Joanna Goral

Age‐dependent decrease in Toll‐like receptor 4‐mediated proinflammatory cytokine production and mitogen‐activated protein kinase expression

Age‐related changes in immunity render elderly individuals more susceptible to infections than the young. Previous work by our laboratory and others showed that macrophages from aged mice are functionally impaired. Macrophages produce proinflammatory cytokines, tumor necrosis factor α (TNF‐α) and interleukin (IL)‐6, when stimulated with lipopolysaccharide (LPS), which signals through Toll‐like receptor‐4 (TLR4) and requires activation of mitogen‐activated protein kinases (MAPKs). We investigated whether aging is associated with alterations in TNF‐α and IL‐6 production and MAPK expression and activation in thioglycollate‐elicited peritoneal macrophages from mice. Kinetics and LPS dose‐responsiveness of macrophage TNF‐α production did not differ by age. Unstimulated macrophages did not differ by age in their cytokine production. However, LPS‐stimulated (100 ng/mL) cultures from aged mice produced 100 ± 30 pg/mL TNF‐α and 6000 ± 2000 pg/mL IL‐6, and those from young mice produced 280 ± 50 pg/mL and 10,650 ± 10 pg/mL, respectively (P<0.05). Likewise, levels of activated MAPKs did not differ by age in unstimulated macrophages, and LPS‐stimulated macrophages from aged mice had <70% activated p38 and c‐jun NH2‐terminal kinase (JNK) than those of young controls. Of particular interest, we observed >25% reduction of total p38 and JNK in macrophages from aged mice relative to young. In addition, surface TLR4 levels did not vary with age. We conclude that macrophages from aged mice exhibited suppressed proinflammatory cytokine production, which correlated with diminished total levels and LPS‐stimulated activation of p38 and JNK. These observations suggest that decreased MAPK expression could be a mechanism responsible for age‐related deterioration of the immune system.

Exposure-dependent effects of ethanol on the innate immune system.

Extensive evidence indicates that ethanol (alcohol) has immunomodulatory properties. Many of its effects on innate immune response are dose dependent, with acute or moderate use associated with attenuated inflammatory responses, and heavy ethanol consumption linked with augmentation of inflammation. Ethanol may modify innate immunity via functional alterations of the cells of the innate immune system. Mounting evidence indicates that ethanol can diversely affect antigen recognition and intracellular signaling events, which include activation of mitogen activated protein kinases, and NFkappaB, mediated by Toll-like receptors, leading to altered inflammatory responses. The mechanism(s) underlying these changes may involve dose-dependent effects of ethanol on the fluidity of cell membrane, resulting in interference with the timely assembly or disassembly of lipid rafts. Ethanol could also modify cell activation by specific interactions with cell membrane molecules.

In Vivo Ethanol Exposure Down-Regulates TLR2-, TLR4-, and TLR9-Mediated Macrophage Inflammatory Response by Limiting p38 and ERK1/2 Activation

Ethanol is known to increase susceptibility to infections, in part, by suppressing macrophage function. Through TLRs, macrophages recognize pathogens and initiate inflammatory responses. In this study, we investigated the effect of acute ethanol exposure on murine macrophage activation mediated via TLR2, TLR4, and TLR9. Specifically, the study focused on the proinflammatory cytokines IL-6 and TNF-α and activation of p38 and ERK1/2 MAPKs after a single in vivo exposure to physiologically relevant level of ethanol followed by ex vivo stimulation with specific TLR ligands. Acute ethanol treatment inhibited IL-6 and TNF-α synthesis and impaired p38 and ERK1/2 activation induced by TLR2, TLR4, and TLR9 ligands. We also addressed the question of whether ethanol treatment modified activities of serine/threonine-specific, tyrosine-specific phosphatases, and MAPK phosphatase type 1. Inhibitors of three families of protein phosphatases did not restore ethanol-impaired proinflammatory cytokine production nor p38 and ERK1/2 activation. However, inhibitors of serine/threonine protein phosphatase type 1 and type 2A significantly increased IL-6 and TNF-α levels, and prolonged activation of p38 and ERK1/2 when triggered by TLR4 and TLR9 ligands. In contrast, with TLR2 ligand stimulation, TNF-α production was reduced, whereas IL-6 levels, and p38 and ERK1/2 activation were not affected. In conclusion, acute ethanol exposure impaired macrophage responsiveness to multiple TLR agonists by inhibiting IL-6 and TNF-α production. Mechanism responsible for ethanol-induced suppression involved inhibition of p38 and ERK1/2 activation. Furthermore, different TLR ligands stimulated IL-6 and TNF-α production via signaling pathways, which showed unique characteristics.

Neurochemical, endocrine and immunological responses to stress in young and old fischer 344 male rats

Two experiments were performed. In the first, a 20 min conditioned emotional response (CER) paradigm was used to compare the neurochemical, endocrine and immunological responses to stress of 7- and 22-month-old Fischer 344 (F344) male rats. In the second, corticosterone levels 20 min following ether stress, and regional brain type I and II corticosterone receptor densities were examined using 7- and 17.5-month-old F344 male rats. Dopamine (DA) metabolism in old nonstressed rats was significantly reduced in the medial frontal cortex, neostriatum, nucleus accumbens and hypothalamus, but not in the amygdala. The CER procedure, nevertheless, increased medial frontal cortical, nucleus accumbens and amygdaloid DA turnover in both the young and old rats. The young and old nonstressed rats did not evidence differences in norepinephrine (NE) and serotonin (5-HT) concentrations. However, stress resulted in a decrease in medial frontal cortical 5-hydroxyindoleacetic acid (5-HIAA) and hypothalamic 5-HT levels in old but not in young animals. These observations suggest age-related differences in the response of central NE and 5-HT systems to stress. Ether and the CER procedure led to exaggerated corticosterone responses in the old rats (17.5 and 22 month, respectively). Hippocampal type I but not type II corticosterone receptors were decreased by 47% in the 17.5-month-old rats. Thus, age-related changes in hippocampal corticosterone receptor types do not occur in unison, and the exacerbated corticosterone response to stress precedes the reported down-regulation of hippocampal type II corticosterone receptors in aged rats. Age-related changes were not observed in the concentrations of corticosterone receptors in other brain regions, or in the prolactin response to stress. The old rats, however, evidenced a reduction in the availability of the renin substrate, angiotensinogen, and in stress-induced renin secretion. Immune function was impaired in the old nonstressed rats, and further compromised by exposure to the CER procedure. In comparison to the young control rats, the old nonstressed rats showed an increased percentage of splenic large granular lymphocytes, reduced splenic natural killer cytotoxicity, and impaired Con-A-stimulated splenic T lymphocyte proliferation. Reductions in T splenic cell proliferation and natural killer cytotoxicity were observed in the young rats subjected to the CER paradigm, but not to the same extent as in the old rats. These observations indicate that aging male F344 rats evidence major alterations in basal central monoamine, endocrine and immune functions, and an increased sensitivity of these systems to stress.

Acute ethanol exposure inhibits macrophage IL-6 production: role of p38 and ERK1/2 MAPK.

Acute ethanol consumption has been linked to an increase in infectious complications in trauma and burn patients. Ethanol modifies production of a variety of macrophage‐derived immunoregulatory mediators. Lipopolysaccharide (LPS), a potent stimulator of inflammatory responses in macrophages, activates several intracellular signaling pathways, including mitogen‐activated protein kinases (MAPK). In the current study, we investigated the effect of acute ethanol exposure on in vivo activation of p38 and extracellularly regulated kinases 1 and 2 (ERK1/2) MAPK in murine macrophages and the corresponding, LPS‐stimulated interleukin (IL)‐6 production. We demonstrated that a single dose of ethanol transiently down‐regulated p38 and ERK1/2 activation levels (3–24 h after treatment) and impaired IL‐6 synthesis. Ethanol‐related reduction in IL‐6 production was not further affected by the presence of inhibitors of p38 and ERK1/2 (SB 202190 and PD 98059, respectively). These results demonstrate that acute ethanol exposure can impair macrophage IL‐6 production and indicate that this effect may result from ethanol‐induced alterations in intracellular signaling through p38 and ERK1/2.

Aberrant acute-phase response in aged interleukin-6 knockout mice

ABSTRACT This study was designed to determine whether the acute-phase response in aged mice is altered by interleukin (IL) 6 deficiency. Young and aged wild-type (WT) and IL-6 knockout (KO) BALB/C female mice were injected with lipopolysaccharide (LPS; 1.5 &mgr;g/g body weight). After 24 h, aged IL-6 KO mice had an improved survival when compared with aged WT mice. Serum levels of IL-6 in aged WT animals given LPS were determined and, as expected, were significantly higher when compared with young LPS-treated WT animals (P < 0.05). Serum levels of the acute-phase protein, serum amyloid A, were 50% lower in aged LPS-treated IL-6 KO mice relative to aged WT mice given LPS (P < 0.001). In contrast, the induction of LPS-binding protein was not affected by age or IL-6 deficiency in LPS-treated animals. Circulating levels of corticosterone were markedly reduced in aged LPS-treated IL-6 KO mice relative to aged WT mice given LPS. These data indicate that IL-6 is an important contributor to the outcome of the acute-phase response of aged individuals challenged with endotoxin. We conclude that the absence of IL-6, a cytokine that contributes to the elevated basal proinflammatory state observed in aging, can improve the ability of aged mice to withstand an otherwise lethal challenge of bacterial endotoxin.

Reduced Levels of Hsp70 Result in a Therapeutic Effect of l5-Deoxyspergualin on Acute Graft-Versus-Host Disease in (DA×LEW)Fl Rats

We have shown previously that increased levels of hsp70, and antibodies reactive with hsp70 parallel the onset and severity of graft-versus-host disease (GVHD) in a parent --> (DA x LEW)F1 rat model. In this study we have assessed the effect of reducing the levels of the 70 kDa heat shock protein (hsp70), on the morbidity and mortality of acute GVHD in (DA x LEW)F1 rats. The reduction was accomplished by the administration of 15-deoxyspergualin (DSG), an immunosuppressive agent which binds to a constitutively expressed member of the 70 kDa heat shock protein family. DSG administered via three different protocols reduced GVHD-associated morbidity. One of the regimens, which consisted of intermittent DSG administration, also significantly reduced GVHD associated mortality. This DSG treatment reduced hsp70 levels in spleen and lymph nodes, inhibited anti-hsp70 antibody production, and diminished the serum levels of IL-2, IFN-gamma, TNF-alpha, and IL-10. IL-4 levels in the serum did not change during GVHD and were not effected by DSG. These results show that the mechanism of DSG immunosuppressive effect in rat GVHD may involve DSGs capacity to bind to hsp70, which in turn may lead to a decrease in levels of circulating anti-hsp70 antibodies, and reduced production of cytokines.

hsp70, hsp32, and grp78 are increased in thermally injured skin with and without antithrombin(human) concentrate infusion.

An acquired deficiency of antithrombin (AT), an anti-inflammatory protein, develops in patients with thermal injuries. Skin thermotolerance is regulated by heat shock protein (hsp) genes. hsp70, hsp32, hsp27, and glucose-regulated protein78 (grp78) were studied in burned and unburned human skin to determine whether correction of the AT deficiency modulated the intensity of expression of these proteins. Fifty-four human skin samples were prepared by Western blot analysis: 11 unburned and 22 burned control skin samples and 7 unburned and 14 burned skin samples from patients treated with AT(Human), or AT(H). The intensity of hsp32 expression in burned AT(H)-treated skin (P < .001) and in burned control skin (P < .01) was significantly increased compared with unburned control skin. The intensity of expression of hsp70 was statistically significant in burned AT(H)-treated skin compared with unburned control skin (P < .02), as was that of grp78 (P < .01). Thermally injured skin with or without AT(H) treatment had an increased expression of hsp70, hsp32, and grp78 compared with unburned control skin.

The effects of amino acids on protein degradation and translocation of non-histone proteins to the nucleus in lymphocytes

Tryptophan, phenylalanine and leucine have two parallel effects in cultured lymphocytes, they inhibit cellular proteolysis and increase the translocation of non-histone proteins to the nucleus. The latter is associated with an increased cellular binding of [3H]actinomycin D, indicating an altered structure of chromatin. The amino acids also inhibit the cellular uptake of [3H]chloroquine, suggesting that inhibited protein degradation is lysosomal. Several amine catabolites of tryptophan and phenylalanine, some of which are known to play a role as biogenic amines, have similar actions, and can explain, at least in part, the effects of their parent amino acids. Fractionation of the nuclear 3H-labeled non-histone proteins according to pH 2.5-6.5 shows that such proteins with a high rate of degradation in untreated cells correspond to the 3H-labeled non-histone proteins with a high rate of translocation in tryptophan treated cells. These data suggest that the degradation and the translocation of the non-histone proteins are linked and that the increased translocation of the non-histone proteins to the nucleus may be the consequence of inhibited lysosomal degradation of these proteins by the amino acids.

Effect of d-fenfluramine on the lymphocyte response of hiv+ humans

The objective of this study was to analyse the effect of d-dexfenfluramine (d-FEN) on the human lymphocyte response, in vitro. Experiments were designed to determine whether d-FEN augments specific human immune parameters associated with protection from opportunistic microbial pathogens and particularly focuses on d-FEN as a means by which to augment the function of CD8+ and CD4+ lymphocytes. Lymphocytes were examined for three reasons: (1) for their ability to inhibit the growth of Candida albicans; (2) for their ability to proliferate in response to a mitogen; and, (3) their cytokine profile (vis., production of IL-2, IFN-gamma and TNF-alpha). Peripheral blood mononuclear cells (PBMC) were obtained from 20 HIV+ patients. The patients were diagnosed as HIV+ within the past 0.5-9 years. d-FEN was found to augment the capacity of CD8+ lymphocytes to inhibit the growth of the opportunistic microbial pathogen, C. albicans. d-FEN enhanced the capacity of CD4+ lymphocytes to proliferate in response to the mitogen, Concanavalin A, and to increase the amount of IL-2 produced by CD4+ and CD8+ lymphocytes from AIDS patients. d-FEN increased the number of CD4+ and CD8+ lymphocytes that produced IFN-gamma from either non-AIDS or AIDS patients and increased the number of AIDS patients CD8+ lymphocytes that produce TNF-alpha. These in vitro data suggest that d-FEN may be effective in enhancing immune function in immunocompromised individuals.