Effects of inflammatory cytokines and phorbol esters on the adhesion of U937 cells, a human monocyte-like cell line, to endothelial cell monolayers and extracellular matrix proteins.

The accumulation of mononuclear phagocytes at sites of chronic inflammation is dependent on an increase in the rate of extravasation of blood-borne monocytes through the vascular endothelium into the connective tissue. Once the monocytes have emigrated into the connective tissue, they may differentiate into tissue macrophages, presumably following interactions with extracellular matrix proteins. To study these processes, we tested the effects of cytokines and phorbol esters on the adhesion of U937 cells, a human monocyte-like cell line, to cultured endothelial cells (EC) and to matrix proteins. In the absence of cytokines, very few of the U937 cells adhered to EC (5% or less in most experiments). When EC were pretreated for optimal periods of time (4-8 hr) with recombinant interleukin-1 alpha (IL-1 alpha), IL-1 beta, tumor necrosis factor-alpha (TNF alpha), or lymphotoxin (LT; also known as TNF-beta), 35-85% of the U937 cells were able to bind. Interferon-gamma (IFN-gamma) and interleukin-2 (IL-2) did not stimulate U937-EC binding, even though IFN-gamma was shown to increase EC adhesiveness for T lymphocytes. Phorbol esters also greatly stimulated U937-EC adhesion but, in this case, the increase was due to an action on the U937 cells. A monoclonal antibody (MAb), 60.3, against the CD11/CD18 family of leukocyte adhesion molecules partially inhibited the adhesion of untreated and phorbol ester-treated U937 cells to noncytokine-treated EC. However, that MAb had no effect on U937 cell binding to TNF-alpha-treated EC. Thus U937 cells use both CD11/CD18-dependent and -independent mechanisms to adhere to EC. In the absence of stimulating agents, only a small proportion of the U937 cells (2-20%) adhered to fibronectin (FN), and almost none bound to either laminin (LN) or gelatin (denatured type I collagen). In the presence of phorbol esters, a much larger proportion of the U937 cells adhered to FN, with only slight increases in the proportion of cells which bound to LN or gelatin. Additional adhesion assays performed in the presence of a pentapeptide containing the amino acid sequence arg-gly-asp (RGD), which is part of one of the cell-binding domains of FN, demonstrated that the RGD-containing peptide almost totally blocked the phorbol ester-induced adhesion of U937 cells to FN. In contrast, the peptide had no inhibitory effect on the phorbol ester-induced binding of U937 cells to EC.     

Characterization and structural analysis of Fc gamma receptors of human monocytes, a monoblast cell line (U937) and a myeloblast cell line (HL-60) by a monoclonal antibody

A monoclonal antibody, FR51, raised against the IgG Fc receptor (Fc gamma R) of the human monoblast cell line U937 was used to analyze the distribution of this antigen on various human cells. This antibody inhibited the binding of human IgG to the Fc gamma R on U937 cells, HL-60 cells and human peripheral blood monocytes. In contrast, the Fc gamma R on human granulocytes (neutrophil cells) and on an Epstein-Barr virus-transformed human lymphoblastoid cell line (Raji) were not recognized, indicated by the failure of blocking the binding of human IgG ligand to the Fc gamma R on these cells. By affinity chromatography of detergent-containing cell free lysates of surface-iodinated U937 cells, HL-60 cells and monocytes, a protein of 70-kDa was isolated. This protein was identified as the Fc gamma R by rebinding the isolated protein to immobilized human IgG. Removal of the carbohydrate moiety with endo-beta-N-acetylglucosaminidase F demonstrated that the receptors consist of a 40-kDa polypeptide. Analysis of the polypeptide patterns obtained by proteolytic digestion of either mature (70-kDa) or deglycosylated (40-kDa) receptors isolated from monocytes, U937 cells and HL-60 cells strongly suggests that the Fc gamma R are identical. The monoclonal antibody FR51 specifically reacts with Fc gamma R on human monocytes, a myeloblast and a monoblast cell line but not with the receptors on a B cell line and neutrophil cells.     

Post-receptor occupancy events in leukocytes during β1 integrin-ligand interactions

Leukocyte adhesion to and subsequent spreading on the endothelium are the initial steps in the migration of these cells to the surrounding tissues. We have investigated the participation of different VLA heterodimers in cell spreading by using the anti-β1 TS2/16 monoclonal antibody (mAb) which induces a conformational change of different VLA integrin receptors, enabling a high-affinity interaction with their ligands. Both VLA-4 and VLA-5 fibronectin (FN), as well as VLA-2 collagen (COL) receptors mediated cell spreading and morphological changes. The spreading of U-937 and α4-transfected K-562 cells was induced in both FN and VCAM-1, suggesting that the morphological changes may be induced by cell-cell as well as cell-extracellular matrix (ECM) interactions. Furthermore, the β-regulated cell spreading on VCAM-1 and COL took place independently of the VLA-5 FN receptor function. The enhancing effect on cell attachment induced by anti-β1 TS2/16 mAb was observed in the presence of different doses of cytochalasin D, whereas cell spreading was abolished. Signal transduction during β1-stimulated integrin-ligand interaction was also investigated. We have found the co-localization of β1 integrins and tyrosine-phosphorylated proteins during the spreading of U-937 and α2- and α4-transfected K-562 cells on both ECM (FN and COL) and cellular (VCAM-1) ligands. Kinetic studies showed that tyrosine phosphorylation was almost coincident with cellular spreading. The tyrosine phosphorylation of polypeptides of 130 kDa and 77 kDa was triggered in U-937 cells by the interaction of FN with the VLA-5 receptor in a high-affinity conformation. However, no signaling was observed by inducing the high-affinity state of receptor in the absence of appropriate ligand. These data suggest that tyrosine kinase activation is a post-receptor occupancy event that might be critical in regulating the adhesive properties of integrins.     

Monoclonal antibody against human macrophages/monocytes and granulocytes

A monoclonal antibody of the IgG2a isotype directed against lymphokine-activated human macrophage cell line (U937) was produced and characterized. By indirect immunofluorescence microscopy or binding assay, this antibody (MaG-1) reacted with human peripheral monocytes, peritoneal macrophages, alveolar macrophages, and peripheral granulocytes but not with lymphocytes, erythrocytes, and platelets. MaG-1 antigen was also expressed by 34% of bone-marrow cells which includes monocytic and granulocytic precursors, whereas lymphoid and erythroid precursor cells were found on MaG-1 negative population. Immunoprecipitation of 125I-labeled U937 cell extract with the Mag-1 antibody under nonreducing and reducing conditions yielded a specific band of 66 kD. Thus, this antibody defines a new human macrophage/monocyte and granulocyte-specific antigen and may be useful for studying differentiation of human macrophages/monocytes.     

Monocyte adhesion to endothelium in simian immunodeficiency virus-induced AIDS encephalitis is mediated by vascular cell adhesion molecule-1/alpha 4 beta 1 integrin interactions.

Because the mechanisms associated with recruitment of monocytes to brain in AIDS encephalitis are unknown, we used tissues from rhesus monkeys infected with simian immunodeficiency virus (SIV) to examine the relative contributions of various adhesion pathways in mediating monocyte adhesion to endothelium from encephalitic brain. Using a modified Stamper and Woodruff tissue adhesion assay, we found that the human monocytic cell lines, THP-1 and U937, and the B cell line, Ramos, preferentially bound to brain vessels from monkeys with AIDS encephalitis. Using a combined tissue adhesion/immunohistochemistry approach, these cells only bound to vessels expressing vascular cell adhesion molecule-1 (VCAM-1). Furthermore, pretreatment of tissues with antibodies to VCAM-1 or cell lines with antibodies to VLA-4 (CD49d) inhibited adhesion by more than 70%. Intercellular adhesion molecule-1 (ICAM-1)/beta 2 integrin interactions were not significant in mediating cell adhesion to the vasculature in encephalitic simian brain using a cell line (JY) capable of binding rhesus monkey ICAM-1. In addition, selectin-mediated interactions did not significantly contribute to cell binding to encephalitic brain as there was no immunohistochemical expression of E-selectin and P-selectin in either normal or encephalitic brain, nor was there a demonstrable adhesive effect from L-selectin using L-selectin-transfected 300.19 cells on simian encephalitic brain. These results demonstrate that using the tissue adhesion assay, THP-1, U937, and Ramos cells bind to vessels in brain from animals with AIDS encephalitis using VCAM-1/alpha 4 beta 1 integrin interactions and suggest that VCAM-1 and VLA-4 may be integral for monocyte recruitment to the central nervous system during the development of AIDS encephalitis.     

Regulation of Fc epsilon receptor expression on a human monoblast cell line U937.

Fc epsilon receptor (Fc epsilon R) expression on several human cell lines (U937, RPMI 8866, HL 60, THP-1, and Molt 4) and its regulation were examined by immunofluorescent analysis using a monoclonal anti-human Fc epsilon R antibody, H107. Phorbol ester (PMA), recombinant gamma interferon (IFN-gamma) and H107 itself enhanced Fc epsilon R expression on a FC epsilon R positive cell line U937, whereas these reagents did not induce FC epsilon R expression on the Fc epsilon R negative cell lines, Molt 4, HL 60 and THP-1. Dexamethasone not only suppressed by 50% the spontaneous Fc epsilon R expression on U937 cells but also completely inhibited the enhancement of their Fc epsilon R expression on U937 cells induced by PMA, IFN-gamma or H107. Dexamethasone caused a little suppression of Fc epsilon R expression by RPMI 8866 cells. The results showed that Fc epsilon R expression on a human monoblast cell line U937 was up- or down-regulated by a variety of physiological or pharmacological agents. These experimental systems provide a good model for the investigation of the regulatory mechanisms of Fc epsilon R expression.     

Enhancement of activation-induced cell death by fibronectin in murine CD4+ CD8+ thymocytes.

Development of T cells in the thymus is achieved through the interactions of thymocytes with their microenvironments. This study focused on the function of fibronectin (FN), a major extracellular matrix molecule in the thymus, in the cell death induced by activation via the T-cell antigen receptor. FN alone did not increase cell death in murine thymocytes above the baseline level, but it significantly enhanced the cell death induced by fixed anti-CD3 monoclonal antibody (mAb), especially when a high concentration of anti-CD3 mAb was used. DNA fragmentation increased in parallel with cell death, indicating that cell death was a result of the apoptosis. Fluorescence-activated cell sorter (FACS) analysis revealed that the activation-induced cell death (AICD) caused by anti-CD3 mAb alone, or by a combination of anti-CD3 mAb and FN, occurred selectively in CD4+ CD8+ thymocytes. Very late activation antigen (VLA)-4 and VLA-5 are two major ligands to FN on thymocytes. The expression of both ligands was investigated at different stages of thymocyte development. VLA-4 was predominantly expressed at the CD4- CD8- stage, and thereafter the expression was reduced, whereas VLA-5 was constantly expressed during maturation. Furthermore, the enhancing effect by FN was inhibited in the presence of the Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) peptide but not in the presence of the connecting segment-1 (CS-1) peptide, suggesting that enhancement of AICD observed in CD4+ CD8+ thymocytes is mediated through VLA-5.     

Fibronectin synthesis by activated T lymphocytes: up-regulation of a surface-associated isoform with signalling function

Fibronectin (FN) is a major constituent of the extracellular matrix. We now provide evidence for a surface-associated isoform of FN that is synthesized by T cells upon activation. The T-cell-derived FN has an unusual splice pattern: an additional domain, EDB, is produced whereas sequences within another domain, IIICS, are spliced out. CS1, the binding domain for very late antigen-4 (VLA-4), however, is still generated. To study the potential function of surface-associated FN its synthesis was down-regulated by an antisense oligonucleotide, then proliferation of T cells was induced by cross-linked anti-CD3. Proliferation was reduced as was expression of CD25. Moreover, when T cells were cultured in high density, the synthetic peptide QILDVPST, corresponding to CS1, inhibited proliferation, as did antibodies to VLA-4. We propose that surface-associated FN is a ligand for VLA-4, which by binding to VLA-4 on an adjacent cell, provides a costimulatory signal, thus sustaining T-cell proliferation.     

Adsorption characteristics of human plasma fibronectin in relationship to cell adhesion

Adsorption of human plasma fibronectin (FN) on nonsulfonated and sulfonated polymer surfaces was studied, by using a polyclonal antiserum to FN and the ELISA method. ELISA signal was recorded as a function of FN concentration in solutions. The concentration dependence of FN binding shows the saturation effect in the range 5-10 microg/mL. ELISA data are discussed in the terms of a self-assembled monolayer and different conformations of the FN molecule.The early adhesion of L1210 cells to polymer surfaces after prior adsorption of FN on these surfaces was studied under static conditions. In the case of FN adsorbed on sulfonated surfaces, the relative number of adhering cells increased with the increase of the interfacial surface tension (i.e., the cell adhesion depends on the surface density of sulfonic groups). However, in the case of FN adsorbed on nonsulfonated surfaces, the relative number of adhering cells was low and independent on the interfacial surface tension.The alpha(5)beta(1)-integrin blocking by a monoclonal antibody resulted in a strong inhibition of the cell adhesion to FN adsorbed on sulfonated polymer surfaces. This indicates that cell adhesion to FN adsorbed on these surfaces is mostly mediated by the alpha(5)beta(1)-integrin. In contrast, in the case of FN adsorbed on nonsulfonated surfaces the cell adhesion was not inhibited by the alpha(5)beta(1)-integrin blocking.     

Expression and functions of very late antigen 1 in inflammatory joint diseases

In the human immune system, very late antigen 1 (VLA-1), a putative collagen receptor, is expressed on the surface of T lymphocytes that have undergone mitogenic or antigenic stimulation. A new VLA-1-specific monoclonal antibody, 1B3.1, was used to probe the expression and function of VLA-1 on T lymphocytes in patients with arthritis. Synovial mononuclear cells from the joints of patients with rheumatoid arthritis or other joint diseases contained 32.9±13.8% 1B3.1-positive cells (42.8±10.4% in patients with rheumatoid arthritis and 28 ± 12.6% in non rheumatoid patients). In the peripheral blood, patients with active rheumatoid arthritis expressed VLA-1 on 11.7±6.0% of their mononuclear cells, compared to 1.9±1.5% in controls (P<0.001). Using dual fluorescence analysis, virtually all the 1B3.1-positive synovial cells were CD3+ T lymphocytes and included both CD4+ and CD8+ T cells. When 1B3.1-expressing synovial mononuclear cells orin vitro activated T lymphocytes were triggered with anti-CD3 antibodies, marked augmentation of their proliferation occurred if they were simultaneously cross-linked with mab 1B3.1. Collagen type IV, a putative ligand of VLA-1, also augmented T-cell proliferation to anti-CD3. The data suggest that the VLA-1 molecule could play an important role in the pathophysiology of arthritis by modulating T-cell activation in these diseases.     

Very late antigen integrins are involved in the adhesive interaction of lymphoid cells to human gingival fibroblasts.

To date, it is still unclear how the trafficking and retention of activated lymphocytes in periodontal lesions are regulated. In this study, we investigated the molecular basis for the adhesive interactions between lymphocytes and human gingival fibroblasts (HGF). Peripheral blood T lymphocytes (PBT) exhibited binding ability, but only when the calls were activated with phorbol 12-myristate 13-acetate (PMA). Among several human cell lines tested, PMA-stimulated Molt-4, a human T-cell leukaemia line, also displayed significant binding ability to HGF. In order to clarify the molecule(s) involved in this cell-cell interaction, a panel of monoclonal antibodies (mAb) was prepared to PMA-activated Molt-4 and one clone, 4-145, was selected on the basis of its ability to block the binding of PMA-activated Molt-4 to HGF. Moreover, 4-145 inhibited the binding of not only activated Molt-4 but also activated PBT and other cell types to HGF. Biochemical and flow cytometric analyses revealed that 4-145 probably recognizes the beta 1 chain of very late antigen (VLA) integrins. Blocking experiments using mAb specific for the alpha-chain of VLA integrins demonstrated the involvement of alpha 4 (VLA-4) and, to a lesser extent, alpha 5 (VLA-5) chains in the adhesive interactions between T cells and HGF. Despite the significant involvement of VLA integrins in the adhesive interaction between PBT and HGF, the binding of PBT to human dermal fibroblasts (HDF) was not abrogated by 4-145, suggesting that HGF and HDF differ in their requirement of VLA integrins for adhesion to activated PBT. Furthermore, the fact that vascular cell adhesion molecule-1 (VCAM-1), one of the ligands of VLA-4, was not detected on HGF by flow cytometry and anti-fibronectin (FN) Ab did not block the adhesive interaction to HGF suggests that not-yet-identified ligand(s) for VLA-4 might be present on HGF.     

Proinflammatory and proapoptotic activities associated with Bordetella pertussis filamentous hemagglutinin

Filamentous hemagglutinin (FHA) is a dominant cell surface-associated Bordetella pertussis adhesin. Recognition that this protein is secreted in significant amounts and that bacterial adhesins may have other activities, prompted an assessment of FHA effects on human macrophages. Incubation of human macrophage-like U937 cells with preparations of FHA resulted in dose-dependent cytotoxicity, with death of 95% of treated cells after 24 h. Based on the use of four independent methods, death of these cells could be largely attributed to apoptosis. FHA-associated apoptosis was also observed in THP-1 macrophage-like cells, fresh human peripheral blood monocyte-derived macrophages (MDM), and BEAS-2B human bronchial epithelial cells. Infection of MDM with wild-type B. pertussis resulted in apoptosis within 6 h, while infection with an FHA-deficient derivative strain was only 50% as effective. FHA-associated cytotoxicity was preceded by host cell secretion of tumor necrosis factor alpha (TNF-alpha), a potential proapoptotic factor. However, pretreatment of cells with a neutralizing anti-TNF-alpha monoclonal antibody inhibited only 16% of the FHA-associated apoptosis. On the other hand, a blocking monoclonal antibody directed against TNF-alpha receptor 1 inhibited FHA-associated apoptosis by 47.7% (P = 0.0001), suggesting that this receptor may play a role in the death pathway activated by FHA. Our in vitro data indicate that secreted and cell-associated FHA elicits proinflammatory and proapoptotic responses in human monocyte-like cells, MDM, and bronchial epithelial cells and suggest a previously unrecognized role for this prominent virulence factor in the B. pertussis-host interaction.     

Dibutyryl cyclic AMP stimulation of a monocyte-like cell line, U937: a model for monocyte chemotaxis and Fc receptor-related functions.

Treatment of the U937 cell line with 1 mM dibutyryl cyclic AMP (Bt2cAMP) resulted in a reduction in cell size and inhibition of DNA synthesis, and morphologically the cells appeared similar to macrophages. Electron micrographs indicated an increase in intracellular apparatus, whilst histochemical studies revealed smaller, denser nuclei and a greater intensity of non-specific esterase staining. Ia-like antigens (HLA-DR and HLA-DC) and complement receptor CR1 were not detected on U937 cells by monoclonal antibodies, nor were they induced by Bt2cAMP. CR3 was present in small amounts on U937 cells, and stimulation with Bt2cAMP increased the expression of this molecule in the cytoplasm and on the cell surface. Leu M3, a monocyte-specific antibody, was weakly reactive on both unstimulated and stimulated cells, whereas transferrin receptors, present on 90% of U937 cells, were lost after 48-hr stimulation with Bt2cAMP. JW6 and NH6, two monoclonal antibodies raised in our laboratory and found to be against immature monocytic antigens, showed decreased expression on stimulation. Monomer IgG binding via Fc receptors decreased on stimulated cells, and a monoclonal antibody (32.2) specific for FcRI confirmed this to be due to a decrease in the number of high-affinity receptors, rather than a decrease in IgG-binding affinity. In contrast, expression of the low-affinity FcRII, monitored by monoclonal antibody IV3, increased dramatically after stimulation. Other functional changes included the production of superoxide anions and the induction of non-specific phagocytosis. Two dimensional gel analysis, of detergent soluble proteins from unstimulated and 48-hr stimulated U937 cells, showed many differences in protein expression. A detailed investigation of these changes will facilitate a better understanding of the molecular mechanisms involved in the differentiation of U937 cells.     

Expression of the fibronectin receptor VLA-5 is regulated during human B cell differentiation and activation.

We examined the expression of VLA-5, a fibronectin receptor, during human B cell development and activation. VLA-5 is a member of the integrin supergene family; VLAs are heterodimers of at least six unique alpha chains sharing a common beta chain; most are involved in cell attachment to extracellular matrix (ECM). A hypothesis of haematopoietic development is that maturing cells leave the bone marrow because of the loss of VLA-5 during differentiation. However, mature B cells are not primarily circulating cells, and the role of ECM receptors in homing to peripheral lymphoid tissue and inflammatory sites is unknown. To examine the expression of VLA-5 during B cell development, cell lines blocked at specific stages of differentiation were evaluated for their synthesis and surface expression of VLA-5 using VLA-5-specific antibody and cDNA probes. VLA-5 mRNA and surface expression were found in the pre-B cell lines, REH and Nall 1, but not in more differentiated Raji cells or in several EBV-transformed peripheral B cell lines. Circulating peripheral B lymphocytes and resting tonsillar and splenic B lymphocytes expressed no VLA-5 by FACS analysis. Interestingly, mRNA and surface expression of VLA-5 were found in SKW, a highly differentiated, IgM-secreting line. In addition, low levels of staining for VLA-5 expression could be demonstrated when tonsillar or peripheral blood B lymphocytes were stimulated by Staphylococcus aureus Cowan (SAC). All cell lines expressed VLA-3 and VLA-4, two other receptors reported to mediate fibronectin binding in some cell types. Thus, our studies provided no evidence for developmental or inflammatory regulation of these receptors. Binding studies, however, demonstrated that adherence of both pre-B REH cells and SKW cells to fibronectin was almost completely inhibited by a monoclonal antibody to VLA-5 alpha. In addition, Raji cells, which lack VLA-5 but express VLA-3 and VLA-4, showed very low level binding to fibronectin. This demonstrates that for some B lymphocytes VLA-5, rather than other possible fibronectin receptors, primarily mediates attachment to fibronectin. These data also suggest that human VLA-5 expression is regulated during B cell development, with expression at a very early stage and then again after activation. This pattern of loss and reacquisition of an ECM receptor may be relevant to normal B cell maturation and to function during immunologic injury.     

Histamine influences the expression of the interleukin-6 receptor on human lymphoid,monocytoid and hepatoma cell lines

Based on our earlier data on the enhancing effect of histamine on the action of interleukin-6 (IL-6), we have studied the molecular mechanisms of these interactions. The effect of histamine was investigated on the binding of¹²⁵I-IL-6 by B lymphoma cell line CESS, monocytoid cell line U937 and hepatoma cell line HepG2. Histamine increases the IL-6 binding by CESS cells and inhibits that by U937 and HepG2 cells. Using H₁ receptor (cetirizin and loderix) and H₂ receptor (cimetidine and ranitidine) specific antagonists, an H₁-dependent stimulation of IL-6 binding by CESS cells was found. In contrast, down-regulation of IL-6 binding by histamine was clearly mediated through H₂ receptors.On U937 cells, using a monoclonal antibody reacting with the 80 kd chain of the human IL-6 receptor, an H₂-receptor mediated inhibition of IL-6 receptor expression was found by FACS analysis.     

Interaction of immune complexes isolated from hepatitis C virus-infected individuals with human cell lines

We investigated the interaction of immune complexes (IC) isolated from hepatitis C virus (HCV)-infected individuals with several cell lines that differentially express Fc receptors, and analyzed viral infection by the presence of HCV RNA sequences. Monocytic (U937 and Monomac-6) and lymphocytic (MOLT-4 and Jurkat) cell lines were incubated with interferon- plus phorbol myristate acetate to stimulate the expression of Fc receptors before addition of IC. Cell interaction with IC was monitored by flow cytometry. Positive cell fluorescence was detected in U937 and Monomac-6 cells [mean fluorescence intensity (MFI) 10.56±0.8 and 11.60±0.8, respectively]. Incubation of cells with monoclonal antibodies against Fc receptors for IgG before addition of IC decreased MFI in both cell lines (U937 2.1±0.5, Monomac-6 4.4±0.8, P<0.001), indicating that cell-IC interaction through these receptors was inhibited. In particular, the blockage of FcRII was responsible for this effect. No binding of IC with either MOLT-4 or Jurkat cell lines was detected, which correlated with a very low Fc receptor expression. HCV RNA sequences were identified in the cells up to 120 h of post incubation with IC. These results suggest that IC can mediate entry of HCV to both U-937 and Monomac-6 cell lines mainly through the FcRII.     

CD147 monoclonal antibodies induce homotypic cell aggregation of monocytic cell line U937 via LFA-1/ICAM-1 pathway

CD147 is a 50 000-60 000 MW glycoprotein of the immunoglobulin superfamily broadly expressed on haemopoietic cell lines and peripheral blood cells. In the present study, six monoclonal antibodies (mAbs) directed against the CD147 protein were generated. The antigen defined by the generated CD147 mAbs is widely expressed on haemopoietic cell lines, peripheral blood cells and is a lymphocyte activation-associated cell surface molecule. The generated CD147 mAbs precipitated a broad protein band from U937 cells of 45 000-65 000 MW under reducing conditions. Functional analysis indicated that the CD147 mAbs markedly induced homotypic cell aggregation of U937 cells, but not K562 cells. The CD147 mAb-induced cell aggregation was inhibited by leucocyte function-antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) mAbs. However, the expression of LFA-1 and ICAM-1 molecules on U937 was not altered by CD147 mAb treatment. The U937 cell aggregation induced by CD147 mAb was also inhibited by ethylenediamine tetra-acetic acid (EDTA), sodium azide and when incubated at 4 degrees. We therefore propose that the binding of CD147 mAb to CD147 molecule, which mimics the natural ligand binding, may generate intracellular signals that activate LFA-1/ICAM-1 intercellular adhesion pathway.     

Preparation of a monoclonal antibody against human monocyte lineage

Preparation and characterization of a monoclonal antibody termed M206 directed to a human monocyte lineage are described. The antibody was produced by somatic cell hybridization between BALB/c spleen cells primed with human histiocytic lymphoma cell line U937 and murine myeloma cell line P₃U₁. The antibody reacted intensely with human peripheral blood monocytes as well as U937 but did not react with peripheral blood T and B cells. Granulocytes were weakly stained with the antibody by indirect immunofluorescence. M206 also reacted intensely with the immature leukemic cells from patients with acute monocytic leukemia but was unreactive with other types of leukemic cells except cells from chronic myelogenous leukemia which showed varied patterns of reactivities. M206 reacted with a single polypeptide chain with a molecular weight of 180,000 on the surface of U937 cells.     

Characterization of the human monocyte high affinity Fc receptor (hu FcRI)

The high affinity Fc receptor (FcRI) of a human monocytic cell line, U937, was further characterized using a previously described murine monoclonal antibody, FcRmAb32. This antibody immunoprecipitated a 70 K cell surface glycoprotein. A solid phase ligand binding assay and a solid phase immunoprecipitation assay were combined to confirm that the 70 K cell surface glycoprotein immunoprecipitated by FcRmAb32 is an IgG binding protein. N-glycanase digestion shows that at least 20% of the relative mobility of the 70 K FcRI glycoprotein is due to N-linked carbohydrate. FcRmAb32 immunoprecipitated a 70 K glycoprotein from biosynthetically labelled U937 cells that co-migrated with the surface iodinated glycoprotein on 2-dimensional gel electrophoresis. A 50 K protein, that is biosynthetically labelled but not accessible to surface iodination, which, bound to control antibodies was also present in FcRmAb32 immunoprecipitates. FcRmAb32 only bound the mature fully glycosylated form of FcRI. The 70 K FcRI was not phosphorylated constitutively nor when U937 cells were stimulated by PMA.     

Expression and functional activity of the very late activation antigen-4 molecule on human natural killer cells in different states of activation

In the present study we describe the expression and functional activity of the alpha4beta1 heterodimer molecule on human natural killer (NK) cells. Flow cytometric analyses showed that fresh and activated NK cells expressed high levels of very late activation antigen-4 (VLA-4) molecules. These cells bound to fibronectin (FN) and to its 38 000-MW proteolytic fragment through the VLA-4 integrin that was blocked with HP2/1 anti-alpha4 monoclonal antibodies (mAbs) and with the FN peptide fragment CS1. No inhibitory effects were observed in the presence of anti-alpha5 mAb, FN peptide fragment CS2 or other irrelevant mAb. Fresh NK cells were unable to aggregate, despite their expression of VLA-4, and only activated (cultured and lymphocyte-activated killer cells) NK cells showed homotypic aggregation with HP1/7 and HP2/4 anti-alpha4 mAb related to cellular activation. These results underline new evidence of how NK cells in different states of activation maintain different constitutive levels of alpha4beta1 integrin activity, and highlight the possibility of a different functional regulation by the cells bearing VLA-4, in the expression of these epitopes and their ability to interact with their ligands.