Purification and partial biochemical characterization of a Mycoplasma fermentans-derived substance that activates macrophages to release nitric oxide, tumor necrosis factor, and interleukin-6

Abstract

Mycoplasmal products may exert a number of diverse in vitro effects on cells of the immune system. A macrophage-activating substance from Mycoplasma fermentans was described in this laboratory and named mycoplasma-derived high-molecular-weight material (MDHM). Using synthesis of nitric oxide by peritoneal cells from endotoxin low-responder mice as an assay system, MDHM was purified as follows. After freeze-thawing of M. fernentans, MDHM activity was sedimented with the membrane fraction. Membranes were delipidated with chloroform-methanol, and MDHM activity was extracted with octyl glucoside. Coextracted proteins were degraded by proteinase K. MDHM was further purified by reversed-phase high-pressure liquid chromatography and eluted in one major and one minor peak of activity. Neither carbohydrates nor amino acids were found as constituents. MDHM had the following properties: it partitioned into the phenol phase upon phenol-water extraction and into the Triton phase after extraction with Triton X-114. MDHM was not inactivated by either phospholipase A2 or triglyceride lipases. However, mild periodate treatment led to a >95% loss of activity. Also, alkaline hydrolysis at 25°C completely abolished MDHM activity with a half-life of 2 min. MDHM activity was spread out over a wide molecular weight range upon sodium dodecyl sulfatepolyacrylamide gel electrophoresis of membranes, whereas after proteinase treatment MDHM activity migrated close to the front. These features of MDHM, taken together, speak in favor of an amphiphilic molecule with a lipid moiety carrying fatty acids in ester linkage and a polyol moiety of unknown character. MDHM was active in the nanogram-per-milliliter range, activating macrophages to release nitric oxide, interleukin-6, and tumor necrosis factor.

Physicochemical characterization and biological activity of a glycoglycerolipid from Mycoplasma fermentans

“…These are glycolipids, lipopeptides (macrophage-activating lipopeptides, MALP), or lipoproteins. The glycoconjugates MALP-I and -II are known to activate macrophages [33] on a TLR-2 and MyD88-dependent pathway [34] and are active down to picomolar concentration [32]. Also, they are strong inducers of cytokines and chemokines.…”

“…LPS reacts essentially to TLR4, while the lipopeptide or protein exhibit TLR2-reactivity. Thus, a possible contamination of the MfGl-II with a lipoprotein or MALP-2 [32], which could explain the cytokine-inducing capacity, can be excluded.…”

Engagement of Toll-like receptors by mycoplasmal superantigen: downregulation of TLR2 by MAM/TLR4 interaction

“…Monoclonal anti-murine or anti-human cytokine antibodies and recombinant cytokine standards were purchased from eBioscience, San Diego, CA. Measurement of NO (NO 2 plus NO 3 -) in cell supernatants was performed as described previously using a modified Griess assay (Sigma, St. Louis, MO) (Muhlradt and Frisch, 1994;Mu et al, 2001).…”

The importance of B‐cells and ecto‐5′nucleotidase in Mycoplasma fermentans infection and the relevance to rheumatoid arthritis

“…38,39 M. fermentans has also been reported to induce a number of cytokines, such as interleukin-1 (IL-1), IL-6, granulocyte colony-stimulating factor, prostaglandins and tumour necrosis factor (TNF). [40][41][42] Christensen et al 43 reported that lymphotoxin, prostaglandin E 2 (PGE 2 ) and (in some people only) TNF would increase the 5 0 N activity of lymphocytes. The most effective was PGE 2 , which gave a threefold increase at the highest concentrations used (3 lM), whereas IL-6 had no effect.…”