Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1997 Sep;65(9):3577–3584. doi: 10.1128/iai.65.9.3577-3584.1997

Serum factors, cell membrane CD14, and beta2 integrins are not required for activation of bovine macrophages by lipopolysaccharide.

T W Jungi 1, H Sager 1, H Adler 1, M Brcic 1, H Pfister 1
PMCID: PMC175509  PMID: 9284122

Abstract

The role of serum factors such as lipopolysaccharide (LPS)-binding protein (LBP) and of macrophage-expressed CD14 and beta2 integrins in the activation of bovine macrophages by LPS was investigated. Macrophage activation was determined by measuring tumor necrosis factor production, NO generation, and upregulation of procoagulant activity by LPS (Escherichia coli O55:B5) at concentrations of 100 pg/ml to 100 ng/ml. The 50% effective dose for LPS was 1 order of magnitude higher than that for activating human macrophages. Macrophages were activated by LPS in the presence of serum or in the presence of albumin demonstrated to be free of LBP. The capacity to react to LPS in the absence of LBP was not due to the acquisition of LBP during a previous culture in serum. It was then established which CD14-specific antibodies block LPS binding to monocytes. Among the CD14-specific antibodies recognizing bovine mononuclear phagocytes (60bca, 3C10, My4, CAM36, VPM65, CMRF31, and TUK4), the first four blocked the binding of LPS-fluorescein isothiocyanate to bovine monocytes at low concentrations. Anti-CD14 antibodies did not block LPS-mediated activation of bovine bone marrow-derived macrophages, monocyte-derived macrophages, and alveolar macrophages. This was observed in experiments in which anti-CD14 concentrations exceeded the 50% inhibitory dose by >30-fold (3C10 and My4) or >300-fold (60bca), as defined in the binding assay described above. Monocyte-derived macrophages from an animal deficient in beta2 integrins and control macrophages were activated by similar concentrations of LPS, suggesting that beta2 integrins are not important bovine LPS receptors. Thus, in bovine macrophages, LPS recognition pathways which are independent of exogenous LBP, of membrane-expressed CD14, and of beta2 integrins may exist.

Full Text

The Full Text of this article is available as a PDF (198.3 KB).

Selected References

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

  1. Adler H., Frech B., Thöny M., Pfister H., Peterhans E., Jungi T. W. Inducible nitric oxide synthase in cattle. Differential cytokine regulation of nitric oxide synthase in bovine and murine macrophages. J Immunol. 1995 May 1;154(9):4710–4718. [PubMed] [Google Scholar]
  2. Adler H., Peterhans E., Jungi T. W. Generation and functional characterization of bovine bone marrow-derived macrophages. Vet Immunol Immunopathol. 1994 Jun;41(3-4):211–227. doi: 10.1016/0165-2427(94)90098-1. [DOI] [PubMed] [Google Scholar]
  3. Adler H., Peterhans E., Nicolet J., Jungi T. W. Inducible L-arginine-dependent nitric oxide synthase activity in bovine bone marrow-derived macrophages. Biochem Biophys Res Commun. 1994 Jan 28;198(2):510–515. doi: 10.1006/bbrc.1994.1075. [DOI] [PubMed] [Google Scholar]
  4. Alderson M. R., Tough T. W., Ziegler S. F., Armitage R. J. Regulation of human monocyte cell-surface and soluble CD23 (Fc epsilon RII) by granulocyte-macrophage colony-stimulating factor and IL-3. J Immunol. 1992 Aug 15;149(4):1252–1257. [PubMed] [Google Scholar]
  5. Bertoni G., Kuhnert P., Peterhans E., Pauli U. Improved bioassay for the detection of porcine tumor necrosis factor using a homologous cell line: PK(15). J Immunol Methods. 1993 Apr 2;160(2):267–271. doi: 10.1016/0022-1759(93)90187-c. [DOI] [PubMed] [Google Scholar]
  6. Cavaillon J. M., Fitting C., Haeffner-Cavaillon N., Kirsch S. J., Warren H. S. Cytokine response by monocytes and macrophages to free and lipoprotein-bound lipopolysaccharide. Infect Immun. 1990 Jul;58(7):2375–2382. doi: 10.1128/iai.58.7.2375-2382.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cohen L., Haziot A., Shen D. R., Lin X. Y., Sia C., Harper R., Silver J., Goyert S. M. CD14-independent responses to LPS require a serum factor that is absent from neonates. J Immunol. 1995 Dec 1;155(11):5337–5342. [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. Dentener M. A., Bazil V., Von Asmuth E. J., Ceska M., Buurman W. A. Involvement of CD14 in lipopolysaccharide-induced tumor necrosis factor-alpha, IL-6 and IL-8 release by human monocytes and alveolar macrophages. J Immunol. 1993 Apr 1;150(7):2885–2891. [PubMed] [Google Scholar]
  11. Dziarski R. Cell-bound albumin is the 70-kDa peptidoglycan-, lipopolysaccharide-, and lipoteichoic acid-binding protein on lymphocytes and macrophages. J Biol Chem. 1994 Aug 12;269(32):20431–20436. [PubMed] [Google Scholar]
  12. Eperon S., Jungi T. W. The use of human monocytoid lines as indicators of endotoxin. J Immunol Methods. 1996 Aug 14;194(2):121–129. doi: 10.1016/0022-1759(96)00073-7. [DOI] [PubMed] [Google Scholar]
  13. Fukuse S., Maeda T., Webb D. R., Devens B. H. A 69-kDa membrane protein associated with lipopolysaccharide (LPS)-induced signal transduction in the human monocytic cell line THP-1. Cell Immunol. 1995 Sep;164(2):248–254. doi: 10.1006/cimm.1995.1168. [DOI] [PubMed] [Google Scholar]
  14. Golenbock D. T., Bach R. R., Lichenstein H., Juan T. S., Tadavarthy A., Moldow C. F. Soluble CD14 promotes LPS activation of CD14-deficient PNH monocytes and endothelial cells. J Lab Clin Med. 1995 May;125(5):662–671. [PubMed] [Google Scholar]
  15. 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]
  16. Hailman E., Lichenstein H. S., Wurfel M. M., Miller D. S., Johnson D. A., Kelley M., Busse L. A., Zukowski M. M., Wright S. D. Lipopolysaccharide (LPS)-binding protein accelerates the binding of LPS to CD14. J Exp Med. 1994 Jan 1;179(1):269–277. doi: 10.1084/jem.179.1.269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hampton R. Y., Golenbock D. T., Penman M., Krieger M., Raetz C. R. Recognition and plasma clearance of endotoxin by scavenger receptors. Nature. 1991 Jul 25;352(6333):342–344. doi: 10.1038/352342a0. [DOI] [PubMed] [Google Scholar]
  18. Haziot A., Ferrero E., Köntgen F., Hijiya N., Yamamoto S., Silver J., Stewart C. L., Goyert S. M. Resistance to endotoxin shock and reduced dissemination of gram-negative bacteria in CD14-deficient mice. Immunity. 1996 Apr;4(4):407–414. doi: 10.1016/s1074-7613(00)80254-x. [DOI] [PubMed] [Google Scholar]
  19. Haziot A., Ferrero E., Lin X. Y., Stewart C. L., Goyert S. M. CD14-deficient mice are exquisitely insensitive to the effects of LPS. Prog Clin Biol Res. 1995;392:349–351. [PubMed] [Google Scholar]
  20. Haziot A., Rong G. W., Lin X. Y., Silver J., Goyert S. M. Recombinant soluble CD14 prevents mortality in mice treated with endotoxin (lipopolysaccharide). J Immunol. 1995 Jun 15;154(12):6529–6532. [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. Ishii Y., Shuyi W., Kitamura S. Soluble CD14 in serum mediates LPS-induced increase in permeability of bovine pulmonary arterial endothelial cell monolayers in vitro. Life Sci. 1995;56(25):2263–2272. doi: 10.1016/0024-3205(95)00216-s. [DOI] [PubMed] [Google Scholar]
  24. Jahr T. G., Sundan A., Lichenstein H. S., Espevik T. Influence of CD14, LBP and BPI in the monocyte response to LPS of different polysaccharide chain length. Scand J Immunol. 1995 Jul;42(1):119–127. doi: 10.1111/j.1365-3083.1995.tb03634.x. [DOI] [PubMed] [Google Scholar]
  25. Jungi T. W. A turbidimetric assay in an ELISA reader for the determination of mononuclear phagocyte procoagulant activity. J Immunol Methods. 1990 Oct 4;133(1):21–29. doi: 10.1016/0022-1759(90)90314-l. [DOI] [PubMed] [Google Scholar]
  26. Jungi T. W., Brcic M., Eperon S. Human macrophages respond to LPS in a serum-independent, CD14-dependent manner. Immunol Lett. 1996 Dec 1;54(1):37–43. doi: 10.1016/s0165-2478(96)02645-4. [DOI] [PubMed] [Google Scholar]
  27. Jungi T. W., Krampe M., Sileghem M., Griot C., Nicolet J. Differential and strain-specific triggering of bovine alveolar macrophage effector functions by mycoplasmas. Microb Pathog. 1996 Dec;21(6):487–498. doi: 10.1006/mpat.1996.0078. [DOI] [PubMed] [Google Scholar]
  28. Jungi T. W., Miserez R., Brcic M., Pfister H. Change in sensitivity to lipopolysaccharide during the differentiation of human monocytes to macrophages in vitro. Experientia. 1994 Feb 15;50(2):110–114. doi: 10.1007/BF01984945. [DOI] [PubMed] [Google Scholar]
  29. Jungi T. W., Thöny M., Brcic M., Adler B., Pauli U., Peterhans E. Induction of nitric oxide synthase in bovine mononuclear phagocytes is differentiation stage-dependent. Immunobiology. 1996 Aug;195(3):385–400. doi: 10.1016/S0171-2985(96)80054-4. [DOI] [PubMed] [Google Scholar]
  30. Keller R., Keist R. Abilities of activated macrophages to manifest tumoricidal activity and to generate reactive nitrogen intermediates: a comparative study in vitro and ex vivo. Biochem Biophys Res Commun. 1989 Nov 15;164(3):968–973. doi: 10.1016/0006-291x(89)91764-6. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Lee J. D., Kato K., Tobias P. S., Kirkland T. N., Ulevitch R. J. Transfection of CD14 into 70Z/3 cells dramatically enhances the sensitivity to complexes of lipopolysaccharide (LPS) and LPS binding protein. J Exp Med. 1992 Jun 1;175(6):1697–1705. doi: 10.1084/jem.175.6.1697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lei M. G., Stimpson S. A., Morrison D. C. Specific endotoxic lipopolysaccharide-binding receptors on murine splenocytes. III. Binding specificity and characterization. J Immunol. 1991 Sep 15;147(6):1925–1932. [PubMed] [Google Scholar]
  34. Lynn W. A., Liu Y., Golenbock D. T. Neither CD14 nor serum is absolutely necessary for activation of mononuclear phagocytes by bacterial lipopolysaccharide. Infect Immun. 1993 Oct;61(10):4452–4461. doi: 10.1128/iai.61.10.4452-4461.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. MacIntyre E. A., Roberts P. J., Jones M., Van der Schoot C. E., Favalaro E. J., Tidman N., Linch D. C. Activation of human monocytes occurs on cross-linking monocytic antigens to an Fc receptor. J Immunol. 1989 Apr 1;142(7):2377–2383. [PubMed] [Google Scholar]
  36. Meylan M., Abegg R., Sager H., Jungi T. W., Martig J. Fallvorstellung: Bovine Leukozyten-Adhäsions-Defizienz (BLAD) in der Schweiz. Schweiz Arch Tierheilkd. 1997;139(6):277–281. [PubMed] [Google Scholar]
  37. Miserez R., Jungi T. W. LPS-induced, but not interferon-gamma-induced procoagulant activity of suspended human macrophages is followed by a refractory state of low procoagulant expression. Thromb Res. 1992 Mar 15;65(6):733–744. doi: 10.1016/0049-3848(92)90112-n. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Pedron T., Girard R., Chaby R. Variation of LPS-binding capacity, epitope expression, and shedding of membrane-bound CD14 during differentiation of human monocytes. J Immunol. 1995 Aug 1;155(3):1460–1471. [PubMed] [Google Scholar]
  40. Rosenberg S. A., Ligler F. S., Ugolini V., Lipsky P. E. A monoclonal antibody that identifies human peripheral blood monocytes recognizes the accessory- cells required for mitogen-induced T lymphocyte proliferation. J Immunol. 1981 Apr;126(4):1473–1477. [PubMed] [Google Scholar]
  41. Schletter J., Brade H., Brade L., Krüger C., Loppnow H., Kusumoto S., Rietschel E. T., Flad H. D., Ulmer A. J. Binding of lipopolysaccharide (LPS) to an 80-kilodalton membrane protein of human cells is mediated by soluble CD14 and LPS-binding protein. Infect Immun. 1995 Jul;63(7):2576–2580. doi: 10.1128/iai.63.7.2576-2580.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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]
  43. Schütt C., Schilling T., Grunwald U., Stelter F., Witt S., Krüger C., Jack R. S. Human monocytes lacking the membrane-bound form of the bacterial lipopolysaccharide (LPS) receptor CD14 can mount an LPS-induced oxidative burst response mediated by a soluble form of CD14. Res Immunol. 1995 Jul-Aug;146(6):339–350. doi: 10.1016/0923-2494(96)81038-8. [DOI] [PubMed] [Google Scholar]
  44. Shuster D. E., Kehrli M. E., Jr, Ackermann M. R., Gilbert R. O. Identification and prevalence of a genetic defect that causes leukocyte adhesion deficiency in Holstein cattle. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):9225–9229. doi: 10.1073/pnas.89.19.9225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sundan A., Ryan L., Brinch L., Espevik T., Waage A. The involvement of CD14 in stimulation of TNF production from peripheral mononuclear cells isolated from PNH patients. Scand J Immunol. 1995 Jun;41(6):603–608. doi: 10.1111/j.1365-3083.1995.tb03613.x. [DOI] [PubMed] [Google Scholar]
  46. Theofan G., Horwitz A. H., Williams R. E., Liu P. S., Chan I., Birr C., Carroll S. F., Mészáros K., Parent J. B., Kasler H. An amino-terminal fragment of human lipopolysaccharide-binding protein retains lipid A binding but not CD14-stimulatory activity. J Immunol. 1994 Apr 1;152(7):3623–3629. [PubMed] [Google Scholar]
  47. 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]
  48. Ulevitch R. J., Tobias P. S. Recognition of endotoxin by cells leading to transmembrane signaling. Curr Opin Immunol. 1994 Feb;6(1):125–130. doi: 10.1016/0952-7915(94)90043-4. [DOI] [PubMed] [Google Scholar]
  49. Werner-Felmayer G., Baier-Bitterlich G., Fuchs D., Hausen A., Murr C., Reibnegger G., Werner E. R., Wachter H. Detection of bacterial pyrogens on the basis of their effects on gamma interferon-mediated formation of neopterin or nitrite in cultured monocyte cell lines. Clin Diagn Lab Immunol. 1995 May;2(3):307–313. doi: 10.1128/cdli.2.3.307-313.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. 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]
  51. Wright S. D., Ramos R. A., Patel M., Miller D. S. Septin: a factor in plasma that opsonizes lipopolysaccharide-bearing particles for recognition by CD14 on phagocytes. J Exp Med. 1992 Sep 1;176(3):719–727. doi: 10.1084/jem.176.3.719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. 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]
  53. 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]
  54. Yang Z., Carter C. D., Miller M. S., Bochsler P. N. CD14 and tissue factor expression by bacterial lipopolysaccharide-stimulated bovine alveolar macrophages in vitro. Infect Immun. 1995 Jan;63(1):51–56. doi: 10.1128/iai.63.1.51-56.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Yang Z., Khemlani L. S., Dean D. F., Carter C. D., Slauson D. O., Bochsler P. N. Serum components enhance bacterial lipopolysaccharide-induced tissue factor expression and tumor necrosis factor-alpha secretion by bovine alveolar macrophages in vitro. J Leukoc Biol. 1994 Apr;55(4):483–488. doi: 10.1002/jlb.55.4.483. [DOI] [PubMed] [Google Scholar]

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

RESOURCES