Martha K. Cathcart

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.

Regulation of Superoxide Anion Production by NADPH Oxidase in Monocytes/Macrophages: Contributions to Atherosclerosis

Monocyte extravasation into the vessel wall has been shown to be a critical step in the development of atherosclerosis. Upon activation, monocytes produce a burst of superoxide anion due to activation of the NADPH oxidase enzyme complex. Monocyte-derived superoxide anion contributes to oxidant stress in inflammatory sites, is required for monocyte-mediated LDL oxidation, and alters basic cell functions such as adhesion and proliferation. We hypothesize that monocyte-derived superoxide anion production contributes to atherosclerotic lesion formation. In this brief review, we summarize our current understanding of the signal transduction pathways regulating NADPH oxidase activation and related superoxide anion production in activated human monocytes. Novel pathways are identified that may serve as future targets for therapeutic intervention in this pathogenic process. The contributions of superoxide anion and NADPH oxidase to atherogenesis are discussed. Future experiments are needed to clarify the exact role of NADPH oxidase-derived superoxide anion in atherogenesis, particularly that derived from 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.

CCR1+/CCR5+ Mononuclear Phagocytes Accumulate in the Central Nervous System of Patients with Multiple Sclerosis

Mononuclear phagocytes (monocytes, macrophages, and microglia) are considered central to multiple sclerosis (MS) pathogenesis. Molecular cues that mediate mononuclear phagocyte accumulation and activation in the central nervous system (CNS) of MS patients may include chemokines RANTES/CCL5 and macrophage inflammatory protein-1alpha/CCL3. We analyzed expression of CCR1 and CCR5, the monocyte receptors for these chemokines, on circulating and cerebrospinal fluid CD14+ cells, and in MS brain lesions. Approximately 70% of cerebrospinal fluid monocytes were CCR1+/CCR5+, regardless of the presence of CNS pathology, compared to less than 20% of circulating monocytes. In active MS lesions CCR1+/CCR5+ monocytes were found in perivascular cell cuffs and at the demyelinating edges of evolving lesions. Mononuclear phagocytes in early demyelinating stages comprised CCR1+/CCR5+ hematogenous monocytes and CCR1-/CCR5- resident microglial cells. In later stages, phagocytic macrophages were uniformly CCR1-/CCR5+. Cultured in vitro, adherent monocytes/macrophages up-regulated CCR5 and down-regulated CCR1 expression, compared to freshly-isolated monocytes. Taken together, these findings suggest that monocytes competent to enter the CNS compartment derive from a minority CCR1+/CCR5+ population in the circulating pool. In the presence of ligand, these cells will be retained in the CNS. During further activation in lesions, infiltrating monocytes down-regulate CCR1 but not CCR5, whereas microglia up-regulate CCR5.

Protein Kinase Cδ Is Required for p47phox Phosphorylation and Translocation in Activated Human Monocytes

Our laboratory is interested in understanding the regulation of NADPH oxidase activity in human monocyte/macrophages. Protein kinase C (PKC) is reported to be involved in regulating the phosphorylation of NADPH oxidase components in human neutrophils; however, the regulatory roles of specific isoforms of PKC in phosphorylating particular oxidase components have not been determined. In this study calphostin C, an inhibitor for both novel PKC (including PKCδ, -ε, -θ, and -η) and conventional PKC (including PKCα and -β), inhibited both phosphorylation and translocation of p47phox, an essential component of the monocyte NADPH oxidase. In contrast, GF109203X, a selective inhibitor of classical PKC and PKCε, did not affect the phosphorylation or translocation of p47phox, suggesting that PKCδ, -θ, or -η is required. Furthermore, rottlerin (at doses that inhibit PKCδ activity) inhibited the phosphorylation and translocation of p47phox. Rottlerin also inhibited O⨪2 production at similar doses. In addition to pharmacological inhibitors, PKCδ-specific antisense oligodeoxyribonucleotides were used. PKCδ antisense oligodeoxyribonucleotides inhibited the phosphorylation and translocation of p47phox in activated human monocytes. We also show, using the recombinant p47phox-GST fusion protein, that p47phox can serve as a substrate for PKCδ in vitro. Furthermore, lysate-derived PKCδ from activated monocytes phosphorylated p47phox in a rottlerin-sensitive manner. Together, these data suggest that PKCδ plays a pivotal role in stimulating monocyte NADPH oxidase activity through its regulation of the phosphorylation and translocation of p47phox.

The oxidation of lipoproteins by monocytes-macrophages. Biochemical and biological mechanisms

The oxidation of lipoproteins has been proposed as a biological process that initiates and accelerates arterial lesion development (1–5). Oxidized lipoproteins accumulate in lesions (6) and may form at other inflammatory sites. Whether the oxidized lipoprotein is an initiator or accelerator of disease is the subject of speculation, debate, and intensive study. Various cellular and biochemical mediators of lipoprotein oxidation in vivo have been proposed, but none has yet been proven to be responsible. Two decades ago we demonstrated that low density lipoprotein (LDL), the plasma level of which correlates with the risk of atherosclerosis, could injure endothelial cells (ECs) in culture (7). The capacity of LDL to injure cells was directly related to the level of LDL oxidation, and we speculated on a possible role for oxidized LDL-mediated endothelial injury in atherogenesis (8, 9). Contemporaneously, Dr. Daniel Steinberg’s group (10, 11) demonstrated that LDL exposed to cultured ECs was altered such that it became a ligand for scavenger receptors. In 1984, both Steinberg’s group and ours (12, 13) demonstrated that the “EC-modified LDL” they had characterized and the “oxidized LDL” we had described were the same entity. Their report highlighted the macrophage recognition of the EC-oxidized lipoprotein, and ours the capacity of EC or smooth muscle cell (SMC)-oxidized LDL to injure cells. These papers introduced the concept that reactive oxygen species from vascular cells could transform LDL, causing it to exhibit dramatically altered composition and atherogenic properties. The first demonstration that certain leukocyte populations could oxidize LDL employed human neutrophils and activated populations of adherent human monocytes, cells well known to generate abundant reactive oxygen species in vitro and in vivo (14). The identity of the cells responsible for the oxidation of LDL that accumulates in lesions is uncertain. Although it is well known that free ferrous or cupric ions catalyze lipid peroxidation reactions in vitro, the presence of free metal ions in vivo is doubted (15). Multiple mechanisms exist in vivo for binding free transition metal ions, rendering them redox-inactive (15–17). In this minireview, we take the position that monocyte-derived macrophages are likely candidates to mediate the in vivo oxidation of lipoproteins, because they are prominent in arterial lesions, known to generate activation-dependent reactive oxygen species, and, unlike EC and SMC (12, 18), capable in vitro of LDL oxidation in media free of metal ion additives. In vitro LDL appears to be oxidized extracellularly without interaction with the LDL receptor (19–21). There are multiple potential pathways through which monocytes-macrophages may promote extracellular LDL oxidation. In this review we evaluate cellular mechanisms (both enzymatic and non-enzymatic) for LDL oxidation. We use the term “monocyte-macrophage” as a shorthand reference to in vitro studies performed on isolated monocytes, macrophages, and monocyte-like cell lines.

Renal cell carcinoma–derived gangliosides suppress nuclear factor-κB 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.

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.

Human monocytes use Rac1, not Rac2, in the NADPH oxidase complex.

Phagocyte NADPH oxidase is critical for defense against pathogens and contributes to inflammatory tissue injury. One component of the NADPH oxidase complex is the small GTP-binding protein Rac. There are two isoforms of Rac, and Rac2 is the predominant isoform in neutrophils and has been shown to be essential for NADPH oxidase activity. In primary human monocytes we report that in contrast to neutrophils, Rac1 is the predominantly expressed isoform. Upon monocyte activation by a variety of agents, we found that Rac1 dissociates from Rho GDP dissociation inhibitor (RhoGDI) and translocates to the membrane. We also found that Rac1 interacts with two other NADPH oxidase components, p67phox and p47phox, upon monocyte activation. These data indicate that Rac1, and not Rac2, is a component of the activated NADPH oxidase in monocytes. This finding suggests that it may be possible to selectively interfere with either monocyte or neutrophil NADPH oxidase activity, thereby selectively targeting chronic versus acute inflammatory processes.

Interleukin-13 induction of 15-lipoxygenase gene expression requires p38 mitogen-activated protein kinase-mediated serine 727 phosphorylation of Stat1 and Stat3.

ABSTRACT Interleukin-13 (IL-13) is a cytokine secreted by Th2 lymphocytes that is capable of inducing expression of 15-lipoxygenase (15-LO) in primary human monocytes. We recently demonstrated that induction of 15-LO requires the activation of Jak2 and Tyk2 kinases and Stats 1, 3, 5, and 6. Since IL-13-induced 15-LO expression was inhibited by H7 (a serine-threonine kinase inhibitor), we predicted that Stat serine phosphorylation may also be crucial for 15-LO expression. In this study, we present evidence indicating that IL-13-induced 15-LO mRNA expression was detectable as early as 1 h by real-time reverse transcription-PCR. We found that IL-13 induced a time-dependent serine phosphorylation of both Stat1 and Stat3, detectable at 15 min after IL-13 treatment. In addition, the activation of p38 mitogen-activated protein kinase (MAPK) was detected in a time-dependent fashion, with peak phosphorylation at 15 min after IL-13 treatment. SB202190, a p38 MAPK-specific inhibitor, markedly inhibited IL-13-induced Stat1 and Stat3 serine phosphorylation as well as DNA binding. Furthermore, treatment of cells with Stat1 or Stat3 decoys significantly impaired IL-13-induced 15-LO expression. Taken together, our results provide the first evidence that IL-13 induces p38 MAPK phosphorylation/activation, which regulates Stat1 and Stat3 serine 727 phosphorylation. Both of these events are important steps in IL-13-induced 15-LO expression in human monocytes.