Regulation of Human Monocyte Functions by Acute Ethanol Treatment: Decreased Tumor Necrosis Factorα, Interleukin-lβ and Elevated Interleukin-10, and Transforming Growth Factor-β Production

We and others have previously shown that even acute ethanol exposure has the capacity to modulate immune functions, particularly monocyte functions. Herein, we tested the hypothesis that acute ethanol treatment inhibits inflammatory, while increasing inhibitory cytokine production in human blood monocytes that, in tum, could contribute to the overall immune abnormalities seen after alcohol use. Our data show that in vitro treatment of blood monocytes with a physiologically relevant dose of alcohol (W mM) results in significantly decreased induction of tumor necrosis factor-α (TNFα) and interleukin (IL)-lβ by bacterial stimulation of either Gram-positive [staphylococcal enterotoxin B (SEB), 1 μg/ml of SEB] or Gram-negative [lipopolysaccharide (LPS), 1 μg/ml of LPS] origin both at the protein and mRNA levels. In contrast, acute ethanol treatment induces monocyte production of mediators with immunoinhibitory potential, including transforming growth factor-β and IL-10. We further show that ethanol not only induces monocyte/macrophage (Mø) IL-10 and transforming growth factor-β, but even augments bacterial (both LPS and SEB) stimulation-induced production of both of these cytokines. IL-10 is a potent inhibitor of Mø TNFα production. We found that ethanol-induced elevation in Mø IL-10 levels contributes to the decreased Mø TNFα production to bacterial challenge in eth-anol-exposed Mø. However, mRNA levels for TNFα are downregu-lated as early as 1.5 hr after ethanol treatment, suggesting that ethanol likely has an IL-10 independent, direct effect on early signaling events of TNF α induction.     

Immunocytochemical localization of follicle stimulating hormone in normal human stomach

Immunoreactive follicle-stimulating hormone (IR-FSH) is detected in sections of formalin-fixed and paraffin-embedded gastric mucosal tissue of normal men, using the immunoperoxidase staining technique and specific antisera to hFSH (NIDDK, NIH). Positive staining for IR-FSH was detected in the parietal cells lining the gastric glands of the intermediate zone. The staining was intracytoplasmic and distributed throughout the cytoplasm. IR-FSH was also found to be present in the basal part of the foveolar epithelium. Stromal tissue and nuclei were devod of the stain. The zymogen cells in the deeper region of the mucosa did not show any detectable staining for IR-FSH. The presence of IR-FSH in gastric mucosa was also detected by radioimmunoassay. Gel chromatography of the gastric tissue extract showed a single peak of FSH immunoreactivity that coeluted with the ¹²⁵I-labled highly purified FSH preparation (NIDDK, NIH). Furthermore, the FSH in the pituitary tissue extract had a chromatographic profile similar to that of IR-FSH from gastric tissue, and ¹²⁵I-FSH labeled highly purified FSH, indicating a close resemblance in their molecular sizes. These results demonstrate that IR-FSH is present in the normal human gastric mucosa. The role of this regulatory petpide in gastric tissue, if any, needs to be investigated.     

Inhibition of NF-kappa B binding correlates with increased nuclear glucocorticoid receptor levels in acute alcohol-treated human monocytes

BACKGROUND: Acute alcohol treatment blocks inflammatory cytokines via inhibition of NF-kappaB in monocytes in the presence of ongoing IkappaBalpha degradation, suggesting regulation of NF-kappaB activation downstream of IkappaBalpha degradation. DNA binding of NF-kappaB has been suggested to be regulated by other nuclear regulatory factors, including the glucocorticoid receptor (GR). Here, we show for the first time that acute alcohol (25 mM) exposure modulates GR activation in monocytes. METHODS: Human peripheral blood monocytes were treated with lipopolysaccharide (LPS) in the presence or absence of alcohol (25 mM) for 1 hour. Nuclear GR levels were estimated by Western blotting and NFkappaB activation was studied in the same extracts by gel shift analysis (EMSA). Cells were stimulated with 1 microM of Dex to be used as positive control for GR activation. GR/GRE binding was also determined in nuclear extracts by EMSA. IkappaBalpha mRNA known to be induced by GR/GRE activation was studied in total RNA extracts by the SuperArray method (SuperArray Inc., Bethesda, MD). RESULTS: LPS is a potent inducer of GR nuclear translocation and GR binding to the glucocorticoid response element (GRE). Acute alcohol treatment both induced (p < 0.05) and augmented (p < 0.05) LPS-stimulated GR nuclear levels. However, alcohol inhibits LPS-induced (nonligand bound) GR/GRE binding activity in monocytes. This inhibition of GR transactivation by alcohol was further confirmed by decreased expression (40%) of a target gene, IkappaBalpha. Thus, alcohol treatment increases nonligand-bound nuclear GR, but inhibits its transactivation function. Ligand-induced GR/GRE binding was decreased in alcohol-treated monocytes. Inhibition of ligand-induced GR/GRE binding by alcohol exposure is likely due to cytoplasmic retention of the GR. CONCLUSIONS: Our results show that acute alcohol exposure inhibits GR in monocytes by differently affecting ligand- and nonligand-induced GR nuclear translocation. These data also suggest that acute alcohol regulates GR activation in monocytes concomitant to inhibition of NF-kappaB activation.     

IKKβ inhibition protects against bone and cartilage destruction in a rat model of rheumatoid arthritis

Objective

The IKK complex regulates NF‐κB activation, an important pathway implicated in the rheumatoid arthritis (RA) disease process. This study was undertaken to assess the efficacy of N‐(6‐chloro‐7‐methoxy‐9H‐β‐carbolin‐8‐yl)‐2‐methylnicotinamide (ML120B), a potent and selective small molecule inhibitor of IKKβ.

Methods

Polyarthritis was induced in rats by injection of Freund's complete adjuvant into the hind footpad. ML120B was administered orally twice daily, either prophylactically or therapeutically. Paw volumes and body weights were measured every 2–3 days throughout the study. We assessed bone erosions by several methods: histologic evaluation, quantitative micro–computed tomography (micro‐CT) imaging analysis, and measurement of type I collagen fragments in the serum. Quantitative polymerase chain reaction was used to evaluate expression of messenger RNA for genes related to inflammation and to bone and cartilage integrity.

Results

Oral administration of ML120B inhibited paw swelling in a dose‐dependent manner (median effective dosage 12 mg/kg twice daily) and offered significant protection against arthritis‐induced weight loss as well as cartilage and bone erosion. We were able to directly demonstrate that NF‐κB activity in arthritic joints was reduced after ML120B administration. Also, we observed that down‐regulation of the NF‐κB pathway via IKKβ inhibition dampened the chronic inflammatory process associated with rat adjuvant‐induced arthritis.

Conclusion

The results of the present study suggest that IKKβ inhibition is an effective therapeutic approach to treat both the inflammation and the bone/cartilage destruction observed in RA. Methods for the determination of serum markers for bone and cartilage destruction, as well as micro‐CT analysis, may aid in predicting and evaluating the therapeutic efficacy of IKKβ inhibition therapy in humans.

     

Bone marrow-derived immune cells mediate sensitization to liver injury in a myeloid differentiation factor 88-dependent fashion

Toll-like receptors (TLRs) expressed on both immune cells and hepatocytes recognize microbial danger signals and regulate immune responses. Previous studies showed that TLR9 and TLR2 mediate Propionibacterium acnes-induced sensitization to lipopolysaccharide-triggered acute liver injury in mice. Ligand-specific activation of TLR2 and TLR9 are dependent on the common TLR adaptor, myeloid differentiation factor 88 (MyD88). Here, we dissected the role of MyD88 in parenchymal and bone marrow (BM)-derived cells in liver sensitization. Using chimeric mice with green fluorescent protein-expressing BM cells, we identified that P. acnes-induced liver inflammatory foci are of BM origin. Chimeras with MyD88-deficient BM showed no inflammatory foci after P. acnes or TLR2+TLR9 challenge, suggesting that recruitment of inflammatory cells to the liver required MyD88 expression in BM-derived cells. Further, selective MyD88 deficiency in parenchymal cells in mice with wild-type BM failed to prevent inflammatory cell infiltration. These results demonstrate that MyD88 in immune cells rather than in liver parenchymal cells plays an important role in inflammatory cell recruitment and liver injury.     

Critical role of toll-like receptors and the common TLR adaptor, MyD88, in induction of granulomas and liver injury

BACKGROUND/AIMS: Toll-like receptors (TLR) recognize pathogens and regulate innate immune activation. Here, we investigated the roles of TLR9 and the common TLR adaptor, MyD88, in liver injury. METHODS: C57BL6, TLR9(-/-), IFNgamma(-/-) or MyD88(-/-) mice were primed with Propionibacterium acnes, TLR9 (CpG) or TLR2 (lipoteichoic acid) ligands followed by LPS challenge. ALT, cytokines and liver histology were assessed. RESULTS: Selective priming through TLR9 but not TLR2 induced granulomas, elevated serum ALT, and sensitized C57BL6 mice to increased LPS-induced serum IL-6, IL-12 and IFNgamma levels. Further, TLR2 and TLR9 ligands synergized in induction of granulomas and sensitization to LPS-induced inflammation. IFNgamma induction by P. acnes, TLR2 and TLR9 ligands required MyD88. In MyD88(-/-) mice P. acnes failed to induce granulomas and both MyD88 and TLR9 deficiency prevented P. acnes-induced sensitization to LPS. Increased mRNA expression of genes of the TLR4 signaling complex (TLR4, CD14, MD-2, and MyD88) and the NADPH complexes (p47phox, p67phox, gp91phox, and p22phox) was induced by priming with P. acnes or TLR9 plus TLR2 suggesting mechanisms for LPS sensitization and liver injury. CONCLUSIONS: TLR9+/-TLR2 activation via MyD88-dependent pathways plays a pivotal role in liver sensitization and granuloma formation.     

A Recent Perspective on Alcohol, Immunity, and Host Defense

Background:  Multiple line of clinical and experimental evidence demonstrates that both acute, moderate, and chronic, excessive alcohol use result in various abnormalities in the functions of the immune system.
Methods:  Medline and Pubmed databases were used to identify published reports with particular interest in the period of 2000–2008 in the subject of alcohol use, infection, inflammation, innate, and adaptive immunity.
Results:  This review article summarizes recent findings relevant to acute or chronic alcohol use-induced immunomodulation and its consequences on host defense against microbial pathogens and tissue injury. Studies with in vivo and in vitro alcohol administration are both discussed. The effects of alcohol on lung infections, trauma and burn injury, liver, pancreas, and cardiovascular diseases are evaluated with respect to the role of immune cells. Specific changes in innate immune response and abnormalities in adaptive immunity caused by alcohol intake are detailed.
Conclusion:  Altered inflammatory cell and adaptive immune responses after alcohol consumption result in increased incidence and poor outcome of infections and other organ-specific immune-mediated effects.     

Acute Alcohol Inhibits the Induction of Nuclear Regulatory Factor κB Activation Through CD14/Toll‐Like Receptor 4, Interleukin‐1, and Tumor Necrosis Factor Receptors: A Common Mechanism Independent of Inhibitory κBα Degradation?

BACKGROUND: Nuclear translocation and DNA binding of the nuclear factor kappaB (NF-kappaB) is an early event in inflammatory cell activation in response to stimulation with bacterial components or cytokines. Cell activation via different receptors culminates in a common pathway leading to NF-kappaB activation and proinflammatory cytokine induction. We have previously shown that acute alcohol inhibits NF-kappaB activation by lipopolysaccharide (LPS) in human monocytes. Here we investigated whether acute alcohol treatment of human monocytes also inhibits NF-kappaB when induced through activation of the interleukin (IL)-1 or tumor necrosis factor (TNF) receptors. METHODS: Human peripheral blood monocytes were treated with LPS, TNFalpha, and IL-1beta in the presence or absence of 25mM alcohol for 1 hr. NF-kappaB activation was determined by electrophoretic mobility shift assays using nuclear extracts. Inhibitory kappaB(alpha) (IkappaB(alpha)) was estimated by Western blotting in cytoplasmic extracts. Chinese hamster ovary cells expressing human CD14 were treated with LPS in the presence or absence of alcohol to study NF-kappaB and IkappaB(alpha) regulation. RESULTS: Our results indicate that acute alcohol inhibits IL-1beta- and TNFalpha-induced NF-kappaB activation. We further show in CD14/toll-like receptor 4-expressing Chinese hamster ovary cells the specificity of alcohol-mediated inhibition of NF-kappaB via the toll-like receptor 4/CD14 receptors. Inhibition of NF-kappaB by acute alcohol was concomitant with decreased levels of the IkappaB(alpha) molecule in the cytoplasm of LPS, IL-1, and TNFalpha-activated monocytes. CONCLUSIONS: These data suggest a unique, IkappaB(alpha)-independent pathway for the inhibition of NF-kappaB activation by acute alcohol in monocytes. Universal inhibition of NF-kappaB by acute alcohol via these various receptor systems suggests a target for the effects of alcohol in the NF-kappaB activation cascade that is downstream from IkappaB(alpha) degradation. Further, these results demonstrate that acute alcohol is a potent inhibitor of NF-kappaB activation by mediators of early (LPS) or late (IL-1, TNF(alpha)) stages of inflammation in monocytes.