TY - JOUR
T1 - Evidence that the p38 MAP kinase pathway is dysregulated in HLA-B27-expressing human monocytic cells
T2 - correlation with HLA-B27 misfolding
AU - Sahlberg, Anna S
AU - Penttinen, Markus A
AU - Heiskanen, Kaisa M
AU - Colbert, Robert A
AU - Sistonen, Lea
AU - Granfors, Kaisa
PY - 2007/8
Y1 - 2007/8
N2 - 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.
AB - 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.
KW - Cell Line
KW - Cysteine/chemistry
KW - Dose-Response Relationship, Drug
KW - Enzyme Inhibitors/pharmacology
KW - Glutamic Acid/chemistry
KW - HLA-B27 Antigen/chemistry
KW - Humans
KW - Monocytes/metabolism
KW - Protein Conformation
KW - Protein Folding
KW - RNA, Small Interfering/pharmacology
KW - Salmonella Infections/microbiology
KW - Salmonella enteritidis/drug effects
KW - Transfection
KW - p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors
U2 - 10.1002/art.22746
DO - 10.1002/art.22746
M3 - Article
C2 - 17665421
SN - 0004-3591
VL - 56
SP - 2652
EP - 2662
JO - Arthritis and Rheumatism
JF - Arthritis and Rheumatism
IS - 8
ER -