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 monocyte/macrophage C2 production and HLA-DR expression by IL-4 (BSF-1) and IFN-gamma

IL-4 was originally described on the basis of its ability to co-stimulate the proliferation of resting B cells treated with anti-IgM. Recently, this cytokine has been shown to have other effects on mast cells, T cells, B cells, and macrophages. We studied the ability of IL-4 to regulate the production of C2 by human monocytes and monocytic cell lines and compared this with stimulation of HLA-DR expression, another recently described activity of IL-4. Responses to IL-4 were compared to IFN-gamma, a cytokine with both activities. IL-4 up-regulated C2 production by human monocytes and this effect was not inhibited by neutralizing anti-IFN-gamma antibody. IL-4 also stimulated C2 production by HL-60 cells that had been pre-treated with vitamin D3 to induce monocytic differentiation. IL-4 did not stimulate C2 production by U937 cells. IFN-gamma, in contrast to IL-4, stimulates C2 production by all three cell types. Although IL-4 increased C2 production by HL-60 cells we could not detect C2 mRNA by Northern blotting. However, co-stimulation of these cells with IL-4 and low concentrations of IFN-gamma resulted in an additive effect on C2 production and a greater increase in C2 mRNA than was seen with IFN-gamma alone. As reported by others, IL-4-stimulated HLA-DR expression by monocytes. In contrast to our findings regarding C2 production, stimulation of HLA-DR expression was inhibited by neutralizing anti-IFN-gamma mAb and IL-4 did not stimulate HLA-DR expression by U937 or HL-60 cells. IFN-gamma stimulated HLA-DR expression by all three cell types. These results identify IL-4 as an additional cytokine able to directly stimulate C2 production by human monocytes and by a monocytic cell line whereas IL-4 stimulation of HLA-DR expression by monocytes appears to be IFN-gamma dependent.     

Induction of monocyte procoagulant activity with OKT3 antibody

OKT3 monoclonal antibody (MoAb), a mouse MoAb against cluster of differentiation 3 (CD3) molecule, induced a large amount of procoagulant activity (PCA) in human peripheral blood mononuclear cells (PBM). The PCA-inducing capability in OKT3 MoAb was abolished by absorption with T lymphocytes or Sepharose-conjugated antibody to mouse IgG. Most of the PCA in PBM was associated with monocytes. There was a dose-dependent increase in PCA when increasing numbers of T cells were added to the monocytes in the presence of OKT3 MoAb. OKT3 MoAb did not induce PCA in either T cells or monocytes alone. T cells pulsed with OKT3 MoAb only in the presence of monocytes could induce PCA in monocytes. Culture supernatants (CS) from PBM stimulated with OKT3 MoAb did not enhance PCA in monocytes; however, it did induce PCA in the human monocyte-like cell line (U937) which differs in some properties from monocytes; this activity could be abolished by the MoAb against human interferon-gamma (IFN-gamma). Nevertheless, neither human IFN-gamma nor interleukin 1 or 2 had significant direct effect in inducing PCA in U937 cells; CS from either monocytes or T cells alone stimulated with OKT3 MoAb did not induce PCA in U937 cells. This apparent discrepancy suggests that there may be factors in CS that induce PCA in U937 cells only in the presence of IFN-gamma. The PCA induced in monocytes or U937 cells was tissue factor-like because of the dependence on coagulation factors V, VII, and X. These observations suggest that OKT3 MoAb is a potent T cell-dependent monocyte PCA inducer and stimulates T cells only in the presence of monocytes. The direct cellular interaction between monocytes and stimulated T cells appears to be necessary to elicit monocyte PCA with OKT3 MoAb stimulation. Thus, monocytes may play a dual role, not only as effector cells, but also as cells that collaborate with T cells after OKT3 MoAb stimulation so as to produce PCA.     

Recombinant granulocyte-macrophage colony-stimulating factor down-regulates expression of IL-2 receptor on human mononuclear phagocytes by induction of prostaglandin E

Recent studies have shown that normal human alveolar macrophages and blood monocytes, as well as HL-60 and U937 monocyte cell lines, newly express IL-2R after stimulation with rIFN-gamma or LPS. In addition, macrophages transiently express IL-2R in vivo during immunologically mediated diseases such as pulmonary sarcoidosis and allograft rejection. We therefore investigated in vitro factors that modulate macrophage expression of IL-2R. IL-2R were induced on normal alveolar macrophages, blood monocytes, and HL-60 cells using rIFN-gamma (24 to 48 h at 240 U/ml), and cells were cultured for an additional 12 to 24 h with rIL-2 (100 U/ml), recombinant granulocyte-macrophage CSF (rGM-CSF, 1000 U/ml), rGM-CSF plus indomethacin (2 X 10(-6) M), PGE2 (0.1 to 10 ng/ml), 1 X 10(-6) M levels of caffeine, theophylline, and dibutyryl cyclic AMP, or medium alone. IL-2R expression was quantitated by cell ELISA (HL-60 cells) or determined by immunoperoxidase staining (alveolar macrophages, blood monocytes, and HL-60 cells), using anti-Tac and other CD25 mAb. PGE production was assayed by RIA. We found greater than 95% of alveolar macrophages, monocytes, and HL-60 cells expressed IL-2R after rIFN-gamma treatment and remained IL-2R+ in the presence of IL-2R or medium alone. By comparison, greater than 95% of cells induced to express IL-2R became IL-2R- after addition of rGM-CSF, and the culture supernatants from GM-CSF-treated cells contained increased levels of PGE. This inhibition of macrophage IL-2R expression by rGM-CSF was blocked by indomethacin, and IL-2R+ macrophages became IL-2R- after addition of PGE2 alone. These findings indicate GM-CSF down-regulates IL-2R expression by human macrophages via induction of PGE synthesis. Moreover, a similar down-regulation of IL-2R expression was seen after stimulation with caffeine, theophylline, or dibutyryl cyclic AMP. Hence, GM-CSF, PGE, and other pharmacologic agents that act to increase intracellular levels of cAMP may play a modulatory role, antagonistic to that of IFN-gamma on cellular expression of IL-2R by human inflammatory macrophages in vivo.     

Interleukin 1 production by the human monocyte cell line U937 requires a lymphokine induction signal distinct from interleukin 2 or interferons

A soluble product from cloned human T lymphocytes is capable of stimulating U937 cells, a line of human monocytes, to produce interleukin 1 (IL 1). We previously reported that U937 cells exposed to T lymphocyte-conditioned medium secrete mononuclear cell factor (MCF), which increases collagenase and prostaglandin E2 production by adherent rheumatoid synovial cells. Whereas structural and functional homologies between lymphocyte-activating factor (LAF, or IL 1) and MCF were described, previous attempts to measure LAF secretion by lymphokine-stimulated U937 cells were unsuccessful. Although the crude supernatants of cultured U937 cells exposed to medium from lectin-stimulated peripheral blood or cloned T lymphocytes contained MCF activity, no LAF activity was detected. After these crude supernatants were chromatographed on Ultrogel AcA54, however, and the fractions were individually assayed for IL 1, MCF and LAF activities were coeluted with apparent m.w. approximately 14,000 to 23,000. The inability to detect LAF activity in the unfractionated medium was accounted for by an inhibitor of lymphocyte proliferation present in fractions of higher m.w. The T lymphocyte product that stimulated U937 cell maturation and monokine production was secreted in response to lectin-stimulation in a dose-dependent fashion. Although we have previously demonstrated that the hormone 1,25-dihydroxyvitamin D3 caused maturational changes in U937 cells, and other investigators have reported effects of alpha and gamma interferon, these changes are dissociable from IL 1 production. Thus, a distinct lymphocyte-derived signal, necessary for the production of IL 1 by U937 cells, can be identified and dissociated from other biologic products that cause "maturational" changes. The detection of LAF activity in U937 cell supernatants requires the removal of an inhibitor of lymphocyte proliferation.     

Gamma interferon augments Fc gamma receptor-mediated dengue virus infection of human monocytic cells.

It has been reported that anti-dengue antibodies at subneutralizing concentrations augment dengue virus infection of monocytic cells. This is due to the increased uptake of dengue virus in the form of virus-antibody complexes by cells via Fc gamma receptors. We analyzed the effects of recombinant human gamma interferon (rIFN-gamma) on dengue virus infection of human monocytic cells. U937 cells, a human monocytic cell line, were infected with dengue virus in the form of virus-antibody complexes after rIFN-gamma treatment. Pretreatment of U937 cells with rIFN-gamma resulted in a significant increase in the number of dengue virus-infected cells and in the yield of infectious virus. rIFN-gamma did not augment dengue virus infection when cells were infected with virus in the absence of anti-dengue antibodies. Gamma interferon (IFN-gamma) produced by peripheral blood lymphocytes from dengue-immune donors after in vitro stimulation with dengue antigens also augmented dengue virus infection of U937 cells. IFN-gamma did not augment dengue virus infections when cells were infected with virus in the presence of F(ab')2 prepared from anti-dengue immunoglobulin G. Human immunoglobulin inhibited IFN-gamma-induced augmentation. IFN-gamma increased the number of Fc gamma receptors on U937 cells. The increase in the percentage of dengue antigen-positive cells correlated with the increase in the number of Fc gamma receptors after rIFN-gamma treatment. These results indicate that IFN-gamma-induced augmentation of dengue virus infection is Fc gamma receptor mediated. Based on these results we conclude that IFN-gamma increases the number of Fc gamma receptors and that this leads to an augmented uptake of dengue virus in the form of dengue virus-antibody complexes, which results in augmented dengue virus infection.     

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.     

Generation of activated killer (AK) cells by recombinant interleukin 2 (rIL 2) in collaboration with interferon-gamma (IFN-gamma)

Activation of human peripheral blood mononuclear cells (PBMC) by interleukin 2 (IL 2) and the role of interferon-gamma (IFN-gamma) in the IL 2-induced activation were investigated. Activated killer (AK) cells against NK-resistant tumor cell lines were induced in the medium containing recombinant IL 2 (rIL 2) and autologous serum without any other stimulating agents. AK activity was induced by doses of rIL 2 as low as 3 U/ml, and reached a maximum at 10(3) U/ml. Incubation of PBMC with rIL 2 resulted in IFN-gamma production and augmented NK activity after 1 day of culture, and in induction of AK cells and proliferative response after 2 days of culture. These results suggested that endogenous IFN-gamma was required for rIL 2-induction of AK cells and proliferative response. To prove this, PBMC were cultured with rIL 2 and rIFN-gamma or were pretreated with rIFN-gamma before culture with rIL 2. Both rIFN-gamma treatments of PBMC augmented rIL 2-induced AK activity and proliferative response. rIL 2-induced IFN-gamma production was also enhanced by the rIFN-gamma pretreatment of PBMC. The addition of anti-IFN-gamma antibody to rIL 2 cultures abrogated the rIL 2-induced NK augmentation, AK generation, and proliferative response in proportion to the decreased amounts of endogenous IFN-gamma detectable in culture. rIFN-gamma and/or rIL 2 cultures of PBMC increased Tac antigen expression on cell surfaces as measured by flow cytometry. Enhanced Tac expression by rIL 2 was abrogated by adding anti-IFN-gamma antibody. These data indicate that: 1) AK generation and IFN-gamma production are mediated by IL 2, and 2) IFN-gamma production may be required for IL 2 induction of AK cells and proliferative response. These finding are consistent with the hypothesis that AK generation involves a collaboration between IL 2 and IFN-gamma, in which IL 2 stimulates PBMC to produce IFN-gamma, which in turn acts as a differentiation signal that may be involved in the IL 2-initiated AK generation and proliferative response.     

Synthesis of C1 inhibitor (C1-INA) by a human monocyte-like cell line, U937

Human monocytes are known to synthesize many of the components of complement, including C1-INA. In this report we demonstrate that the human monocyte-like cell line U937 is also capable of synthesizing functional C1-INA. This was shown in several ways, including 1) incorporation of tritiated amino acids into antigenic C1-INA, immunoprecipitation, and detection by fluorography; 2) a sensitive ELISA, which allowed quantitation of antigenic C1-INA in cell lysates, and 3) a C2-dependent hemolytic assay in which the functional activity of U937 C1-INA was assayed. Data from the ELISA indicate that U937 cells contain between 2.1 to 12.8 ng of C1-INA per 1 X 10(6) cells. Furthermore, fluorescence-activated cell sorter analysis revealed that approximately 16% of U937 cells carry C1-INA as a surface bound antigen. Other proteins found to be synthesized by U937 cells include C1r, C8, and possibly alpha-2-macroglobulin. These results suggest that the U937 cell line could be a convenient and valuable model for the study of monocyte C1-INA synthesis and physiology.     

Comparative analysis of the priming effect of human interferon-gamma, -alpha, and -beta on synergism with muramyl dipeptide analog for anti-tumor expression of human blood monocytes

Freshly isolated human peripheral blood monocytes from healthy volunteers were not cytotoxic to allogeneic A375 melanoma cells, but they were activated to the cytotoxic state by incubation in vitro with either des-methyl muramyl dipeptide (norMDP; minimal effective dose, 0.5 micrograms/ml) or recombinant human interferon-gamma (rIFN-gamma; minimal effective dose, 1 U/ml). A combination of subthreshold concentrations of these agents (norMDP, 0.5 micrograms/ml; rIFN-gamma, 10 U/ml) also induced significant cytotoxicity, indicating that the effects of norMDP and rIFN-gamma in monocyte activation are synergistic. Natural human IFN-gamma (nIFN-gamma) and norMDP also had similar synergistic effects. Pretreatment of rIFN-gamma with anti-IFN-gamma antibody completely inhibited its synergistic effect with norMDP in monocyte activation. Because pretreatment of rIFN-gamma and norMDP with polymyxin B did not interfere with their effects in monocyte activation, the preparations were not contaminated with lipopolysaccharide. Moreover, because pretreatment of monocyte monolayers with anti-Leu-11b antibody (anti-natural killer (NK) cell antibody) and complement did not interfere with the synergistic effects of norMDP and rIFN-gamma, whereas pretreatment with anti-Leu-M1 antibody (anti-monocyte antibody) caused complete inhibition of their effects, the observed tumor cytotoxicity of monocyte-rich monolayers was probably not due to a small number of adherent NK cells, but to the stimulation of the monocytes. Natural and recombinant IFN-alpha and IFN-beta at concentrations of greater than or equal to 100 U/ml also induced tumoricidal activity of monocytes, but unlike IFN-gamma, their effects were additive with norMDP, and they had less priming effect than IFN-gamma when they were added before norMDP to monocytes. These findings suggest that recombinant human IFN-gamma has much more synergistic potential with norMDP than IFN-alpha or IFN-beta, and this synergism of rIFN-gamma and norMDP for monocyte activation could be of clinical value in treatment of disseminated malignant diseases, because these compounds are readily available at standardized concentrations.     

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.     

Spontaneous cytotoxicity and tumor necrosis factor production by peripheral blood monocytes from AIDS patients

Peripheral blood monocytes (PBM) from AIDS patients have exhibited defects in some but not all of the immune functions yet tested. This study has examined the capacity of AIDS PBM to lyse tumor target cells as well as their ability to secrete TNF. Untreated PBM from AIDS patients were significantly cytotoxic to U937 target cells and responded to IFN-gamma pretreatment with augmented cytotoxicity. Both the spontaneous and IFN-gamma-stimulated cytotoxic activity was significantly (p less than 0.01) higher than that observed with normal PBM. The cytotoxic activity depended on the E:T ratio used and was higher in AIDS PBM at all ratios tested (10:1 to 40:1). Because TNF has been implicated in macrophage cell-mediated cytotoxicity, we examined whether the elevated cytotoxic activity of AIDS PBM was associated with an increase in TNF production. Supernatants from PBM cultured overnight with or without IFN-gamma were tested in a bioassay measuring cytotoxicity against U937 target cells as well as in an RIA specific for TNF. Supernatants derived from either unstimulated or IFN-gamma-treated AIDS PBM exhibited significantly higher levels of cytotoxicity than supernatants from normal macrophages. Both normal and AIDS PBM produced higher levels of cytotoxic factors in response to IFN-gamma. As determined by the RIA, AIDS PBM spontaneously released high levels of TNF whereas little TNF was produced by normal PBM. Treatment with IFN-gamma augmented the level of TNF production in both AIDS and normal PBM. These results demonstrate that PBM from AIDS patients have undergone in vivo activation as manifested by both cytotoxicity against tumor target cells and production of TNF. Target cell lysis by both AIDS PBM and their supernatants was inhibited by monoclonal anti-rTNF, suggesting that the increase in PBM cell-mediated cytotoxicity was caused by an increase in TNF production. The significance of these findings in the pathogenesis of the disease is discussed.     

Differing macrophage and lymphocyte roles in resistance to Legionella pneumophila infection.

Similar to guinea pig macrophages and human monocytes, macrophages from the peritoneal cavity of thioglycolate pretreated A/J mice are permissive for growth of Legionella pneumophila. In contrast, macrophages from BDF1 mice are not permissive for L. pneumophila. Lymphocytes from A/J and BDF1 mice proliferated in response to Legionella Ag but guinea pig lymphocytes did not. Also, splenocyte cultures from A/J mice treated with either Con A or Legionella vaccine produced supernatants which induced A/J macrophages to restrict Legionella growth, but guinea pig splenocyte culture supernatants obtained after stimulation with L. pneumophila vaccine did not induce Legionella growth restriction activity by guinea pig macrophages. Murine rIFN-gamma but not rIFN-alpha markedly inhibited growth of Legionella in A/J mouse macrophages and monoclonal anti-IFN-gamma antibody neutralized the anti-Legionella activity of culture supernatants from A/J mouse splenocytes responding to Legionella Ag. From these data, IFN-gamma appears to be an important factor in anti-Legionella activity of Ag-activated mouse splenocyte culture supernatants. Cyclosporin A, when given to either A/J or BDF1 mice, reduced the proliferation responses of splenocytes to T cell mitogens and also decreased the IFN production of A/J spleen cells to Legionella Ag. In addition, drug treatment decreased the resistance of A/J mice to Legionella infection as shown by an increase in the number of viable bacteria in the liver. However, injection of drug treated mice with lymphokine-rich splenocyte culture supernatant reconstituted the resistance of these animals. These results suggest an important role for lymphocyte activation and lymphokine production in the resistance of A/J mice to Legionella infection. The greater resistance of BDF1 mice, however, may result from nonpermissive macrophages and responsive lymphocytes. In the case of guinea pigs, susceptibility to Legionella infections may result from both the permissive nature of the macrophages and the relatively unresponsive nature of the lymphocytes in these animals.     

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 interferongamma (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 thes cells. We cultured both cell lines for 48 hours in the presence and absence of IFN-gamma (10000 units/ml)n 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°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, <0.3ng/5 × 10⁶ cells. Peripheral blood mononuclear phagocytes from normal volumteers, cultured and challenged in vitro at under identical conditions, released 11.3 ± 2.9 ng LTB₄ and 2.0 ± 1.5 ng LTC₄/10⁶ 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.     

Plasminogen activator production by human monocytes. I. Enhancement by activated lymphocytes and lymphocyte products.

Human monocytes, but not nylon wool column-nonadherent lymphocytes, produce plasminogen activator. The activity is found only in association with intact cells. Exposure of monocytes to activated lymphocytes or to lymphokine-rich supernatants enhances monocyte plasminogen activator production. The assay allows assessment of baseline and activated human monocyte function.     

Regulation of CD93 cell surface expression by protein kinase C isoenzymes

Human CD93, also known as complement protein 1, q subcomponent, receptor (C1qRp), is selectively expressed by cells with a myeloid lineage, endothelial cells, platelets, and microglia and was originally reported to be involved in the complement protein 1, q subcomponent (C1q)-mediated enhancement of phagocytosis. The intracellular molecular events responsible for the regulation of its expression on the cell surface, however, have not been determined. In this study, the effect of protein kinases in the regulation of CD93 expression on the cell surface of a human monocyte-like cell line (U937), a human NK-like cell line (KHYG-1), and a human umbilical vein endothelial cell line (HUV-EC-C) was investigated using four types of protein kinase inhibitors, the classical protein kinase C (cPKC) inhibitor Go6976, the novel PKC (nPKC) inhibitor Rottlerin, the protein kinase A (PKA) inhibitor H-89 and the protein tyrosine kinase (PTK) inhibitor herbimycin A at their optimum concentrations for 24 hr. CD93 expression was analyzed using flow cytometry and glutaraldehyde-fixed cellular enzyme-linked immunoassay (EIA) techniques utilizing a CD93 monoclonal antibody (mAb), mNI-11, that was originally established in our laboratory as a CD93 detection probe. The nPKC inhibitor Rottlerin strongly down-regulated CD93 expression on the U937 cells in a dose-dependent manner, whereas the other inhibitors had little or no effect. CD93 expression was down-regulated by Go6976, but not by Rottlerin, in the KHYG-1 cells and by both Rottlerin and Go6976 in the HUV-EC-C cells. The PKC stimulator, phorbol myristate acetate (PMA), strongly up-regulated CD93 expression on the cell surface of all three cell-lines and induced interleukin-8 (IL-8) production by the U937 cells and interferon-gamma (IFN-gamma) production by the KHYG-1 cells. In addition, both Go6976 and Rottlerin inhibited the up-regulation of CD93 expression induced by PMA and IL-8 or IFN-gamma production in the respective cell-lines. Whereas recombinant tumor necrosis factor-alpha (rTNF-alpha) slightly up-regulated CD93 expression on the U937 cells, recombinant interleukin-1beta (rIL-1beta), recombinant interleukin-2 (rIL-2), recombinant interferon-gamma (rIFN-gamma) and lipopolysaccharide (LPS) had no effect. Taken together, these findings indicate that the regulation of CD93 expression on these cells involves the PKC isoenzymes.     

Activation of mouse peritoneal macrophages in vitro and in vivo by interferon-gamma

To determine the role of IFN-gamma in the activation of resident mouse peritoneal macrophages, crude macrophage-activating lymphokines were incubated with a monoclonal anti-murine IFN-gamma antibody. This treatment abolished the capacity of mitogen-induced lymphokines to enhance either H2O2 release or activity against the intracellular protozoa Toxoplasma gondii and Leishmania donovani. All macrophage-activating factor detected by these assays was also removed by passing the lymphokines over a Sepharose column to which the monoclonal anti-IFN-gamma antibody had been coupled. Therefore, pure murine rIFN-gamma was tested both in vitro and in vivo as a single activating agent. After 48 hr of pretreatment in vitro with 0.01 to 1 antiviral U/ml, macrophage H2O2-releasing capacity was enhanced an average of 6.4-fold; half-maximal stimulation was induced by 0.03 U/ml. Resident macrophages infected with T. gondii half-maximally inhibited parasite replication after 24 hr of preincubation with 0.14 U/ml of rIFN-gamma, and near complete inhibition was achieved by pretreatment with 100 U/ml. Half-maximal leishmanicidal activity was induced by 0.08 U/ml of rIFN-gamma, and 67 to 75% of intracellular L. donovani amastigotes were killed after macrophages were preincubated with 10 to 100 U/ml. Eighteen hours after parenteral injection of rIFN-gamma, peritoneal macrophages displayed a dose-dependent enhancement of H2O2-releasing capacity and antiprotozoal activity. Half-maximal enhancement required 85 to 250 U or rIFN-gamma given i.p. Peritoneal macrophages were also activated by rIFN-gamma injected i.v. and intramuscularly. These results suggest that, in the mouse model, IFN-gamma is likely to be a primary factor within mitogen-induced lymphokines responsible for activating macrophage oxidative metabolism and antiprotozoal activity, and indicate that rIFN-gamma is a potent activator of these effector functions both in vitro and in vivo. These findings provide a rationale for evaluating rIFN-gamma in the treatment of systemic intracellular infections, and indicate that murine models are appropriate for such studies.     

In vitro and in vivo activation of human mononuclear phagocytes by interferon-gamma. Studies with normal and AIDS monocytes

To determine the potential immunotherapeutic role of interferon-gamma (IFN-gamma) as a mononuclear phagocyte-activating agent, we examined the effector cell function of peripheral blood monocytes from healthy donors and acquired immunodeficiency syndrome (AIDS) patients after either in vitro and/or in vivo treatment with recombinant (r) IFN-gamma. When assayed immediately after a 24-hr in vitro pulse with 300 U/ml, normal and AIDS monocytes behaved similarly with little augmentation of their intrinsically high levels of H2O2 release and activity against Toxoplasma gondii; in contrast, activity toward the more resistant intracellular pathogen, Leishmania donovani, was appreciably enhanced by rIFN-gamma. In addition, upon testing 4 to 6 days after in vitro pulsing, both normal and AIDS monocytes showed clear evidence of persistent activation in all three assays. The capacity of IFN-gamma to similarly activate monocytes in vivo was confirmed in all ten treated AIDS patients by examining cells before and after 24-hr infusions of 0.03 and 0.5 mg of rIFN-gamma/square meter (M2) of body surface area. For postinfusion monocytes tested after 1 day in culture, H2O2 release and antitoxoplasma activity were essentially unchanged, but antileishmanial effects were augmented. After 5 to 7 days in culture, monocytes from treated patients showed 3.2- to 5.9-fold increases in H2O2-releasing capacity and increases of 49 to 68% and 35 to 61% in intracellular activity against T. gondii and L. donovani, respectively. These results indicate that the human monocyte can be induced by rIFN-gamma to express signs of both immediate and persistent activation and suggest that, as a direct activator of mononuclear phagocytes, rIFN-gamma may also have potential as an immunotherapeutic agent for patients with intracellular infections.     

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.     

Modulation of surface antigens of a human monocyte cell line, U937, during incubation with T lymphocyte-conditioned medium: detection of T4 antigen and its presence on normal blood monocytes

The human monocyte line, U937, derived from an individual with histiocytic lymphoma, undergoes morphological and functional changes when incubated with medium conditioned by lectin-stimulated cloned human T lymphocytes. Using monoclonal antibodies and flow cytometry, we therefore analyzed alterations in surface components that might accompany these morphological changes, in comparison with components present on normal blood monocytes. The U937 cells possess three surface antigens in common with blood monocytes, detected with OKM1, 4F2, and anti-monocyte.2 (the last monocyte specific). DR antigen was not detectable on U937 cells with three anti-DR framework antibodies but was detected on blood monocytes. Unexpectedly, OKT4, a monoclonal antibody to T4 antigen previously believed to be restricted to helper T lymphocytes, also reacted with U937 cells. Six monoclonal antibodies to other epitopes on T4 also reacted with U937 cells. None of these could be inhibited by blocking of Fc receptors. T4 with its various epitopes were also expressed on normal human blood monocytes. Other lymphocyte surface markers (T3, T8, T6) and fibronectin were not detectable on U937 cells or monocytes. An individual, whose lymphocytes lacked the epitope detected with OKT4 but had epitopes detected with OKT4 A, B, C, and D, had monocytes with identical reactivity, evidence that the T4 on monocytes and lymphocytes are products of the same structural gene. Stimulation of U937 cells for 24 hours with supernatants from Con A-stimulated T lymphocyte clones caused an increase in expression of OKM1 and Fc receptor activity and a decrease in expression of T4, consistent with a more mature phenotype of blood monocytes. Although the function of the T4 molecule is unknown, it is notable that it is displayed by two cells of distinct lineage which interact in the response to soluble antigens.