significant difference from starting wet weight (P < 0.05).

Figure 7

Figure 7

Model of MMP, macrophage, and chondrocyte interactions in HD resorption. Activated macrophages (small gray circles) produced TNF-α, which requires the production of MMP-7 to become soluble (sTNF-α). Soluble TNF-α induces chondrocytes (larger dark circles) to produce MMP-3. MMP-3 production by disc chondrocytes is required for the release of a macrophage chemoattractant and the subsequent macrophage infiltration of the disc. Degradation of the disc (cleared area) is mediated by the infiltration of the macrophages.

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