Mechanisms of serum potentiation of GM-CSF production by human airway smooth muscle cells

Abstract

Inflammation and vascular leakage are prevalent in asthma. This study aimed to elucidate the mechanisms involved in serum potentiation of cytokine-induced granulocyte macrophage colony stimulating factor (GM-CSF) production by human airway smooth muscle cells and to identify possible factors responsible. Serum-deprived cells at low density were stimulated with TNF-α and IL-1β for 24 h. Human AB serum (10%), inhibitors of RNA and protein synthesis or specific signaling molecules, or known smooth muscle mitogens were then added for 24 h. Culture supernatants were analyzed for GM-CSF levels, and cells were harvested to assess viability, cell cycle progression, GM-CSF-specific mRNA content, and p38 phosphorylation. Serum potentiated GM-CSF release when added before, together with (maximal), or after the cytokines. The potentiation involved both new GM-CSF-specific mRNA production and protein synthesis. The mitogens IGF, PDGF, and thrombin all potentiated GM-CSF release, and neutralizing antibodies for EGF, IGF, and PDGF reduced the serum potentiation. Inhibitor studies ruled as unlikely the involvement of p70S6kinase and the MAPK p42/p44, two signaling pathways implicated in proliferation, and the involvement of the MAPK JNK, while establishing roles for p38 MAPK and NF-κB in the potentiation of GM-CSF release. Detection of significant p38 phosphorylation in response to serum stimulation, through Western blotting, further demonstrated the involvement of p38. These studies have provided evidence to support p38 being targeted to interrupt the cycle of inflammation, vascular leakage and cytokine production in asthma.

Inhibition of Tumor Necrosis Factor-α-Inducible Inflammatory Genes by Interferon-γ Is Associated with Altered Nuclear Factor-κB Transactivation and Enhanced Histone Deacetylase Activity

“…Recent evidence using newly developed pharmacological inhibitors identified the necessary NF-B-activating kinase, IKK␤, as a potential new target for treatment of inflammatory responses in asthma (Birrell et al, 2005). We and others showed that NF-B is activated by TNF␣ or IL-1␤ in ASM cells and represents an essential transcription factor involved in the regulation of VCAM-1 (Issa et al, 2006), ICAM-1 (Amrani et al, 1999), CXC chemokine growth-related oncogene protein-␣ (Issa et al, 2006), and granulocyte macrophage-colony-stimulating factor (Lalor et al, 2004). Through the use of BMS-345541, a potent IKK␤ inhibitor (Burke et al, 2003), we demonstrated here that NF-B also played a central role in IFN␥-sensitive, TNF␣-inducible genes IL-6, IL-8, and eotaxin.…”

Asthmatic airway smooth muscle CXCL10 production: mitogen-activated protein kinase JNK involvement

“…Total cell lysates were prepared (19) and subjected to SDS-PAGE gel electrophoresis and Western blotting. The Iκ-Bα and phosphorylated p65 bands were detected using Phospho-NF-κB p65 (Ser276) and Iκ-Bα antibodies diluted 1/1,000 and 1/500, respectively.…”

“…Growth-arrested ASM cells were treated with PD98059 (30 μM), SB203580 (10 μM), SP600125 (10 μM), or the negative control SB202474 (10 μM), as previously reported (19, 29). The cells were treated with the inhibitors or their vehicle (0.1% vol/vol DMSO) 45 min before and during stimulation with the cytokines for 24 h.…”

“…Growth-arrested ASM cells were treated with SP600125 (10 μM) or vehicle (0.1% vol/vol DMSO) 45 min before and during cytomix stimulation for 0, 5, 10, 30, and 60 min. Total cell lysates were prepared (19, 29) and, together with prestained standards and JNK controls, subjected to SDS-PAGE gel electrophoresis and Western blotting. Total JNK and phosphorylated JNK bands were visualized using SAPK/JNK and phospho-SAPK/JNK (Thr183/Tyr185) antibodies (diluted 1/1,000), respectively.…”

Airway smooth muscle CXCR3 ligand production: regulation by JAK-STAT1 and intracellular Ca2+

“…ASMCs in six-well plates were treated with agents or vehicle as described above and stimulated with cytomix for 2.5, 5, 10, and 30 min. Whole cell lysates were prepared as previously described (30). Each sample (25 l) was subjected to SDS-PAGE and Western blot analysis.…”