Potent synergistic effect of IL-3 and TNF on matrix metalloproteinase 9 generation by human eosinophils

Abstract

TNF (designated as TNF-α under previous nomenclature) is the preeminent activator of MMP-9 generation from a variety of cells including eosinophils. We have previously established that TNF strongly synergizes with IFN-γ and IL-4 for eosinophil synthesis of Th1- and Th2-type chemokines respectively. Thus, we sought to determine if TNF-induced synthesis of MMP-9 would be enhanced by the presence of Th1, Th2, or the eosinophil-associated common beta chain (βc) cytokines. Human blood eosinophils were cultured with TNF alone or in combination with either IFN-γ, IL-4, IL-3, IL-5, or GM-CSF. Concentrations and activities of MMP-9 in eosinophil culture supernates were measured by ELISA and gelatin zymography, mRNA transcription and stabilization by quantitative real-time PCR, and signaling events by immunoblotting and intracellular flow cytometric analysis. Individually, TNF, GM-CSF, or IL-3, but not IL-4 or IFN-γ, induced relatively small (<0.2 ng/ml) but statistically significant quantities of MMP-9. Remarkable synergistic synthesis of MMP-9 (ng/ml levels) occurred in response to TNF plus IL-3, GM-CSF or IL-5, in the order of IL-3>GM-CSF>IL-5. Zymography revealed that eosinophils release MMP-9 in its pro-form. Eosinophil stimulation with the combination of IL-3 plus TNF led to increased steady-state levels of MMP-9 mRNA, prolonged mRNA stabilization, and enhanced activation of ERK1/2 phosphorylation. Inhibition of NF-κB, MEK kinase, or p38 MAP kinase, but not JNK signaling pathways, diminished IL-3/TNF-induced MMP-9 mRNA and protein production. Thus, the synergistic regulation of eosinophil MMP-9 by IL-3 plus TNF likely involves cooperative interaction of multiple transcription factors downstream from ERK, p38, and NF-κB activation as well as post-transcriptional regulation of MMP-9 mRNA stabilization. Our data indicate that within microenvironments rich in βc-family cytokines and TNF, eosinophils are an important source of proMMP-9 and highlight a previously unrecognized role for synergistic interaction between TNF and βc-family cytokines, particularly IL-3, for proMMP-9 synthesis.

Figure 1. βc-family cytokines plus TNF-induced eosinophil proMMP-9

(A) Concentrations of total MMP-9 in supernates from eosinophils (2×10⁶ cells/ml) stimulated 72 h (n=11 subjects, p<0.05, verses *medium, ^†TNF, or ^‡respective single cytokine). (B) Kinetics of MMP-9 generation by eosinophils (2×10⁶ cells/ml) cultured for 3, 24, 48, or 72 h with medium alone, TNF plus IL-5, GM-CSF, or IL-3, respectively (p<0.05, verses *medium, ^†TNF, or ^‡respective single cytokine).

Figure 1. βc-family cytokines plus TNF-induced eosinophil proMMP-9

(A) Concentrations of total MMP-9 in supernates from eosinophils (2×10⁶ cells/ml) stimulated 72 h (n=11 subjects, p<0.05, verses *medium, ^†TNF, or ^‡respective single cytokine). (B) Kinetics of MMP-9 generation by eosinophils (2×10⁶ cells/ml) cultured for 3, 24, 48, or 72 h with medium alone, TNF plus IL-5, GM-CSF, or IL-3, respectively (p<0.05, verses *medium, ^†TNF, or ^‡respective single cytokine).

Figure 2. Early (1 h) and late (72 h) release of MMP-9 by eosinophils

(A and C) Concentrations of total MMP-9 (pg/ml of supernate) or (B and D) gelatinase activity in 20 µl supernate from eosinophils (2×10⁶ cells/ml) stimulated with TNF ± IL-3 (10 ng/ml) for 1 or 72 h in medium containing human serum albumin or fetal bovine serum. Recombinant proMMP-9 (MW=92 kDa) was used as a positive control in lane 1 of the zymograms. (Representative of 2 subjects.)

Figure 3. IL-3 plus TNF-induced eosinophil MMP-9 mRNA

Eosinophils were cultured with medium (circles), IL-3 (down triangles), TNF (up triangles), or IL-3 plus TNF (diamonds). MMP-9 mRNA levels were determined by qPCR and normalized to β-glucuronidase and expressed as fold change (2-ΔΔCt). Data are mean ± SEM of 4–5 subjects (closed symbols) or two subjects (open circles). p<0.05, versus *medium, ^†IL-3, or ^‡TNF.

Figure 4. Effect of IL-3 plus TNF on MMP-9 mRNA stability

Eosinophils were cultured for 15 h with IL-3 (down triangles), TNF (up triangles), or IL-3 plus TNF (diamonds). Cells were harvested at 0, 30, 60, and 120 min after addition of actinomycin D, and MMP-9 mRNA was quantified by qPCR. Data were normalized to β-glucuronidase and expressed as the % of mRNA remaining compared to T0. Data are mean ± SEM of 3 subjects.

Figure 5. Signaling cascades initiated by IL-3 plus TNF

(A) Representative immunoblots and (B) immunoblot densitometric analysis (n=3 subjects; p<0.05, versus *medium or ^†IL-3) of phosphorylated MAP kinases, phospho-STAT5, and IκBα in eosinophils cultured for 5 min. (C) Representative three-dimensional histograms for detection of NF-κB phospho-p65 by flow cytometric analysis. Eosinophils were cultured for 5, 15, and 30 min with medium alone (no color), IL-3 (light gray), TNF (dark gray), or IL-3 plus TNF (black). (D) Summarized median channel fluorescence (MCF) from 4 individual subjects are expressed as the mean ± SEM.

Figure 5. Signaling cascades initiated by IL-3 plus TNF

(A) Representative immunoblots and (B) immunoblot densitometric analysis (n=3 subjects; p<0.05, versus *medium or ^†IL-3) of phosphorylated MAP kinases, phospho-STAT5, and IκBα in eosinophils cultured for 5 min. (C) Representative three-dimensional histograms for detection of NF-κB phospho-p65 by flow cytometric analysis. Eosinophils were cultured for 5, 15, and 30 min with medium alone (no color), IL-3 (light gray), TNF (dark gray), or IL-3 plus TNF (black). (D) Summarized median channel fluorescence (MCF) from 4 individual subjects are expressed as the mean ± SEM.

Figure 6. Effect of MAP kinase and NF-κB inhibitors on IL-3 plus TNF-induced eosinophil MMP-9

Eosinophils were preincubated for 1 h as indicated with the MEK kinase inhibitor U0126 or its inactive analogue U0124, the p38 MAP kinase inhibitor SB203580 or its inactive analogue SB202474, the JNK inhibitor II or its inactive analogue, or the NF-κB inhibitor BAY 11-7082 and then stimulated with IL-3/TNF. (A) MMP-9 protein at 24 h (mean ± SEM of 5 subjects; p<0.05, versus *IL-3/TNF alone or ^†its inactive analogue). (B) MMP-9 mRNA at 6 h (average of 2 subjects).

Figure 6. Effect of MAP kinase and NF-κB inhibitors on IL-3 plus TNF-induced eosinophil MMP-9

Eosinophils were preincubated for 1 h as indicated with the MEK kinase inhibitor U0126 or its inactive analogue U0124, the p38 MAP kinase inhibitor SB203580 or its inactive analogue SB202474, the JNK inhibitor II or its inactive analogue, or the NF-κB inhibitor BAY 11-7082 and then stimulated with IL-3/TNF. (A) MMP-9 protein at 24 h (mean ± SEM of 5 subjects; p<0.05, versus *IL-3/TNF alone or ^†its inactive analogue). (B) MMP-9 mRNA at 6 h (average of 2 subjects).