Regulation of Fc gamma receptor expression and phagocytosis of a human monoblast cell line U937. Participation of cAMP and protein kinase C in the effects of IFN-gamma and phorbol ester

We investigated the positive and negative effects of IFN-gamma, PMA, dibutyryl cAMP (Bt2cAMP), dexamethasone and transforming growth factor-beta (TGF-beta) on Fc gamma R subtype expression and phagocytosis of a human monoblast cell line, U937. IFN-gamma increased and Bt2cAMP decreased Fc gamma RI expression determined by a mAb 32.2, whereas PMA and Bt2cAMP increased Fc gamma RII expression determined by a mAb IV-3. Phagocytosis was measured microscopically by counting ingested aggregated human IgG- or BSA-treated ox E (Eo'-IgG or Eo'-BSA). IFN-gamma increased the phagocytosis of Eo'-IgG but not that of Eo'-BSA, and PMA increased the phagocytosis of both Eo'-IgG and Eo'-BSA. Bt2cAMP decreased both basal and IFN-gamma- and PMA-augmented phagocytosis of U937 cells. Dexamethasone also inhibited both basal and IFN-gamma-augmented Fc gamma RI expression and PMA-augmented Fc gamma RII expression and phagocytosis, but did not affect IFN-gamma-augmented phagocytosis of Eo'-IgG. The augmentation of phagocytosis of Eo'-IgG by IFN-gamma thus seems to be due mainly to the increased internalizing process rather than to increased Fc gamma RI expression. TGF-beta slightly decreased Fc gamma R expression. In a study of the participation of protein kinase C (PK-C), it was found that H-7, a PK-C inhibitor, did not inhibit either IFN-gamma- or PMA-enhanced Fc gamma RI and Fc gamma RII expression, respectively, and 1-oleoyl-2-acetylglycerol and N-(6-phenylhexyl)-5-chloro-1-naphthalenesulfonamide, both PK-C activators, did not show any apparent increase in Fc gamma R expression and phagocytosis. These results show that Fc gamma RI and Fc gamma RII expression on U937 cells is regulated by different mechanisms and that IFN-gamma and PMA play their roles in Fc gamma R expression and phagocytosis by different pathways. It is possible that cAMP but not PK-C plays an important role in the regulation of Fc gamma R expression and phagocytosis.     

Regulation of Fc gamma receptor subtype expression on a human eosinophilic leukemia cell line EoL-3: participation of cAMP and protein kinase C in the effects of interferon-gamma and phorbol ester.

We investigated the effects of interferon-gamma (IFN-gamma), phorbol myristate acetate (PMA), and dibutyryl cAMP (Bt2cAMP) on Fc gamma R subtype expression on a human eosinophilic leukemia cell line, EoL-3. Unstimulated EoL-3 cells expressed Fc gamma RII as determined by monoclonal antibody (mAb) IV-3, whereas there was little or no Fc gamma RI and Fc gamma RIII expression as determined by mAbs 32.2 and 3G8, respectively. IFN-gamma induced Fc gamma RI expression, and Bt2 cAMP, which did not induce Fc gamma RI expression by itself, showed an additive effect on IFN-gamma-induced Fc gamma RI expression. Fc gamma RII expression was augmented by IFN-gamma, PMA, and Bt2 cAMP. Bt2 cAMP also showed an additive effect on IFN-gamma-augmented Fc gamma RII expression. Fc gamma RIII expression could be induced only by IFN-gamma plus Bt2 cAMP. H-7, a protein kinase C (PK-C) inhibitor, suppressed the enhancement of Fc gamma R subtype expression induced by these reagents. These results show that Fc gamma R subtype expression on EoL-3 cells is regulated differently in each subtype and that cAMP and PK-C play important roles in the regulation.     

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.     

Interferon-gamma-induced epithelial ICAM-1 expression and monocyte adhesion. Involvement of protein kinase C-dependent c-Src tyrosine kinase activation pathway.

Interferon-gamma (IFN-gamma) induced intercellular adhesion molecule-1 (ICAM-1) expression in human NCI-H292 epithelial cells, as shown by enzyme-linked immunosorbent assay and immunofluorescence staining. The enhanced ICAM-1 expression resulted in increased adhesion of U937 cells to NCI-H292 cells. Tyrosine kinase inhibitors (genistein or herbimycin), Src family inhibitor (PP2), or a phosphatidylinositol-phospholipase C inhibitor (U73122) attenuated the IFN-gamma-induced ICAM-1 expression. Protein kinase C (PKC) inhibitors (staurosporine or Ro 31-8220) also inhibited IFN-gamma-induced response. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a PKC activator, stimulated ICAM-1 expression; this effect was inhibited by tyrosine kinase or Src inhibitor. ICAM-1 promoter activity was enhanced by IFN-gamma and TPA in cells transfected with pIC339-Luc, containing the downstream NF-kappaB and gamma-activated site (GAS) sites, but not in cells transfected with GAS-deletion mutant, pIC135 (DeltaAP2). Electrophoretic gel mobility shift assay demonstrated that GAS-binding complexes in IFN-gamma-stimulated cells contained STAT1alpha. The IFN-gamma-induced ICAM-1 promoter activity was inhibited by tyrosine kinase inhibitors, a phosphatidylinositol-phospholipase C inhibitor, or PKC inhibitors, and the TPA-induced ICAM-1 promoter activity was also inhibited by tyrosine kinase inhibitors. Cotransfection with a PLC-gamma2 mutant inhibited IFN-gamma- but not TPA-induced ICAM-1 promoter activity. However, cotransfection with dominant negative mutants of PKCalpha or c-Src inhibited both IFN-gamma- and TPA-induced ICAM-1 promoter activity. The ICAM-1 promoter activity was stimulated by cotransfection with wild type PLC-gamma2, PKCalpha, c-Src, JAK1, or STAT1. An immunocomplex kinase assay showed that both IFN-gamma and TPA activated c-Src and Lyn activities and that these effects were inhibited by staurosporine and herbimycin. Thus, in NCI-H292 epithelial cells, IFN-gamma activates PLC-gamma2 via an upstream tyrosine kinase to induce activation of PKC-alpha and c-Src or Lyn, resulting in activation of STAT1alpha, and GAS in the ICAM-1 promoter, followed by initiation of ICAM-1 expression and monocyte adhesion.     

Differential regulation of the low affinity Fc receptor for IgE (Fc epsilon R2/CD23) and the IL-2 receptor (Tac/p55) on eosinophilic leukemia cell line (EoL-1 and EoL-3)

Two types of activation Ag, low affinity FcR for IgE (Fc epsilon R2)/CD23 and IL-2R (Tac/p55), were expressed and differently regulated on human eosinophilic leukemia cell lines (EoL-1 and EoL-3). Because the binding of IgE on EoL-3 cells was completely inhibited by H107 (anti-Fc epsilon R2/CD23 mAb) but not by irrelevant mAb, essentially all the low affinity Fc epsilon R2 on EoL-3 seemed to be the Fc epsilon R2/CD23 molecules. Both IL-4 and IFN-gamma enhanced the surface expression of Fc epsilon R2, whereas IL-1, IL-2, and IL-5 showed no effects, as determined by surface staining with anti-Fc epsilon R2 antibody (H107). In contrast to Fc epsilon R2 up-regulation, IL-4 and IFN-gamma showed a differential effect on the regulation of IL-2R (Tac/p55). Whereas IFN-gamma up-regulated the receptor expression of IL-2R/Tac, IL-4 did not. The result suggests that these lymphokines are involved in the different aspects of the activation pathway of the eosinophils. The possible role of Fc epsilon R2 and IL-2R on the function of eosinophils in allergic reaction is discussed.     

Induction by IL 1 and interferon-gamma: tissue distribution, biochemistry, and function of a natural adherence molecule (ICAM-1)

ICAM-1 is a cell surface glycoprotein originally defined by a monoclonal antibody (MAb) that inhibits phorbol ester-stimulated leukocyte aggregation. Staining of frozen sections and immunofluorescence flow cytometry showed intercellular adhesion molecule-1 (ICAM-1) is expressed on non-hematopoietic cells such as vascular endothelial cells, thymic epithelial cells, certain other epithelial cells, and fibroblasts, and on hematopoietic cells such as tissue macrophages, mitogen-stimulated T lymphocyte blasts, and germinal center dendritic cells in tonsils, lymph nodes, and Peyer's patches. ICAM-1 staining on vascular endothelial cells is most intense in T cell areas in lymph nodes and tonsils showing reactive hyperplasia. ICAM-1 is expressed in low amounts on peripheral blood leukocytes. Phorbol ester-stimulated differentiation of myelomonocytic cell lines greatly increases ICAM-1 expression. ICAM-1 expression on dermal fibroblasts is increased threefold to fivefold by either interleukin 1 (IL 1) or interferon-gamma at 10 U/ml over a period of 4 or 10 hr, respectively. The induction is dependent on protein and mRNA synthesis and is reversible. ICAM-1 displays Mr heterogeneity in different cell types with a Mr of 97,000 on fibroblasts, 114,000 on the myelomonocytic cell line U937, and 90,000 on the B lymphoblastoid cell JY. ICAM-1 biosynthesis involves a Mr approximately 73,000 intracellular precursor. The non-N-glycosylated form resulting from tunicamycin treatment has a Mr of 55,000. ICAM-1 isolated from phorbol myristic acetate (PMA) stimulated U937 and from fibroblasts yields an identical major product of Mr = 60,000 after chemical deglycosylation. ICAM-1 MAb interferes with the adhesion of phytohemagglutinin blasts, and the adhesion of the cell line SKW3 to human dermal fibroblast cell layers. Pretreatment of fibroblasts but not lymphocytes with ICAM-1 MAb, and of lymphocytes but not fibroblasts with lymphocyte function-associated antigen 1 MAb inhibits adhesion. Intercellular adhesion is increased by prior exposure of fibroblasts to IL 1, and correlates with induction of ICAM-1.     

Purified intercellular adhesion molecule-1 (ICAM-1) is a ligand for lymphocyte function-associated antigen 1 (LFA-1)

Lymphocyte function-associated antigen 1 (LFA-1) is a leukocyte cell surface glycoprotein that promotes intercellular adhesion in immunological and inflammatory reactions. It is an alpha beta complex that is structurally related to receptors for extracellular matrix components, and thus belongs to the integrin family. ICAM-1 (intercellular adhesion molecule-1) is a distinct cell surface glycoprotein. Its broad distribution, regulated expression in inflammation, and involvement in LFA-1-dependent cell-cell adhesion have suggested that ICAM-1 may be a ligand for LFA-1. We have purified ICAM-1 and incorporated it into artificial supported lipid membranes. LFA-1+ but not LFA-1- cells bound to ICAM-1 in the artificial membranes, and the binding could be specifically inhibited by anti-ICAM-1 treatment of the membranes or by anti-LFA-1 treatment of the cells. The cell binding to ICAM-1 required metabolic energy production, an intact cytoskeleton, and the presence of Mg2+ and was temperature dependent, characteristics of LFA-1- and ICAM-1-dependent cell-cell adhesion.     

Lysis of human keratinocytes by allogeneic HLA class I-specific cytotoxic T cells. Keratinocyte ICAM-1 (CD54) and T cell LFA-1 (CD11a/CD18) mediate enhanced lysis of IFN-gamma-treated keratinocytes.

Exposure of human KC to IFN-gamma increases their susceptibility to lysis by CTL. The mechanism of this enhanced lysis was investigated by analyzing interactions of IFN-gamma-treated and nontreated cultured KC with allogeneic class I-specific CTL clones. rIFN-gamma treatment augmented KC lysis in a time- and dose-dependent manner. Increased lysis of IFN-KC was detected after only 2 h of IFN-gamma treatment and was maximal by 12 h. Enhanced lysis of IFN-KC was Ag-specific, inasmuch as nonantigenic IFN-KC were not lysed either directly or as bystanders during the lysis of antigenic KC. Parallel immunofluorescence and cytotoxicity assays of KC treated with IFN-gamma for various intervals revealed a direct correlation between the degree of increased KC lysis and levels of cell surface ICAM-1 (CD54), but not of specific alloantigen or beta 2-microglobulin. Lysis of nontreated KC was blocked by mAb against class I or CD3, but not by mAb against ICAM-1 or LFA-1. In contrast, lysis of IFN-KC was partially inhibited by anti-ICAM-1 or anti-LFA-1 mAb, but resisted inhibition by anti-class I mAb except in the presence of anti-ICAM-1. These results indicate that both ICAM-1/LFA-1 and Ag/CD3-TcR interactions are important for Ag-specific lysis of IFN-KC, whereas lysis of nontreated KC depends on Ag/CD3-TcR but not ICAM-1/LFA-1 interactions. Equivalent inhibition of IFN-KC lysis by mAb against ICAM-1 or LFA-1 suggests that ICAM-1 is the only LFA-1 ligand involved in enhanced IFN-KC lysis. Furthermore, enhanced CTL lysis of KC after short-term IFN-gamma treatment can be explained solely on the basis of ICAM-1 induction, because all of the increase in specific lysis associated with IFN-gamma treatment could be blocked by mAb that block ICAM-1/LFA-1 interactions.     

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.     

IFN-gamma and transforming growth factor-beta 1 differently regulate fibronectin and laminin receptors of human differentiating monocytic cells.

The regulation of extracellular matrix (ECM) protein receptor expression was followed in the human promonocytic cell line U937 before and after stimulation either with PMA or various cytokines implicated in monocytopoiesis. On undifferentiated U937 cells, alpha-chains of very late Ag (VLA)-4, VLA-5, and VLA-6 were constitutively expressed whereas alpha-chains of VLA-2 (alpha 2) and vitronectin receptor (alpha V) were not. Maturation of U937 cells with PMA resulted in a marked decrease in alpha 4 expression (25% of control by day 5), and a small but significant increase in the expression of alpha 2 and alpha v over 4 days of stimulation. Unstimulated U937 cells attached to fibronectin (FN) but not to laminin (LM), collagens I/IV-coated surfaces. After PMA stimulation, U937 cells exhibited enhanced adherence on FN and expressed the ability to adhere to LM. PMA stimulation also promoted U937 spreading both on FN and LM. Adhesion on FN all along the maturation pathway was specifically and totally inhibited by anti-alpha 5 mAb but not by anti-alpha 4 mAb. Anti-beta 1, anti-alpha 6, anti-alpha 2, and anti-alpha v mAb, as well as Tyr-Ile-Gly-Ser-Arg and Arg-Gly-Asp synthetic peptides from LM, had no effect on adhesion of PMA-stimulated cells on LM, implying that U937 cell adherence to LM is mediated through hitherto distinct receptors. In the presence of rIFN-gamma, differentiating U937 cells did not adhere to LM and lost the capacity to bind to FN. Loss of adhesion to FN was correlated with the concomitant decrease in the expression of alpha 4 and alpha 5 integrin subunits. In contrast, TGF-beta 1 mimicked most of the effects of PMA by enhancing the attachment of maturating U937 cells on FN through alpha 5 receptors and by promoting adherence to LM. TGF-beta 1 stimulation also promoted U937 cell spreading on both FN- and LM-coated surfaces. The data suggest that inflammatory cytokines such as IFN-gamma and TGF-beta 1 may be critically important in the homing of monocytic cells at sites of inflammation by modulating cell-surface expression of ECM receptors.     

Phorbol myristate acetate-activated keratinocytes stimulate proliferation of resting peripheral blood mononuclear lymphocytes via a MHC-independent, but protein kinase C- and intercellular adhesion molecule-1-dependent, mechanism.

Recent attention has focused on the role keratinocytes (KC) may play in the induction of T cell-mediated inflammatory responses in skin, particularly because KC, when activated by immunologic stimuli, express MHC class II Ag and secrete immunomodulatory cytokines. We tested the capacity of normal human KC that were stimulated with PMA to induce PBMC proliferation. PMA-treated, but not untreated, KC induced proliferation of allogeneic as well as autologous PBMC; in addition, when purified CD4+ or CD8+ T cells were used as responders, each subset proliferated. PBMC proliferation was not due to direct action of PMA on PBMC, nor to contamination of KC cultures with Langerhans cells (LC) or dermal APC. Pretreatment with different protein kinase C inhibitors abrogated the capacity of PMA-stimulated KC to induce proliferation. Paraformaldehyde-fixed PMA-KC stimulated PBMC proliferation, whereas supernatants from PMA-treated KC failed to do so, indicating that a membrane-associated activity on PMA-KC contributes to the induction of PBMC proliferation. PMA induced intercellular adhesion molecule-1 (ICAM-1) expression on KC; furthermore, mAb against ICAM-1 or against its ligand lymphocyte function-associated Ag (LFA-1) (CD11a/CD18) significantly, but incompletely, reduced the stimulatory capacity of PMA-treated KC, indicating that ICAM-1/LFA-1 interaction contributed to PBMC proliferation. IFN-gamma or TNF-alpha also induced ICAM-1 on KC, but these KC failed to stimulate proliferation, suggesting that PMA induces additional signals from KC, which act in concert with ICAM-1 to promote proliferation. Finally, mAb against HLA-ABC or HLA-DR did not inhibit proliferation. We conclude that PMA can activate KC to stimulate T cell proliferation in a MHC-independent fashion. This activation is mediated by protein kinase C and in part by the induction of ICAM-1 expression on KC.     

Influence of recombinant IL-4, IFN-alpha, and IFN-gamma on the production of human IgE-binding factor (soluble CD23)

This study documents the influence of rIL-4, IFN-gamma, and IFN-alpha on the production of IgE-BF and the expression of lymphocyte receptor for IgE or CD23 Ag (Fc epsilon R II) by human mononuclear cells. IL-4 increases the secretion of IgE-binding factor (BF) by highly purified B lymphocytes, adherent cells, and U937 monoblastic cells. The effect of IL-4 on purified B cells is augmented by costimulating the cells with F(ab')2 anti-IgM. IFN-gamma, IL-2, IL-1-alpha, or IL-1 beta and the low m.w. B cell growth factor have no effect on IgE-BF production by purified B cells even when they are used in combination with anti-IgM. Stimulation of purified T cells with IL-4 or IL-4 plus PMA leads to the production of very small amounts of IgE-BF that might well be derived from the contaminating non-T cells. IFN-gamma increases IgE-BF synthesis by unfractionated PBMC, T cell-depleted PBMC, adherent cells, and U937 cells suggesting that it induces monocytes to release IgE-BF, IFN-gamma suppresses the IL-4-induced Fc epsilon R II expression and IgE-BF production by highly purified B cells but not by PBMC or their T cell-depleted fractions. IFN-alpha inhibits IgE-BF production by IFN-gamma-stimulated PBMC and by IL-4-stimulated cells suggesting that it exerts its effect on B cells and on monocytes. Moreover IFN-alpha suppresses the IL-4-induced expression of Fc epsilon R II on B cells. Both IFN-alpha and IFN-gamma suppress the synthesis of IgE by PBMC in response to IL-4. Taken collectively the results indicate that: 1) IL-4 induces IgE-BF production by both B cells and monocytes, 2) IFN-gamma stimulates IgE-BF synthesis by monocytes but suppresses its production by IL-4-stimulated B cells, and finally 3) IFN-alpha inhibits IgE-BF synthesis in response to either IFN-gamma or IL-4.     

Characterization of accessory cell costimulation of Th1 cytokine synthesis.

We studied the capacity of macrophage and B cell lines to provide a costimulatory signal that enhances synthesis of IFN-gamma and IL-2 by mouse Th1 clones stimulated with suboptimal doses of immobilized anti-CD3 antibody. The J774 macrophage line and the CH27 B lymphoma line had the greatest costimulatory activity and routinely increased IL-2 production by 10-fold to 100-fold. Other macrophage and B cell lines had less activity and T cell lines were unable to costimulate. The J774 and CH27 lines did not costimulate IL-4 production by a Th2 clone and had only a small effect on IL-2 production by T cell hybridomas. The process of costimulation was fixation-sensitive, contact-dependent and did not involve stable cytokines present in the T cell/accessory cell conditioned media. Neutralizing antibodies for IL-1, IL-6, and TNF failed to inhibit costimulation. Antibodies to the LFA-1/ICAM-1 pair of adhesion molecules also failed to inhibit. Costimulation of IL-2 production by accessory cells was found to have a unidirectional species restriction: mouse accessory cells costimulated mouse and human IL-2-producing T cells, but human U937 cells induced with PMA were effective only for human T cells. The results indicate that accessory cells can significantly regulate Th1 effector function at the level of cytokine production.     

Troglitazone suppresses cell growth of myeloid leukemia cell lines by induction of p21WAF1/CIP1 cyclin-dependent kinase inhibitor.

In a human eosinophilic leukemia cell line, EoL-1, cell proliferation was suppressed by 2-day treatment with troglitazone. EoL-1 cells treated with troglitazone were arrested and maintained in the G0/G1 phase in the cell cycle. This suppression correlated with the up-regulation of mRNA for p21WAF1/CIP1 cyclin-dependent kinase (Cdk) inhibitor. The inhibitory effects of troglitazone on cell proliferation and expression of p21 mRNA were observed in a human myelomonocytic cell line, U937, and a human myelomonoblastic cell line, KPB-M15. In addition, in EoL-1 cells, p21 protein was induced by troglitazone treatment and the induction was inhibited by protein synthesis inhibitor, cycloheximide. These data suggest that troglitazone inhibits cell proliferation in myeloid leukemia cell lines at least in part by induction of p21 Cdk inhibitor.     

IL-4 induces LFA-1 and LFA-3 expression on Burkitt's lymphoma cell lines. Requirement of additional activation by phorbol myristate acetate for induction of homotypic cell adhesions

LFA-1 and LFA-3 expression is absent or low on Burkitt's lymphoma cell lines and low on the EBV-transformed B cell line UD61. Incubation of cells of BL2 and of UD61 with various concentrations of IL-4 resulted in induction of LFA-1 and LFA-3 expression in a dose dependent fashion. This effect was already observed after 16 h of incubation whereas maximal expression was obtained after 72 h. Induction of LFA-1 and LFA-3 expression seemed to be specific for IL-4, because IL-1, IL-2, IL-3, IFN-alpha, IFN-gamma and a low m.w. B cell growth factor were ineffective. LFA-1 and LFA-3 induction by IL-4 was blocked specifically by an anti-IL-4 antiserum. Induction of LFA-1 expression by IL-4 was furthermore confirmed at the specific LFA-1 beta-chain mRNA level. IL-4 was unable to induce LFA-1 expression on EBV-transformed lymphoblastoid cell lines of two LFA-1-deficient patients. BL2 grows as single cells, but induction of LFA-1 and LFA-3 expression by IL-4 was insufficient to induce homotypic cell adhesions and required PMA as a second signal. PMA alone did not induce LFA-1 antigen expression and was unable to induce adhesions between BL2 cells in the absence of IL-4 in 22 h assays. Addition of PMA to BL2 cells that expressed LFA-1 Ag upon incubation with IL-4 resulted in aggregate formation within 30 min. Adhesions between BL2 cells induced by IL-4 in combination with PMA were blocked by anti-LFA-1 beta or anti-LFA-1 alpha-chains mAb. In addition, these mAbs dispersed preformed aggregates of BL2 cells. Our results indicate that IL-4 can induce the adhesion molecules LFA-1 and LFA-3 on B cell lines, but that an additional activation signal provided by PMA was required for the induction of homotypic cell adhesions.     

U937 and THP-1 cells do not release LTB4, LTC4, or LTD4 in response to A23187

U937 and THP-1 cells possess some characteristics of human mononuclear phagocytes, cells which synthesize and release LTB4, LTC4, and LTD4. Incubation of these cells with recombinant human interferon-gamma (IFN-gamma) or Phorbol Myristate Acetate (PMA) induces a more differentiated cell state. We hypothesized that U937 and THP-1 cells would release LTB4, LTC4, and LTD4 in response to stimulation with the non-physiologic agonist, calcium ionophore A23187 and that preincubation with IFN-gamma or PMA might alter leukotriene release by these cells. We cultured both cell lines for 48 hours in the presence and absence of IFN-gamma (1000 units/ml) and for 120 hours in the presence and absence of PMA (160 nM) and then challenged them with A23187 (5uM) for 30 minutes at 37 degrees C. The supernatants were deproteinated and assayed by RIA for LTB4 and LTC4 and by RP-HPLC for LTB4, LTC4, and LTD4. Neither U937 nor THP-1 cells released quantities of leukotrienes detectable by RIA, less than 0.3ng/5 X 10(6) cells. Peripheral blood mononuclear phagocytes from normal volunteers, cultured and challenged in vitro at under identical conditions, released 11.3 +/- 2.9 ng LTB4 and 2.0 +/- 1.5 ng LTC4/10(6) viable monocytes. The lack of leukotriene production by U937 and THP-1 cells was not altered by preincubation for 48 hours with IFN-gamma (n = 3) nor by preincubation with PMA for 120 hours (n = 3). We conclude 1) U937 and THP-1 cells do not appear to be appropriate in vitro models for the examination of leukotriene release from normal mononuclear phagocytes. 2) Pre-incubation of U937 and THP-1 cells with IFN-gamma or PMA under the conditions tested, does not induce the ability of these cell lines to release leukotrienes.     

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

Intercellular adhesion molecule-1 (ICAM-1) on the surface of cultured umbilical vein and saphenous vein endothelial cells was upregulated between 2.5- and 40-fold by rIL-1, rTNF, LPS and rIFN gamma corresponding to up to 5 X 10(6) sites/cell. Endothelial cell ICAM-1 was a single band of 90 kD in SDS-PAGE. Purified endothelial cell ICAM-1 reconstituted into liposomes and bound to plastic was an excellent substrate for both JY B lymphoblastoid cell and T lymphoblast adhesion. Adhesion to endothelial cell ICAM-1 in planar membranes was blocked completely by monoclonal antibodies to lymphocyte function associated antigen-1 (LFA-1) or ICAM-1. Adhesion to artificial membranes was most sensitive to ICAM-1 density within the physiological range found on resting and stimulated endothelial cells. Adhesion of JY B lymphoblastoid cells, normal and genetically LFA-1 deficient T lymphoblasts and resting peripheral blood lymphocytes to endothelial cell monolayers was also assayed. In summary, LFA-1 dependent (60-90% of total adhesion) and LFA-1-independent basal adhesion was observed and the use of both adhesion pathways by different interacting cell pairs was increased by monokine or lipopolysaccharide stimulation of endothelial cells. The LFA-1-dependent adhesion could be further subdivided into an LFA-1/ICAM-1-dependent component which was increased by cytokines and a basal LFA-1-dependent, ICAM-1-independent component which did not appear to be affected by cytokines. We conclude that ICAM-1 is a regulated ligand for lymphocyte-endothelial cell adhesion, but at least two other major adhesion pathways exist.     

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.     

A human intercellular adhesion molecule (ICAM-1) distinct from LFA-1

Homotypic adhesion by phorbol ester-stimulated lymphocytes requires LFA-1 and Mg+2 and does not involve like-like interactions between LFA-1 molecules on adjacent cells. The latter finding suggested that a second molecule, distinct from LFA-1, also participates in LFA-1-dependent adhesion. The identification of such a molecule was the object of this investigation. After immunization with LFA-1-deficient EBV-transformed lymphoblastoid cells, a MAb was obtained that inhibits phorbol ester-stimulated aggregation of LFA-1+ EBV lines. This MAb defines a novel cell surface molecule, which is designated intercellular adhesion molecule 1 (ICAM-1). ICAM-1 is distinct from LFA-1 in both cell distribution and structure. In SDS-PAGE, ICAM-1 isolated from JY cells is a single chain of Mr = 90,000. As shown by MAb inhibition, ICAM-1 participates in phorbol ester-stimulated adhesion reactions of B lymphocyte and myeloid cell lines and T lymphocyte blasts. However, aggregation of one T lymphocyte cell line (SKW-3) was inhibited by LFA-1 but not ICAM-1 MAb. It is proposed that ICAM-1 may be a ligand in many, but not all, LFA-1-dependent adhesion reactions.