Influence of synthetic antiendotoxin peptides on lipopolysaccharide (LPS) recognition and LPS-induced proinflammatory cytokine responses by cells expressing membrane-bound CD14

Abstract

Lipopolysaccharides (LPS) are proinflammatory bacterial products implicated in the pathogenesis of gram-negative sepsis and septic shock. Polymyxin B (PMB), a cyclic, cationic peptide antibiotic, inhibits biological activities of LPS through high-affinity binding to the lipid A moiety. Small synthetic peptides have been designed to mimic the primary and secondary structures of PMB to determine structural requirements for binding and detoxification of lipid A and to assess possible therapeutic potential. The purpose of this study was to compare and contrast the endotoxin-neutralizing activities of two synthetic antiendotoxin peptides (SAEP-2 and SAEP-4), PMB, and an LPS core-specific monoclonal antibody (MAb), WN1 222-5, based on their abilities to inhibit CD14-mediated target cell uptake of fluorescein isothiocyanate (FITC)-conjugated LPS, detected by flow cytometry and confocal microscopy, and LPS-induced production of the proinflammatory cytokines, interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha), as measured by bioassays. PMB and SAEP-4 produced dose-dependent inhibition of FITC-LPS uptake by CD14-transfected Chinese hamster ovary fibroblasts (CHO-CD14 cells) and by human peripheral blood mononuclear cells. The anti-LPS MAb, WN1 222-5, also blocked LPS uptake by these cells and synergized with PMB and SAEP-4. LPS-induced IL-6 release was inhibited by PMB, SAEP-4, and MAb WN1 222-5, and these inhibitory activities were additive or synergistic. LPS-induced TNF-alpha release by PBMC was also inhibited by PMB and SAEP-4 alone and in combination with anti-LPS MAb. SAEP-2, in contrast, produced comparatively minor decrements in cellular uptake of LPS and LPS-induced cytokine responses, and did so only in the absence of serum, while a nonsense peptide exerted no discernible inhibitory effect on LPS uptake or LPS-induced cytokine expression in the presence or absence of serum. Thus, PMB and SAEP-4, like the LPS-reactive MAb, WN1 222-5, block proinflammatory activities of LPS in part by preventing LPS recognition by membrane-bound CD14-expressing target cells. Differences in peptide structure, however, like those exemplified by SAEP-2 and SAEP-4, may differentially affect the endotoxin-neutralizing potency of these peptides despite similar binding activity against lipid A, reflecting possible differences in peptide solubility or peptide regulation of intracellular signal transduction.

FIG. 1

Amino acid sequences of peptides employed in this study. DAB, α,γ-diaminobutyric acid; X, 6-methyl/heptanoyloctanoyl; Thr, threonine; Phe, phenylalanine; Leu, leucine; Lys, lysine; Cys, cysteine.

FIG. 2

Inhibitory effects of various peptides on the uptake of FITC-conjugated E. coli O111:B4 LPS by CHO-CD14 cells (left side of figure) and by human PBMC (right side of figure) in the presence of different concentrations of NHS. FITC-conjugated LPS (1 μg/ml) was preincubated with peptides, in the presence or absence of NHS, at 37°C for 15 min, and the mixture was then added to cells and incubated at 37°C for an additional 30 min. Cell-associated fluorescence was measured by flow cytometry, and the data are expressed as the MFI. Panels A to D document the influence of PMB, SAEP-4, SAEP-2, and “nonsense peptide,” respectively, on the cell uptake of LPS. The P value shown in parentheses below each horizontal axis indicates the statistical significance, determined by two-way ANOVA, of differences in MFI produced by the material specified for that axis. N.S., not significant (P > 0.05).

FIG. 3

Inhibitory effects of various peptides, alone or in combination with LPS core-specific MAb (WN1 222-5) on the uptake of FITC-conjugated E. coli O111:B4 LPS by CHO-CD14 cells (left side of figure) and by PBMC (right side of figure). See Fig. 2 legend for experimental procedures, except that NHS was held constant in this experiment at 10% (vol/vol) and WN1 222-5 was introduced at various concentrations as shown. Panels A to D document the influence of PMB, SAEP-4, SAEP-2, and “nonsense peptide,” respectively, on the cell uptake of LPS. The P values shown in parentheses below each horizontal axis indicate the statistical significance, as determined by two-way ANOVA, of differences in MFI produced by the material specified for that axis. N.S., not significant (P > 0.05).

FIG. 4

Inhibitory effects of various peptides on LPS-induced IL-6 production by CHO-CD14 cells (left side of figure) and by human PBMC (right side of figure) in the presence of different concentrations of NHS. E. coli O111:B4 LPS at 1 μg/ml was preincubated with peptides in the presence or absence of NHS at 37°C for 15 min, and the mixture was then added to cells and incubated at 37°C for an additional 2 h in an atmosphere containing 5% CO₂. The supernatants were collected and IL-6 concentration measured by using the B9 cell proliferation assay. The data shown are representative of those obtained in three similar experiments. Panels A to D document the influence of PMB, SAEP-4, SAEP-2, and “nonsense peptide,” respectively, on LPS-induced IL-6 release. The P values shown in parentheses below each horizontal axis indicate the statistical significance, as determined by two-way ANOVA, of differences in IL-6 concentration produced by the material specified for that axis. N.S., not significant (P > 0.05).

FIG. 5

Inhibitory effect of various peptides, alone or in combination with LPS core-specific MAb (WN1 222-5), on LPS-induced IL-6 production by CHO-CD14 cells (left side of figure) and by human PBMC (right side of figure). See Fig. 4 legend for experimental procedures, except that in this experiment the concentration of NHS was held constant at 10% (vol/vol), and the concentration of WN1 222-5 was varied as indicated. The data shown are representative of those obtained in three similar experiments. Panels A to D document the influence of PMB, SAEP-4, SAEP-2, and “nonsense peptide,” respectively, on LPS-induced IL-6 release. The P values shown in parentheses below each horizontal axis indicate the statistical significance, as determined by two-way ANOVA, of differences in IL-6 concentration produced by the material specified for that axis. N.S., not significant (P > 0.05).

FIG. 6

Inhibitory effects of various peptides, alone or in combination with LPS core-specific MAb (WN1 222-5), on LPS-induced TNF-α production by human PBMC. See Fig. 4 legend for experimental procedures, except for the following: (i) in this experiment the concentration of NHS was held constant at 10% (vol/vol), and the concentration of WN1 222-5 was varied as indicated, and (ii) supernatant TNF-α concentrations were measured by using the L929 cell cytotoxicity assay. The P values shown in parentheses below each horizontal axis indicate the statistical significance, as determined by two-way ANOVA, of differences in TNF-α concentration produced by the material specified for that axis. N.S., not significant (P > 0.05).

FIG. 7

Laser scanning confocal microscopic images of representative CHO-CD14 cells exposed to 1 μg of FITC-conjugated E. coli O111:B4 LPS per ml in the presence or absence of various peptides and LPS core-reactive MAb WN1 222-5. LPS was preincubated at 37°C for 15 min, with or without peptide and/or anti-LPS MAb, in the presence of 10% (vol/vol) NHS. The mixture was then added to CHO-CD14 cells, followed by further incubation at 37°C for 30 min. Panels A to H show reaction mixtures containing, in addition to FITC-LPS, 10% NHS, and cells, the following reactants: A, none; B, PMB; C, SAEP-2; D, SAEP-4; E, “nonsense peptide”; F, anti-LPS MAb; G, PMB plus anti-LPS MAb; and H, SAEP-4 plus anti-LPS MAb. Single cells averaging 15 μm in diameter appear in each panel. A representative cell is shown for each specified set of conditions, selected from among at least 50 randomly observed cells. The experiment was repeated twice with similar results. Fluorescence intensity is color coded on a linear scale as follows: red > yellow > green > blue > black (background).