Mycobacteriology Laboratory, William S. Middleton Memorial Veterans ... by toxic deep-rough-chemotype lipopolysaccharide (ReLPS) of Escherichia coli in a.
INFECTION AND IMMUNITY, Apr. 1989, p. 1336-1338 0019-9567/89/041336-03$02.00/0 Copyright © 1989, American Society for Microbiology
Vol. 57, No. 4
Diphosphoryl Lipid A from Rhodopseudomonas sphaeroides ATCC 17023 Blocks Induction of Cachectin in Macrophages by Lipopolysaccharide KUNI TAKAYAMA,l .* NILOFER QURESHI,"2 BRUCE BEUTLER,3 AND THEO N. KIRKLAND4'5 Mycobacteriology Laboratory, William S. Middleton Memorial Veterans Hospital, Madison, Wisconsin 537051; Department of Bacteriology, College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin, 537062; Howard Hughes Medical Institute Research Laboratories, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 752353; and Division of Infectiouis Diseases, Veterans Administration Medical Center,4 and
Department of Pathology and Medicine, University of California,5 San Diego, California 92161 Received 7 November 1988/Accepted 10 January 1989
Purified diphosphoryl lipid A (DPLA) obtained from the nontoxic lipopolysaccharide of Rhodopseudomonas sphaeroides ATCC 17023 was shown to block the induction of cachectin (tumor necrosis factor) in the RAW 264.7 macrophage cell line by toxic deep-rough-chemotype lipopolysaccharide (ReLPS) of Escherichia coli in a concentration-dependent manner. The ReLPS-to-DPLA mass ratios of 1:10 and 1:100 (when 1.0 ng of ReLPS per ml was used) gave 55 and 95% inhibitions, respectively, of the induction of cachectin. Since the structure of the DPLA from R. sphaeroides is so similar to that of the lipid A moiety of the toxic ReLPS from E. coli, we suggest that this inhibition could have been due to competitive binding by DPLA to the active sites on the macrophage. This DPLA could become a useful reagent to study the nature of lipopolysaccharide/lipid A binding in macrophages and perhaps other responding cells. The lipopolysaccharide (LPS) obtained from Rhodopseudomonas sphaeroides ATCC 17023 was reported by Strittmatter et al. (21) to be nontoxic. The complete structure of the LPS from this source has now been established (14, 18, 19). The structure of the lipid A moiety of the LPS from R. sphaeroides is strikingly similar to that of the lipid A of toxic enterobacterial and Salmonella LPS (9, 22). The only two major differences noted are the presence of a 3-ketodecanoate instead of a 3-hydroxytetradecanoate at the 2 position (R4) and a \7-tetradecenoate instead of a tetradecanoate in acyloxyacyl linkage at the 2' position (R2) of the glucosamine disaccharide of the R. sphaeroides lipid A (Fig. 1). Because of this close resemblance in structures, we wondered whether this nontoxic LPS or its derivative could serve as an antagonist in the activation of macrophages by the toxic LPS. For these experiments, we chose the diphosphoryl lipid A (DPLA) prepared from the LPS of R. sphaeroides to be the antagonist because it can be easily obtained in a highly purified form and it is similar to the toxic DPLA from the LPS of Salmonella typhimurium (25). The LPS from R. sphaeroides grown at 26°C, prepared by a previously described method (14), was hydrolyzed with 0.02 M sodium acetate (pH 2.5) at 100°C for 90 min. The free acid of DPLA was fractionated on Silica Gel H thin-layer plates (500 p.m thick; 20 by 20 cm) at a load of 4.0 mg per plate, using the solvent system chloroform-methanol-water-concentrated ammonium hydroxide (50:25:4:2, vol/vol). The DPLA band was visualized with iodine vapor and recovered from the plate. It was characterized as the tetramethyl derivative by plasma desorption mass spectrometry. It is nontoxic based on the chicken embryo lethality test (50% chicken embryo lethal dose, >20 p.g; K. Takayama, N. Qureshi, and K. R. Myers, unpublished results); its structure is shown in Fig. 1. For the agonist to activate the RAW 264.7 murine macro*
phage cell line, we chose the toxic deep-rough-chemotype LPS (ReLPS) from Escherichia coli D31m4, which was recently purified and characterized (15). We found that the DPLA from R. sphaeroides blocked the induction of cachectin (tumor necrosis factor [TNF]) by the RAW 264.7 cells. This is the first clear example of an analog of lipid A showing strong antagonism against a toxic agonist in the induction of cachectin. The immunoblot method was used to quantitate TNF production by RAW 264.7 murine macrophage cells. RAW 264.7 cells (4, 16) were seeded in 24-well plates (Nunc) at a density of 3 x 105 cells per well in Dulbecco modified Eagle medium supplemented with 5% fetal calf serum. After 12 h, cell monolayers were washed twice with 1 ml of serum-free medium and then left covered with 2 p1 of the same medium. An aqueous suspension of DPLA or ReLPS or both was then added to the final concentration indicated. The cells were incubated for 12 h, after which the medium was removed for measurement of TNF by immunoblotting. A 100-p.1 portion of medium was mixed with 100 p1I of sodium dodecyl sulfate-containing sample buffer, heated to 100°C for 5 min, and subjected to electrophoresis in a 10 to 15% polyacrylamide gradient gel. Proteins were then transferred to nitrocellulose electrophoretically, and TNF was visualized by the use of a rabbit anti-mouse TNF polyclonal serum (3, 5) applied at a 1:100 dilution, followed by alkaline phosphataseconjugated goat anti-rabbit immunoglobulin G (Bio-Rad Laboratories). The toxic ReLPS from E. coli caused the induction of cachectin by RAW 264.7 cells at all concentrations tested (1 to 100 ng/ml) (Fig. 2). Optimal induction occurred at 10 ng of ReLPS per ml. The DPLA of R. sphaeroides was not able to induce the formation of cachectin at 1 to 1,000 ng/ml. We observed only slight induction at 104 ng/ml. When the DPLA of R. sphaeroides was added together with 10 ng of ReLPS per ml, we observed definite inhibition in induction at 103 ng of DPLA per ml (ReLPS-to-DPLA mass ratio of 1:100). This
Corresponding author. 1336
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NOTES
TABLE 1. Induction of TNF by ReLPS, lack of induction by DPLA from R. sphaeroides, and blocking of induction by pretreatment with DPLA, using RAW 264.7 cells and the indicator cell line L929 Dilution for 50% killing'
Treatment (ng/ml)
ReLPS 0.1 1 10 100 DPLA 10 100 1,000 10,000
(R2) FIG. 1. Structure of the predominant form of DPLA obtained from the LPS of R. sphaeroides ATCC 17023, where R1 and R3 are 3-hydroxydecanoate, R2 is zA7-tetradecanoyloxytetradecanoate, and R4 is 3-ketotetradecanoate. The cis or trans configuration of the double bond in the A7-tetradecanoate is not established.
inhibition was probably maximal at
