Skip to main content
NCBI home page
Clinical and Experimental Immunology logoLink to Clinical and Experimental Immunology
. 1993 Sep;93(3):456–463. doi: 10.1111/j.1365-2249.1993.tb08201.x

A novel flow cytometric assay for the quantification of adhesion of subsets within a heterogeneous cell population; analysis of lymphocyte function-associated antigen-1 (LFA-1)-mediated binding of bone marrow-derived primary tumour cells of patients with multiple myeloma.

E J Ahsmann 1, R J Benschop 1, T D de Gruyl 1, J A Faber 1, H M Lokhorst 1, A C Bloem 1
PMCID: PMC1554908  PMID: 8103718

Abstract

In a previous study the expression of the adhesion molecule LFA-1 on tumour cells in patients suffering from multiple myeloma (MM) was correlated with growth of the malignant plasma cells in vivo. Here we describe a novel in vitro flow cytometric adhesion assay (FCAA) which, based on scatter and fluorescence properties, was used to analyse the contribution of the LFA-1/intercellular adhesion molecule-1 (ICAM-1) adhesion pathway in the binding of bone marrow (BM)-derived LFA-1-positive primary tumour cells of patients with MM to interferon-gamma (IFN-gamma)-activated, ICAM-1-positive, human venous umbilical endothelial cells (huVEC) in vitro. To validate the FCAA, cells from different myeloma cell lines were labelled with the fluorescent dye CFDA or stained for CD38 expression, and LFA-1-mediated adhesion to IFN-gamma-activated endothelial cells was quantified. Results obtained with the FCAA were compared with a conventional adhesion assay employing 51Cr-labelled cells. Statistical analysis revealed that both assays gave similar results. This allowed analysis of the contribution of LFA-1 to the adhesive potential of malignant plasma cells in bone marrow mononuclear cells (BMMC) from MM patients to IFN-gamma-activated endothelial cells. The results prove that LFA-1 expressed on bone marrow-derived plasma cells from MM patients can be used for cellular adhesion to ICAM-1 expressed on adherent growing cells, and are suggestive for a role of the LFA-1/ICAM-1 adhesion pathway in the pathophysiology of MM. The FCAA described in this study is a generally applicable assay, allowing analysis of the interaction of distinct subpopulations with in vitro grown adherent cells of different origin.

Full text

PDF
456

Selected References

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

  1. Ahsmann E. J., Lokhorst H. M., Dekker A. W., Bloem A. C. Lymphocyte function-associated antigen-1 expression on plasma cells correlates with tumor growth in multiple myeloma. Blood. 1992 Apr 15;79(8):2068–2075. [PubMed] [Google Scholar]
  2. Anderson K. C., Jones R. M., Morimoto C., Leavitt P., Barut B. A. Response patterns of purified myeloma cells to hematopoietic growth factors. Blood. 1989 May 15;73(7):1915–1924. [PubMed] [Google Scholar]
  3. Barker H. F., Hamilton M. S., Ball J., Drew M., Franklin I. M. Expression of adhesion molecules LFA-3 and N-CAM on normal and malignant human plasma cells. Br J Haematol. 1992 Jul;81(3):331–335. doi: 10.1111/j.1365-2141.1992.tb08236.x. [DOI] [PubMed] [Google Scholar]
  4. Benschop R. J., de Smet M. B., Bloem A. C., Ballieux R. E. Adhesion of subsets of human blood mononuclear cells to endothelial cells in vitro, as quantified by flow cytometry. Scand J Immunol. 1992 Dec;36(6):793–800. doi: 10.1111/j.1365-3083.1992.tb03141.x. [DOI] [PubMed] [Google Scholar]
  5. Bloemen P., Moldenhauer G., van Dijk M., Schuurman H. J., Bloem A. C. Multiple ICAM-1 (CD54) epitopes are involved in homotypic B-cell adhesion. Scand J Immunol. 1992 May;35(5):517–523. doi: 10.1111/j.1365-3083.1992.tb03250.x. [DOI] [PubMed] [Google Scholar]
  6. Butcher E. C. Leukocyte-endothelial cell recognition: three (or more) steps to specificity and diversity. Cell. 1991 Dec 20;67(6):1033–1036. doi: 10.1016/0092-8674(91)90279-8. [DOI] [PubMed] [Google Scholar]
  7. Caligaris-Cappio F., Bergui L., Gregoretti M. G., Gaidano G., Gaboli M., Schena M., Zallone A. Z., Marchisio P. C. 'Role of bone marrow stromal cells in the growth of human multiple myeloma. Blood. 1991 Jun 15;77(12):2688–2693. [PubMed] [Google Scholar]
  8. Cornbleet P. J., Gochman N. Incorrect least-squares regression coefficients in method-comparison analysis. Clin Chem. 1979 Mar;25(3):432–438. [PubMed] [Google Scholar]
  9. Durie B. G. Staging and kinetics of multiple myeloma. Semin Oncol. 1986 Sep;13(3):300–309. [PubMed] [Google Scholar]
  10. Dustin M. L., Springer T. A. Lymphocyte function-associated antigen-1 (LFA-1) interaction with intercellular adhesion molecule-1 (ICAM-1) is one of at least three mechanisms for lymphocyte adhesion to cultured endothelial cells. J Cell Biol. 1988 Jul;107(1):321–331. doi: 10.1083/jcb.107.1.321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gallin J. I., Clark R. A., Kimball H. R. Granulocyte chemotaxis: an improved in vitro assay employing 51 Cr-labeled granulocytes. J Immunol. 1973 Jan;110(1):233–240. [PubMed] [Google Scholar]
  12. Hamilton M. S., Ball J., Bromidge E., Franklin I. M. Surface antigen expression of human neoplastic plasma cells includes molecules associated with lymphocyte recirculation and adhesion. Br J Haematol. 1991 May;78(1):60–65. doi: 10.1111/j.1365-2141.1991.tb04383.x. [DOI] [PubMed] [Google Scholar]
  13. Honn K. V., Tang D. G. Adhesion molecules and tumor cell interaction with endothelium and subendothelial matrix. Cancer Metastasis Rev. 1992 Nov;11(3-4):353–375. doi: 10.1007/BF01307187. [DOI] [PubMed] [Google Scholar]
  14. Hynes R. O. Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 1992 Apr 3;69(1):11–25. doi: 10.1016/0092-8674(92)90115-s. [DOI] [PubMed] [Google Scholar]
  15. Iwato K., Kawano M., Asaoku H., Tanabe O., Tanaka H., Kuramoto A. Separation of human myeloma cells from bone marrow aspirates in multiple myeloma and their proliferation and M-protein secretion in vitro. Blood. 1988 Aug;72(2):562–566. [PubMed] [Google Scholar]
  16. Kawano M. M., Huang N., Tanaka H., Ishikawa H., Sakai A., Tanabe O., Nobuyoshi M., Kuramoto A. Homotypic cell aggregations of human myeloma cells with ICAM-1 and LFA-1 molecules. Br J Haematol. 1991 Dec;79(4):583–588. doi: 10.1111/j.1365-2141.1991.tb08085.x. [DOI] [PubMed] [Google Scholar]
  17. Kawano M., Hirano T., Matsuda T., Taga T., Horii Y., Iwato K., Asaoku H., Tang B., Tanabe O., Tanaka H. Autocrine generation and requirement of BSF-2/IL-6 for human multiple myelomas. Nature. 1988 Mar 3;332(6159):83–85. doi: 10.1038/332083a0. [DOI] [PubMed] [Google Scholar]
  18. Kawano M., Tanaka H., Ishikawa H., Nobuyoshi M., Iwato K., Asaoku H., Tanabe O., Kuramoto A. Interleukin-1 accelerates autocrine growth of myeloma cells through interleukin-6 in human myeloma. Blood. 1989 Jun;73(8):2145–2148. [PubMed] [Google Scholar]
  19. Klein B., Zhang X. G., Jourdan M., Content J., Houssiau F., Aarden L., Piechaczyk M., Bataille R. Paracrine rather than autocrine regulation of myeloma-cell growth and differentiation by interleukin-6. Blood. 1989 Feb;73(2):517–526. [PubMed] [Google Scholar]
  20. Lo S. K., Van Seventer G. A., Levin S. M., Wright S. D. Two leukocyte receptors (CD11a/CD18 and CD11b/CD18) mediate transient adhesion to endothelium by binding to different ligands. J Immunol. 1989 Nov 15;143(10):3325–3329. [PubMed] [Google Scholar]
  21. McFaul S. J., Bowman P. D. Quantitation of polymorphonuclear leukocyte adherence to endothelial cells by electronic particle size discrimination. J Immunol Methods. 1990 Jul 3;130(2):171–176. doi: 10.1016/0022-1759(90)90045-w. [DOI] [PubMed] [Google Scholar]
  22. Nemunaitis J., Andrews D. F., Mochizuki D. Y., Lilly M. B., Singer J. W. Human marrow stromal cells: response to interleukin-6 (IL-6) and control of IL-6 expression. Blood. 1989 Nov 1;74(6):1929–1935. [PubMed] [Google Scholar]
  23. Nordan R. P., Potter M. A macrophage-derived factor required by plasmacytomas for survival and proliferation in vitro. Science. 1986 Aug 1;233(4763):566–569. doi: 10.1126/science.3726549. [DOI] [PubMed] [Google Scholar]
  24. Prieto J., Beatty P. G., Clark E. A., Patarroyo M. Molecules mediating adhesion of T and B cells, monocytes and granulocytes to vascular endothelial cells. Immunology. 1988 Apr;63(4):631–637. [PMC free article] [PubMed] [Google Scholar]
  25. Roldán E., Brieva J. A. Terminal differentiation of human bone marrow cells capable of spontaneous and high-rate immunoglobulin secretion: role of bone marrow stromal cells and interleukin 6. Eur J Immunol. 1991 Nov;21(11):2671–2677. doi: 10.1002/eji.1830211105. [DOI] [PubMed] [Google Scholar]
  26. Shimizu Y., Newman W., Tanaka Y., Shaw S. Lymphocyte interactions with endothelial cells. Immunol Today. 1992 Mar;13(3):106–112. doi: 10.1016/0167-5699(92)90151-V. [DOI] [PubMed] [Google Scholar]
  27. Spierenburg G. T., Polak-Vogelzang A. A., Bast B. J. Indicator cell lines for the detection of hidden mycoplasma contamination, using an adenosine phosphorylase screening test. J Immunol Methods. 1988 Nov 10;114(1-2):115–119. doi: 10.1016/0022-1759(88)90162-7. [DOI] [PubMed] [Google Scholar]
  28. Springer T. A. Adhesion receptors of the immune system. Nature. 1990 Aug 2;346(6283):425–434. doi: 10.1038/346425a0. [DOI] [PubMed] [Google Scholar]
  29. Terstappen L. W., Johnsen S., Segers-Nolten I. M., Loken M. R. Identification and characterization of plasma cells in normal human bone marrow by high-resolution flow cytometry. Blood. 1990 Nov 1;76(9):1739–1747. [PubMed] [Google Scholar]
  30. Tiemeyer M., Swiedler S. J., Ishihara M., Moreland M., Schweingruber H., Hirtzer P., Brandley B. K. Carbohydrate ligands for endothelial-leukocyte adhesion molecule 1. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1138–1142. doi: 10.1073/pnas.88.4.1138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Van Camp B., Durie B. G., Spier C., De Waele M., Van Riet I., Vela E., Frutiger Y., Richter L., Grogan T. M. Plasma cells in multiple myeloma express a natural killer cell-associated antigen: CD56 (NKH-1; Leu-19). Blood. 1990 Jul 15;76(2):377–382. [PubMed] [Google Scholar]
  32. Van Riet I., De Waele M., Remels L., Lacor P., Schots R., Van Camp B. Expression of cytoadhesion molecules (CD56, CD54, CD18 and CD29) by myeloma plasma cells. Br J Haematol. 1991 Nov;79(3):421–427. doi: 10.1111/j.1365-2141.1991.tb08050.x. [DOI] [PubMed] [Google Scholar]
  33. Wakkers P. J., Hellendoorn H. B., Op de Weegh G. J., Heerspink W. Applications of statistics in clinical chemistry. A critical evaluation of regression lines. Clin Chim Acta. 1975 Oct 15;64(2):173–184. doi: 10.1016/0009-8981(75)90199-0. [DOI] [PubMed] [Google Scholar]
  34. Zhang X. G., Bataille R., Jourdan M., Saeland S., Banchereau J., Mannoni P., Klein B. Granulocyte-macrophage colony-stimulating factor synergizes with interleukin-6 in supporting the proliferation of human myeloma cells. Blood. 1990 Dec 15;76(12):2599–2605. [PubMed] [Google Scholar]
  35. Zimmerman G. A., Prescott S. M., McIntyre T. M. Endothelial cell interactions with granulocytes: tethering and signaling molecules. Immunol Today. 1992 Mar;13(3):93–100. doi: 10.1016/0167-5699(92)90149-2. [DOI] [PubMed] [Google Scholar]
  36. de Fougerolles A. R., Springer T. A. Intercellular adhesion molecule 3, a third adhesion counter-receptor for lymphocyte function-associated molecule 1 on resting lymphocytes. J Exp Med. 1992 Jan 1;175(1):185–190. doi: 10.1084/jem.175.1.185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. de Fougerolles A. R., Stacker S. A., Schwarting R., Springer T. A. Characterization of ICAM-2 and evidence for a third counter-receptor for LFA-1. J Exp Med. 1991 Jul 1;174(1):253–267. doi: 10.1084/jem.174.1.253. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Clinical and Experimental Immunology are provided here courtesy of British Society for Immunology

RESOURCES