Potent synergistic effect of IL-3 and TNF on matrix metalloproteinase 9 generation by human eosinophils.
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.
Allergy, Pulmonary and Critical Care Medicine Division, Department of Medicine, University of Wisconsin School of Medicine and Public Health, Madison, WI 53792, USA. eak@medicine.wisc.edu
Elizabeth A B
EA
Kelly
Lin Ying
LY
Liu
Stephane
S
Esnault
Beatriz Helena
BH
Quinchia Johnson
Nizar N
NN
Jarjour
D016207:Cytokines / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D004804:Eosinophils / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D007377:Interleukin-3 / Q000378:metabolism*; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D016207:Cytokines; D007377:Interleukin-3; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D048049:Extracellular Signal-Regulated MAP Kinases; D020780:Matrix Metalloproteinase 9