Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1997 Jun;65(6):2272–2277. doi: 10.1128/iai.65.6.2272-2277.1997

Saturable CD14-dependent binding of fluorescein-labeled lipopolysaccharide to human monocytes.

A Troelstra 1, P Antal-Szalmas 1, L A de Graaf-Miltenburg 1, A J Weersink 1, J Verhoef 1, K P Van Kessel 1, J A Van Strijp 1
PMCID: PMC175315  PMID: 9169763

Abstract

We used rough lipopolysaccharide (ReLPS) to construct a fluorescein-labeled LPS (FITC-LPS) with a very high labeling efficiency that bound to isolated human monocytes in a CD14-dependent fashion and that in this respect behaved indistinctively from native LPS. The CD14-dependent binding could be inhibited either by a 1,000-fold excess of unlabeled LPS or by polymyxin B, bactericidal/permeability-increasing protein, cationic protein 18, or soluble CD14. Although this FITC-LPS preparation no longer possessed the ability to prime neutrophils for the production of reactive oxygen species or to stimulate human monocytes to produce tumor necrosis factor, activation of the Limulus amoebocyte lysate cascade was comparable to activation by native LPS. Binding to monocytes was enhanced by human pooled serum (HPS) or LPS-binding protein (LBP) for LPS concentrations up to 100 ng/ml and was completely CD14 dependent. For LPS concentrations exceeding 100 ng/ml, binding was still partially CD14 dependent, but not HPS or LBP dependent. CD14-dependent association of LPS with monocytes was shown to be totally saturable. In conclusion, we found an HPS- or LBP-dependent binding of FITC-LPS to monocytes that was CD14 dependent at up to 100 ng of LPS per ml, and saturation of binding was shown.

Full Text

The Full Text of this article is available as a PDF (149.7 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bazil V., Horejsí V., Baudys M., Kristofová H., Strominger J. L., Kostka W., Hilgert I. Biochemical characterization of a soluble form of the 53-kDa monocyte surface antigen. Eur J Immunol. 1986 Dec;16(12):1583–1589. doi: 10.1002/eji.1830161218. [DOI] [PubMed] [Google Scholar]
  2. Bone R. C. The pathogenesis of sepsis. Ann Intern Med. 1991 Sep 15;115(6):457–469. doi: 10.7326/0003-4819-115-6-457. [DOI] [PubMed] [Google Scholar]
  3. Brade H., Galanos C. A method to detect 2-keto-3-deoxyoctanat and related compounds on pherograms and chromatograms. Anal Biochem. 1983 Jul 1;132(1):158–159. doi: 10.1016/0003-2697(83)90440-2. [DOI] [PubMed] [Google Scholar]
  4. Brade H., Galanos C., Lüderitz O. Differential determination of the 3-Deoxy-D-mannooctulosonic acid residues in lipopolysaccharides of Salmonella minnesota rough mutants. Eur J Biochem. 1983 Mar 1;131(1):195–200. doi: 10.1111/j.1432-1033.1983.tb07249.x. [DOI] [PubMed] [Google Scholar]
  5. Cooper N. R., Morrison D. C. Binding and activation of the first component of human complement by the lipid A region of lipopolysaccharides. J Immunol. 1978 Jun;120(6):1862–1868. [PubMed] [Google Scholar]
  6. Corrales I., Weersink A. J., Verhoef J., van Kessel K. P. Serum-independent binding of lipopolysaccharide to human monocytes is trypsin sensitive and does not involve CD14. Immunology. 1993 Sep;80(1):84–89. [PMC free article] [PubMed] [Google Scholar]
  7. Couturier C., Haeffner-Cavaillon N., Caroff M., Kazatchkine M. D. Binding sites for endotoxins (lipopolysaccharides) on human monocytes. J Immunol. 1991 Sep 15;147(6):1899–1904. [PubMed] [Google Scholar]
  8. Din Z. Z., Mukerjee P., Kastowsky M., Takayama K. Effect of pH on solubility and ionic state of lipopolysaccharide obtained from the deep rough mutant of Escherichia coli. Biochemistry. 1993 May 4;32(17):4579–4586. doi: 10.1021/bi00068a014. [DOI] [PubMed] [Google Scholar]
  9. Frey E. A., Miller D. S., Jahr T. G., Sundan A., Bazil V., Espevik T., Finlay B. B., Wright S. D. Soluble CD14 participates in the response of cells to lipopolysaccharide. J Exp Med. 1992 Dec 1;176(6):1665–1671. doi: 10.1084/jem.176.6.1665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gegner J. A., Ulevitch R. J., Tobias P. S. Lipopolysaccharide (LPS) signal transduction and clearance. Dual roles for LPS binding protein and membrane CD14. J Biol Chem. 1995 Mar 10;270(10):5320–5325. doi: 10.1074/jbc.270.10.5320. [DOI] [PubMed] [Google Scholar]
  11. Haeffner-Cavaillon N., Chaby R., Cavaillon J. M., Szabó L. Lipopolysaccharide receptor on rabbit peritoneal macrophages. I. Binding characteristics. J Immunol. 1982 May;128(5):1950–1954. [PubMed] [Google Scholar]
  12. 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]
  13. Haziot A., Rong G. W., Bazil V., Silver J., Goyert S. M. Recombinant soluble CD14 inhibits LPS-induced tumor necrosis factor-alpha production by cells in whole blood. J Immunol. 1994 Jun 15;152(12):5868–5876. [PubMed] [Google Scholar]
  14. Haziot A., Rong G. W., Silver J., Goyert S. M. Recombinant soluble CD14 mediates the activation of endothelial cells by lipopolysaccharide. J Immunol. 1993 Aug 1;151(3):1500–1507. [PubMed] [Google Scholar]
  15. Heumann D., Gallay P., Barras C., Zaech P., Ulevitch R. J., Tobias P. S., Glauser M. P., Baumgartner J. D. Control of lipopolysaccharide (LPS) binding and LPS-induced tumor necrosis factor secretion in human peripheral blood monocytes. J Immunol. 1992 Jun 1;148(11):3505–3512. [PubMed] [Google Scholar]
  16. Heumann D., Gallay P., Betz-Corradin S., Barras C., Baumgartner J. D., Glauser M. P. Competition between bactericidal/permeability-increasing protein and lipopolysaccharide-binding protein for lipopolysaccharide binding to monocytes. J Infect Dis. 1993 Jun;167(6):1351–1357. doi: 10.1093/infdis/167.6.1351. [DOI] [PubMed] [Google Scholar]
  17. Hirata M., Shimomura Y., Yoshida M., Morgan J. G., Palings I., Wilson D., Yen M. H., Wright S. C., Larrick J. W. Characterization of a rabbit cationic protein (CAP18) with lipopolysaccharide-inhibitory activity. Infect Immun. 1994 Apr;62(4):1421–1426. doi: 10.1128/iai.62.4.1421-1426.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Horwitz A. H., Williams R. E., Nowakowski G. Human lipopolysaccharide-binding protein potentiates bactericidal activity of human bactericidal/permeability-increasing protein. Infect Immun. 1995 Feb;63(2):522–527. doi: 10.1128/iai.63.2.522-527.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kirkland T. N., Finley F., Leturcq D., Moriarty A., Lee J. D., Ulevitch R. J., Tobias P. S. Analysis of lipopolysaccharide binding by CD14. J Biol Chem. 1993 Nov 25;268(33):24818–24823. [PubMed] [Google Scholar]
  20. Kitchens R. L., Munford R. S. Enzymatically deacylated lipopolysaccharide (LPS) can antagonize LPS at multiple sites in the LPS recognition pathway. J Biol Chem. 1995 Apr 28;270(17):9904–9910. doi: 10.1074/jbc.270.17.9904. [DOI] [PubMed] [Google Scholar]
  21. Kitchens R. L., Ulevitch R. J., Munford R. S. Lipopolysaccharide (LPS) partial structures inhibit responses to LPS in a human macrophage cell line without inhibiting LPS uptake by a CD14-mediated pathway. J Exp Med. 1992 Aug 1;176(2):485–494. doi: 10.1084/jem.176.2.485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Krüger C., Schütt C., Obertacke U., Joka T., Müller F. E., Knöller J., Köller M., König W., Schönfeld W. Serum CD14 levels in polytraumatized and severely burned patients. Clin Exp Immunol. 1991 Aug;85(2):297–301. doi: 10.1111/j.1365-2249.1991.tb05722.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Landmann R., Zimmerli W., Sansano S., Link S., Hahn A., Glauser M. P., Calandra T. Increased circulating soluble CD14 is associated with high mortality in gram-negative septic shock. J Infect Dis. 1995 Mar;171(3):639–644. doi: 10.1093/infdis/171.3.639. [DOI] [PubMed] [Google Scholar]
  24. Larrick J. W., Hirata M., Balint R. F., Lee J., Zhong J., Wright S. C. Human CAP18: a novel antimicrobial lipopolysaccharide-binding protein. Infect Immun. 1995 Apr;63(4):1291–1297. doi: 10.1128/iai.63.4.1291-1297.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Larrick J. W., Hirata M., Zheng H., Zhong J., Bolin D., Cavaillon J. M., Warren H. S., Wright S. C. A novel granulocyte-derived peptide with lipopolysaccharide-neutralizing activity. J Immunol. 1994 Jan 1;152(1):231–240. [PubMed] [Google Scholar]
  26. Meager A., Parti S., Leung H., Peil E., Mahon B. Preparation and characterization of monoclonal antibodies directed against antigenic determinants of recombinant human tumour necrosis factor (rTNF). Hybridoma. 1987 Jun;6(3):305–311. doi: 10.1089/hyb.1987.6.305. [DOI] [PubMed] [Google Scholar]
  27. Pollack M., Espinoza A. M., Guelde G., Koles N. L., Wahl L. M., Ohl C. A. Lipopolysaccharide (LPS)-specific monoclonal antibodies regulate LPS uptake and LPS-induced tumor necrosis factor-alpha responses by human monocytes. J Infect Dis. 1995 Sep;172(3):794–804. doi: 10.1093/infdis/172.3.794. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Schütt C., Schilling T., Grunwald U., Schönfeld W., Krüger C. Endotoxin-neutralizing capacity of soluble CD14. Res Immunol. 1992 Jan;143(1):71–78. doi: 10.1016/0923-2494(92)80082-v. [DOI] [PubMed] [Google Scholar]
  30. Shnyra A., Hultenby K., Lindberg A. A. Role of the physical state of Salmonella lipopolysaccharide in expression of biological and endotoxic properties. Infect Immun. 1993 Dec;61(12):5351–5360. doi: 10.1128/iai.61.12.5351-5360.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Skelly R. R., Munkenbeck P., Morrison D. C. Stimulation of T-independent antibody responses by hapten-lipopolysaccharides without repeating polymeric structure. Infect Immun. 1979 Feb;23(2):287–293. doi: 10.1128/iai.23.2.287-293.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Takayama K., Mitchell D. H., Din Z. Z., Mukerjee P., Li C., Coleman D. L. Monomeric Re lipopolysaccharide from Escherichia coli is more active than the aggregated form in the Limulus amebocyte lysate assay and in inducing Egr-1 mRNA in murine peritoneal macrophages. J Biol Chem. 1994 Jan 21;269(3):2241–2244. [PubMed] [Google Scholar]
  33. 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]
  34. Troelstra A., Giepmans B. N., Van Kessel K. P., Lichenstein H. S., Verhoef J., Van Strijp J. A. Dual effects of soluble CD14 on LPS priming of neutrophils. J Leukoc Biol. 1997 Feb;61(2):173–178. doi: 10.1002/jlb.61.2.173. [DOI] [PubMed] [Google Scholar]
  35. Ulevitch R. J. Recognition of bacterial endotoxins by receptor-dependent mechanisms. Adv Immunol. 1993;53:267–289. doi: 10.1016/s0065-2776(08)60502-7. [DOI] [PubMed] [Google Scholar]
  36. Ulmer A. J., Feist W., Heine H., Kirikae T., Kirikae F., Kusumoto S., Kusama T., Brade H., Schade U., Rietschel E. T. Modulation of endotoxin-induced monokine release in human monocytes by lipid A partial structures that inhibit binding of 125I-lipopolysaccharide. Infect Immun. 1992 Dec;60(12):5145–5152. doi: 10.1128/iai.60.12.5145-5152.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Warner S. J., Savage N., Mitchell D. Characteristics of lipopolysaccharide interaction with human peripheral-blood monocytes. Biochem J. 1985 Dec 1;232(2):379–383. doi: 10.1042/bj2320379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Weersink A. J., Van Kessel K. P., Torensma R., Van Strijp J. A., Verhoef J. Binding of rough lipopolysaccharides (LPS) to human leukocytes. Inhibition by anti-LPS monoclonal antibody. J Immunol. 1990 Jul 1;145(1):318–324. [PubMed] [Google Scholar]
  39. Wilde C. G., Seilhamer J. J., McGrogan M., Ashton N., Snable J. L., Lane J. C., Leong S. R., Thornton M. B., Miller K. L., Scott R. W. Bactericidal/permeability-increasing protein and lipopolysaccharide (LPS)-binding protein. LPS binding properties and effects on LPS-mediated cell activation. J Biol Chem. 1994 Jul 1;269(26):17411–17416. [PubMed] [Google Scholar]
  40. 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]
  41. Wright S. D., Tobias P. S., Ulevitch R. J., Ramos R. A. Lipopolysaccharide (LPS) binding protein opsonizes LPS-bearing particles for recognition by a novel receptor on macrophages. J Exp Med. 1989 Oct 1;170(4):1231–1241. doi: 10.1084/jem.170.4.1231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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]
  43. van Deventer S. J., Buller H. R., ten Cate J. W., Sturk A., Pauw W. Endotoxaemia: an early predictor of septicaemia in febrile patients. Lancet. 1988 Mar 19;1(8586):605–609. doi: 10.1016/s0140-6736(88)91412-2. [DOI] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES