Isolation and partial purification of macrophage- and dendritic cell-activating components from Mycoplasma 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 degrees 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.

FIG. 1.

TX-114 extraction of M. arthritidis indicates activity is restricted to the detergent phase. Following detergent extraction of washed pellets of virulent strain 158p10p9 (see Materials and Methods), aqueous and detergent phases were assayed for induction of TNF-α by RAW 264.7 cells after 24 h. LPS-free NS was similarly treated with TX-114, and the phases were also tested. LPS (10 ng/ml) was a positive control. Mean values ± SEM of triplicate determinations are shown.

FIG. 2.

Effect of polymyxin B on M. arthritidis extracts. Polymyxin B (10 μg/ml final concentration) was incubated with extract (Ex) dilutions from virulent strain 158p10p9 at 200, 40, and 8 ng/ml for 1 h at 37°C prior to being added to RAW 264.7 cells for induction of TNF-α. LPS (100 ng/ml) and lipid A (100 ng/ml) were used as positive controls to show the inhibitory effects of polymyxin B on activation by these inducers.

FIG. 3.

Comparative activities of TX-114 extracts of virulent versus avirulent M. arthritidis strains. The detergent phases from TX-114 extraction of avirulent strain 158, MAV-1-infected 158L3-1a, and highly virulent 158p10p9 were compared in a dose-response experiment for TNF-α production by RAW 264.7 cells. Results are expressed as means ± SEM (see text for P values). Three different preparations of strains 158 and 158p10p9 and two of 158L3-1a were tested with similar results.

FIG. 4.

Nitric oxide production by TX-114 extracts from virulent versus avirulent M. arthritidis strains. Extracts from strains 158, 158L3-1a, and 158p10p9 were incubated at various concentrations with RAW 264.7 cells for 48 h, and supernatant NO content was measured after conversion to nitrite and nitrate. Mean results from three experiments are shown (see text for P values).

FIG. 5.

Activation of DC by extracts from virulent versus avirulent strains of M. arthritidis. XS52 cells were treated with 1 μg/ml of extracts from all three strains of M. arthritidis. After 24 h, cells were harvested and stained with fluorescein isothiocyanate-conjugated anti-class II MHC or anti-CD40 antibodies or with phycoerythrin-conjugated anti-B7-1 or -B7-2 antibodies (open histograms). Isotype-matched control antibodies (filled histograms) were included in all experiments (see Materials and Methods). Flow cytometry data from a representative experiment of three are shown. (See text for P values.)

FIG. 6.

Effect of PK on activity of M. arthritidis 158p10p9 extracts. Extracts (Ex) at 1 μg/ml (A) or 0.08 μg/ml (B) were treated for 1 h at 37°C with 5 μg/ml SPK or 5 μg/ml agarose-bound (PKA) enzyme. The soluble enzyme was deactivated by 100°C heat or the agarose-bound enzyme was removed by centrifugation prior to assay for TNF-α on RAW 264.7 cells.

FIG. 7.

Up-regulation of CD25 by CHO cells transfected with TLR2 or TLR4 in response to 158p10p9 extracts. CHO cells with the TLR2 or TLR4 transgenes together with CD14 were exposed to M. arthritidis 158p10p9 extracts for 18 h at 37°C. CD25 up-regulation, a measure of TLR activation, was quantitated by flow cytometry. MALP-2 was used as a positive control for activation of TLR2, and LPS was used as a control for activation of TLR4. FITC, fluorescein isothiocyanate.

FIG. 8.

IL-6 production in vitro and in vivo induced by M. arthritidis extracts in macrophages from TLR2^+/+ and TLR2^−/− mice. (A) Macrophages from C57BL/6 and C57BL/6 TLR2 KO mice. Resident peritoneal macrophages were harvested from individual mice, and suspensions were treated in duplicate with various doses of 158p10p9 extracts, MALP-2 (100 ng/ml), lipid A (100 ng/ml), or NS alone. Supernatants were harvested at 18 h and assayed in triplicate for IL-6 production. Mean values ± SEM are shown. (B and C) Three mice each of strains C57BL/6 and C57BL/6 TLR2 KO received an intravenous injection of 158 or 158p10p9 extracts (5 μg/mouse) and were exsanguinated 90 min later, and the resulting sera were tested for (B) IL-6 and (C) TNF-α. Mean results from three mice with triplicate assays ± SEM are shown.

FIG. 9.

Role of CD14 in macrophage activation by M. arthritidis 158p10p9 TX-114 extracts. THP-1 cells were pretreated with anti-human CD14 (10 μg/ml) for 1 h at 37°C, after which extracts at 1 and 0.2 μg/ml (final concentration) were added. After incubation for a further 18 h at 37°C, culture supernatants were harvested and analyzed for TNF-α. Lipid A and MALP-2, both at 0.1 μg/ml, were also tested. Results are means ± SEM.

FIG. 10.

(A) Activity of TX-114 detergent extracts separated by SDS-PAGE. After PAGE, (lipo)proteins were transferred to a nitrocellulose membrane, and after extraction of 2-mm strips in dimethyl sulfoxide followed by precipitation with sodium carbonate buffer, they were assayed for induction of TNF-α production by RAW 264.7 cells. A region of the gel which contained no extract was also transferred and used as a negative control (NT). There were at least three potent macrophage-activating components (arrowheads). (B) Reverse-phase gradient chromatography of M. arthritidis 158p10p9 extracts. Detergent-phase extracts from TX-114 fractionation were loaded onto a 15 RPC column and eluted with a 0 to 100% isopropanol gradient. Fractions were assayed for TNF-α production by RAW 264.7 cells. Duplicate suspensions were assayed for each fraction, and triplicate wells were tested during the enzyme-linked immunosorbent assay.