Assessing effects of an antigen complex from opportunistic bacteria on activation of CD54 receptor expression by human promonocytic U937 cell line

Opportunistic pathogens play an important role in development of respiratory infections, causing high morbidity and mortality rates. A promising approach is the use of antigen-based drugs from opportunistic bacteria that stimulate the immune system. Search for appropriate assay techniques and biomarkers of immune response is an important task in their development. Potential stimulation of myeloid cells is of particular interest for these purposes. The U937 cell line is widely used as a model for analyzing the functional activity of monocytes and macrophages, being derived from human histiocytic lymphoma cells and exhibiting promonocyte characteristics, The CD54 (ICAM-1) molecule is a surface glycoprotein that functions as an adhesion receptor and plays a key role in targeted migration of leukocytes from bloodstream to inflamed tissues. Under the inflammatory stimulation, expression of ICAM-1 increases significantly on the surface of immune cells, making it a potential activation marker in certain components of innate immune system, including U937 cell model. This study considered activation of CD54 (ICAM-1) receptor expression in human U937 promonocytic cells under the influence of two different antigen complex from opportunistic pathogens, supplied with immunostimulatory copolymer, or without its addition. The test antigen complex included antigens from four species of opportunistic bacteria: Escherichia coli, Proteus vulgaris, Staphylococcus aureus, Klebsiella pneumoniae. As an additional immunostimulating agent, a copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone hydrochloride was used. Flow cytometry was applied in order to assess CD54 (ICAM- 1) expression on U937 cells, The cells were stimulated with peptidoglycan from S. aureus (positive control), and with mentioned variants of the antigen complex form opportunistic bacteria. The cell activity was evaluated using the activation coefficients, which reflected the increase in CD54 expression. The activation coefficient for positive controls was considered significant if it reached, at least, 50% (activation of cells by the reference antigen), whereas cell activation by the studied bacterial antigen complexes was considered significant at an activation coefficient of at least 30%. It was shown that both variants and the positive control induced a dose-dependent increase in CD54 expression. The optimal stimulation concentration was 25 µg/mL. These effects confirmed ability of the antigen complex from opportunistic pathogens to activate monocyte-macrophage cells and enhance the innate immune response. The obtained results emphasize the importance of using ICAM-1 as a marker of immune cell activation. The tested method seems to be promising in order to test effectiveness of drugs based on opportunistic bacteria.