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Transient exposure of macrophages to P38 map kinase inhibition conditions cell responses through MAPK activated protein kinase-2 regulation
  1. Nasser Malik1,
  2. Margaret Lees1,
  3. Vivienne Moradi1,
  4. Stefan Laufer2,
  5. Georg Schett3,
  6. Michael Burnet4,
  7. Michael Seed1
  1. 1Centre for Experimental Medicine & Rheumatology, William Harvey Research Institute, Queen Mary's School of Medicine & Dentistry, London, UK
  2. 2Institute of Pharmacy, University of Tuebingen, Tuebingen, Germany
  3. 3Division of Rheumatology, University of Erlangen-Nuremberg, Erlangen, Germany
  4. 4Synovo GmbH, Tuebingen, Germany

Abstract

Background and objectives Previous work by the authors suggests that selective p38 mitogen-activated protein kinase (MAPK) inhibitors, SB203580 and ML3403, have inhibitory effects on adherent accessory cells such as macrophages that last up to 120 h after the drugs are washed off, whereas no such effect is seen in lymphocytes.1 This raises the question as to whether p38 MAPK signalling remains inhibited after washout or whether there is a long term priming effect on the macrophages. To answer this, intracellular p38 MAPK activity of human macrophages transiently exposed to the p38 MAPK inhibitor, SB203580, was determined with a substrate phosphorylation assay for different time-points after washout. Tumour necrosis factor α (TNFα) synthesis was also assayed.

Methods U937 cells, a human monocytic cell line, were differentiated to the macrophage phenotype with PMA (80 nM) for 48 h (PMA-U937 cells). The cells were trypsinised and plated in 12 well plates at 5×105/well. After overnight serum starvation, the cells were incubated with SB203580 for 2 h before being washed four times and rested for various time-points. Cells were then stimulated with 100 ng/ml LPS for 30 min and supernatants were collected for TNFα measurement by ELISA and whole cell lysates were prepared for western analysis of downstream targets of p38, transcription factors ATF-2 and MK-2.

Results PMA-induced differentiation of U937 cells to macrophages was confirmed by extracellular staining with macrophage markers (CD16 and CD51/61) and flow cytometry. LPS stimulated TNFα production by PMA-U937 cells, which was completely inhibited by SB203580. LPS stimulated a concentration-dependent increase in the phosphorylation of ATF-2 and MK-2. However, only phosphorylation of MK-2 was inhibited by SB203580. Washing the cells after drug incubation restored the ability to stimulate phospho-MK-2 in the macrophages shortly after washout (1 min and 2 h), however the inhibition of MK-2 phosphorylation by p38 MAPK was reasserted 4 h after drug withdrawal. Phospho-ATF-2 remained unaffected throughout, indicating messenger selectivity.

Conclusions This data confirms that p38 inhibition has a selective effect on accessory cells that persists after washout, and that this could be regulated through MAPK activated protein kinase-2. In addition, SB203580 preconditioning appears to have a biphasic effect on macrophages, with inhibition of phospho-MK-2 when the drug is present, no inhibition shortly after washout as would be expected, but with an unexpected secondary inhibition of P38 MAPK phosphorylation of MK-2 at longer time-points. Since preconditioning is not witnessed in T lymphocytes, the inhibition of p38 MAPK may have different anti-inflammatory actions depending on the time elapsed after drug administration.

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