- Top of page
- Materials and methods
The triacylated lipid A partial structure OM-174 was characterized in detail using a variety of physical and biological techniques. OM-174 aggregates adopt the micellar HI structure. The temperature (Tc) of the gel to liquid-crystalline phase transition of the hydrocarbon chains is 0 °C, from which high fluidity of the acyl chains at 37 °C can be deduced. The molecular area of a single OM-174 molecule at a surface pressure of 30 mN·m−1 is 0.78 ± 0.04 nm2. Conformational analyses, using IR spectroscopy, of the behavior of the various functional groups of OM-174 as compared with hexa-acyl lipid A suggest altered hydration of the phosphate charges and unusually strong hydration of the ester groups, which is probably related to the high accessibility of these groups to water in the micellar aggregate structure. OM-174 was shown to intercalate into a phospholipid membrane corresponding to the macrophage membrane within seconds in the presence, and within minutes to hours in the absence, of LPS-binding protein. In the Limulus amebocyte lysate assay, the triacyl lipid A is more than 105-fold less active than hexa-acyl lipid A, but only 10-fold less active in inducing IL-6 in human mononuclear cells, and equally active in inducing NO production in murine macrophages. These findings are used to explain the mechanism of the lipid A-induced cell activation.
Lipopolysaccharides (LPSs), particularly amphiphiles forming the lipid matrix of the outer leaflet of the outer membrane of Gram-negative bacteria , are also called endotoxins because of their ability to induce a variety of biological effects in mammalian cells . Their lipid moiety, called lipid A, consists of a diglucosamine saccharide phosphorylated in positions 1 and 4′ and is acylated, in Enterobacteriaceae, with six or seven acyl chains in either amide or ester linkage with the diglucosamine backbone. Lipid A is known to represent the ‘endotoxic principle’ of LPS because it can induce the same spectrum of endotoxin reactions as its parent LPS .
The physical or physicochemical behavior of aqueous suspensions of LPS and lipid A may be important in their biological action. Physicochemical characteristics include: (a) the critical micellar concentration (CMC), which is the concentration above which, on further addition of lipids to the bulk solution, no further increase in monomer concentration takes place; (b) the shape and size of the lipid aggregates above the CMC; (c) the mobility of the hydrocarbon chains (fluidity) within the aggregates; (d) the dependence of all these parameters on pH, concentration of univalent and bivalent cations, and temperature. We have previously investigated free lipid A preparations from Escherichia coli and Salmonella minnesota under near-physiological conditions and could relate particular physicochemical characteristics to their biological activity [4–6]. In this study, the results of measurements performed with the triacyl lipid A OM-174 are presented, which differs from E. coli lipid A by the absence of the acyl chains linked directly through ester bonds to the diglucosamine backbone. This compound has been shown to be a candidate for an effective anticancer treatment [7,8]. We have found that OM-174 differs from E. coli lipid A in many physicochemical characteristics, but is still able to express cytokine-inducing activity. These findings are interpreted according to our model of cell activation, which presumes the intercalation of endotoxin monomers into the target cell membrane.
- Top of page
- Materials and methods
OM-174 is a triacylated partial structure of the hexa-acylated natural lipid A. It has been reported to induce regression of tumors in rats bearing established colon tumors [7,8]. Furthermore, OM-174 was found not to be directly toxic to tumor cells, but the observed effects involved the host-mediated antitumor reaction.
Despite the fact that the backbone, a 1,4′-bisphosphorylated β(16)-linked diglucosamine, is identical for OM-174 and lipid A, their overall physicochemical behavior, as well as biological reactivity, is very different, as indicated by the Tc, the fluidity at the physiological temperature (37 °C), the supramolecular conformation of aggregates, and the molecular area within a monolayer. These parameters are, of course, not independent. For example, a reduction in the number of acyl chains leads to a decrease in Tc from 43 °C for the hexa-acyl lipid A to 30 °C for a penta-acyl structure and 15 °C for a tetra-acyl partial structure  down to 0 °C for the triacyl OM-174. Concomitantly, the supramolecular structures at 37 °C changes from an inverted cubic (hexa-acyl lipid A ), through a lamellar (tetra-acyl lipid A, unpublished data) to a micellar HI (OM-174) structure. The decrease in the number of acyl chains and the concomitant change in the supramolecular conformation are accompanied by the ability to bind increasing amounts of water in the interface region. Furthermore, the CMC, which can only be approximated for hexa-acyl lipid A to be lower than 0.1 µm, can be estimated for OM-174 to lie around 0.1 mm (unpublished results). The different physicochemical parameters, thus, vary systematically when going from the hexa-acyl to the triacyl compound.
The expression of biological activity of a given endotoxin is a very complex function of its physicochemical characteristics. It was found that a prerequisite for the induction of cytokine production in monocytes/macrophages is the tendency of the endotoxin molecules to adopt, at least partially, a non-lamellar inverted supramolecular structure implying a conical–concave shape for the individual molecules [22–24]. Endotoxins fulfilling this basic condition can be further distinguished by different acyl chain fluidities at 37 °C (corresponding to different Tc values), i.e. samples with the greatest fluidity at 37 °C exhibit highest biological activity and vice versa [22,23]. Great fluidity at 37 °C per se, however, is not necessarily related to biological activity; for example, lipid A from Rhodobacter capsulatus is largely inactive but has very fluid acyl chains . Whether endotoxin activity results more from the interaction of aggregates or from that of monomers with target cell membranes is a matter of controversy [26–29]. Irrespective of this, we have shown that the molecular shape of the lipid A moiety is a determinant of biological activity. Thus, the individual lipid A molecules in non-lamellar aggregate structures have a non-cylindrical conical–concave shape and may cause strong disturbance in the host cell membrane, for instance in the direct vicinity of a signal-transducing protein. It is known that the lipid A molecules either intercalate by themselves (hydrophobic interaction; unpublished results), or with the help of serum proteins such as LBP , into the target cell membranes. For both processes, the number of endotoxin monomers at a given concentration is important for the ability to intercalate into cell membranes. The ‘residence time’ of monomers within micellar structures is of the order of 10−4 s, whereas for lamellar structures this value is ≈ 10+4 s ; no data are available for inverted structures. Consequently, in the concentration range used in the biological test systems, all OM-174 aggregates are rapidly dissolved, and the free monomers may readily intercalate into the cell membranes of monocytes/macrophages. This is demonstrated in Fig. 8, which shows that OM-174 is incorporated in the presence of LBP, but also intercalates on its own, into a cell membrane corresponding to the composition of the macrophage membrane on a relatively short timescale. Lipid A and LPS do not intercalate in the absence of LBP at comparable or even higher concentrations . This is apparently related to the fact that samples with lamellar or inverted structures are stable, at least on a timescale of days, their disaggregation and subsequent transport into membranes being catalysed by proteins such as LBP and CD14 [30–33].
Biological activity of OM-174 on murine and human monocytes/macrophages should also be discussed in this context. Triacyl lipid A is a strong inducer of NO (Fig. 9) and a moderate inducer of IL-6 production (Fig. 10). These results may be understood in view of the ability of OM-174 to intercalate into target cell membranes as described above and by its preference for non-lamellar aggregate structures, although micellar rather than inverted, leading to a conical–convex shape of the individual molecules. These conical molecules may exert mechanical stress on the putative signaling protein, as proposed earlier for conical–concave shaped hexa-acyl lipid A .
Also the lack of LAL activity of OM-174 (Table 1) may be seen in the light of its unusual physicochemical characteristics. It has been shown that LPS monomers activate the enzymatic LAL reaction cascade to a much smaller extent  than aggregates at comparable concentrations. In the LAL activity measurements, for the reasons discussed above, OM-174 is present mainly in the monomeric state, whereas LPS is present in the aggregated state.
Our findings seem to suggest that widely accepted general ideas on the molecular requirements for endotoxic molecules to express biological activity should be revised. It was thought that full biological activity is expressed by a lipid A molecule consisting of a hexa-acylated, bisphosphorylated β1,6-linked d-GlcN disaccharide [2,34]. This was based on observations in various different test systems, in particular cytokine-inducing capacity, LAL activity, pyrogenicity, and lethal toxicity in animal models. All these activities are usually observed for endotoxically active compounds, but not for inactive compounds, except for LAL activity, which is sometimes found for otherwise inactive substances . Here, we describe an endotoxin partial structure with high cytokine-inducing capacity but almost no LAL activity.
In this context, the results of Funatogawa et al.  should be mentioned. These authors found cytokine-inducing capacity for a synthetic triacyl monosaccharide lipid A partial structure (GLA-60), although the applied lipid concentration was two orders of magnitude higher (10 µg·mL−1) than that of hexa-acyl lipid A (100 ng·mL−1). This has never previously been observed for a monosaccharide lipid A partial structure (see review ). From these results and the findings presented here for OM-174, it should be emphasized, that in vivo studies on pyrogenicity and lethal toxicity should also be included to provide further valuable information on structural requirements for the different aspects of endotoxicity.
In summary, we have characterized a triacyl lipid A partial structure, OM-174, which has the ability to induce cytokine responses but has very low LAL activity. These observations have been interpreted in the light of the molecular and supramolecular characteristics that we have determined, to give insights into the nature of the biologically active unit of lipid A and endotoxin. The findings of biological activity for the triacyl lipid A partial structure OM-174 in terms of its capacity to induce NO and IL-6 production in monocytes/macrophages, in relation to its physicochemical characteristics and, in particular to respective data for hexa-acyl lipid A, emphasize the necessity for comprehensive analysis, as presented here, for an understanding of endotoxin reactions.