Evidence that human Fc gamma receptor IIA (CD32) subtypes are not receptors for oxidized LDL.

Several lines of evidence suggest that clearance of oxidized LDL (oxLDL) immune complexes by macrophage IgG Fc receptors (Fc gamma Rs) plays a role in atherogenesis. Ox-LDL may also be cleared directly by Fc gamma Rs, as shown for murine Fc gamma RII-B2. In humans, the homologous Fc gamma R is Fc gamma RIIA (CD32), which is abundantly expressed on monocytes and macrophages and shares 60% sequence identity with murine Fc gamma RII-B2. As murine Fc gamma RII-B2 and human Fc gamma RIIA also share similar IgG ligand-binding properties, the purpose of this study was to test the hypothesis that human CD32 is a receptor for oxLDL. For these studies we used transfected Chinese hamster ovary (CHO) cells, monocytes, and cell lines that functionally express either of two Fc gamma RIIA subtypes (R131 or H131) and assayed binding or degradation of several preparations of oxLDL. The integrity of all oxLDL preparations was checked by studying their ability to react with CHO cells expressing human type I scavenger receptors and by other characteristics of lipoprotein oxidation. Although we showed that each preparation of oxLDL could recognize class A or class B scavenger receptors, we did not detect any differences in the binding or degradation of any type of oxLDL preparation among control versus CHO cell transfectants. Using monocytes that express Fc gamma RIIA and CD36, we showed that the binding of oxLDL was inhibited by antibodies to CD36, but not by Fc gamma RIIA antibodies. Thus, the data do not support the hypothesis that human Fc gamma RIIA is by itself a receptor for oxLDL. We conclude that human CD32 can mediate uptake of lipoprotein immune complexes, but does not mediate uptake of oxLDL in the absence of anti-oxLDL antibodies. OxLDL may interact with human mononuclear phagocytes directly via other types of receptors, such as class A and class B scavenger receptors or CD68.

HIV-1-infected monocytes and monocyte-derived macrophages are impaired in their ability to produce superoxide radicals.

Monocytes and monocyte-derived macrophages play a key role in immune defense against pathogenic organisms. Superoxide anion production is a key mechanism by which phagocytes kill pathogens. We sought to determine whether human immunodeficiency virus-infected monocytes and monocyte-derived macrophages are compromised in their ability to produce the superoxide anion following stimulation with phorbol myristate acetate (PMA) or after cross-linking the type I Fc receptor for IgG (Fc gamma RI). Fc gamma RI was cross-linked by the binding of monoclonal antibody 197, which reacts with an epitope of Fc gamma RI via its Fc region. Monocytes and monocyte-derived macrophages obtained from seronegative donors were infected in vitro with human immunodeficiency virus-1JR-FL and used in effector assays that measured superoxide anion production by the reduction of nitroblue tetrazolium. Reduced nitroblue tetrazolium was measured spectrophotometrically and by microscopy in which the percentage of cells containing intracellular deposits of the dye was assessed. By spectrophotometric measurement, we found that human immunodeficiency virus-infected monocytes and monocyte-derived macrophages produced less superoxide anion following either phorbol myristate acetate stimulation or Fc gamma RI cross-linking than uninfected cells from the same donor. Using microscopy we saw no difference in the percentage of infected and uninfected macrophages containing intracellular deposits of nitroblue tetrazolium suggesting that human immunodeficiency virus-infected macrophages produce less superoxide anion on a per cell basis than uninfected macrophages. Activation of human immunodeficiency virus-infected monocytes with interferon-gamma for 72 h prior to stimulation with phorbol myristate acetate or monoclonal antibody 197 increased their ability to reduce nitroblue tetrazolium. These findings suggest that impairment in the production of reactive oxygen intermediates may, in some cases, contribute to the pathogenesis of human immunodeficiency virus infection and the acquired immunodeficiency syndrome.

Inhibition of HIV-1 infectivity by low molecular weight heparin. Results of in vitro studies and a pilot clinical trial in patients with advanced AIDS.

Several sulfated polysaccharides have been shown to have anti-HIV activity in vitro. However, many of these compounds are not suited for use in vivo because they present an increased risk of bleeding or cannot be administered chronically. We tested the anti-HIV effects of low molecular weight heparin (LMW-heparin) (Enoxaparin) in vitro using a model system of HIV infectivity because LMW-heparin can be given to patients on a long-term basis with little risk. In vitro, LMW-heparin was shown to inhibit HIV-1 production from a T cell lymphoma line (H9) and phytohemagglutinin-stimulated lymphoblasts. Inhibition of infectivity was dose dependent at concentrations achievable in vivo. We then performed a pilot clinical trial in 13 patients with advanced AIDS of 6 months of chronic, self-administered Enoxaparin given in standard prophylactic doses. CD4 counts appeared to stabilize or increase in most patients during the first 3 months of treatment, then remained stable or declined after 6 months. There was no appreciable change in serum p24 levels. There was no evidence of drug toxicity and no bleeding episodes. These findings demonstrate that a commercially available, relatively non-toxic form of LMW-heparin is a potent inhibitor of HIV-1 production in cultured cells and that it is feasible to treat patients with AIDS with LMW-heparin on a long-term basis. Definitive clinical trials of LMW-heparins and related compounds as experimental anti-viral agents in patients with HIV infection are indicated.

Variation in the binding of 125I-labeled interferon-beta ser to cellular receptors during growth of human renal and bladder carcinoma cells in vitro.

Studies of various established human bladder and renal carcinoma cell lines cultured in vitro demonstrated the presence of specific, saturable, high affinity binding sites for 125I-labeled human interferon Beta ser IFN-beta ser). This recombinant produced interferon labeled with approximately one atom of 125I/molecule of IFN expressed minimal or no loss of antiviral activity. A single class of binding sites (1000-2000/cell) with an affinity constant of 10(10)-10(11) L/M was measured at 4 degrees C for cells exhibiting widely different sensitivity to the antiproliferative effect of IFN-beta ser. Major fluctuations in the binding of 125I-labeled IFN-beta ser to cellular receptors were observed during in vitro proliferation of four of five cell lines examined. A significant decrease (P less than 0.001) in specific binding was observed 48 h after cultures were established. Cell cycle analysis suggested that within the first 24 h and in the very late log and stationary phase of growth of ACHN (human renal carcinoma) cells, variations in the binding of 125I-labeled IFN-beta ser were partially attributable to binding fluctuations during the mitotic cycle. The 2- to 3-fold decline 24 h following plating of ACHN cells corresponded to a 70% decrease in the number of cells in G0-G1. T24 (human transitional cell carcinoma) and ACHN cells, synchronized by serum starvation, demonstrated increased binding of 125I-labeled IFN-beta ser 4-16 h following serum replenishment. This increase in receptor binding occurred prior to the onset of DNA and protein synthesis and was followed by a decline immediately prior to cell division. Binding site analysis indicated that the increased binding prior to DNA synthesis was due to a 5- to 6-fold increase in receptor affinity for the radiolabeled ligand. After an initial 40% decline in receptors per cell following serum stimulation, receptor concentration remained essentially unchanged. Induction of 2',5'-oligoadenylate synthetase in ACHN cells and antiproliferative activity in RT112, RT4, T24 (human transitional cell carcinoma), and ACHN cells by IFN-beta ser decreased significantly 48 h following plating. These changes in the biological activity of this interferon corresponded to growth related fluctuations in the IFN-beta ser binding.

Synergistic antiproliferative effects of human recombinant alpha 54- or beta ser-interferon with gamma-interferon on human cell lines of various histogenesis.

The antiproliferative effects of human interferon (IFN), IFN-gamma, IFN-alpha 54, and IFN-beta ser, alone and in combination, were assessed on 10 human cell lines. All IFNs were produced by recombinant technology and purified to homogeneity. In all cell lines except one, the addition of IFN-gamma to either IFN-alpha 54 or IFN-beta ser resulted in a synergistic antiproliferative effect, regardless of individual IFN sensitivities or tissue of origin. The only exception was a normal diploid fibroblast cell in which an additive antiproliferative effect occurred with combination IFN treatment. A malignant fibrosarcoma cell line of similar mesenchymal origin demonstrated a synergistic antiproliferative effect. One cell line was sensitive to both type I and type II IFN alone. Three cell lines were relatively resistant to IFN-gamma but not to IFN-beta ser, one was relatively resistant to IFN-beta ser but not to IFN-gamma, and the remainder were resistant to both IFN-gamma and IFN-beta ser or IFN-alpha 54. The addition of IFN-alpha 54 to IFN-beta ser in the SKCO 1 cell line resulted in an antagonistic interaction. Timing experiments with RT112 cells indicate that IFN-gamma and IFN-beta ser need not be in the media at the same time for the synergistic effect to occur. Combinations of type I and type II IFN thus resulted in synergistic antiproliferative effect for transformed human cells of various histogenesis, including some with a relative resistance to one or both IFN types.

Antitumor effects of polyribonucleotides for mouse transitional cell carcinoma enhanced by cyclophosphamide.

Mouse bladder tumor (MBT-2), derived from a carcinogen-induced transitional cell carcinoma of the bladder, has proven a useful model for study of pathogenesis and prediction of cytotoxic drug sensitivity of human bladder carcinoma. To define optimal conditions for activity of the potent interferon inducer polyriboinosinic-polyribocytidylic acid [poly(I) X poly(C)] in this model, studies of dose, timing, and combinations with a cytotoxic drug were initiated. Poly(I) X poly(C) inhibited MBT-2 growth when 10(5) or 10(6) tumor cells were implanted. Tumor growth reduction was relatively more pronounced in mice inoculated with higher numbers of MBT-2 cells (10(6] than in mice inoculated with an intermediate dose (10(5] or small dose (10(4]. In mice inoculated with 10(5) MBT-2 tumor cells, poly(I) X poly(C) (2.5 or 10 mg/kg i.p.) on Days 5 to 19 every other day reduced tumor size markedly. It had no effect, however, on tumor incidence or the time of their first detection. Treatment for a shorter period (alternate days from Days 11 to 19) resulted in less inhibition of tumor growth. Once treatment was discontinued, tumors grew progressively. Polyriboadenylic:polyribouridylic acid [poly(A) X poly(U)] (10 mg/kg) which inhibited tumor growth but to a lesser degree than poly(I) X poly(C) induced lower, less sustained levels of serum interferon. Cyclophosphamide, injected i.p. on Day 1, resulted in inhibition of tumor incidence and growth in direct proportion to the dose administered (25 to 200 mg/kg), but it was curative only at greater than or equal to 30% lethal doses. When combined with poly(I) X poly(C) (2.5 or 10 mg/kg), cyclophosphamide (50 mg/kg) had an additive antitumor effect. Optimal inhibition of MBT-2 tumor growth occurred by combining cyclophosphamide (100 mg/kg) with poly(I) X poly(C) (2.5 mg/kg); eight of 14 mice were tumor free on Day 60.

Augmented antiproliferative effects of interferons at elevated temperatures against human bladder carcinoma cell lines.

The in vitro antiproliferative effects of interferons (IFN) against the human bladder carcinoma cell lines T24, RT4, HT1197, and 647V were evaluated at temperatures ranging from 37-41 degrees. At 37 degrees, the antiproliferative activities of IFN, either naturally produced or produced by recombinant DNA technology, were different against different cell lines. An increase in temperature markedly enhanced the antimitotic effect of IFN for all cells. For example, T24 cells grown at 37 degrees and treated with 200 units naturally produced IFN-alpha or IFN-beta per ml for 7 days were inhibited 50 to 60%. No change in cell proliferation occurred in untreated T24 cells grown at 39.5 degrees. Treatment with 200 units IFN-alpha or IFN-beta per ml at 39.5 degrees inhibited these cells 80 to 90%. Similar results were obtained with IFN produced by recombinant DNA technology and purified to homogeneity. Colony formation by the RT4 cell line, at 37 degrees, was decreased less than 10% with 200 units IFN-alpha per ml and 63% by 200 units IFN-beta per ml. At 39.5 degrees, colony formation by untreated RT4 cells was inhibited 48%. Treatment with IFN-beta at 39.5 degrees did not result in an enhancement of the antiproliferative effect; however, treatment with IFN-alpha enhanced the inhibition from less than 10% to 98%. These results suggest that a supraadditive relationship exists between antiproliferative effects of IFN and temperature elevation. The differences seen between IFN-alpha and IFN-beta may be due to the different stabilities of these two molecules. In order to probe the mechanism of the enhanced antiproliferative effect, activity of an IFN-induced enzyme, 2'-5'-oligoadenylate synthetase, was measured. IFN-alpha treatment resulted in significantly greater 2'-5'-oligoadenylate synthetase induction at 39.5 degrees than at 37 degrees. Thus, two cellular effects resulting from IFN were augmented by increased temperature.

Antiproliferative activities of interferons against human bladder carcinoma cell lines in vitro.

The antiproliferative effect of interferons against 5 human bladder carcinoma cell lines, RT112, T24, RT4, 647V and HT1197, was determined in vitro. Each of these human bladder carcinoma cell lines except 647V was sensitive to human interferons in liquid media. The antiproliferative effect of interferons was observed only upon continuous exposure, not after 1 hour. Partially purified, naturally produced interferon beta was more inhibitory of cell growth than naturally produced interferon alpha. Interferon alpha 54, 76, 61, 6L and 1 purified to homogeneity were as effective as naturally produced, partially pure interferon alpha. Although interferon beta, produced by recombinant DNA technology and purified to homogeneity, was not equivalent in effectiveness to naturally produced interferon beta, its antiproliferative activity was greater than interferon alpha 54 for 3 of 4 cell lines tested. Antimitotic effects may underlie, at least in part, the potential therapeutic activity of interferons for bladder carcinoma.

In vitro and in vivo effects of levamisole on monocyte chemotaxis in normal donors and patients with colorectal carcinoma.

Levamisole, a synthetic phenylimidazothiozole, was found to enhance chemotaxis of monocytes from both normal individuals and those with colorectal carcinoma. The optimum concentration in vitro varied among individuals but occurred most consistently at 200 micrograms/ml (approximately 10(-3) M). Overall relative increase in monocyte chemotaxis in 123 individuals was 29.3 +/- 4.2% (SE; p less than 0.0001 by paired t test comparison of individual baseline to stimulated values). The frequency of significant augmentation was the same in both patient and normal groups (30-45% of individuals tested). However, the degree of enhancement in vitro was greatest in patients with colorectal carcinoma. Twenty-nine volunteers ingested levamisole to evaluate in vivo effects on monocyte chemotaxis; significant increase occurred in 22. Both normal individuals and patients were stimulated to a similar degree and frequency. The immunomodulatory effects of levamisole may result from effects on monocyte function.