Evidence That the p38 MAP Kinase Pathway Is Dysregulated in HLA-B27-expressing Human Monocytic Cells: Correlation With HLA-B27 Misfolding

Arthritis Rheum. 2007 Aug;56(8):2652-62. doi: 10.1002/art.22746.

Abstract

Objective: To investigate the cause of the enhanced intracellular replication of Salmonella enteritidis in HLA-B27-transfected U937 human monocytic cells and the contribution of HLA-B27 heavy chain (HC) misfolding.

Methods: U937 monocytic cell transfectants stably expressing pSV2neo resistant vector (mock), wild-type HLA-B27, or mutated HLA-B27 HCs with amino acid substitutions in the B pocket were differentiated, infected with S enteritidis, and treated with signaling pathway inhibitors or specific p38 small interfering RNA (siRNA). The numbers of living intracellular bacteria were determined with the colony-forming unit method. To visualize S enteritidis, the bacteria were transformed with green fluorescent protein, and studied by microscopy.

Results: Treatment with the p38 MAPK inhibitors or with p38 siRNA enhanced the replication of S enteritidis in U937 transfectants, whereas the other inhibitors had no effect. In mock-transfected cells and in cells expressing the mutated B27 HCs in which the misfolding had been corrected, p38 inhibitors impaired their ability to resist the replication of bacteria (mock, B27.A2B, B27.E45M, and B27.C67A). In contrast, the number of intracellular bacteria was not significantly increased in p38 inhibitor-treated cells expressing misfolded B27 HCs (B27g, B27cDNA, and B27.H9F).

Conclusion: Our results show that p38 activity plays a crucial role in controlling intracellular S enteritidis in U937 cells. Enhanced replication of bacteria in B27-expressing cells requires that the HCs contain glutamic acid at position 45 and cysteine at position 67. Furthermore, in transfectants expressing misfolded B27 HCs, p38 inhibition had no significant effect on bacterial replication, suggesting that in these cells, the p38 pathway may not function properly.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Cysteine / chemistry
  • Dose-Response Relationship, Drug
  • Enzyme Inhibitors / pharmacology
  • Glutamic Acid / chemistry
  • HLA-B27 Antigen* / chemistry
  • HLA-B27 Antigen* / genetics
  • HLA-B27 Antigen* / metabolism
  • Humans
  • Monocytes / metabolism*
  • Monocytes / microbiology
  • Protein Conformation
  • Protein Folding*
  • RNA, Small Interfering / pharmacology
  • Salmonella Infections / microbiology
  • Salmonella enteritidis / drug effects
  • Salmonella enteritidis / growth & development
  • Salmonella enteritidis / pathogenicity*
  • Transfection
  • p38 Mitogen-Activated Protein Kinases / antagonists & inhibitors
  • p38 Mitogen-Activated Protein Kinases / metabolism*

Substances

  • Enzyme Inhibitors
  • HLA-B27 Antigen
  • RNA, Small Interfering
  • Glutamic Acid
  • p38 Mitogen-Activated Protein Kinases
  • Cysteine