Robert T. Cook

Chronic ethanol consumption by mice results in activated splenic T cells

Previous studies have shown that T cells from human alcoholics overexpress activation or memory markers such as human leukocyte antigen‐DR, CD45RO, CD57, and CD11b and may have reduced levels of CD62L. In those studies, we demonstrated that the increased CD57+ T cell population rapidly produces interferon‐γ (IFN‐γ) and tumor necrosis factor α, independent of a second signal requirement, consistent with an increased effector T cell population. In contrast to the length of alcohol abuse by human alcoholics, most work with mice has involved 2‐week ethanol exposures or less, which result in decreased IFN‐γ responses. In the present work, we have evaluated C57Bl/6 or BALB/c mice, which were administered 20% w/v ethanol in water for 3–13 weeks. In these mice, rapid cytoplasmic IFN‐γ expression by T cells after stimulation through the T cell receptor was significantly increased versus normals. Studies of surface‐activation markers showed that T cells from chronically ethanol‐fed mice had reduced CD62L expression and an increased percentage of CD44hi T cells. The CD44hi subset was largely second signal‐independent for secreted IFN‐γ and interleukin (IL)‐4 production at early times after stimulation. The enriched T cells of chronic ethanol mice secreted more IFN‐γ and IL‐4 than controls and equivalent IL‐2 at early times after stimulation (6–24 h). The overall results support the concept that in humans and mice, chronic alcohol exposure of sufficient duration results in T cell activation or sensitization in vivo and an increased percentage of the effector/memory subset.

Activated CD-8 cells and HLA DR expression in alcoholics without overt liver disease

Lymphocytes from alcoholics without liver disease were immunophenotyped by flow cytometry immediately after admission for detoxication and again after 4 to 10 days of abstinence. We found a small but significant elevation of T lymphocytes at admission compared to controls and decreases in the numbers of B cells and natural killer cells in many patients. A significant elevation of activated T cells was confirmed. The ratio of activated T cells to activated non-T cells was also substantially increased, but declined slightly during early withdrawal. The increase in activated T cells was due mostly to increased numbers of activated CD8hi cells. These activation changes did not revert toward normal as quickly as the other changes and may represent an indication of immune damage at a preclinical stage. An additional finding of interest was a substantial decrease in the expression of HLA DR on CD4+ and non-T cells. The significance of this decrease is not known, but we speculate that it may result in a decline in the efficiency of antigen presentation.

Alcohol and inflammation and immune responses: summary of the 2006 Alcohol and Immunology Research Interest Group (AIRIG) meeting

The 11th annual meeting of the Alcohol and Immunology Research Interest Group was held at Loyola University Medical Center, Maywood, Illinois on November 17, 2006. The Alcohol and Immunology Research Interest Group meeting is held annually to exchange new findings and ideas that arise from ongoing research examining the effects of alcohol intake on the immune system. The event consisted of five sessions, two of which featured plenary talks from invited speakers, two with oral presentations from selected abstracts, and a final poster session. Participants presented new data on a variety of topics including the effects of ethanol on key cells of the immune system (neutrophils, dendritic cells, NK cells), B cell responses, the capacity to clear infectious agents, and the barrier functions of skin, lung, and intestine.

Loss of the CD5+ and CD45RAhi B cell subsets in alcoholics

Chronic alcoholics are frequently immunodeficient, have polyclonal hypergammaglobulinaemia, and often have autoantibodies. Recent work in other diseases has shown that functional distinctions of possible relevance to autoimmunity and immunodeficiency can be found among the B cell subsets defined by differential expression of the surface markers CD5 and CD45RA. Therefore, we have evaluated the CD5,CD45RA B cell subsets of both chronic alcoholics without evidence of active liver disease (AWLD), and alcoholics admitted for acute alcoholic liver disease (ALD). Mean B cell numbers were normal in AWLD, but significantly reduced in ALD. Analysis of B cells by three‐colour flow cytometry in 20 patients and 29 controls revealed a sharp decrease in the percentage of alcoholics’ B cells which were CD5+, 37.6% versus 16.3%, P<0.00001; absolute CD5+ B cell numbers were similarly reduced (58.9 cells/μl versus 20.9; P=0.0012). In addition to the loss of CD5+ B cells, there was a reduction in the percentage of B cells which are CD5−CD45RAhi, leaving many patients with a B cell profile which was predominantly CD19+CD5−CD45RAlo. This subset appears phenotypically similar to the IgM‐producing CD5−CD45RAlo subset described by others, and may be enriched for autoantibody‐producing cells. One outlier patient was an ALD with 61% of B cells which were CD5+, which also is a profile consistent with increased autoantibody production.

Chronic Alcohol Consumption Increases the Severity of Murine Influenza Virus Infections

Respiratory infections with both seasonal as well as potential pandemic Influenza viruses represent a significant burden on human health. Furthermore, viruses such as Influenza are increasingly recognized as important etiologic agents in community acquired pneumonia. Within the U.S. alone, ∼12.9 million people are heavy drinkers and chronic abuse of alcohol is known to increase the risk and severity of community acquired pneumonia. Given the lack of knowledge regarding Influenza disease in this population, we determined the effects of chronic alcohol consumption on Influenza virus infection. Herein, we report that mice exposed to chronic ethanol have sharp increases in morbidity, mortality, and pulmonary virus titers relative to controls. These increases in influenza severity correspond with inhibited pulmonary influenza-specific CD8 T cell responses. Further, chronic ethanol consumption results in an enhanced pulmonary lesion severity, similar to that recently described for pandemic influenzas. Together, our results suggest that chronic alcohol consumption may increase the risk for severe influenza virus infections by altering the pulmonary inflammatory environment and CD8 T cell response.

Fetal exposure to ethanol has long-term effects on the severity of influenza virus infections.

Alcohol use by pregnant women is a significant public health issue despite well-described risks to the fetus including physical and intellectual growth retardation and malformations. Although clinical studies are limited, they suggest that in utero alcohol exposure also results in significant immune deficiencies in naive neonates. However, little is known about fetal alcohol exposure (FAE) effects on adult infections. Therefore, to determine the long-term effects of FAE on disease susceptibility and the adult immune system, we infected FAE adult mice with influenza virus. In this study, we demonstrate that mice exposed to ethanol during gestation and nursing exhibit enhanced disease severity as well as increased and sustained pulmonary viral titers following influenza virus infection. Secondary exposure to alcohol as an adult further exacerbates these effects. Moreover, we demonstrate that FAE mice have impaired adaptive immune responses, including decreased numbers of virus-specific pulmonary CD8 T cells, a decreased size and frequency of pulmonary B cell foci, and reduced production of influenza-specific Ab following influenza infection. Together, our results suggest that FAE induces significant and long-term defects in immunity and susceptibility to influenza virus infection and that FAE individuals could be at increased risk for severe and fatal respiratory infections.

TH1 cytokine response of CD57+ T-cell subsets in healthy controls and patients with alcoholic liver disease

Patients with chronic inflammatory diseases, including Crohns disease and rheumatoid arthritis, as well as those with certain viral infections, and patients who are transplant recipients or who have certain hematologic malignancies have been observed to have CD57+ T cell expansion in both CD4+ and CD8+ subsets. We have reported previously that alcoholic patients also have CD57+ T cell expansion. Because many alcoholics become seriously deficient in cell-mediated immunity, it is of interest to determine whether the expanded CD57+ subsets can respond to stimulation with normal T helper cell subtype 1 (TH1) cytokine production. We report evaluation of the CD57 T-cell subsets of patients with alcoholic liver disease (ALD) with the use of cytoplasmic staining after stimulation through the T-cell receptor (TCR). The CD57+ subsets of the T cells of both healthy individuals and patients with ALD express significantly higher amounts of cytoplasmic tumor necrosis factor-alpha (TNF-) and interferon-gamma (IFN-) after 6 h of stimulation than do the CD57- subsets. This increased production can persist up to 46 h of continuous stimulation. Under these assay conditions, very little cytoplasmic interleukin (IL)-4 is observed in the T cells of either healthy control subjects or patients with ALD. Measurement of cytokine secretion by sort-purified CD57 T-cell subsets with the use of enzyme-linked immunosorbent assay (ELISA) shows that the CD57+ T-cell subset produces 18- to 30-fold more TNF- and IFN-, respectively, than does the CD57- subset in the first 12 h of stimulation. This response requires only stimulation through the TCR for the CD57+ subset, whereas significant secretion by the CD57- subset requires added IL-2 or anti-CD28 antibody. These results are consistent with the concept of the CD57+ T-cell subset as a differentiated effector cell and demonstrate that patients with ALD who are not drinking at the time of evaluation have normal or increased immediate TH1 T-cell responses.

Chronic ethanol induces inhibition of antigen-specific CD8+ but not CD4+ immunodominant T cell responses following Listeria monocytogenes inoculation.

Chronic ethanol consumption results in immunodeficiency. Previous work with chronic ethanol‐fed mice has shown reduced splenic weight and cellularity, including reduced numbers of CD8+ T cells. However, antigen‐specific CD8+ and CD4+ T cell responses in chronic ethanol‐fed mice have been studied relatively little. We have used an attenuated Listeria monocytogenes strain DPL 1942 (LM ΔactA) to inoculate mice and subsequently used CD4+ and CD8+ immunodominant peptides of LM to measure the CD4+ and CD8+ T cell responses after chronic ethanol exposure. We found no major differences between control and ethanol‐fed mice in the kinetics and persistence of antigen‐specific CD4+ T cells in response to an immunodominant LM peptide, as measured by intracellular IFN‐γ staining. In contrast to CD4+ responses, three methods of in vitro antigen presentation indicated that the primary response of CD8+ T cells to several different epitopes was reduced significantly in mice chronically fed ethanol. Antigen‐specific CD8+ T cells were also reduced in chronic ethanol‐fed mice during the contraction phase of the primary response, and memory cells evaluated at 29 and 60 days after inoculation were reduced significantly. BrdU proliferation assays showed that in vivo proliferation of CD8+ T cells was reduced in ethanol‐fed mice, and IL‐2‐dependent in vitro proliferation of naive CD8+ T cells was also reduced. In conclusion, these results suggest that antigen‐specific CD4+ T cell responses to LM are affected little by chronic ethanol consumption; however, antigen‐specific CD8+ T cell responses are reduced significantly, as are in vivo and in vitro proliferation. The reduction of antigen‐specific CD8+ T cells may contribute strongly to the immunodeficiency caused by ethanol abuse.

Chronic Ethanol Consumption Decreases Murine Langerhans Cell Numbers and Delays Migration of Langerhans Cells as Well as Dermal Dendritic Cells

BACKGROUND Chronic alcoholics experience increased incidence and severity of infections, the mechanism of which is incompletely understood. Dendritic cells (DC) migrate from peripheral locations to lymph nodes (LN) to initiate adaptive immunity against infection. Little is known about how chronic alcohol exposure affects skin DC numbers or migration. METHODS Mice received 20% EtOH in the drinking water for up to 35 weeks. Baseline Langerhans cell (LC) and dermal DC (dDC) numbers were enumerated by immunofluorescence (IF). LC repopulation after inflammation was determined following congenic bone marrow (BM) transplant and ultraviolet (UV) irradiation. Net LC loss from epidermis was determined by IF following TNF-alpha or CpG stimulation. LC and dDC migration into LN was assessed by flow cytometry following epicutaneous FITC administration. RESULTS Chronic EtOH consumption caused a baseline reduction in LC but not dDC numbers. The deficit was not corrected following transplantation with non-EtOH-exposed BM and UV irradiation, supporting the hypothesis that the defect is intrinsic to the skin environment rather than LC precursors. Net loss of LC from epidermis following inflammation was greatly reduced in EtOH-fed mice versus controls. Ethanol consumption for at least 4 weeks led to delayed LC migration into LN, and consumption for at least 8 weeks led to delayed dDC migration into LN following epicutaneous FITC application. CONCLUSIONS Chronic EtOH consumption causes decreased density of epidermal LC, which likely results in decreased epidermal immunosurveillance. It also results in altered migratory responsiveness and delayed LC and dDC migration into LN, which likely delays activation of adaptive immunity. Decreased LC density at baseline appears to be the result of an alteration in the skin environment rather than an intrinsic LC defect. These findings provide novel mechanisms to at least partially explain why chronic alcoholics are more susceptible to infections, especially those following skin penetration.

Fractionation and preliminary characterization of a low molecular weight bovine hepatic inhibitor of DNA synthesis in regenerating rat liver

Abstract A heterogenous low molecular weight inhibitor of DNA synthesis in regenerating rat liver has been further fractionated by silica gel chromatography. The material is shown to contain a subfraction which stimulates isotope uptake in regenerating rat liver in vivo, as well as a strongly inhibitory subfraction which inhibits DNA synthesis up to 95%. The inhibitor fraction is effective both in vivo and in isolated rat liver nuclei from regenerating rat liver, whereas the stimulatory fraction is effective only in vivo. Other measurements of interest indicate that the unfractionated inhibitor preparation is not hepatotoxic, nor does it depress isotope uptake by the liver. These attributes are consistent with, but do not prove, the presence of a hepatic chalone.