Steven H. Zuckerman

Transcriptional inhibition of endotoxin-induced monokine synthesis following heat shock in murine peritoneal macrophages

The role of heat shock proteins (HSP) during the inflammatory response has been controversial. The effect of heat shock (HS) on the synthesis of the monokines tumor necrosis factor (TNF) and interleukin 1 (IL‐1) by endotoxin–stimulated thioglycollate–elicited peritoneal macrophages was investigated. HS was deemed to have affected macrophages if a 70 kD HSP appeared on SDS gels; identity of this protein as the highly conserved HSP70 was then confirmed by immunoprecipitation. Maximal increases in HSP70 were apparent 2–5 h after HS at 45° for 12 min. Synthesis of HSP70 was no longer detected 24 h post HS. A reciprocal relationship between HSP70 and TNF was apparent in kinetic studies. TNF was not detected in culture supernatants if macrophages were endotoxin–stimulated 2 to 6 h after HS; however, the same stimulation 24 h later induced significant TNF secretion. RNA analysis of HS and non–HS macrophage cultures demonstrated a 60–fold reduction in TNF message in the HS macrophages 1 h after endotoxin stimulation. TNF mRNA levels remained depressed at 6 h while the HSP70 message had increased 30–fold. The ability of HS macrophages to ingest antibody–coated erythrocytes was not significantly affected following heat treatment. Macrophage response to HS can be said to inhibit transcription of inducible monokines while retaining other macrophage functions.

Interferon-γ Induces Downregulation of Tangier Disease Gene (ATP-Binding-Cassette Transporter 1) in Macrophage-Derived Foam Cells

Cholesterol efflux is a fundamental process that serves to mitigate cholesterol accumulation and macrophage foam cell formation. Recently, we reported that cholesterol efflux to high density lipoprotein subfraction 3 was reduced by interferon-gamma (IFN-gamma) and that this decrease was associated with an increase in acyl coenzyme A:cholesterol acyltransferase (ACAT) expression. In the present study, although treatment of murine peritoneal macrophages with IFN-gamma resulted in a 2-fold decrease in HDL-mediated cholesterol efflux, efflux to lipid-free apolipoprotein A-I was reduced >4-fold and approached basal levels. This decrease was associated with a 3- to 4-fold reduction in ATP-binding-cassette transporter 1 (ABC1) mRNA content, the gene responsible for the defect in Tangier disease. Consistent with the reduction in cholesterol and phospholipid efflux in Tangier fibroblasts, downregulation of ABC1 expression by IFN-gamma also resulted in reduced phosphatidylcholine and sphingomyelin efflux to apolipoprotein A-I. Whereas foam cells had a 3-fold increase in ABC1 mRNA, the decrease in ABC1 message levels by IFN-gamma was observed in foam cells and control macrophages. This effect of IFN-gamma was independent of general macrophage activation (inasmuch as similar changes were not detected with granulocyte-macrophage colony-stimulating factor) and was not observed with other ABC transporters (inasmuch as the expression of the transporter in antigen processing was upregulated 4-fold in these same cells). Therefore, by decreasing cholesterol efflux through pathways that include the upregulation of ACAT and the downregulation of ABC1, IFN-gamma can shift the equilibrium between macrophages and foam cells and thus impact the progression of an atherosclerotic lesion.

Gamma interferon: a central mediator in atherosclerosis

Abstract.Atherosclerosis is a chronic inflammatory disease of the vasculature with lesions developing in the arterial wall, frequently in the coronary and carotid arteries. The interaction between macrophages and lymphocytes within the atherosclerotic lesion microenvironment exemplifies a site where both innate and adaptive immunity contribute towards disease progression. As gamma interferon (IFN-γ), the classic macrophage activating factor, has been localized to atherosclerotic lesions, this review will focus on its contribution to plaque pathology and will finally consider how current therapies, as exemplified by HMG CoA reductase inhibitors or statins, may impact this process beyond lipid lowering, in part by inhibiting IFN-γ dependent processes. IFN-γ sources within the atheroma as well as receptors, signaling pathways and its effects on macrophages as well as on vascular smooth muscle and endothelial cells will be considered. Therapeutic interventions targeting molecular events associated with IFN-γ signaling offer novel approaches to the treatment of atherosclerosis.

Inhibition of LDL oxidation and myeloperoxidase dependent tyrosyl radical formation by the selective estrogen receptor modulator raloxifene (LY139481 HCL)

Cellular oxidation of protein and lipoproteins is believed to contribute to the pathology associated with both acute and chronic inflammatory processes. Enzymatic, myeloperoxidase and lipoxygenase, and non- enzymatic oxidation of low density lipoprotein, LDL, has been implicated in foam cell formation and the progression of atherosclerotic changes within the arterial wall. In the present study, the in vitro protective role of the selective estrogen receptor modulator, raloxifene, in these oxidant triggered processes has been investigated. Raloxifene, as with estrogen was observed to inhibit both copper mediated LDL oxidation as well as the cellular modification of LDL by murine peritoneal macrophages. Raloxifene was, however, a more potent inhibitor of LDL oxidation than 17 beta-estradiol. The inhibition of macrophage LDL modification by raloxifene was not due to a non-specific effect on all effector functions as phagocytosis of opsonized yeast was comparable with control macrophage cultures. In addition to the protective effects on LDL oxidation, raloxifene also inhibited tyrosyl radical formation catalyzed by myeloperoxidase. The inhibition of myeloperoxidase activity was observed for both the isolated enzyme and in phorbol ester stimulated murine peritoneal neutrophils. In contrast, raloxifene was a weaker inhibitor of horseradish peroxidase. These results demonstrate a potential protective role for raloxifene as an anti-oxidant in in vitro assays designed to evaluate oxidant mediated radical formation and tissue damage.

Endotoxin tolerance: In vivo regulation of tumor necrosis factor and interleukin-1 synthesis is at the transcriptional level

Endotoxin tolerance is associated with a decreased production of many inflammatory mediators following stimulus-induced desensitization. The mechanisms involved in the regulation of tumor necrosis factor (TNF) and interleukin-1 (IL-1) beta expression were investigated in peritoneal macrophages from endotoxin tolerant mice. The absence of a serum TNF peak in tolerant mice correlated with a significant reduction in both TNF and IL-1 beta mRNA accumulation in macrophages from these animals. The reduction in both IL-1 and TNF mRNA was consistent with a decrease in transcriptional activity by nuclear run-on assays for both cytokines and a reduced amount of the nuclear-associated transcription factor NF-KB. Therefore, the hyporesponsive state associated with endotoxin tolerance is characterized by transcriptional regulation of both TNF and IL-1 synthesis.

Neuroendocrine regulation of in vivo cytokine production and effects: I. In vivo regulatory networks involving the neuroendocrine system, interleukin-1 and tumor necrosis factor-α

We have investigated the in vivo regulatory network involving the neuroendocrine system, interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF). Adrenalectomy or hypophysectomy shifted the sensitivity curve to lipopolysaccharide (LPS)-induced lethal shock as well as TNF- and IL-1-induced deaths. Serum levels of IL-1 or TNF were altered in adrenalectomized or hypophysectomized mice following in vivo stimulation with LPS when compared to appropriate sham-operated control mice. Exogenous administration of either IL-1 or TNF could induce increases in serum corticosterone in sham-operated mice. Finally, treatment of adrenalectomized mice with corticosterone or dexamethasone could inhibit the induction of serum IL-1 and TNF and modified the pattern of these cytokine-induced deaths. Dexamethasone was more effective in these conditions than the natural glucocorticoid, corticosterone. Taken together, these data provide in vivo evidence for a feedback system involving the neuroendocrine axis (hypothalamus, pituitary and adrenal glands) leading to corticosterone production and subsequent regulation and/or modulation of IL-1 or TNF levels or activity.

Cytokine regulation of macrophage apo E secretion: opposing effects of GM-CSF and TGF-β

Biosynthesis of apolipoprotein (apo) E has been previously demonstrated to be regulated in macrophages by intracellular free cholesterol levels as well as by macrophage activating factors. In this report, the regulation of apo E secretion by cytokines detected within atherosclerotic lesions has been investigated. Granulocyte macrophage-colony stimulating factor (GM-CSF) stimulated macrophages had a 3-5-fold reduction in apo E secretion, comparable to that observed for gamma interferon (IFN gamma), while tumor necrosis factor alpha (TNF alpha) and interleukin 1 beta (IL-1 beta) resulted in a 2-fold decrease. In contrast to the reduction in apo E secretion by these cytokines, transforming growth factor beta (TGF-beta) stimulated macrophages secreted 3-fold greater amounts of apo E than controls. The reduced secretion of apo E by GM-CSF was reversible, heat labile, dose dependent, maximal 48 h after cytokine exposure and was coincident with an increase in fibronectin secretion. The opposing effects of GM-CSF and TGF-beta on apo E secretion were consistent with similar changes detected in apo E mRNA levels. Cytokine effects on apo E secretion in cholesterol loaded macrophages were also investigated and found to be similar to the non-loaded cells with GM-CSF decreasing and TGF-beta increasing apo E secretion. The observed differences in apo E secretion did not correlate with any significant changes in either cellular cholesterol distribution in the non-cholesterol loaded macrophages or in basal ACAT activity. In addition to changes in apo E secretion, cytokine treated macrophages pulsed with [14C]oleate and acetylated LDL for 2-6 h had a 2-fold increase (GM-CSF) or decrease (TGF-beta) in cholesterol esterification. Therefore, GM-CSF and TGF-beta mediated changes in apo E secretion may occur through a mechanism independent of changes in cellular free cholesterol levels. These results suggest that cytokines expressed within an atheroma may play an important role in the modulation of macrophage mediated reverse cholesterol transport.

A novel microtiter plate assay for the quantitation of procoagulant activity on adherent monocytes, macrophage and endothelial cells

The interaction of monocytes, macrophages and endothelial cells with inflammatory agents induces cell surface changes resulting in the activation of the coagulation cascade. A large volume 96 well plate microtiter assay has been developed which permits the quantitation of procoagulant activity on endotoxin stimulated cells without requiring the use of purified coagulation factors. Procoagulant activity is measured through a two stage amidolytic assay using commercially available Proplex as a source of factors VII and X and the chromogenic substrate S2222. The assay is rapid, linear, and sensitive as procoagulant activity can be detected on as few as 5 X 10(3) monocytes or macrophages and 3 X 10(2) endothelial cells.

Estriol: A potent regulator of TNF and IL-6 expression in a murine model of endotoxemia

The increased incidence of autoimmune disease in premenopausal women suggests the involvement of sex steroids in the pathogenesis of these disease processes. The effects of estrogen on autoimmunity and inflammation may involve changes in the secretion of inflammatory mediators by mononuclear phagocytes. Estradiol, for example, has been reported to regulate TNF, IL-6, IL-1 and JE expression. In the present study the effects of the estrogen agonist, estriol, on cytokine expression have been investigated in mice administered a sublethal lipopolysaccharide, LPS, challenge. Pretreatment of mice with pharmacologic doses of estriol, 0.4–2 mg/kg, resulted in a significant increase in serum TNF levels in both control and autoimmune MRL/lpr mice, following LPS challenge. This increase in TNF over the placebo group was blocked by the estrogen antagonist tamoxifen. Estriol treated mice also exhibited a rapid elevation in serum IL-6 levels following LPS challenge with the peak increase occurring 1 hr post LPS. This contrasted with the placebo group in which maximal serum IL-6 levels were detected at 3 hrs post challenge. This shift in the kinetics of IL-6 increase by estriol was inhibited by tamoxifen. The estriol mediated effects on TNF and IL-6 serum levels were consistent with the changes in TNF and IL-6 mRNA observed ex vivo in elicited peritoneal macrophages. Macrophage cultures from estriol treated animals however, did not demonstrate significant differences from the placebo group for TNF or NO secretion following in vitro LPS challenge. These results suggest that the estrogen agonist estriol can have significant quantitative, TNF, and kinetic, IL-6, effects on inflammatory monokines produced in response to an endotoxin challenge.

Induction of endothelial cell/macrophage procoagulant activity: synergistic stimulation by gamma interferon and granulocyte-macrophage colony stimulating factor.

Inflammatory mediators such as endotoxin can stimulate the expression of procoagulant activity on both endothelial cells and macrophages while the monokines Interleukin 1, IL-1, and Tumor Necrosis Factor, TNF, induce procoagulant activity on endothelial cells. Incubation of murine peritoneal macrophages with suboptimal concentrations of endotoxin results in a two fold increase in procoagulant activity. Macrophages incubated with gamma interferon, IFN gamma, or Granulocyte-Macrophage Colony Stimulating Factor, GM-CSF, for 16 hours prior to endotoxin stimulation demonstrated a synergistic increase in procoagulant activity. A synergistic increase in procoagulant activity was also observed with primary cultures of human umbilical cord endothelial cells incubated with recombinant human IFN gamma for 16 hours prior to endotoxin, TNF, or IL-1 stimulation. Human GM-CSF had no stimulatory effect on endotoxin or monokine induced endothelial cell procoagulant activity. The augmentation of macrophage and endothelial cell procoagulant activity by IFN gamma and GM-CSF may provide a novel explanation for the role of these cytokines in acute and chronic inflammation.