Skip to main content
NCBI home page
Immunology logoLink to Immunology
. 1995 Apr;84(4):653–661.

The anti-lipid A monoclonal antibody E5 binds to rough gram-negative bacteria, fixes C3, and facilitates binding of bacterial immune complexes to both erythrocytes and monocytes.

M A Seelen 1, P Athanassiou 1, W A Lynn 1, P Norsworthy 1, M J Walport 1, J Cohen 1, K A Davies 1
PMCID: PMC1415161  PMID: 7790040

Abstract

Treatment of patients with septic shock using monoclonal antibodies (mAbs) to endotoxin is still controversial. Clinical trials of E5, one of the mAbs directed against the lipid A moiety of lipopolysaccharide (LPS), are currently in progress. The mechanisms of action of this, and other antibodies under clinical evaluation, are, however, poorly understood. In this study we examined in vitro the ways in which E5 interacted with Gram-negative bacteria, complement, erythrocytes and monocytes. By fluorescence-activated cell sorter (FACS) analysis we showed direct, dose-dependent binding of E5 to Escherichia coli (E. coli) and Salmonella minnesota (S. minnesota). Antibody binding to S. minnesota was enhanced by treatment with the beta-lactam antibiotic amoxycillin, but not by treatment with the aminoglycoside gentamicin. Immune complexes formed between E5 and both species of Gram-negative bacteria activated both classical and alternative complement pathways, but only in the case of S. minnesota did this facilitate binding to erythrocyte CR1 and monocyte CR3. Bacterial C3b and iC3b fixation by E5 was quantified using specific mAbs. These observations suggest that E5 may enhance bacterial clearance in several ways: (1) by facilitating direct complement fixation; (2) by facilitating the binding of opsonized bacteria to cells of the mononuclear phagocyte system; (3) by enabling bacteria to bind to erythrocyte CR1 (CD35), allowing safe carriage in the circulation to the fixed macrophages of the liver and spleen; (4) by acting synergistically with beta-lactam antibiotics.

Full text

PDF
653

Images in this article

656Figure 3
on p.656
657Figure 4
on p.657

Selected References

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

  1. Bogard W. C., Jr, Dunn D. L., Abernethy K., Kilgarriff C., Kung P. C. Isolation and characterization of murine monoclonal antibodies specific for gram-negative bacterial lipopolysaccharide: association of cross-genus reactivity with lipid A specificity. Infect Immun. 1987 Apr;55(4):899–908. doi: 10.1128/iai.55.4.899-908.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Braude A. I., Douglas H. Passive immunization against the local Shwartzman reaction. J Immunol. 1972 Feb;108(2):505–512. [PubMed] [Google Scholar]
  3. Chalfin D. B., Holbein M. E., Fein A. M., Carlon G. C. Cost-effectiveness of monoclonal antibodies to gram-negative endotoxin in the treatment of gram-negative sepsis in ICU patients. JAMA. 1993 Jan 13;269(2):249–254. [PubMed] [Google Scholar]
  4. Chia J. K., Pollack M., Guelde G., Koles N. L., Miller M., Evans M. E. Lipopolysaccharide (LPS)-reactive monoclonal antibodies fail to inhibit LPS-induced tumor necrosis factor secretion by mouse-derived macrophages. J Infect Dis. 1989 May;159(5):872–880. doi: 10.1093/infdis/159.5.872. [DOI] [PubMed] [Google Scholar]
  5. Clas F., Euteneuer B., Stemmer F., Loos M. Interaction of fluid phase C1/C1q and macrophage membrane-associated C1q with gram-negative bacteria. Behring Inst Mitt. 1989 Jul;(84):236–254. [PubMed] [Google Scholar]
  6. Danner R. L., Elin R. J., Hosseini J. M., Wesley R. A., Reilly J. M., Parillo J. E. Endotoxemia in human septic shock. Chest. 1991 Jan;99(1):169–175. doi: 10.1378/chest.99.1.169. [DOI] [PubMed] [Google Scholar]
  7. Davies K. A., Erlendsson K., Beynon H. L., Peters A. M., Steinsson K., Valdimarsson H., Walport M. J. Splenic uptake of immune complexes in man is complement-dependent. J Immunol. 1993 Oct 1;151(7):3866–3873. [PubMed] [Google Scholar]
  8. 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]
  9. Fishelson Z. Complement-related proteins in pathogenic organisms. Springer Semin Immunopathol. 1994;15(4):345–368. doi: 10.1007/BF01837365. [DOI] [PubMed] [Google Scholar]
  10. Fisher C. J., Jr, Zimmerman J., Khazaeli M. B., Albertson T. E., Dellinger R. P., Panacek E. A., Foulke G. E., Dating C., Smith C. R., LoBuglio A. F. Initial evaluation of human monoclonal anti-lipid A antibody (HA-1A) in patients with sepsis syndrome. Crit Care Med. 1990 Dec;18(12):1311–1315. doi: 10.1097/00003246-199012000-00001. [DOI] [PubMed] [Google Scholar]
  11. Fraker P. J., Speck J. C., Jr Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1,3,4,6-tetrachloro-3a,6a-diphrenylglycoluril. Biochem Biophys Res Commun. 1978 Feb 28;80(4):849–857. doi: 10.1016/0006-291x(78)91322-0. [DOI] [PubMed] [Google Scholar]
  12. Greenman R. L., Schein R. M., Martin M. A., Wenzel R. P., MacIntyre N. R., Emmanuel G., Chmel H., Kohler R. B., McCarthy M., Plouffe J. A controlled clinical trial of E5 murine monoclonal IgM antibody to endotoxin in the treatment of gram-negative sepsis. The XOMA Sepsis Study Group. JAMA. 1991 Aug 28;266(8):1097–1102. [PubMed] [Google Scholar]
  13. Joiner K. A., Goldman R., Schmetz M., Berger M., Hammer C. H., Frank M. M., Leive L. A quantitative analysis of C3 binding to O-antigen capsule, lipopolysaccharide, and outer membrane protein of E. coli 0111B4. J Immunol. 1984 Jan;132(1):369–375. [PubMed] [Google Scholar]
  14. Joiner K. A., Hammer C. H., Brown E. J., Cole R. J., Frank M. M. Studies on the mechanism of bacterial resistance to complement-mediated killing. I. Terminal complement components are deposited and released from Salmonella minnesota S218 without causing bacterial death. J Exp Med. 1982 Mar 1;155(3):797–808. doi: 10.1084/jem.155.3.797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Krieger J. I., Fletcher R. C., Siegel S. A., Fearon D. T., Neblock D. S., Boutin R. H., Taylor R. P., Daddona P. E. Human anti-endotoxin antibody HA-1A mediates complement-dependent binding of Escherichia coli J5 lipopolysaccharide to complement receptor type 1 of human erythrocytes and neutrophils. J Infect Dis. 1993 Apr;167(4):865–875. doi: 10.1093/infdis/167.4.865. [DOI] [PubMed] [Google Scholar]
  16. Lachmann P. J., Oldroyd R. G., Milstein C., Wright B. W. Three rat monoclonal antibodies to human C3. Immunology. 1980 Nov;41(3):503–515. [PMC free article] [PubMed] [Google Scholar]
  17. Loos M., Clas F. Antibody-independent killing of gram-negative bacteria via the classical pathway of complement. Immunol Lett. 1987 Feb;14(3):203–208. doi: 10.1016/0165-2478(87)90102-7. [DOI] [PubMed] [Google Scholar]
  18. Morrison D. C., Ryan J. L. Endotoxins and disease mechanisms. Annu Rev Med. 1987;38:417–432. doi: 10.1146/annurev.me.38.020187.002221. [DOI] [PubMed] [Google Scholar]
  19. Newman S. L., Mikus L. K. Deposition of C3b and iC3b onto particulate activators of the human complement system. Quantitation with monoclonal antibodies to human C3. J Exp Med. 1985 Jun 1;161(6):1414–1431. doi: 10.1084/jem.161.6.1414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Overbeek B. P., Schellekens J. F., Lippe W., Dekker B. A., Verhoef J. Carumonam enhances reactivity of Escherichia coli with mono- and polyclonal antisera to rough Escherichia coli J5. J Clin Microbiol. 1987 Jun;25(6):1009–1013. doi: 10.1128/jcm.25.6.1009-1013.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Parent J. B., Gazzano-Santoro H., Wood D. M., Lim E., Pruyne P. T., Trown P. W., Conlon P. J. Reactivity of monoclonal antibody E5 with endotoxin. II. Binding to short- and long-chain smooth lipopolysaccharides. Circ Shock. 1992 Sep;38(1):63–73. [PubMed] [Google Scholar]
  22. Romulo R. L., Palardy J. E., Opal S. M. Efficacy of anti-endotoxin monoclonal antibody E5 alone or in combination with ciprofloxacin in neutropenic rats with Pseudomonas sepsis. J Infect Dis. 1993 Jan;167(1):126–130. doi: 10.1093/infdis/167.1.126. [DOI] [PubMed] [Google Scholar]
  23. Savill J. S., Wyllie A. H., Henson J. E., Walport M. J., Henson P. M., Haslett C. Macrophage phagocytosis of aging neutrophils in inflammation. Programmed cell death in the neutrophil leads to its recognition by macrophages. J Clin Invest. 1989 Mar;83(3):865–875. doi: 10.1172/JCI113970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Schiller N. L., Hatch R. A., Joiner K. A. Complement activation and C3 binding by serum-sensitive and serum-resistant strains of Pseudomonas aeruginosa. Infect Immun. 1989 Jun;57(6):1707–1713. doi: 10.1128/iai.57.6.1707-1713.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Teng N. N., Kaplan H. S., Hebert J. M., Moore C., Douglas H., Wunderlich A., Braude A. I. Protection against gram-negative bacteremia and endotoxemia with human monoclonal IgM antibodies. Proc Natl Acad Sci U S A. 1985 Mar;82(6):1790–1794. doi: 10.1073/pnas.82.6.1790. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Tonoli M., Davies K. A., Norsworthy P. J., Cohen J., Walport M. J. The anti-lipid A antibody HA-1A binds to rough gram-negative bacteria, fixes complement and facilitates binding to erythrocyte CR1 (CD35). Clin Exp Immunol. 1993 May;92(2):232–238. doi: 10.1111/j.1365-2249.1993.tb03385.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wood D. M., Parent J. B., Gazzano-Santoro H., Lim E., Pruyne P. T., Watkins J. M., Spoor E. S., Reardan D. T., Trown P. W., Conlon P. J. Reactivity of monoclonal antibody E5 with endotoxin. I. Binding to lipid A and rough lipopolysaccharides. Circ Shock. 1992 Sep;38(1):55–62. [PubMed] [Google Scholar]
  28. Wortel C. H., von der Möhlen M. A., van Deventer S. J., Sprung C. L., Jastremski M., Lubbers M. J., Smith C. R., Allen I. E., ten Cate J. W. Effectiveness of a human monoclonal anti-endotoxin antibody (HA-1A) in gram-negative sepsis: relationship to endotoxin and cytokine levels. J Infect Dis. 1992 Dec;166(6):1367–1374. doi: 10.1093/infdis/166.6.1367. [DOI] [PubMed] [Google Scholar]
  29. Ziegler E. J., Fisher C. J., Jr, Sprung C. L., Straube R. C., Sadoff J. C., Foulke G. E., Wortel C. H., Fink M. P., Dellinger R. P., Teng N. N. Treatment of gram-negative bacteremia and septic shock with HA-1A human monoclonal antibody against endotoxin. A randomized, double-blind, placebo-controlled trial. The HA-1A Sepsis Study Group. N Engl J Med. 1991 Feb 14;324(7):429–436. doi: 10.1056/NEJM199102143240701. [DOI] [PubMed] [Google Scholar]
  30. Ziegler E. J., McCutchan J. A., Douglas H., Braude A. I. Prevention of lethal pseudomonas bacteremia with epimerase-deficient E. coli antiserum. Trans Assoc Am Physicians. 1975;88:101–108. [PubMed] [Google Scholar]
  31. Ziegler E. J., McCutchan J. A., Fierer J., Glauser M. P., Sadoff J. C., Douglas H., Braude A. I. Treatment of gram-negative bacteremia and shock with human antiserum to a mutant Escherichia coli. N Engl J Med. 1982 Nov 11;307(20):1225–1230. doi: 10.1056/NEJM198211113072001. [DOI] [PubMed] [Google Scholar]

Articles from Immunology are provided here courtesy of British Society for Immunology

RESOURCES