Interleukin-4 suppression of interleukin-1-induced transcription of collagenase (MMP-1) and stromelysin 1 (MMP-3) in human synovial fibroblasts

Abstract

Objective: To determine the effects of interleukin-4 (IL-4) on IL-1 induction of collagenase (matrix metalloproteinase 1 [MMP-1]) and stromelysin-1 (MMP-3) in human synovial fibroblasts.

Methods: Northern blot analysis was performed to determine the effects of IL-4 on IL-1 induction of MMP messenger RNA (mRNA). MMP protein levels were determined by enzyme-linked immunosorbent assay, and prostaglandin E2 (PGE2) levels were measured by enzyme immunoassay. Run-on transcription assays and transient transfection experiments were performed to determine whether the effects of IL-4 occur at the level of transcription. Activator protein 1 (AP-1) binding was assessed by electrophoretic mobility shift assay.

Results: Northern blot analysis revealed that coincubation of synovial fibroblasts with IL-1 and IL-4 resulted in a significant decrease in both collagenase and stromelysin mRNA levels compared with IL-1 alone, with a concomitant decrease in MMP protein levels. This inhibition is dose dependent, with an IC50 (50% inhibition concentration) for both MMPs of approximately 0.3 ng of IL-4 per ml, and is at least somewhat selective, since IL-1 induction of c-fos mRNA is not affected. Nuclear run-on experiments and transient transfection studies demonstrated that the suppression of IL-1-induced collagenase and stromelysin expression by IL-4 occurs at least in part at the transcriptional level, and that binding of transcription factor AP-1 is not affected. Although IL-1-induced levels of PGE2 are reduced by IL-4, exogenous addition of PGE2 does not abrogate the inhibitory effects of IL-4 on MMP expression.

Conclusion: IL-4 inhibits IL-1 induction of both collagenase and stromelysin, as well as PGE2 production, in human synovial fibroblasts. The inhibition occurs at least in part at the level of transcription, does not affect binding of transcription factor AP-1, and appears to involve a mechanism that is independent of the ability of IL-4 to inhibit production of PGE2.

Figure 1

Interleukin-4 (IL-4) inhibits IL-1 induction of collagenase (Coll) and stromelysin (Stro) mRNA in human synovial fibroblasts (HSF). Total RNA was isolated at the indicated time points after addition of 100 ng of IL-1/ml alone or in combination with 10 ng of IL-4/ml to cultures of human synovial fibroblasts. A, Northern blots were hybridized to cDNA probes corresponding to collagenase (matrix metalloproteinase 1 [MMP-1]), stromelysin 1 (MMP-3), and GAPDH. B, Blots were quantitated by scanning densitometry and normalized to levels of GAPDH. Shown are data from 3 independent experiments with RNA isolated from HSF.

Figure 2

Interleukin-4 (IL-4) inhibits IL-1 induction of collagenase and stromelysin mRNA in rheumatoid arthritis synovial fibroblasts. Total RNA was isolated 6 hours after addition of 100 ng of IL-1/ml alone or in combination with 10 ng of IL-4/ml to a single culture of rheumatoid arthritis synovial fibroblasts. A Northern blot was hybridized and quantitated as described in Figure 1.

Figure 3

Interleukin-4 (IL-4) inhibits the IL-1 induction of collagenase and stromelysin proteins. Conditioned medium was harvested from human synovial fibroblast (HSF) cultures incubated for 24 hours with 100 ng of IL-1β/ml alone or in the presence of 10 ng of IL-4/ml. Levels of collagenase (A) and stromelysin (B) were measured in triplicate by enzyme-linked immunosorbent assay. For stromelysin, HSF cultures derived from 2 different individuals were used.

Figure 4

Interleukin-l (IL-I) induction of c-fos mRNA is not affected by IL-4. The same Northern blots described in Figure 1 were rehybridized with a cDNA probe corresponding to c-fos. Shown are data from 3 independent experiments.

Figure 5

Interleukin-4 (IL4) inhibition of IL-1-induced matrix metalloproteinase mRNA is dose dependent. Total RNA was isolated from human synovial fibroblast cultures 6 hours after addition of 100 ng of IL-1β/ml alone or in the presence of the indicated doses of IL-4. Shown are data from 3 independent experiments. The IC₅₀(50% inhibition concentration) calculated from these data is ~0.3 ng of IL-4/ml.

Figure 6

Interleukin-4 (IL-4) inhibition of IL-1-induccd matrix metalloproteinase expression occurs, at least in part, at the transcriptional level. A, Nuclei were isolated 6 hours after incubation of human synovial fibroblasts with 100 ng of IL-1β/ml alone or in the presence of 10 ng of IL4/ml. Nuclear run-on transcription assays were performed and the rcsulting RNA was hybridized to filters containing cDNA corresponding to collagenase, stromelysin, and GAPDH. Results were quantitated by densitometric scanning and normalized to GAPDH. The average of 2 independent experiments is shown. B, Human foreskin fibroblasts were transiently transfected with a luciferase reporter construct containing a 2-kb fragment of the human stromelysin promoter, along with SVβ-galactosidase as a control for transfection efficiency. Cytokines were added 16 hours after transfection, and cells were harvested 24 hours later. Shown are data from 2 independent experiments performed in triplicate, normalized to levels of β-galactosidase, and expressed relative to the control.

Figure 7

Interleukin-4 (IL-4) inhibits IL-1-induced production of prostaglandin E₂ (PGE₂) in human synovial fibroblasts (HSF). Levels of PGE₂ were measured in triplicate in conditioned media from HSF cultures treated with the appropriate cytokine(s) for 6 hours. HSF cultures isolated from 2 different individuals were used.

Figure 8

Interleukin-4 (IL-4) inhibition of IL-1-induced expression of matrix metalloproteinase is independent of prostaglandin E₂ (PGE₂). Indicated amounts (ng/ml) or PGE₂ (PGE) were added to cultures of human synovial fibroblasts simultaneously with 100 ng of IL-1β/ml and 10 ng of IL-4/ml. Total RNA was isolated after 6 hours, and Northern blots were hybridized with cDNA probes corresponding to collagenase, stromelysin, and GAPDH. The blot was quantitated and normalized to GAPDH as described abovc. Shown are data from 3 independent experiments for collagenase and 2 for stromelysin.

Figure 9

Binding of transcription factor activator protein 1(AP-1) is not affected by interleukin-4 (IL-4). Nuclear extracts were isolated from human synovial fibroblast cultures treated for 1hour with 100 ng of IL-1β/ml, 10 ng IL-4/m1, or both IL-1 and IL-4, as well as from control cultures. Binding of 5 μg of nuclear extract to a ³²P-labeled oligo(dT) probe corresponding to the AP-1 site in the stromelysin promoter was determined by electrophoretic mobility shift assay. P = probe alone, without nuclear extract, C = control, without interleukin.