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. 1996 Sep;64(9):3592–3600. doi: 10.1128/iai.64.9.3592-3600.1996

Potent CD14-mediated signalling of human leukocytes by Escherichia coli can be mediated by interaction of whole bacteria and host cells without extensive prior release of endotoxin.

S S Katz 1, K Chen 1, S Chen 1, M E Doerfler 1, P Elsbach 1, J Weiss 1
PMCID: PMC174268  PMID: 8751904

Abstract

How invading microorganisms are detected by the host has not been well defined. We have compared the abilities of Escherichia coli and lipopolysaccharides (LPS) purified from these bacteria to prime isolated neutrophils for phorbol myristate acetate-stimulated arachidonate release, to trigger respiratory burst in 1% blood, and to increase steady-state levels of tumor necrosis factor alpha mRNA in whole blood. In all three assays, bacteria were > or = 10-fold more potent than equivalent amounts of LPS and could trigger maximal cellular responses at ratios as low as one bacterium per 20 to 200 leukocytes. Both E. coli and LPS-triggered responses were enhanced by LPS-binding protein and inhibited by an anti-CD14 monoclonal antibody and the bactericidal/permeability-increasing protein (BPI). However, whereas O polysaccharide did not affect the potency of isolated LPS, intact E. coli carrying long-chain LPS (O111:B4) was less potent than rough E. coli (J5). Furthermore, material collected by filtration or centrifugation of bacteria incubated under conditions used to trigger arachidonate release or chemiluminescence was 5- or 30-fold less active, respectively, than whole bacterial suspensions. Extracellular BPI (not bound to bacteria) inhibited bacterial signalling, but BPI bound to bacteria was much more potent. Taken together, these findings indicate that E. coli cells can strongly signal their presence to human leukocytes not only by shedding LPS into surrounding fluids but also by exposing endotoxin at or near their surface during direct interaction with host cells.

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Selected References

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  1. Dedrick R. L., Conlon P. J. Prolonged expression of lipopolysaccharide (LPS)-induced inflammatory genes in whole blood requires continual exposure to LPS. Infect Immun. 1995 Apr;63(4):1362–1368. doi: 10.1128/iai.63.4.1362-1368.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Doerfler M. E., Weiss J., Clark J. D., Elsbach P. Bacterial lipopolysaccharide primes human neutrophils for enhanced release of arachidonic acid and causes phosphorylation of an 85-kD cytosolic phospholipase A2. J Clin Invest. 1994 Apr;93(4):1583–1591. doi: 10.1172/JCI117138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. ELBEIN A. D., HEATH E. C. THE BIOSYNTHESIS OF CELL WALL LIPOPOLYSACCHARIDE IN ESCHERICHIA COLI. I. THE BIOCHEMICAL PROPERTIES OF A URIDINE DIPHOSPHATE GALACTOSE 4-EPIMERASELESS MUTANT. J Biol Chem. 1965 May;240:1919–1925. [PubMed] [Google Scholar]
  4. Elsbach P. Antibiotics from within: antibacterials from human and animal sources. Trends Biotechnol. 1990 Jan;8(1):26–30. doi: 10.1016/0167-7799(90)90127-j. [DOI] [PubMed] [Google Scholar]
  5. Elsbach P., Weiss J., Levy O. Integration of antimicrobial host defenses: role of the bactericidal/permeability-increasing protein. Trends Microbiol. 1994 Sep;2(9):324–328. doi: 10.1016/0966-842x(94)90449-9. [DOI] [PubMed] [Google Scholar]
  6. Elsbach P., Weiss J. Prospects for use of recombinant BPI in the treatment of gram-negative bacterial infections. Infect Agents Dis. 1995 Jun;4(2):102–109. [PubMed] [Google Scholar]
  7. Elsbach P., Weiss J. The bactericidal/permeability-increasing protein (BPI), a potent element in host-defense against gram-negative bacteria and lipopolysaccharide. Immunobiology. 1993 Apr;187(3-5):417–429. doi: 10.1016/S0171-2985(11)80354-2. [DOI] [PubMed] [Google Scholar]
  8. Galanos C., Lehmann V., Lüderitz O., Rietschel E. T., Westphal O., Brade H., Brade L., Freudenberg M. A., Hansen-Hagge T., Lüderitz T. Endotoxic properties of chemically synthesized lipid A part structures. Comparison of synthetic lipid A precursor and synthetic analogues with biosynthetic lipid A precursor and free lipid A. Eur J Biochem. 1984 Apr 16;140(2):221–227. doi: 10.1111/j.1432-1033.1984.tb08090.x. [DOI] [PubMed] [Google Scholar]
  9. Gallay P., Carrel S., Glauser M. P., Barras C., Ulevitch R. J., Tobias P. S., Baumgartner J. D., Heumann D. Purification and characterization of murine lipopolysaccharide-binding protein. Infect Immun. 1993 Feb;61(2):378–383. doi: 10.1128/iai.61.2.378-383.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Galloway S. M., Raetz C. R. A mutant of Escherichia coli defective in the first step of endotoxin biosynthesis. J Biol Chem. 1990 Apr 15;265(11):6394–6402. [PubMed] [Google Scholar]
  11. Gazzano-Santoro H., Parent J. B., Grinna L., Horwitz A., Parsons T., Theofan G., Elsbach P., Weiss J., Conlon P. J. High-affinity binding of the bactericidal/permeability-increasing protein and a recombinant amino-terminal fragment to the lipid A region of lipopolysaccharide. Infect Immun. 1992 Nov;60(11):4754–4761. doi: 10.1128/iai.60.11.4754-4761.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Golenbock D. T., Hampton R. Y., Raetz C. R., Wright S. D. Human phagocytes have multiple lipid A-binding sites. Infect Immun. 1990 Dec;58(12):4069–4075. doi: 10.1128/iai.58.12.4069-4075.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Grunwald U., Oeser G., Schröder H. D., Ritscher D., Schütt C. Bindungsmodalitäten von LPS an CD14. Immun Infekt. 1992 Jul;20(3):86–87. [PubMed] [Google Scholar]
  14. Haziot A., Chen S., Ferrero E., Low M. G., Silber R., Goyert S. M. The monocyte differentiation antigen, CD14, is anchored to the cell membrane by a phosphatidylinositol linkage. J Immunol. 1988 Jul 15;141(2):547–552. [PubMed] [Google Scholar]
  15. Haziot A., Tsuberi B. Z., Goyert S. M. Neutrophil CD14: biochemical properties and role in the secretion of tumor necrosis factor-alpha in response to lipopolysaccharide. J Immunol. 1993 Jun 15;150(12):5556–5565. [PubMed] [Google Scholar]
  16. Howard C. J., Glynn A. A. The virulence for mice of strains of Escherichia coli related to the effects of K antigens on their resistance to phagocytosis and killing by complement. Immunology. 1971 May;20(5):767–777. [PMC free article] [PubMed] [Google Scholar]
  17. Ingalls R. R., Golenbock D. T. CD11c/CD18, a transmembrane signaling receptor for lipopolysaccharide. J Exp Med. 1995 Apr 1;181(4):1473–1479. doi: 10.1084/jem.181.4.1473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jack R. S., Grunwald U., Stelter F., Workalemahu G., Schütt C. Both membrane-bound and soluble forms of CD14 bind to gram-negative bacteria. Eur J Immunol. 1995 May;25(5):1436–1441. doi: 10.1002/eji.1830250545. [DOI] [PubMed] [Google Scholar]
  19. Landmann R., Scherer F., Schumann R., Link S., Sansano S., Zimmerli W. LPS directly induces oxygen radical production in human monocytes via LPS binding protein and CD14. J Leukoc Biol. 1995 Mar;57(3):440–449. doi: 10.1002/jlb.57.3.440. [DOI] [PubMed] [Google Scholar]
  20. Levy O., Ooi C. E., Elsbach P., Doerfler M. E., Lehrer R. I., Weiss J. Antibacterial proteins of granulocytes differ in interaction with endotoxin. Comparison of bactericidal/permeability-increasing protein, p15s, and defensins. J Immunol. 1995 May 15;154(10):5403–5410. [PubMed] [Google Scholar]
  21. Mannion B. A., Kalatzis E. S., Weiss J., Elsbach P. Preferential binding of the neutrophil cytoplasmic granule-derived bactericidal/permeability increasing protein to target bacteria. Implications and use as a means of purification. J Immunol. 1989 Apr 15;142(8):2807–2812. [PubMed] [Google Scholar]
  22. Marra M. N., Thornton M. B., Snable J. L., Wilde C. G., Scott R. W. Endotoxin-binding and -neutralizing properties of recombinant bactericidal/permeability-increasing protein and monoclonal antibodies HA-1A and E5. Crit Care Med. 1994 Apr;22(4):559–565. doi: 10.1097/00003246-199404000-00009. [DOI] [PubMed] [Google Scholar]
  23. Marra M. N., Wilde C. G., Collins M. S., Snable J. L., Thornton M. B., Scott R. W. The role of bactericidal/permeability-increasing protein as a natural inhibitor of bacterial endotoxin. J Immunol. 1992 Jan 15;148(2):532–537. [PubMed] [Google Scholar]
  24. Marra M. N., Wilde C. G., Griffith J. E., Snable J. L., Scott R. W. Bactericidal/permeability-increasing protein has endotoxin-neutralizing activity. J Immunol. 1990 Jan 15;144(2):662–666. [PubMed] [Google Scholar]
  25. Mattsby-Baltzer I., Lindgren K., Lindholm B., Edebo L. Endotoxin shedding by enterobacteria: free and cell-bound endotoxin differ in Limulus activity. Infect Immun. 1991 Feb;59(2):689–695. doi: 10.1128/iai.59.2.689-695.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mészáros K., Parent J. B., Gazzano-Santoro H., Little R., Horwitz A., Parsons T., Theofan G., Grinna L., Weickmann J., Elsbach P. A recombinant amino terminal fragment of bactericidal/permeability-increasing protein inhibits the induction of leukocyte responses by LPS. J Leukoc Biol. 1993 Dec;54(6):558–563. doi: 10.1002/jlb.54.6.558. [DOI] [PubMed] [Google Scholar]
  27. Ooi C. E., Weiss J., Doerfler M. E., Elsbach P. Endotoxin-neutralizing properties of the 25 kD N-terminal fragment and a newly isolated 30 kD C-terminal fragment of the 55-60 kD bactericidal/permeability-increasing protein of human neutrophils. J Exp Med. 1991 Sep 1;174(3):649–655. doi: 10.1084/jem.174.3.649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ooi C. E., Weiss J., Elsbach P., Frangione B., Mannion B. A 25-kDa NH2-terminal fragment carries all the antibacterial activities of the human neutrophil 60-kDa bactericidal/permeability-increasing protein. J Biol Chem. 1987 Nov 5;262(31):14891–14894. [PubMed] [Google Scholar]
  29. Peterson P. K., Gekker G., Hu S., Sheng W. S., Anderson W. R., Ulevitch R. J., Tobias P. S., Gustafson K. V., Molitor T. W., Chao C. C. CD14 receptor-mediated uptake of nonopsonized Mycobacterium tuberculosis by human microglia. Infect Immun. 1995 Apr;63(4):1598–1602. doi: 10.1128/iai.63.4.1598-1602.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Raetz C. R. Biochemistry of endotoxins. Annu Rev Biochem. 1990;59:129–170. doi: 10.1146/annurev.bi.59.070190.001021. [DOI] [PubMed] [Google Scholar]
  31. Rietschel E. T., Brade H. Bacterial endotoxins. Sci Am. 1992 Aug;267(2):54–61. doi: 10.1038/scientificamerican0892-54. [DOI] [PubMed] [Google Scholar]
  32. Rietschel E. T., Kim Y. B., Watson D. W., Galanos C., Lüderitz O., Westphal O. Pyrogenicity and immunogenicity of lipid A complexed with bovine serum albumin or human serum albumin. Infect Immun. 1973 Aug;8(2):173–177. doi: 10.1128/iai.8.2.173-177.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rietschel E. T., Kirikae T., Schade F. U., Mamat U., Schmidt G., Loppnow H., Ulmer A. J., Zähringer U., Seydel U., Di Padova F. Bacterial endotoxin: molecular relationships of structure to activity and function. FASEB J. 1994 Feb;8(2):217–225. doi: 10.1096/fasebj.8.2.8119492. [DOI] [PubMed] [Google Scholar]
  34. Rietschel E. T., Kirikae T., Schade F. U., Ulmer A. J., Holst O., Brade H., Schmidt G., Mamat U., Grimmecke H. D., Kusumoto S. The chemical structure of bacterial endotoxin in relation to bioactivity. Immunobiology. 1993 Apr;187(3-5):169–190. doi: 10.1016/S0171-2985(11)80338-4. [DOI] [PubMed] [Google Scholar]
  35. Rietschel E. T., Schade U., Jensen M., Wollenweber H. W., Lüderitz O., Greisman S. G. Bacterial endotoxins: chemical structure, biological activity and role in septicaemia. Scand J Infect Dis Suppl. 1982;31:8–21. [PubMed] [Google Scholar]
  36. Schumann R. R., Leong S. R., Flaggs G. W., Gray P. W., Wright S. D., Mathison J. C., Tobias P. S., Ulevitch R. J. Structure and function of lipopolysaccharide binding protein. Science. 1990 Sep 21;249(4975):1429–1431. doi: 10.1126/science.2402637. [DOI] [PubMed] [Google Scholar]
  37. Somerville J. E., Jr, Cassiano L., Bainbridge B., Cunningham M. D., Darveau R. P. A novel Escherichia coli lipid A mutant that produces an antiinflammatory lipopolysaccharide. J Clin Invest. 1996 Jan 15;97(2):359–365. doi: 10.1172/JCI118423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tesh V. L., Duncan R. L., Jr, Morrison D. C. The interaction of Escherichia coli with normal human serum: the kinetics of serum-mediated lipopolysaccharide release and its dissociation from bacterial killing. J Immunol. 1986 Aug 15;137(4):1329–1335. [PubMed] [Google Scholar]
  39. Tesh V. L., Morrison D. C. The interaction of Escherichia coli with normal human serum: factors affecting the capacity of serum to mediate lipopolysaccharide release. Microb Pathog. 1988 Mar;4(3):175–187. doi: 10.1016/0882-4010(88)90068-x. [DOI] [PubMed] [Google Scholar]
  40. Tesh V. L., Morrison D. C. The physical-chemical characterization and biologic activity of serum released lipopolysaccharides. J Immunol. 1988 Nov 15;141(10):3523–3531. [PubMed] [Google Scholar]
  41. Tobias P. S., Soldau K., Gegner J. A., Mintz D., Ulevitch R. J. Lipopolysaccharide binding protein-mediated complexation of lipopolysaccharide with soluble CD14. J Biol Chem. 1995 May 5;270(18):10482–10488. doi: 10.1074/jbc.270.18.10482. [DOI] [PubMed] [Google Scholar]
  42. Tobias P. S., Soldau K., Kline L., Lee J. D., Kato K., Martin T. P., Ulevitch R. J. Cross-linking of lipopolysaccharide (LPS) to CD14 on THP-1 cells mediated by LPS-binding protein. J Immunol. 1993 Apr 1;150(7):3011–3021. [PubMed] [Google Scholar]
  43. Tobias P. S., Soldau K., Ulevitch R. J. Identification of a lipid A binding site in the acute phase reactant lipopolysaccharide binding protein. J Biol Chem. 1989 Jun 25;264(18):10867–10871. [PubMed] [Google Scholar]
  44. Tobias P. S., Soldau K., Ulevitch R. J. Isolation of a lipopolysaccharide-binding acute phase reactant from rabbit serum. J Exp Med. 1986 Sep 1;164(3):777–793. doi: 10.1084/jem.164.3.777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Ulevitch R. J., Tobias P. S. Receptor-dependent mechanisms of cell stimulation by bacterial endotoxin. Annu Rev Immunol. 1995;13:437–457. doi: 10.1146/annurev.iy.13.040195.002253. [DOI] [PubMed] [Google Scholar]
  46. Van Dijk W. C., Verbrugh H. A., van der Tol M. E., Peters R., Verhoef J. Role of Escherichia coli K capsular antigens during complement activation, C3 fixation, and opsonization. Infect Immun. 1979 Aug;25(2):603–609. doi: 10.1128/iai.25.2.603-609.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Vosbeck K., Tobias P., Mueller H., Allen R. A., Arfors K. E., Ulevitch R. J., Sklar L. A. Priming of polymorphonuclear granulocytes by lipopolysaccharides and its complexes with lipopolysaccharide binding protein and high density lipoprotein. J Leukoc Biol. 1990 Feb;47(2):97–104. doi: 10.1002/jlb.47.2.97. [DOI] [PubMed] [Google Scholar]
  48. Weiss J., Elsbach P., Shu C., Castillo J., Grinna L., Horwitz A., Theofan G. Human bactericidal/permeability-increasing protein and a recombinant NH2-terminal fragment cause killing of serum-resistant gram-negative bacteria in whole blood and inhibit tumor necrosis factor release induced by the bacteria. J Clin Invest. 1992 Sep;90(3):1122–1130. doi: 10.1172/JCI115930. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Weiss J., Inada M., Elsbach P., Crowl R. M. Structural determinants of the action against Escherichia coli of a human inflammatory fluid phospholipase A2 in concert with polymorphonuclear leukocytes. J Biol Chem. 1994 Oct 21;269(42):26331–26337. [PubMed] [Google Scholar]
  50. Williams P., Lambert P. A., Brown M. R., Jones R. J. The role of the O and K antigens in determining the resistance of Klebsiella aerogenes to serum killing and phagocytosis. J Gen Microbiol. 1983 Jul;129(7):2181–2191. doi: 10.1099/00221287-129-7-2181. [DOI] [PubMed] [Google Scholar]
  51. Wright S. D., Detmers P. A., Aida Y., Adamowski R., Anderson D. C., Chad Z., Kabbash L. G., Pabst M. J. CD18-deficient cells respond to lipopolysaccharide in vitro. J Immunol. 1990 Apr 1;144(7):2566–2571. [PubMed] [Google Scholar]
  52. Wright S. D., Jong M. T. Adhesion-promoting receptors on human macrophages recognize Escherichia coli by binding to lipopolysaccharide. J Exp Med. 1986 Dec 1;164(6):1876–1888. doi: 10.1084/jem.164.6.1876. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Wright S. D., Levin S. M., Jong M. T., Chad Z., Kabbash L. G. CR3 (CD11b/CD18) expresses one binding site for Arg-Gly-Asp-containing peptides and a second site for bacterial lipopolysaccharide. J Exp Med. 1989 Jan 1;169(1):175–183. doi: 10.1084/jem.169.1.175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Wright S. D. Multiple receptors for endotoxin. Curr Opin Immunol. 1991 Feb;3(1):83–90. doi: 10.1016/0952-7915(91)90082-c. [DOI] [PubMed] [Google Scholar]
  55. Wright S. D., Ramos R. A., Hermanowski-Vosatka A., Rockwell P., Detmers P. A. Activation of the adhesive capacity of CR3 on neutrophils by endotoxin: dependence on lipopolysaccharide binding protein and CD14. J Exp Med. 1991 May 1;173(5):1281–1286. doi: 10.1084/jem.173.5.1281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Wright S. D., Ramos R. A., Tobias P. S., Ulevitch R. J., Mathison J. C. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science. 1990 Sep 21;249(4975):1431–1433. doi: 10.1126/science.1698311. [DOI] [PubMed] [Google Scholar]
  57. Wurfel M. M., Hailman E., Wright S. D. Soluble CD14 acts as a shuttle in the neutralization of lipopolysaccharide (LPS) by LPS-binding protein and reconstituted high density lipoprotein. J Exp Med. 1995 May 1;181(5):1743–1754. doi: 10.1084/jem.181.5.1743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Wurfel M. M., Kunitake S. T., Lichenstein H., Kane J. P., Wright S. D. Lipopolysaccharide (LPS)-binding protein is carried on lipoproteins and acts as a cofactor in the neutralization of LPS. J Exp Med. 1994 Sep 1;180(3):1025–1035. doi: 10.1084/jem.180.3.1025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Yu B., Wright S. D. Catalytic properties of lipopolysaccharide (LPS) binding protein. Transfer of LPS to soluble CD14. J Biol Chem. 1996 Feb 23;271(8):4100–4105. doi: 10.1074/jbc.271.8.4100. [DOI] [PubMed] [Google Scholar]

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