Biosynthesis of the third component of complement (C3) by the human monocytic-cell line U-937. Induction by phorbol myristate acetate.

Phorbol myristate acetate (PMA)-stimulated human monocyte-like cells (U-937) were found to synthesize the third component of complement (C3), as shown by enzyme-linked immunosorbent assay and immunoprecipitation from [35S]methionine-labelled culture supernatants. C3 synthesis occurred at a rate of about 160 ng of C3/24 h per 10(6) cells on day 7 after addition of PMA; it was blocked by cycloheximide treatment and was restored after removal of the inhibitor. SDS/polyacrylamide-gel-electrophoretic analysis of the immunoprecipitated protein showed that the size and subunit structure of the newly synthesized C3 were identical with those of plasma C3, and that a single-chain intracellular precursor was present in the cell lysates. Haemolytic assays showed that the synthesized C3 fully expressed functional activity in early culture within 4 h. After longer culture, a loss of haemolytic activity was observed. The possibility that newly secreted C3 is cleaved by U-937 cells themselves was suggested.     

Thrombospondin production and thrombospondin-mediated adhesion in U937 cells

U937 cells have low levels of surface thrombospondin (TSP) under control conditions but express higher levels after treatment for 1 day with 100 nM phorbol myristate acetate (PMA). Increased surface expression is due, in part, to increased biosynthesis. Untreated U937 cells do not adhere to TSP-coated plastic culture dishes but adhere strongly to TSP after stimulation with PMA. Untreated U937 cells also adhere weakly to endothelial cell monolayers while PMA-treated U937 cells attach strongly to monolayers of rat pulmonary artery endothelial cells. Endothelial cell adhesion appears to be mediated, in part, by TSP since antibodies to TSP partially inhibit.     

Characterization of human monocytic cell line, U937, in taking up acetylated low-density lipoprotein and cholesteryl ester accumulation. A flow cytometric and HPLC study

The uptake of LDL and acetylated LDL and the ability of cholesteryl ester accumulation by cells of a human monocytic cell line, U937, has been characterized by flow cytometric assay using a fluorescent probe, DiI, and by high-performance liquid chromatography (HPLC). The increase of mean fluorescence intensity of U937 incubated with DiI-labeled lipoproteins demonstrates that this cell line could incorporate DiI-AcLDL, as well as DiI-labeled LDL. Competition and saturation studies indicate that the manner of taking up DiI-AcLDL is receptor-mediated. While differentiated U937 incubated with 16 nM phorbol myristate acetate for 24 h took up little DiI-AcLDL, HPLC analysis confirmed that intracellular free and esterified cholesterols significantly increase in the U937 cells incubated with AcLDL or LDL. The ability of mouse peritoneal macrophage to abundantly accumulate at least five kinds of cholesteryl ester were also shown in this analysis. In contrast, in U937 cells, free fatty acids are incorporated into various substances rather than into cholesteryl esters (as revealed by HPLC analysis), so that the cholesterol in AcLDL taken up by U937 cells is not synthesized into cholesteryl esters to any great extent.     

Spontaneous production of a suppressor factor by a human macrophage-like cell line U937. II. Suppression of antigen- and mitogen-induced blastogenesis, IL 2 production and IL 2 receptor expression in T lymphocytes

U937, a human macrophage-like cell line, spontaneously produces a factor which inhibited blastogenic responses of human blood T lymphocytes stimulated with tuberculin-purified protein derivative (PPD) or phytohemagglutinin (PHA). We investigated the mechanism of suppressor action of the U937 factor. The U937 suppressor factor inhibited interleukin 2 (IL 2) production by human blood T lymphocytes stimulated with PPD or PHA. IL 1 did not overcome the inhibitory action of the U937 factor on PPD-induced IL 2 production by human blood T lymphocytes. The U937 factor also inhibited the production of IL 2 by a human leukemic cell line, JURKAT, stimulated with PHA. The U937 suppressor factor interfered with the expression of Tac antigen (IL 2 receptor) on PPD- or PHA-stimulated blood T lymphocytes. The inhibitory activity of the U937 factor on Tac expression was not affected by the addition of IL 2 or a crude lymphokine-containing T cell supernatant. Tac expression was more sensitive than IL 2 production to inhibition by U937-conditioned medium. The U937 suppressor factor was precipitable by 33 to 67% saturated ammonium sulfate and was inactivated at pH 2 or pH 11. Sephacryl S-200 Gel filtration analysis of U937 culture supernatants revealed that the inhibitory activities for blastogenesis, IL 2 production, and Tac expression co-purified in fractions with an apparent m.w. between 67,000 and 130,000. These data indicate that U937 spontaneously produces a macromolecular suppressive factor with major locus of action on the production of IL 2 and the expression of the IL 2 receptor.     

Spontaneous production of a suppressor factor by the human macrophage-like cell line U937. I. Suppression of interleukin 1, interleukin 2, and mitogen-induced blastogenesis in mouse thymocytes

The human macrophage-like cell line U937 spontaneously produced a nondialyzable factor that inhibited interleukin 1 (IL 1), interleukin 2 (IL 2), and phytohemagglutinin (PHA)-induced blastogenesis in mouse thymocytes. The suppression by U937 supernatant factor occurred independently of the concentration of IL 1 or PHA, indicating that it was noncompetitive. The U937 suppressor factor was not cytotoxic for thymocytes, nor did it affect the spontaneous proliferation of T lymphoblastoid cell lines and U937. Physicochemical characterization showed that the U937 suppressor factor was nondialyzable, partially inactivated by heat treatment (56 degrees C), ammonium sulfate (67% saturation) precipitable, sensitive to pH 2.5, and resistant to freeze-thawing. Molecular weight of the factor inhibiting co-mitogenic IL 1 activity was approximately 85,000, as estimated by gel filtration. The U937 cell line may provide a model for the study of mechanisms and mediators of immunosuppression by mononuclear phagocytes.     

Respiratory syncytial virus infection of human mononuclear phagocytes stimulates synthesis of platelet-activating factor

Production of platelet-activating factor 1-O-alkyl-2-acetyl-sn-glycero-3- phosphocholine (PAF), a potent mediator of inflammation, by mononuclear phagocytes varies with their stage of cellular differentiation and the nature of the eliciting stimulus. The human monocytic cell line U937 can be induced to differentiate to a macrophage-like cell following phorbol myristate acetate exposure, and after differentiation, these cells efficiently support replication of respiratory syncytial virus (RSV). U937 cells induced to differentiate with phorbol myristate acetate demonstrated a time-dependent decrease in PAF synthesis. RSV infection of these differentiated U937 cells caused a sustained stimulation of PAF synthesis that paralleled viral replication and was dependent on infectious virus. Virus increased the activity of lyso-PAF:acetyl-CoA acetyl-transferase (PAF acetyltransferase) in cell lysates, thus enhancing the anabolic pathway of PAF synthesis without altering the activity of PAF acetylhydrolase, which regulates PAF catabolism. RSV infection of human monocytes also caused a marked increase in [3H] monocytes also caused to uninfected monocytes. Thus, virus infection serves as a novel stimulus to induce PAF synthesis in human mononuclear phagocytes and suggests that increased PAF production may have a critical role in the inflammatory response to RSV.     

Enzyme immunoassay of HBeAg employing beta-D-galactosidase

An enzyme-linked immunosorbent assay (ELISA) system for hepatitis B e antigen (HBeAg) was developed employing beta-D-galactosidase conjugated with antibody to HBeAg (anti-HBe) and using m-maleimidobenzoyl-N-hydroxysuccinimide ester as the coupling reagent. The experimental conditions for quantitative assay of HBeAg were determined. The presence of rheumatoid factor in test sera did not affect the results. This assay system is more sensitive than the micro-Ouchterlony method and as sensitive as radioimmunoassay. The use of beta-D-galactosidase for ELISA in the field of virology is recommended.     

LPS-stimulated release of prostaglandin E and thromboxane B2 from the U937 cell line

Human monocytes are known to metabolize arachidonic acid (AA) and to release prostaglandins upon stimulation. Previous data indicate that in vitro maturation and differentiation of monocytes result in alteration of this property with greatly diminished response to stimulators of release of prostaglandin E (PGE) and thromboxane B2 (TxB2) occurring after cells have been cultured. To further study the effects of differentiation on human monocyte AA metabolism, a model system was established based upon the human histiocytic cell line U937. Among tested stimulants, which included opsonized zymosan, complement fragment C3b, phorbol myristate acetate (PMA), calcium ionophore A23187, and concanavalin A, it was found that Escherichia coli lipopolysaccharide (LPS) was unique in that it stimulated increased release of TxB2 from U937 cells. The effect of the phorbol ester PMA, a compound commonly used to induce differentiation of U937, on the ability of U937 to respond to LPS was examined. Following 48 hr of treatment with PMA, U937 became capable of releasing both PGE and TxB2 in response to small doses of LPS. As previously observed for human monocytes, the release of PGE was delayed for several hours following stimulation and failed to reach maximal cumulative levels in culture until 24-48 hr following stimulation. In contrast to human monocytes, PMA-induced U937 were capable of maintaining their responsiveness to LPS for several days. Thus, the U937 cell line provides a useful model for study of the effects of differentiation of human mononuclear phagocytes on their ability to metabolize AA, and for the effects of LPS on histiocytic tumor cell prostaglandin release.     

Nuphar alkaloids with immediately apoptosis-inducing activity from Nuphar pumilum and their structural requirements for the activity

The methanolic extract and its alkaloid fraction from the rhizomes of Nuphar pumilum showed cytotoxic effects on human leukemia cell (U937), mouse melanoma cell (B16F10), and human fibroblast (HT1080). Dimeric sesquiterpene thioalkaloids with the 6-hydroxyl group (6-hydroxythiobinupharidine, 6,6'-dihydroxythiobinupharidine, 6-hydroxythionuphlutine B) showed substantial cytotoxic activity at a concentration of 10 microM, but dimeric sesquiterpene thioalkaloids lacking the 6-hydroxyl group (thiobinupharidine, thionuphlutine B, 6'-hydroxythionuphlutine B, neothiobinupharidine, thionuphlutine B beta-sulfoxide, and neothiobinupharidine beta-sulfoxide) and monomeric sesquiterpene alkaloids (nupharidine, 7-epideoxynupharidine, and nupharolutine) showed weak activity. Next, apoptosis-inducing activity of a principal active constituent, 6-hydroxythiobinupharidine, on U937 was examined using morphological observation and DNA fragmentation assay (TUNEL method). Apoptosis of U937 was immediately observed within 1 h after treatment of 6-hydroxythiobinupharidine at 2.5-10 microM.     

Lymphokine and phorbol (PMA) regulation of complement (C2) synthesis using U937

Human monocytes synthesize large amounts of the second complement component (C2) after incubation with a T-lymphocyte product called monocyte complement stimulator (MCS). The human monocyte-like cell line, U937, also synthesizes C2 and can be stimulated to increase this synthesis by lymphokine-rich culture supernates. Additionally, phorbol myristate acetate (PMA), an agent which induces maturational changes in other macrophage-like cell lines, also stimulates C2 synthesis by U937 cells. Lymphokine and PMA stimulation of C2 secretion by U937 are both reversibly inhibitable by cycloheximide. At optimal concentrations for stimulation of C2 synthesis, PMA inhibits [³H]thymidine incorporation by U937 indicating that increased C2 is not due to increased numbers of U937 cells.     

Regulation of the growth and differentiation of a human monocytic cell line by lymphokines. I. Induction of superoxide anion production and chemiluminescence

The U937 human monocytic cell line was studied to determine its ability to generate a respiratory burst after stimulation with phorbol myristate acetate (PMA) or opsonized zymosan. U937 cells cultured in normal medium produced virtually no superoxide anion or chemiluminescence in response to either stimulus. In contrast, U937 cells cultured in medium containing soluble factors from activated lymphocytes produced significant O2- and chemiluminescence when stimulated with PMA or opsonized zymosan. The chemiluminescence in response to PMA was maximal in U937 cells precultured with these soluble factors for 3 days, whereas maximal responsiveness to opsonized zymosan was not observed until 5 to 6 days of lymphokine exposure. Although this ability to generate a respiratory burst persisted for a number of days in U937 cells that were subsequently recultured in normal medium, this responsiveness was gradually lost in the continued absence of these factors. The data indicate that the U937 monocytic cell line can be activated or induced to differentiate by soluble factors released by activated lymphocytes. In the process, these cells acquire the ability to generate a respiratory burst. The U937 cell line may serve as a useful model for the study of the ontogeny and regulation of the respiratory burst during human monocytic differentiation.     

Protective activity of adult T cell leukemia-derived factor (ADF) against tumor necrosis factor-dependent cytotoxicity on U937 cells.

Adult T cell leukemia-derived factor (ADF) is a human homologue of thioredoxin with many biologic functions including IL-2R induction, growth promotion, thiol-dependent reducing activity, and radical scavenging activity. The regulatory effect of ADF on the cytotoxic activity of TNF was examined by using a human histiocytic lymphoma cell line, U937. When U937 cells were preincubated with recombinant ADF (rADF) (0.1-100 micrograms/ml) at 37 degrees C for 30 min, TNF-dependent cytotoxicity on U937 cells was markedly inhibited. This inhibitory effect was as high as 95% in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (rADF 100 micrograms/ml) and 85% in the 51Cr-releasing assay (rADF 10 micrograms/ml). After pretreatment of U937 cells with IFN-gamma to augment the sensitivity to TNF, an inhibitory effect of rADF was also found. When U937 cells were washed after preincubation with rADF, resistance to TNF-dependent cytotoxicity was still observed, indicating that rADF inhibited the sensitivity of U937 to TNF-dependent cytotoxicity rather than modifying TNF molecules. Scatchard analysis of TNF receptors on U937 cells using 125I-TNF showed that rADF modulated neither the density nor the affinity of the cell membrane significantly. rADF also reduced the cytotoxicity induced by anti-Fas IgM mAb which shows cytotoxicity quite similar to TNF. rADF (10 micrograms/ml) reduced 90% of the cytotoxicity by anti-Fas IgM mAb, without a detectable change either in Fas Ag expression (MFI 58.1 vs 53.3) or in the degradation of anti-Fas IgM mAb as determined by flow cytometric analysis. These findings indicated that the rADF-induced resistance to the cytotoxic effect of TNF and anti-Fas mAb was not related to the modulation of the TNF receptor or Fas Ag.     

Time dependent production of cytokines and eicosanoids by human monocytic leukaemia U937 cells; effects of glucocorticosteroids

In the present study the human monoblast cell line U937 has been used as a model to study the function of human mononuclear phagocytes in asthma. The kinetics of the production of eicosanoids and cytokines, which are thought to play a role in the pathogenesis of asthma, were studied. In addition, the effects of glucocorticosteroids were investigated, as these drugs are of great importance for the treatment of asthmatic patients. After stimulation with phorbol-12 myristate acetate (PMA) for 24 h, U937 cells were cultured in the absence or presence of lipopolysaccharide (LPS: 1 and 5 microg ml(-1)) and glucocorticosteroids (budesonide, fluticasone propionate and prednisolone: 10(-11), 10(-9) and 10(-7) M) for 96 h. The production of interleukin-1beta (IL-1beta), interleukin-6 (IL-6), prostaglandin E2 (PGE2) and thromboxane B2 (TxB2) gradually increased in time after stimulation with LPS, whereas the transient production of tumor necrosis factor alpha (TNF-alpha) reached its maximum between 6 and 12 h. Interferon-gamma (IFN-gamma), interleukin-10 (IL-10) and leukotriene B4 (LTB4) were not detectable. All three glucocorticosteroids (budesonide, fluticasone propionate and prednisolone) completely inhibited the production of both eicosanoids and cytokines. The production of eicosanoids was more sensitive to these glucocorticoids than the production of cytokines. The observed differences in the kinetics of the production of eicosanoids and cytokines stress the importance of time course experiments in studies on the effect of drugs on mononuclear cells.     

Decrease in CD93 (C1qRp) expression in a human monocyte-like cell line (U937) treated with various apoptosis-inducing chemical substances

Human CD93, a receptor for complement component 1, subcomponent q phagocytosis (C1qRp), has been shown to be selectively expressed by cells of a myeloid lineage and was originally reported to be involved in the C1q-mediated enhancement of phagocytosis in innate and adaptive immune responses. The modulation of CD93 expression has been investigated in various cells, particularly in granulocytes and monocytes . We previously reported that a protein kinase C activator (PKC), phorbol myristate acetate (PMA), effectively up-regulated CD93 expression on several cultured cell lines and that its regulation was mainly controlled by a PKC delta-isoenzyme. However, the expression pattern of CD93 in myeloid cells with apoptotic properties remains poorly understood. In this study, we examined the modulation of CD93 expression on a human monocyte-like cell line (U937) treated with various apoptosis-inducing chemical substances : an RNA-synthesis inhibitor, actinomycin D (ActD); a DNA topoisomerase I inhibitor, camptothecin (CPT); a protein-synthesis inhibitor, cycloheximide (CHX); a DNA topoisomerase II inhibitor, etoposide (EPS); and a DNA-synthesis inhibitor, mitomycin C (MMC). Apoptosis was monitored using two-color flow cytometry with Annexin V and 7-amino actinomycin D (7AAD). The above-mentioned substances sufficiently induced the early and late stages of apoptosis, identified as Annexin V positive (+)/7AAD negative (-) cells and Annexin V positive (+)/7AAD positive (+) cells, respectively, in U937 cells after 6 hr of treatment. The modulation of CD93 expression on U937 cells during the early stage of apoptosis, gated as Annexin V positive (+)/7AAD negative (-) cells, was then investigated using a CD93 mAb (mNI-11), originally established in our laboratories, and flow cytometry using a fluorescence-activated cell sorter (FACS). The mean fluorescence intensity (MFI) of the cells that stained positive for CD93 mAb (mNI-11) among the treated U937 cells showed a dramatic decrease in expression. In addition, the expressions of HLA-class I (HLA-A, B, C), HLA-class II (HLA-DR), CD18 (lymphocyte function-associated antigen-1 beta; LFA-1beta) and CD54 (intercellular adhesion molecule-1; ICAM-1) were also markedly decreased on the treated U937 cells identified as Annexin V positive (+)/7AAD negative (-) cells (early stage of apoptosis). Interestingly, the expression patterns of CD93 on the U937 cells treated with the above-mentioned chemical substances closely resembled those of HLA-class I (HLA-A, B, C). An immunoblotting analysis showed that the expression of a surface antigen (molecular size, about 97 kDa) targeted by the CD93 mAb (mNI-11) on the U937 cells treated with various apoptosis-inducing chemical substances had clearly decreased. On the other hand, an enzyme-linked immunoassay (EIA) showed that although PMA-treated U937 cells had strongly secreted soluble CD93 (sCD93) into the culture supernatant, the secretion of sCD93 in the culture supernatant of the U937 cells treated with the above-mentioned chemical substances was not enhanced, compared with that of untreated U937 cells. Importantly, however , the U937 cells with apoptotic properties induced by various apoptosis-inducing chemical substances also rapidly (in 30 min) and strongly secreted sCD93 into the culture supernatant in the presence of PMA. Taken together, these findings indicate that the expression of the CD93 molecule identified by CD93 mAb (mNI-11) is dramatically decreased on U937 cells with apoptotic properties, and that the decrease in CD93 expression on U937 cells treated with apoptosis-inducing chemical substances may be a good model for analyzing the regulation of CD93 expression on apoptotic myeloid cells.     

Specific activation by concanavalin A of the superoxide anion generation capacity during U937 differentiation

Phorbol myristate acetate (PMA) induces changes in the human monocyte-macrophage-like cell line U937 which reflect cellular differentiation. PMA prompted the expression of the superoxide anion (O2-) generating capacity in U937 upon appropriate stimulation. A highly specific stimulation by Concanavalin A (Con A) of O2- release was observed in PMA-differentiated U937 cells, which exceeded in 10-20 times that obtained with Con A-stimulated monocytes and neutrophils. These results indicate that a highly specific machinery required for Con A stimulation, practically absent in mature monocytes and neutrophils, is synthesized during PMA-induced U937 differentiation. A novel cytochrome b putatively involved in O2- generation was detected in U937 cells. This cytochrome b content was increased during PMA-induced cell differentiation, although no linear correlation was found between capability to produce O2- by macrophage-like U937 cells and their content of cytochrome b.     

Topological localization of plasminogen activator inhibitor type 2

BACKGROUND: Plasminogen activator inhibitor type 2 (PAI-2) is a member of the serine protease inhibitor (SERPIN) superfamily and forms stable complexes with urokinase type plasminogen activator (uPA). uPA can be found on the cell surface attached to its specific receptor (uPAR), allowing for controlled degradation of the extracellular matrix by the activation of plasminogen into plasmin. The aim of this study was to evaluate if PAI-2 could also be detected on the cell surface, providing a means of regulating the activity of cell surface uPA. METHODS: Intact or permeabilized cell lines or human peripheral blood leukocytes were assayed by flow cytometry for cell surface uPA or PAI-2. Plasma membrane-enriched preparations prepared from Jurkat, HaCaT, THP-1, U937, or MM6 cells were assayed by enzyme-linked immunosorbent assay (ELISA) or Western blotting for PAI-2 antigen. RESULTS: By flow cytometry, cell surface PAI-2 was not detected on monocytes from human peripheral blood, MM6, or HaCaT cells. Cell surface PAI-2 was only detected very weakly on the surface of U937 cells. In contrast, PAI-2 could be detected in all of these cells when fixed and permeabilized. By ELISA, PAI-2 was very abundant in the cytosol-enriched preparations of U937, MM6, and HaCaT cells, but was present in lower amounts in the plasma membrane-enriched preparations. By Western blotting, monomeric nonglycosylated PAI-2, but not uPA/PAI-2 complexes, could be detected in the cytosol and plasma membrane-enriched preparations. CONCLUSIONS: These results indicate that PAI-2 cannot be detected on the surface of PAI-2-expressing cells, and confirm that PAI-2 is predominantly a cytosolic protein.     

Autogenous production of interferon-beta switches on HLA genes during differentiation of histiocytic lymphoma U937 cells

The expression of class I HLA genes was measured during the in vitro differentiation of human U937 lymphoma cells towards macrophages. Following the onset of differentiation by phorbol myristate acetate the levels of cytoplasmic mRNA that hybridized with a [32P]HLA-B cDNA probe increased by a factor of nine. Elevation in HLA mRNA accumulation was followed by an increase in the rate of synthesis of HLA proteins and also by a dramatic increase in class I HLA cell surface antigen expression, as shown by cytofluorimetric analysis. The elevation in HLA mRNA and surface antigens could be prevented by adding antibodies against human interferon-beta (IFN-beta) to the culture medium at the onset of differentiation. Interferon antiviral activity was detected in the medium of differentiated U937 cells. The same anti-IFN-beta antibodies prevented the increase in (2'-5')oligo(A) synthetase activity which also takes place in differentiating U937 cells. Accumulation of the IFN-induced (2'-5')oligo(A) synthetase in U937 cells is preceded by an increase in its specific 1.6-kb mRNA as shown by hybridization to cloned (2'-5')-oligo(A) synthetase cDNA. The enzyme was preferentially found in the nuclear fraction of differentiating U937 cells. We suggest that an autogenous production of interferon-beta by the differentiating cells, switches on expression of the class I HLA genes as well as that of the (2'-5')oligo(A) synthetase.     

Regulated expression of the Mac-1, LFA-1, p150,95 glycoprotein family during leukocyte differentiation

The regulation of Mac-1, LFA-1, and p150,95 expression during leukocyte differentiation was examined. LFA-1 was present on almost all cell types studied. Both Mac-1 and p150,95 were present on the more mature cells of the myelomonocytic series, but only p150,95 was detected on some B cell lines and cloned cytotoxic T lymphocytes. Phorbol myristate acetate (PMA) stimulation of B chronic lymphocytic leukemia cells dramatically increased p150,95 expression. The resultant Mac-1, LFA-1, p150,95 phenotype resembled hairy cell leukemia, a B cell plasmacytoid leukemia. The promonocytic cell line U937 and the promyeloblastic cell line HL-60 expressed only LFA-1. Monocytic differentiation of U937 cells was stimulated by PMA, and induced the concomitant expression of Mac-1 and p150,95, with more p150,95 induced than Mac-1. Granulocyte/macrophage colony-stimulating factor (GM-CSF) stimulation of U937 cells gave similar results. PMA-stimulated monocytic differentiation of the HL-60 cell line also induced expression of both Mac-1 and p150,95. The number of p150,95 molecules on PMA-stimulated U937 and HL-60 cells were 5 X 10(5) and 3 X 10(5), respectively. Retinoic acid stimulated myeloid differentiation of HL-60 cells and induced expression of both Mac-1 and p150,95. These cells acquired a Mac-1, LFA-1, p150,95 profile that resembled that of granulocytes, with more Mac-1 than p150,95 induced. GM-CSF stimulation of HL-60 cells induced a similar Mac-1 and p150,95 phenotype. The contributions of Mac-1, LFA-1, and p150,95 to aggregation of PMA-differentiated U937 cells were assessed. Monoclonal antibodies to the beta subunit and the LFA-1 alpha subunit, but not those to p150,95 or Mac-1 alpha subunit, inhibited this homotypic adherence.