Isolation and Partial Purification of Macrophage- and Dendritic Cell-Activating Components fromMycoplasma arthritidis: Association with Organism Virulence and Involvement with Toll-Like Receptor 2

Abstract

Mycoplasma arthritidis induces toxicity, arthritis, and dermal necrosis in mice. Virulence factors include a superantigen and membrane adhesins and possibly also a bacteriophage component. Here we compare the biological properties of Triton X-114 extracts derived from avirulent and virulent M. arthritidis strains. Macrophage cell lines and resident peritoneal macrophages were used to assess inflammatory potential as indicated by production of tumor necrosis factor alpha, interleukin-6, and/or nitric oxide. The activity resided exclusively within the hydrophobic detergent phase, was unaffected by heat treatment at 100°C for 30 min, and was resistant to proteinase K digestion, suggesting involvement of a lipopeptide. Contamination of extracts with endotoxin or superantigen was excluded. Extracts of the more virulent strain had higher activity than did those of the avirulent strain. Using CHO cells expressing Toll-like receptor 2 (TLR2) or TLR4, both with transfected CD14, we showed that extracts activated these cells via TLR2 but not by TLR4. Also, macrophages from C57BL/6 TLR2 ؊/؊ mice failed to respond to the extracts, whereas those from TLR2 ؉/؉ cells did respond. The preparations from the virulent strain of M. arthritidis were also more potent in activating dendritic cells, as evidenced by up-regulation of major histocompatibility complex class II, CD40, B7-1, and B7-2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent elution of gel slices revealed the presence of three active moieties which corresponded to molecular masses of approximately 24, 28, and 40 kDa. Three active components were also found by reverse-phase chromatography. We suggest that macrophage activation by M. arthritidis could play a significant role in the inflammatory response induced in the host by this organism.

A Microbial TLR2 Agonist Imparts Macrophage-Activating Ability to Apolipoprotein A-1

“…1B). These results were similar to those obtained previously using TX-114 detergent extraction (6). OGex was again subjected to SDS-PAGE, and the gel was stained with zinc.…”

“…Serum was collected from two to three individual mice 90 min after i.v. injection of each agonist or control (6). Cytokines were determined by ELISA kits from eBioscience or BD Pharmingen.…”

“…It also possesses two distinct adhesins and a bacteriophage, all of which are thought to contribute to disease (5). Recently, cell-associated, macrophage and dendritic cell-activating, TLR2-dependent moieties were found in M. arthritidis, the most active of which had a molecular mass of 25-30 kDa (6). Subsequent work has characterized a distinct 41-kDa lipoprotein (A. Hasebe, H. Mu, L. Washburn, F. Chan, N. Pennock, M. Taylor, and B. Cole, submitted for publication) that also possesses TLR2-dependent bioactivity and that is related to the Mlp family of lipoproteins of M. arthritidis described by Washburn et al (7).…”

“…In this study, we purify and characterize the 28-kDa bioactive component described previously (6) and show, unexpectedly, that it is an apolipoprotein A-1 (apoA-1)-like molecule that acquires bioactivity by associating with mycoplasma cells. The TLR2-dependent bioactive apoA-1 could also be generated by the direct binding of bioactive lipopeptides derived from Division of Rheumatology, Department of Internal Medicine, University of Utah School of Medicine, Salt Lake City, UT 84132 a known macrophage-activating lipoprotein to horse serum-derived apoA-1 (HS apoA-1).…”

“…In view of our findings on multiple TLR usage by several agonists present in the arthritogenic and toxigenic M. arthritidis species (4,6), the present work was initiated to more fully characterize these moieties. In this study, we purify and characterize the 28-kDa bioactive component described previously (6) and show, unexpectedly, that it is an apolipoprotein A-1 (apoA-1)-like molecule that acquires bioactivity by associating with mycoplasma cells.…”