Identification of the physiologically active state of the mycobacterial glycolipid trehalose 6,6'-dimycolate and the role of fibrinogen in the biologic activities of trehalose 6,6'-dimycolate monolayers

When administered in the oil phase of an emulsion, the mycobacterial glycolipid trehalose 6,6'-dimycolate (TDM) acts as an adjuvant, elicits acute inflammation and granuloma formation, and is a pathogen inducing massive pulmonary hemorrhages. TDM forms cylindrical micelles in aqueous media and stable monolayers on hydrophobic surfaces. TDM monolayers bind proteins avidly whereas TDM micelles do not. We report that all of the biologic effects of TDM can be reproduced by monomolecular films of the glycolipid on the surface of 5.7 ± 1.5 μm diameter hydrophobic beads. In contrast, TDM micelles are biologically inert. The magnitude of the inflammatory response to TDM-coated beads depends on the total surface area covered with a monolayer of the glycolipid and on the surface concentration of TDM. For a given surface area, the infiltration of polymorphonuclear leukocytes and macrophages is greatest when beads are coated with TDM near the equilibrium spreading density, which is 2.5 x 10^-3 molecule/A². The biologic effects of TDM are intimately associated with the ability of the monolayer to bind fibrinogen as evidenced by the following: (1) TDM micelles do not bind proteins and are biologically inert. (2) TDM monolayers exposed to plasma-but not TDM monolayers expoded to serum-elicit maximal biologic activity. (3) Plasma fibrinogen adsorbs to the TDM monolayer to the exclusion of other plasma proteins. (4) If the adsorption of fibrinogen is blocked in vivo by preincubation of TDM monolayers with an extraneous protein such as ovalbumin or bovine serum albumin, the biologic activities of the monolayer are temporarily mitigated. Hydrolysis in situ of fibrinogen into fibrin might be a necessary event for expressing the activities of the TDM monolayer because preincubation with thrombin or with peritoneal exudate cell supernatants containing thrombin-like activity decreases the activity of TDM monolayers coated with fibrinogen. Additional support is provided by the observations that i.p. injections of TDM-coated beads generate procoagulant activity in peritoneal exudates, as well as massive pulmonary hemorrhages. We suggest that the active principle of adjuvant preparations containing TDM is a TDM monolayer to which fibrinogen has been adsorbed.