Project leader: KAISA GRANFORS
National Public Health Institute, Department of Bacterial and Inflammatory Diseases, Kiinanmyllynkatu 13, 20520 Turku. Tel: 358-2-331 6623, kaisa.granfors@ktl.fi

Researchers:
- MD Markus Penttinen, University of Turku, National Public Health Institute, markus.penttinen@utu.fi
- MSc student Anna Sahlberg, National Public Health Institute, anna.sahlberg@utu.fi
- MSc Sanna Vähämiko, National Public Health Institute, sanna.vahamiko@utu.fi
- MSc Ildico Bacso, National Public Health Institute, ildico.bacso@utu.fi
- MSc Marja Ruuska, National Public Health Institute, marja.ruuska@utu.fi
- PhD Shichao Ge, National Public Health Institute, ge.shichao@ktl.fi

Key words: HLA-B27, salmonella, yersinia, reactive arthritis

Results

Reactive arthritis (ReA) develops after certain infections (eg. Salmonella, Yersinia) especially in HLA-B27 positive persons. Pathogenetic mechanisms of this complication and how HLA-B27 confers disease susceptibility have remained unknown. We have earlier shown that causative microbes/microbial antigens are not eliminated effectively in HLA-B27 positive persons developing ReA, as Salmonella and Yersinia antigens occur in blood cells from these patients for years. Another characteristic for cells from HLA-B27 positive persons is their exaggregated inflammatory responses to lipopolysaccharide (LPS) stimulation. These two characteristics we have also observed in our in vitro model with transfected monocytic cell lines. The elimination of Salmonella and Yersinia is impaired in HLA-B27-transfected U937 monocytic cells compared to the corresponding HLA-B27 negative control cells. Upon stimulation with Salmonella LPS HLA-B27 expressing cells show stronger response than HLA-B27 negative cells seen as enhanced activation of NF-kB and enhanced secretion of TNF-a. Because LPS is known to be present in the inflamed joints of patients with ReA, the enhanced inflammatory response of HLA-B27 positive cells upon LPS stimulation offers an explanation for the role of HLA-B27 in the development of ReA. These two phenomena seen in our in vitro model cannot be related to antigen presentation, but other mechanisms must be behind them.

To reveal the exact mechanism behind impaired elimination of Salmonella in HLA-B27 positive U937 monocytic cells we investigated the interaction between HLA-B27-expressing U937 monocytes and Salmonella enteritidis in more detail. We also investigated whether the composition of the B pocket of HLA-B27 contributes to the development of the modulatory effects caused by HLA-B27 in collaboration with Prof. Robert Colbert. Increased amounts of intracellular Salmonella enteritidis were observed at 24 hours after infection in the cells expressing misfolding forms of HLA-B27 molecule. In contrast, the cells expressing mutated forms of B27 heavy chains where misfolding is partially or completely corrected were able to resist the replication of Salmonella enteritidis, suggesting that features related to the amino acid composition of B pocket of B27 heavy chain may be responsible for the defect in B27-expressing cells. Our results also show that p38 MAP kinase activity plays a crucial role in controlling intracellular replication of Salmonella enteritidis in U937 cells. The inhibition of p38 activity leads to increased bacterial replication in all the transfectants used, but most remarkably in transfectants that do not misfold, suggesting that the cells expressing misfolding B27 heavy chains may display a relative defect in p38 activation.

Furthermore, HLA-B27-expressing cells showed also enhanced TNF-a production upon LPS stimulation and disturbed regulation of anti-inflammatory cytokine interleukin (IL)-10. Mutagenesis studies revealed that the folding efficiency of HLA-B27 heavy chain determines the severity of the effects caused by HLA-B27, as in the cells expressing strongly misfolding form of HLA-B27, the regulation of IL-10 and TNF-a was more severely disrupted than in the cells expressing wild type HLA-B27.

Our results indicate that the expression of HLA-B27 modulates interaction between monocyte-macrophages and ReA-triggering bacteria in vitro. Such modulatory effects may have important implications in the pathogenesis of ReA, and spondyloarthropathies in general, in vivo.

Selected publications:

Penttinen MA, Heiskanen KM, Mohapatra R, DeLay ML, Colbert RA, Sistonen L, Granfors K. Enhanced intracellular replication of Salmonella enteritidis in HLA-B27-expressing human monocytic cells: dependency on glutamic acid at position 45 in the B pocket of HLA-B27. Arthritis Rheum 2004: 50: 2255-2263.

Vähämiko S, Penttinen MA, Granfors K. Etiology and pathogenesis of reactive arthritis: Role of non-antigen presenting effects of HLA-B27. Arthritis Research & Therapy 2005: 7: 136-141.
 

An abstract of the reserach plan (January 2003)