Glenn F. Evans

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.

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.

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.

Peroxisome proliferator-activated receptor α, γ coagonist LY465608 inhibits macrophage activation and atherosclerosis in apolipoprotein E knockout mice

The apolipoprotein F (apoF) knockout mouse has provided an approach to the investigation of the effect of both cellular and humoral processes on atherosclerotic lesion progression. In the present study, pharmacologic modulation of both interferon gamma (IFNγ)-inducible macrophage effector functions, and atherosclerotic lesions in the apoE knockout mouse were investigated using the peroxisome proliferator-activated receptor (PPAR) α,γ coagonist LY465608. LY465608 inhibited, in a concentration-dependent manner, IFNγ induction of both nitric oxide synthesis and the β2 integrin CD11a in elicited peritoneal macrophages from apoE knockout mice. Similar effects were observed ex vivo following 10 d of treating mice with 10 mg/kg of LY465608. Treatment of apoE knockout mice for 18 wk with LY465608 resulted in a statistically significant 2.5-fold reduction in atherosclerotic lesion area in en face aorta preparations. These effects were apparent in the absence of any reduction in total serum cholesterol or in lipoprotein distribution. Finally, treatment of apoE knockout mice with established atherosclerotic disease resulted in a modest but not statistically significant decrease in aortic lesional surface area. These results demonstrate the utility of PPAR coagonists in reducing the progression of the atherosclerotic lesion.

Exogenous glucocorticoids increase macrophage secretion of apo E by cholesterol-independent pathways

Macrophage apo E synthesis and secretion has been previously demonstrated to be regulated by intracellular free cholesterol levels and is decreased by cytokines and other inflammatory stimuli associated with macrophage activation. In a recent study, the opposing effects of TGF beta and GM-CSF were reported with the former increasing and the latter decreasing apo E secretion and apo E mRNA levels. In an attempt to further understand the mechanisms by which TGF beta increased apo E expression in mouse peritoneal macrophages, the present study was performed to determine whether pharmacological agents could up-regulate apo E secretion by a mechanism independent of intracellular free cholesterol levels. Agents which resulted in increased apo E secretion were subdivided based on their effects on cAMP elevation. In addition to TGF beta, dexamethasone resulted in significant increases in apo E secretion. The 2-4-fold enhancement in apo E secretion by both TGF beta and dexamethasone occurred without concomitant changes in intracellular cAMP or free cholesterol. Other agents which increased apo E secretion included cholera toxin and 8-bromo-cAMP. While these agents did not affect intracellular cholesterol levels, cholera toxin did increase macrophage cAMP. The changes in apo E secretion by dexamethasone and 8-bromo-cAMP were associated with elevations in apo E mRNA. Dexamethasone-treated macrophages had 6-fold increases in apo E mRNA by 48 h when compared with control macrophages. Macrophages stimulated with 8-bromo-cAMP for 48 h demonstrated a more modest but statistically significant (P < 0.001) 2.2-fold increase. Similar effects of dexamethasone, cholera toxin, TGF beta, and 8-bromo-cAMP on apo E secretion were also apparent in macrophage-derived foam cells. In addition to increasing apo E secretion in macrophages and foam cells, dexamethasone and 8-bromo-cAMP inhibited the down-regulation of apo E secretion mediated by LPS and GM-CSF. Finally, the increased apo E secretion by exogenous glucocorticoids or TGF beta was not species specific as similar effects were observed in rabbit peritoneal macrophages. Therefore, while macrophage activation results in decreased apo E synthesis, macrophages exposed to anti-inflammatory agents including dexamethasone, TGF beta, or following cAMP elevation demonstrate increased apo E secretion by a cholesterol-independent mechanism.

LY2228820 Dimesylate, a Selective Inhibitor of p38 Mitogen-activated Protein Kinase, Reduces Angiogenic Endothelial Cord Formation in Vitro and in Vivo

Background: Angiogenesis is a critical process for tumor growth and survival. Results: LY2228820 dimesylate, a p38 MAPK-specific inhibitor, or shRNA knockdown of p38α, MK2, or HSP27 reduced angiogenic cord formation. Conclusion: p38 MAPK modulated soluble factors released from stromal and tumor cells and reduced their downstream signaling in endothelial cells. Significance: Antiangiogenic activity of LY2228820 dimesylate may lead to anti-tumor growth effects. LY2228820 dimesylate is a highly selective small molecule inhibitor of p38α and p38β mitogen-activated protein kinases (MAPKs) that is currently under clinical investigation for human malignancies. p38 MAPK is implicated in a wide range of biological processes, in particular those that support tumorigenesis. One such process, angiogenesis, is required for tumor growth and metastasis, and many new cancer therapies are therefore directed against the tumor vasculature. Using an in vitro co-culture endothelial cord formation assay, a surrogate of angiogenesis, we investigated the role of p38 MAPK in growth factor- and tumor-driven angiogenesis using LY2228820 dimesylate treatment and by shRNA gene knockdown. p38 MAPK was activated in endothelial cells upon growth factor stimulation, with inhibition by LY2228820 dimesylate treatment causing a significant decrease in VEGF-, bFGF-, EGF-, and IL-6-induced endothelial cord formation and an even more dramatic decrease in tumor-driven cord formation. In addition to involvement in downstream cytokine signaling, p38 MAPK was important for VEGF, bFGF, EGF, IL-6, and other proangiogenic cytokine secretion in stromal and tumor cells. LY2228820 dimesylate results were substantiated using p38α MAPK-specific shRNA and shRNA against the downstream p38 MAPK effectors MAPKAPK-2 and HSP27. Using in vivo models of functional neoangiogenesis, LY2228820 dimesylate treatment reduced hemoglobin content in a plug assay and decreased VEGF-A-stimulated vascularization in a mouse ear model. Thus, p38α MAPK is implicated in tumor angiogenesis through direct tumoral effects and through reduction of proangiogenic cytokine secretion via the microenvironment.

Histone deacetylase inhibition results in decreased macrophage CD9 expression.

Histone deacetylase (HDAC) inhibitors have been demonstrated to regulate myeloid cell differentiation. In the present study the effects of the HDAC inhibitor trichostatin A (TSA) on the tetraspanin cell surface antigen CD9 were determined in primary murine macrophages. TSA inhibited CD9 protein and message expression and was optimal by 48 h. TSA did not induce similar effects on other surface markers and resulted in a modest increase or no effect on CD54 and CD11b, respectively. These effects were concentration dependent and concomitant with increased histone H4 acetylation. While interferon-gamma (IFN-gamma) and TSA had similar effects on CD9 expression, transcriptional profiling demonstrated significant differences in the genes activated by these stimuli. Notably CD14 message was down-regulated by IFN-gamma while increased by TSA. These results demonstrate that HDAC inhibition may modulate macrophage function in part through changes in the expression of membrane proteins associated with matrix interactions.

Expression of CD54 (intercellular adhesion molecule-1) and the beta 1 integrin CD29 is modulated by a cyclic AMP dependent pathway in activated primary rat microglial cell cultures.

Microglial cell activation plays a central role in acute and chronic inflammatory processes associated with neurodegeneration. As macrophage activation is generally associated with the up-regulation of specific surface antigens, the expression of CD54, and CD29 were evaluated on CD11b positive neonatal rat microglial cell cultures by flow cytometry. These cells when exposed to lipopolysaccharide, LPS, and gamma interferon, IFNγ, exhibited a 2–3 fold increase in CD54 expression, an increase in CD29 and no change in CD11b. Maximal increases in CD54 and CD29 staining on CD11b positive microglial cells were apparent 20–24 h after LPS and IFNγ while nitrite production reflecting inducible nitric oxide synthase activity, continued to increase. The increases in CD29 and CD54 staining were inhibited in a dose dependent manner by agents which increased intracellular cAMP levels including 100 μM 8-bromoadenosine 3′:5′-cyclic monophosphate but not 8-bromoadenosine monophosphate, the phosphodiesterase inhibitor isobutyl methylxanthine and by direct activation of adenylate cyclase with forskolin. Concomitant with the dose dependent decreases in CD29 and CD54 staining were increases in intracellular cAMP and reduced TNF secretion. These results suggest that regulation of CD29 and CD54 expression on activated microglial cells involves a cAMP dependent pathway.