Phorbol esters induce differentiation of U‐937 human promonocytic cells in the absence of LFA‐1/ICAM‐1‐mediated intercellular adhesion

Abstract

Intercellular adhesions which occur during the mononuclear phagocyte differentiation are predominantly mediated by the lymphocyte-function-associated antigen-1 (LFA-1) family and the intercellular-adhesion molecule-1 (ICAM-1) which is a ligand for LFA-1. Thus, differentiation of U-937 promonocytic cells induced by phorbol esters occurs concomitantly with intercellular LFA-l/ICAM-1 -dependent cluster formation. Since these homotypic adhesions can be inhibited by monoclonal antibodies (mAb) directed to either LFA-1 or ICAM-1, we have analyzed whether the lack of cell -cell adhesions impairs the differentiation process. Treatment of U-937 cells with the phorbol ester 12-0-tetradecanoylphorbol 13-acetate in the presence of mAb to LFA-1 or ICAM-1 antigens yielded cells free from homotypic adhesions but differentiated as evidenced by their decreased proliferation and enhanced capacity for generation of superoxide anion. In addition, expression of the C D l l c antigen was increased, whereas the transferrin receptor disappeared from the cell surface. Vimentin gene transcription was also greatly augmented as opposed to a clear diminution in the levels of c-myc and ornithine decarboxylase transcripts. These results clearly demonstrate that phorbol esters can induce differentiation of monocytic cells independently of cell -cell adhesion.Intercellular adhesions play an important role in many immunological functions. Many of the adhesion phenoma of leucocytes are mediated by the lymphocyte-function-associated antigen 1 (LFA-1) family and the intercellular-adhesion molecule 1 (ICAM-1, CD54) molecule, which is a ligand for LFA-1 [I -61. These molecules are involved in antigen presentation [2, 7, 81, T cell effector functions [9-121, cooperation between B and T lymphocytes [I31 and chemotaxis of myeloid cells [I, 14, 151. The LFA-1 family of antigens is composed of three structurally related glycoproteins, LFA-1 (CD1 la), Mol (CDllb) and p150,95 (CDllc) [16]. These three antigens are noncovalently linked a : j heterodimers which have distinct a subunits of 170, 165 and 150 kDa, respectively, and share a common j subunit of 95 kDa (CD18) [16,17].In mononuclear phagocytes, the regulation of cell -cell adhesion is an important aspect of their function and development. Thus, monocytes initiate diapedesis by adherence to the endothelium. This adhesion to endothelial cells allows the monocytes to exit from blood vessels by passing between adjacent endothelial cells. At the same time that these adhesion processes take place, monocytes maturate into tissue macrophages. Also, upon antigen challenge, there is a massive infiltration of macrophages showing homotypic (macrophage -macrophage) and heterotypic (macrophage -T-cell) interactions. These cellular associations can lead to granuloma Several markers for monocytic differentiation of myeloid cell lines have been used, such as an arrest in their proliferative capacity [23 -25, 281, variations in the levels of specific gene transcripts [26, 29 -331, changes in the expression of specific...

Induction of LFA-1-mediated homotypic adhesions in promonocytic U-937 cells occurs independently of cell differentiation

“…Using the human promonocytic cell line U937 as an experimental model of differentiation, we show that extensive LFA-l/ICAM-l-dependent in teractions can occur in the absence of monocytic differ entiation. This conclusion is supported by the findings that (a) PMA is able to differentiate U937 cells into macrophage-like cells in the absence of LFA-1 /ICAM -1-dependent cellular interactions [19]; and (b) in CD18 deficient patients, LFA-1 adhesion independent func tions of phagocytes are all normal, including shape changes, binding of chemotactic compounds (FMLP) and oxygen radical generation to soluble stimuli [26].…”

“…Also, the differentiation of U-937 cells induced with PMA is characterized by an important decrease in the levels of c-myc and OrnDC as well as the marked increase of the levels of CD18 and vimentin transcripts [19,24]. In contrast, the treatment of U-937 cells with NKI-L16 has no effect on the levels of any of these gene transcripts (Fig.…”

“…Cellular proliferation was measured as previously described [19], Flow cytometry analysis was performed with an EPICS-CS flow cy tome ter (Coulter Cientifica, Mostoles, Spain) using logarithmic amplifiers. For anti gen expression analysis, cells were incubated with the corresponding mAb for 30 min at 4°C.…”

“…RNA blot analyses were performed basically as de scribed [19]. The probes used were: the 1.5 kb Cla IEcoRl fragment of pMC413rC plasmid [21], which con tains the 3rd exon of human c-myc\ the 1.1 kb human vimentin-specific Xhol fragment of L3A 7A plasmid [22]; the 1.5 kb OrnDC-specific Xhol fragment of OD-821 plasmid (a generous gift from Dr. L. Kaczmareck, Varsow, Poland); and the 1 kb £o?RI fragment from the cDNA of the human CD 18 antigen [23].…”

Activation of protein kinase C relays distinct signaling pathways in the same cell type: differentiation and caspase-mediated apoptosis

“…In this context, spontaneously apoptotic U937 cells from exponentially growing cultures demonstrate increased PKC-beta and reduced PKC-zeta expression indicating that the expression of specific PKC isoenzymes is modulated during apoptosis. 18 Although other work has hypothesized that phorbol ester-induced monocytic differentiation can occur independently of cell-to-cell adhesion in human U937 cells, 19,20 the present data clearly demonstrate a strong functional relationship between phorbol ester-mediated adherence, integrin expression and subsequent monocytic differentiation. This relationship is also substantiated by the findings that TPA-treated U937 cells which remain in suspension are unable to differentiate and do not induce p21 WAF/CIP/ sdi-1 .…”