Dual Ca2+ requirement for optimal lipid peroxidation of low density lipoprotein by activated human monocytes.

The oxidative modification of LDL seems a key event in atherogenesis and may participate in inflammatory tissue injury. Our previous studies suggested that the process of LDL oxidation by activated human monocytes/macrophages required O2- and activity of intracellular lipoxygenase. Herein, we studied the mechanisms involved in this oxidative modification of LDL. In this study, we used the human monocytoid cell line U937 to examine the role of Ca2+ in U937 cell-mediated lipid peroxidation of LDL. U937 cells were activated by opsonized zymosan. Removal of Ca2+ from cell culture medium by EGTA inhibited U937 cell-mediated peroxidation of LDL lipids. Therefore, Ca2+ influx and mobilization were examined for their influence on U937 cell-mediated LDL lipid peroxidation. Ca2+ channel blockers nifedipine and verapamil blocked both Ca2+ influx and LDL lipid peroxidation by activated U937 cells. The inhibitory effects of nifedipine and verapamil were dose dependent. TMB-8 and ryanodine, agents known to prevent Ca2+ release from intracellular stores, also caused a dose-dependent inhibition of LDL lipid peroxidation by activated U937 cells while exhibiting no effect on Ca2+ influx. Thus, both Ca2+ influx through functional calcium channels and Ca2+ mobilization from intracellular stores participate in the oxidative modification of LDL by activated U937 cells. 45Ca2+ uptake experiments revealed profound Ca2+ influx during the early stages of U937 cell activation, however, the Ca2+ ionophore 4-bromo A23187 was unable to induce activation of U937 cells and peroxidation of LDL lipids. Release of intracellular Ca2+ by thapsigargin only caused a suboptimal peroxidation of LDL lipids. Our results indicate that although increases in intracellular Ca2+ levels provided by both influx and intracellular Ca2+ mobilization are required, other intracellular signals may be involved for optimal peroxidation of LDL lipids by activated human monocytes.

Lipoxygenase contributes to the oxidation of lipids in human atherosclerotic plaques.

Oxidized LDL is present in human atherosclerotic lesions, but the mechanisms responsible for oxidation in vivo have not been definitively demonstrated. Circumstantial evidence has implicated the enzyme 15-lipoxygenase as a contributor to the formation of oxidized lipids in this disease. To assess whether oxidized lipids are indeed formed by the action of 15-lipoxygenase on polyunsaturated fatty acids (PUFAs) in vivo, we have used a sensitive and specific method (chiral phase HPLC) to analyze the lipid oxidation products present in human atherosclerotic lesions. Human 15-lipoxygenase is an omega-6 lipoxygenase that has previously been shown to oxidize esterified PUFA in a stereospecific manner, forming predominantly cholesteryl hydroperoxy-octadecadienoate (13(S)-HPODE) from cholesteryl linoleate substrate in LDL. This property allows its activity to be distinguished from nonenzymatic oxidation, which results in the formation of equal quantities of the S and R stereoisomers of the same oxidation product. A total of 80 specimens of human atherosclerotic plaque were analyzed. Esterified, oxidized linoleate was purified from human atherosclerotic lesions and from LDL oxidized by copper, and the chirality of these oxidation products was compared. There was significantly greater stereospecificity of oxidation in the oxidized linoleate from human atherosclerotic lesions. Even greater stereospecificity was detected in the HPODE derived from cholesteryl ester, purified from human lesions. Cholesteryl HPODE is the primary oxidation product from cholesteryl linoleate, the major esterified PUFA that accumulates in atherosclerotic vessels. Cholesteryl HPODE and its reduced form, cholesteryl hydroxy-octadecadienoate, were detected in all lesions analyzed. Neither the stereospecificity of oxidation nor the percentage of available substrate oxidized to primary oxidation products was correlated with the stage of disease of the lesions examined. We conclude that 15-lipoxygenase contributes to the formation of oxidized lipids in human atherosclerotic lesions.

Renal cell carcinoma-derived gangliosides suppress nuclear factor-kappaB activation in T cells.

Activation of the transcription factor nuclear factor-kappaB (NFkappaB) is impaired in T cells from patients with renal cell carcinomas (RCCs). In circulating T cells from a subset of patients with RCCs, the suppression of NFkappaB binding activity is downstream from the stimulus-induced degradation of the cytoplasmic factor IkappaBalpha. Tumor-derived soluble products from cultured RCC explants inhibit NFkappaB activity in T cells from healthy volunteers, despite a normal level of stimulus-induced IkappaBalpha degradation in these cells. The inhibitory agent has several features characteristic of a ganglioside, including sensitivity to neuraminidase but not protease treatment; hydrophobicity; and molecular weight less than 3 kDa. Indeed, we detected gangliosides in supernatants from RCC explants and not from adjacent normal kidney tissue. Gangliosides prepared from RCC supernatants, as well as the purified bovine gangliosides G(m1) and G(d1a), suppressed NFkappaB binding activity in T cells and reduced expression of the cytokines IL-2 and IFN-gamma. Taken together, our findings suggest that tumor-derived gangliosides may blunt antitumor immune responses in patients with RCCs.

Generation of a lipid-like cytotoxin from human CD16+ natural killer cells.

Supernatants from unstimulated CD16+ natural killer (NK) cells or from CD16+ NK cells cocultured with K562 tumor cells (to generate NK cytotoxic factor) were both cytotoxic to target cells. Interleukin 2 stimulation of the CD16+ NK cells in the absence of tumor cell stimulation resulted in supernatants which mediated an increased cytotoxicity as compared to the unstimulated supernatants. The cytotoxic activity was recovered in the chloroform fraction of a Bligh-Dyer lipid extraction suggesting that the toxic moiety in the CD16+ NK cell-derived supernatants might be a lipid. Separation of the cytotoxic supernatants into Mr less than 10,000 and Mr greater than 10,000 fractions revealed that the Mr less than 10,000 fraction of both supernatants had no detectable protein but retained cytotoxicity equal to that of the matched unfractionated supernatant. For convenience, we refer to this lipid-like cytotoxin in the Mr less than 10,000 fraction of the supernatants from unstimulated CD16+ NK cells as lipotoxin (LTX) and the cytotoxin in the Mr less than 10,000 fraction of supernatant from interleukin 2 stimulated CD16+ NK cells as LTX*. Increasing concentrations of LTX and LTX* caused a dose related increase in cytotoxicity. Both LTX and LTX* mediated killing as early as 18 h and their cytotoxicity was not significantly affected by heating at 56 degrees for 2 h or by freezing and thawing. Heating at 63 degrees C resulted in a decrease in cytotoxic activity of 10 to 20%. The less than 10,000 dalton fraction of supernatants from both unstimulated and interleukin 2 stimulated CD3- cells (a crude NK cell population) mediated greater cytotoxicity than the CD3+ cell supernatants, and the majority of cytotoxicity from the CD3- cell supernatants was recovered in this fraction. Thus, NK cells were more efficient producers of the lipid-like cytotoxin than T-cells but whether LTX made by NK cells can also be made by T-cells remains to be determined. We propose that lipotoxin: (a) coexists with protein cytotoxins in NK cell supernatant preparations; (b) mediates significant cytotoxicity when separated from proteinaceous cytotoxins; (c) is responsible for the spontaneously secreted cytotoxic activity observed by others; (d) is distinct from previously reported proteinaceous cytotoxins, e.g., NK cytotoxic factor, tumor necrosis factor alpha, and cytolysin/perforin; (e) accounts for the lipophilic nature of cytotoxic factor activity in NK cell supernatants; and (f) causes the cytotoxic activity observed in a small molecular weight fraction of stimulated NK cell supernatants.

Initial studies of the molecular organization of the cell-substrate adhesion site.

Using selective extraction reagents and non-penetrating probes, studies have been initiated on the molecular organization of substrate-attached material, adhesion sites which pinch off from the cell surface of normal Balb/c 3T3 or SV40-transformed Balb/c 3T3 (SVT2) cells and which remain bound to the serum-coated substrate during EGTA-mediated detachment of cells. Extraction of SVT2 adhesion sites with non-ionic detergents resulted in (a) only small amounts of leucine-radiolabeled protein and glucosamine-radiolabeled polysaccharide being solubilized; (b) selective solubilization of 80% of the adhesion site actin, and (c) solubilization of 95% of the phospholipid from these membranous pools. ATP in combination with potassium chloride extracted 60% of the actin. The 3T3 and SVT2 adhesion site proteins which are accessible to lactoperoxidase-catalyzed iodination were also determined. Many of the serum-derived proteins, bound to the substrate, were iodinated during iodination treatment of serum-coated or substrate-attached material-coated substrates, whereas the cellular proteins in the adhesion sites were not iodinated even though they were present in larger quantity as revealed by Coomassie blue staining. Iodination of cells, followed by their EGTA-mediated detachment and reattachment to fresh serum-coated substrates, indicated that the principal iodinated cell surface component deposited in new adhesion sites is the large external transformation-sensitive glycoprotein (even though large external transformation-sensitive glycoprotein is not the only principal iodinated cell surface component of these cells). These studies further establish the selective enrichment in this adhesive material of specific cell surface components and indicate that they are tenaciously bound at the interface between the serum coating and the undersurface of the adhesion site membranous pools.

Monocytes and neutrophils oxidize low density lipoprotein making it cytotoxic.

Free radicals are believed to be involved in leukocyte induced tissue injury. The present studies were performed to determine whether low density lipoprotein (LDL) might serve as a mediator of tissue injury after leukocyte induced free radical oxidation of LDL. Our results show that incubation of LDL with monocytes or polymorphonuclear leukocytes (PMN) leads to oxidation of the lipoprotein rendering it toxic to proliferating fibroblasts. Monocyte activation enhances these effects. Butylated hydroxytoluene (BHT), vitamin E (vit E) and glutathione (GSH) virtually prevent the oxidation of LDL and the formation of cytotoxic LDL, indicating that these alterations are mediated by leukocyte-derived free radicals. This is the first demonstration that short-lived free radicals emanating from phagocytic cells could mediate cell injury through the action of a stable cytotoxin formed by the oxidation of LDL. The fact that lipoproteins can transfer a cytotoxic effect from leukocytes to proliferating cells reveals a pathway for cell destruction which may have implications in atherosclerotic plaque progression, macrophage mediated toxicity to tumor cells and tissue injury by inflammatory processes.

Lipoxygenases and atherosclerosis: protection versus pathogenesis.

15 lipoxygenase (15LO) is a lipid-oxidizing enzyme that is considered to contribute to the formation of oxidized lipids in atherosclerotic lesions. Monocyte-macrophages are the key cells that express 15LO in atherosclerotic lesions. In this review, we examine the evidence for 15LO involvement in atherogenesis and explore and evaluate the potential mechanisms whereby 15LO may contribute to the oxidation of LDL by monocyte-macrophages. We also describe several possible pro- versus anti-atherogenic functions that may be mediated by various products of 15LO lipid oxidation. Central pathways involved in regulating 15LO expression and activity that may serve as future targets for intervention and regulation of this enzyme are also reviewed.

A multi-step isolation scheme for obtaining CD16+ human natural killer cells.

A multi-step isolation scheme capitalizing on negative selection protocols is described for obtaining an enriched population of CD16+ human natural killer (NK) cells. The isolation scheme consists of incubating peripheral blood mononuclear cells (MNC) on nylon wool, rosetting the nylon wool non-adherent cells with sheep red blood cells (SRBCs) for 1 h at 29 degrees C and then utilizing a 'panning' technique to remove CD3+, non-rosetting cells. The final working cell population contained 70-80% CD16+ cells, 15% CD2+ cells, 1-3% CD3+ cells, 5-7% SIg+ cells and no detectable MO2+ cells. In comparing the final NK cell population from the multi-step isolation protocol to NK cells obtained by the Percoll density gradient centrifugation technique, the multistep method: (1) yielded a higher percentage of CD16+ cells, (2) mediated a greater degree of cytotoxicity at a 25:1 E:T ratio, and (3) contained fewer contaminating monocytes/macrophages (none were detectable). In addition, the multi-step scheme allowed recovery of 30% of the total CD16+ cells present compared to only 7% recovered by the Percoll density gradient technique. Pretreatment of the enriched NK cells, obtained from the multi-step scheme, with interleukin-2 (3.5 and 7.0 U/ml of activity) resulted in an increase in NK cell-mediated cytotoxicity. In addition, these cells were as effective at synthesizing the cytotoxin, NKCF, at a 25:1 E:T ratio as at 50:1 and 100:1 E:T ratios. This multi-step isolation scheme consistently yields a high percentage of CD16+ NK cells and thus may greatly facilitate studies on the mechanism(s) involved in NK cell-mediated cytotoxicity and may further the study of the cytotoxins involved.

Partial purification and characterization of B cell growth factor constitutively secreted by human T-cell hybridoma.

Human T cell hybridomas were established by fusion of PHA-activated PBL with the 8-azaguanine resistant human T-leukemic cell line CEM-CM3. High levels of B cell growth factor (BCGF) activity were detected in the supernatants of hybridoma C8-2B2 and its subclones. Hybridoma C8-2B2, in addition to the Leu 3a, also expressed the OKT11 surface marker which was not detectable on the parent CEM-CM3 cells. BCGF from the culture supernatant was purified by combined use of salt fractionation and gel filtration to 36.6 fold with 23.9% recovery of activity. The BCGF produced by hybridoma C8-2B2 has a molecular weight range of 16,000-20,000 in two major electrophoretically different forms with pI values of 6.4 and 7.4.

Selective suppression of lymphokine production by human hybridoma suppressor factor (HSF).

We have made a human thymus cell hybridoma that secretes an immunosuppressive monoclonal lymphokine, referred to as hybridoma suppressor factor (HSF). This factor modulates the function of CD4+ cells suppressing their IL-2 production and suppressing PWM-induced B cell differentiation into Ig producing cells. Here we have examined the effect of HSF on the generation of T cell-derived lymphokines that regulate B cell growth and differentiation as well as the expression of other proteins involved in the control of T cell growth i.e., the p55 chain of the IL-2R and the transferrin receptor (TFR). HSF suppressed IFN-gamma activity produced by mitogen-stimulated PBMC without affecting the generation of lymphokines responsible for BCGF and BCDF activities. Additionally, HSF did not inhibit the expression of either IL-2R (p55) or TFR by activated T cells in spite of causing the suppression of IL-2 production. This evidence was further supported by experiments in which HSF selectively suppressed the accumulation of IL-2 mRNA without affecting IL-2R (p55) mRNA expression in mitogen-stimulated PBMC. The selective action of HSF may help to clarify the regulatory mechanisms involved in lymphokine gene expression as well as provide a way by which immune responses involved in autoimmunity and transplant rejection may be interrupted.

Human hybridoma suppressor factor (HSF) inhibits IL2 production in addition to suppressing immunoglobulin production.

The human thymus cell hybridoma, 8E-24, secretes a potent immunosuppressive factor(s), HSF, which inhibits polyclonal immunoglobulin (Ig) production. Our current studies reveal that this suppression is monocyte dependent in that its suppressive activity for Ig production was not observed in monocyte-depleted lymphocyte cultures but was restored by addition of monocytes. The requirement for monocytes was equally satisfied by autologous or allogeneic monocytes or monocyte conditioned media. Although the suppression mediated by HSF required the presence of monocytes, the mechanism for monocyte participation appears to be different from that reported for suppression mediated by soluble immune response suppressor (SIRS) in that general oxygen free radical scavengers did not inhibit this activity. Further information on the mechanism of action of HSF was obtained from studies on its suppressive effect on the phytohemagglutinin(PHA)-induced proliferative response. In this system HSF significantly suppressed PHA induced interleukin 2(IL2) production of peripheral blood mononuclear cells (PBMC) in a dose dependent fashion without inhibiting the proliferative response of CTLL-20 target cells to IL2. Interleukin 1 (IL1) production by monocyte cultures was also not suppressed by HSF. These results indicate that HSF interferes with IL2 production and not its induction by IL1 or its interaction with the IL2 receptor. To investigate the role of HSF-induced IL2 suppression in the pokeweed mitogen (PWM) antibody synthesis assay, the time course of IgG, IgM, IL1, and IL2 production of PWM stimulated PBMC cultures was examined. Results showed that the peak of IL2 production occurred on the second day of culture and was significantly suppressed by HSF while IL1 production was not affected during the seven day culture period. Similar suppression of IL2 and IgM production was observed in cultures of B cells and T4+ cells. Reconstitution of the IL2 levels in these cultures with recombinant IL2 completely restored antibody production. These results suggest that HSF in the presence of monocytes modulates the function of T4+ cells by inhibiting IL2 production. The inhibition of IL2 production by HSF appears to be responsible for the suppression of antibody production.

Predominance of esterified hydroperoxy-linoleic acid in human monocyte-oxidized LDL.

Low density lipoprotein that was oxidized by activated human monocytes was analyzed to determine the identity of oxidized fatty acids present and the conditions required for their formation. The oxidized lipids were also analyzed under conditions allowing preservation of their oxidation state. Using reversed-phase high performance liquid chromatography (HPLC) analysis of native and saponified lipid extracts of oxidized low density lipoprotein (LDL), we found that the major fatty acid oxidation product was esterified hydroperoxyoctadecadienoic acid (HPODE), the oxidized product of the most abundant polyunsaturated fatty acid in human LDL, linoleic acid. Although some esterified hydroxyoctadecadienoic acid (HODE) was also detected, the reduction of HPODE to HODE did not appear to be monocyte-dependent. Essentially all of the HPODE was found to be esterified with the majority being esterified to cholesterol followed by phospholipids and generally following the abundance of esterified linoleic acid within the lipid classes. The percent of cholesteryl linoleate converted to cholesteryl HPODE and cholesteryl HODE at the end of the 24-h incubation was determined to be approximately 13.5%. The formation of oxidized esterified linoleic acid in the LDL was shown to require immunological activation of the human monocytes, a previously observed requirement for general LDL oxidation in this culture system. The oxidized esterified linoleic acid was present in the supernatant with the LDL and was not cell-associated. HPODE formation on LDL was prevented by including superoxide dismutase (SOD) or eicosatetraynoic acid (ETYA) during the 24-h coincubation of activated monocytes with LDL whereas indomethacin was without effect. The analysis of the lipid oxidation products in oxidized LDL can provide insight into the mechanisms involved in oxidation of LDL by activated human monocytes.

Assessment of 5-lipoxygenase involvement in human monocyte-mediated LDL oxidation.

Lipoxygenase (LO) activity has been implicated in the process by which activated human monocytes oxidize normal human low density lipoprotein (LDL) and render it toxic to target cells. Here we examined the role of 5-LO in activated monocyte-mediated LDL modification. Five putative inhibitors of 5-LO (A63162, CGS8515, PF5901, RG6866, and MK886) were used to determine if they prevented activated monocytes from oxidizing LDL. Only RG6866, A63162, and CGS8515 inhibited monocyte-mediated LDL oxidation. Nonspecific effects of these drugs on LDL oxidation by activated monocytes were examined. RG6866 and A63162 were both found to be general antioxidants at their effective concentrations. CGS8515 was toxic at its effective concentration. A63162, CGS8515, and RG6866 also inhibited 15-LO activity in vitro. MK886 and PF5901 did not exhibit the nonspecific effects above and did not inhibit monocyte-mediated LDL oxidation, whereas both MK886 and PF5901 inhibited production of 5-LO metabolites by activated monocytes at concentrations that had no effect on LDL oxidation by the activated monocytes. Since neither of these agents inhibited LDL oxidation, we conclude that 5-LO is not involved in human monocyte oxidation of LDL. The possibility that a cellular 12- or 15-LO is involved in human monocyte-mediated LDL oxidation remains to be evaluated.

Protein kinase C activity is required for lipid oxidation of low density lipoprotein by activated human monocytes.

Our previous studies have shown that human monocytes can oxidize native low density lipoprotein (LDL) and transform it to a cytotoxin. We also found that intracellular Ca2+ levels are integrally involved in lipid oxidation of LDL by activated monocytes. In these studies, we investigated the protein kinase C (PKC) signaling pathway for its contribution to the process of monocyte oxidation of LDL lipids. We found substantial protein phosphorylation induced upon monocyte activation. Pharmacologic inhibition of PKC activity with the PKC inhibitors H-7 (1-100 microM), calphostin C (1-10 microM), and GF109203X (0.1-10 microM) caused a dose-dependent inhibition of cellular protein phosphorylation, including that of several previously identified PKC substrates. These inhibitors of PKC activity also substantially inhibited LDL lipid oxidation by activated monocytes. This inhibition was correlated with a profound suppression of superoxide anion production by these cells. In contrast, inhibition of cAMP-dependent protein kinase activity altered neither monocyte-mediated LDL lipid oxidation nor O2- production by activated monocytes. Delaying the addition of PKC inhibitors until after the peak production of O2-, which occurs during the respiratory burst, still resulted in inhibition of LDL lipid oxidation, suggesting roles for PKC in both early and late events. To corroborate these findings using other approaches, we used phorbol 12-myristate 13-acetate to down-regulate PKC activity and also used antisense oligonucleotides as specific PKC inhibitors. Results of both types of studies support the conclusion that PKC activity is required for activated monocytes to oxidize LDL lipids. Thus, PKC activation in this system is essential, one critical pathway regulated by PKC activity is the production of O2-, and continued PKC activity is required for optimal oxidation of LDL lipids.

Suppression of polyclonal immunoglobulin production by a soluble factor produced by a human thymus hybridoma.

A human thymus cell hybridoma was established using thymus cells obtained from a patient with common variable hypogammaglobulinemia and associated thymoma. This hybridoma secreted a suppressor factor for polyclonal antibody synthesis. Supernatants of this hybrid showed 40-80% suppression of both IgM and IgG synthesis by pokeweed mitogen-stimulated human peripheral blood lymphocytes. Hybridoma supernatants were suppressive for immunoglobulin production only if added within the initial 48 h of the seven-day culture period. Suppression of antibody production by the hybridoma supernatant was prevented by preabsorption with T lymphocytes. Further, the suppressor factor was shown to inhibit antibody production in reconstructed cultures containing T4+ cells and B cells, yet the suppression could be abrogated by increasing the number of T4+ cells. The hybrid supernatant had no affect on the proliferation of human mononuclear cells in response to pokeweed mitogen, lipopolysaccharide, concanavalin A or alloantigen but inhibited phytohemagglutinin-induced proliferation. The target cell population for the inhibition of phytohemagglutinin responsiveness was shown to be a T4+ lymphocyte (helper inducer T cell). These results suggest that thymus hybridoma cells can produce immunoregulatory products that act through the modulation of T4+ lymphocyte function. To our knowledge this is the first human thymus cell hybridoma to be reported. Studies on such cell lines may provide important information on immunoregulatory thymic factors.

Oxygen free radical generation and regulation of proliferative activity of human mononuclear cells responding to different mitogens.

We have compared various mitogenic stimuli for their ability to induce hydrogen peroxide (H2O2) and superoxide anion (O2-) production by PBMC and the effect of these reactive oxygen species and hydroxyl radical (OH.) has been assessed on proliferation. Our results show that pokeweed mitogen (PWM) stimulated PBMC to release H2O2 which interfered with proliferation since inclusion of catalase enhanced PBMC thymidine uptake. In contrast, phytohemagglutinin (PHA) and monoclonal antibody to CD3 (alpha CD3) did not induce PBMC to generate H2O2. O2- release by PBMC, which is readily induced by phorbol myristate acetate (PMA), did not occur when the cells were stimulated with PWM, PHA, or alpha CD3. In correlation, the O2- scavenger enzyme superoxide dismutase (SOD) had no effect on the proliferative response of the cells to the same mitogens, whereas it impaired the thymidine uptake of PMA-stimulated PBMC. A regulatory role for OH. was implied by studies using a battery of OH scavengers known to inhibit PMA-stimulated PBMC proliferation. OH. scavengers markedly inhibited the lymphoblastic transformation of alpha CD3-stimulated cells but had little or no effect on PHA- and PWM-stimulated PBMC. Thus, one manner by which PBMC proliferation is regulated is through oxygen free radical production which varies depending on the type of mitogenic stimulus.

Subtractive antibody to a human immunosuppressive lymphokine affinity isolates a suppressive factor and blocks its function.

Hybridoma suppressor factor(s) (HSF), secreted by a human thymus hybridoma (8E-24) established in this laboratory, suppresses Ig as well as IL-2 synthesis by peripheral blood mononuclear cells (PBMC). To aid in the characterization of this lymphokine, we prepared a subtractive antibody to HSF using the products of the hybridoma parent cell line to generate antibodies to irrelevant proteins. The concentrated supernatant fluid of the hybridoma parent cell line (CEM) was used to generate rabbit antibodies and titers of anti-CEM were monitored by enzyme immunoassay (EIA). Next, to remove factors shared by both the parent cell line and hybridoma, the concentrated supernatant fluid of 8E-24 (crude HSF) was passed over an immunoaffinity column, composed of protein A beads coupled to anti-CEM. The 'subtracted' HSF, termed partially purified HSF, was shown to be suppressive in vitro and was then used to prepare a second rabbit antisera. Using partially purified HSF as antigen, the presence of specific antibody was monitored by EIA. This antibody (anti-HSF) was used to prepare another immunoaffinity column by covalently coupling this antibody to protein A beads. Factors bound and then eluted from this affinity column were shown to inhibit IL-2 production by PBMC in a manner similar to HSF. Specific activity of the affinity purified HSF was 50 times that of partially purified HSF. Furthermore, the suppressive activity of affinity purified HSF was abrogated in the presence of anti-HSF. Western blot analysis performed on the concentrated crude HSF, using both the anti-HSF and anti-CEM antibodies, revealed the presence of several bands that selectively reacted with anti-HSF and not anti-CEM. Each of these bands were present in the affinity purified HSF. Of particular interest due to their similar size to the suppressive agent are a band at 12 kDa that reacts selectively with anti-HSF and is detected in crude and affinity purified HSF and a band at 10 kDa. In summary, this protocol resulted in the detection and separation of hybridoma specific proteins within the predicted size range of the suppressive lymphokine.

Selective inhibition of cytosolic phospholipase A2 in activated human monocytes. Regulation of superoxide anion production and low density lipoprotein oxidation.

Our previous studies have shown that monocyte activation and release of O-2 are required for monocyte-mediated low density lipoprotein (LDL) lipid oxidation. We have also found that intracellular Ca2+ levels and protein kinase C activity are requisite participants in this potentially pathogenic process. In these studies, we further investigated the mechanisms involved in the oxidation of LDL lipids by activated human monocytes, particularly the potential contributions of the cytosolic phospholipase A2 (cPLA2) signaling pathway. The most well-studied cPLA2, has a molecular mass of 85 kDa and has been reported to be regulated by both Ca2+ and phosphorylation. We found that cPLA2 protein levels and cPLA2 enzymatic activity were induced upon activation of human monocytes by opsonized zymosan. Pharmacologic inhibition of cPLA2 activity by AACOCF3, which has been reported to be a specific inhibitor of cPLA2 as compared with sPLA2, caused a dose-dependent inhibition of cPLA2 enzymatic activity and LDL lipid oxidation by activated human monocytes, whereas sPLA2 activity was not affected. To corroborate these findings, we used specific antisense oligonucleotides to inhibit cPLA2. We observed that treatment with antisense oligonucleotides caused suppression of both cPLA2 protein expression and enzymatic activity as well as monocyte-mediated LDL lipid oxidation. Furthermore, antisense oligonucleotide treatment caused a substantial inhibition of O-2 production by activated human monocytes. In parallel experimental groups, cPLA2 sense oligonucleotides did not affect cPLA2 protein expression, cPLA2 enzymatic activity, O-2 production, or monocyte-mediated LDL lipid oxidation. These studies support the proposal that cPLA2 activity is required for activated monocytes to oxidize LDL lipids.

In vitro knockout of human p47phox blocks superoxide anion production and LDL oxidation by activated human monocytes.

We previously reported that superoxide dismutase (SOD) blocked human monocyte oxidation of LDL and therefore concluded that superoxide anion (O(2)(.-)) was required for oxidation. Others, however, have suggested that SOD may inhibit by mechanisms alternative to the dismutation of O(2)(.-). This study definitively addresses the involvement of O(2)(.-) in monocyte oxidation of LDL. Using an antisense ODN designed to target p47phox mRNA, we found that treatment of monocytes with antisense ODN caused a substantial and selective decrease in expression of p47phox protein, whereas sense ODN was without effect. Corresponding functional assays demonstrated that antisense ODN inhibited production of O(2)(.-). As sense ODN caused no inhibition of O(2)(.-) production, these results suggested that inhibition of p47phox expression caused reduction in O(2)(.-) production. Evaluation of the contribution of O(2)(.-) production to monocyte-mediated oxidation of LDL lipids confirmed that O(2)(.-) production is required for LDL lipid oxidation as antisense ODN treatment significantly inhibited LDL oxidation whereas sense ODN treatment caused no inhibition. This is the first report of the reduction of NADPH oxidase activity in intact human monocytes by directly targeting the mRNA of a significant member of this enzyme complex. Our results provide convincing data that O(2)(.-) is indeed required for monocyte-mediated LDL oxidation.