Alcohol consumption is associated with alterations in macrophage responses to interferon-gamma and infection by Salmonella typhimurium.

Abuse of ethanol (EtOH) by human beings and administration of EtOH to experimental animals has been shown to be associated with a suppression of the immune system. Consumption of EtOH has also been associated with an increased incidence and severity of infections of human beings and experimental animals, which has been attributed to the immunosuppression associated with EtOH consumption. It has been shown that EtOH also affects the function of macrophages (MØ), which are important effector cells in the innate and adaptive immune responses to infectious agents. The present studies were designed to investigate the effects of EtOH on MØ function with an animal model of EtOH consumption. The experiments reported in this paper were done with inflammatory MØ and were designed to determine the effects of EtOH on the ability of inflammatory MØ to respond to interferon-gamma (IFN-gamma) to control the intracellular growth of Salmonella typhimurium, as well as the production of proinflammatory cytokines and nitric oxide. The ability of MØ from EtOH-fed mice to respond to bacterial endotoxin (lipopolysaccharide (LPS)) and IFN-gamma was also evaluated. MØ isolated from EtOH-fed mice did not respond as well to IFN-gamma as MØ isolated from control mice as measured by control of S. typhimurium, as well as tumor necrosis factor (TNF) and nitric oxide production. Interleukin (IL)-6 production was not affected. Activation of MØ from EtOH-fed mice with LPS and IFN-gamma produced levels of nitric oxide and TNF only slightly less than the levels seen in MØ from control mice, but a significant decrease in IL-6 was seen when MØ from EtOH-fed mice were stimulated with this combination. Flow cytometric analyses showed that IFN-gamma receptor expression was not affected by EtOH. Together the data presented in this paper show that consumption of EtOH is associated with changes in inflammatory MØ responses to IFN-gamma.

Initiation of alcoholic fatty liver and hepatic inflammation with a specific recall immune response in alcohol-consuming C57Bl/6 mice.

Whether immunological responses are involved in initiation and progression of alcoholic liver disease is unclear. We describe a mouse model of alcoholic liver injury characterized by steatosis and hepatic inflammation initiated by a recall immune response. Mice immune to Listeria monocytogenes fed a liquid diet containing ethanol and challenged with viable bacteria developed steatosis within 24 h and, at a later time, elevated serum alanine aminotransferase levels, indicating more liver damage in this group. Listeria antigen also induced steatosis and increased serum alanine aminotransferase levels in immune ethanol-consuming mice. The production of tumour necrosis factor by a recall immune response in this model is a major, but not the only, component in initiation of alcoholic liver disease.

Alcohol consumption by C57BL/6 mice is associated with depletion of lymphoid cells from the gut-associated lymphoid tissues and altered resistance to oral infections with Salmonella typhimurium.

Studies were done to test whether ethanol (ETOH) consumption alters resistance to mucosal and systemic infections by Salmonella typhimurium. S. typhimurium-immune and -nonimmune mice were fed 1 of 3 diets (an ETOH-containing liquid diet, an isocaloric liquid diet equal in volume to that of the ETOH-treated group, or laboratory chow) in a pair-feeding design and were infected orally or intravenously with S. typhimurium. The number of bacteria in spleen and liver and the effect of ETOH feeding and infection on the number of lymphoid cells in the gut-associated lymphoid tissues (GALT) were determined. ETOH feeding resulted in profound loss of GALT lymphoid cells and an increased number of Salmonella organisms in the intestines, liver, and spleen of infected nonimmune, but not of immune, mice. These data show that ETOH consumption in this model impairs host defense mechanisms that control mucosal infections and inhibits the mechanisms that control levels of bacteria in the central organs.

Effect of adrenalectomy on ethanol-associated changes in lymphocyte cell numbers and subpopulations in thymus, spleen, and gut-associated lymphoid tissues.

Consumption of ethanol (ETOH) by experimental animals and human beings is associated with elevated serum levels of corticosteroids. One of the most robust findings associated with ETOH consumption is a loss of lymphocytes from thymus and spleen, as well as from peripheral lymphoid organs to include mesenteric lymph nodes and Peyer's patches, which are lymphoid organs associated with the gastrointestinal tract. To study the role of corticosteroids in loss of cells from thymus, spleen, and gut-associated lymphoid organs, adrenalectomized (ADX) or intact C57Bl/6 mice were fed a liquid diet containing ETOH (to supply 36% of calories as ETOH) or an isocaloric control diet with a pair-feeding protocol. Loss of lymphocytes from all lymphoid organs was associated closely with serum corticosterone levels in both ETOH-fed and pair-fed groups. ETOH-fed ADX animals showed much less cell loss than did ETOH-fed intact animals. However, there was still an association between ETOH consumption and cell loss when cell loss in ETOH-fed ADX animals was compared with that in ADX pair-fed and ADX chow-fed groups. In both intact and ADX animals ETOH consumption was associated with a loss of immature (CD4(+) and CD8(+)) cells from the thymus. These data lead to the suggestion that corticosteroids are responsible for most of the cell loss from thymus, spleen, mesenteric lymph nodes, and Peyer's patches in association with ETOH consumption. Some cell loss, however, is independent of corticosteroids. The data presented here also support the suggestion that cell loss from lymphoid organs could be the result of nutritional factors.

Influence of ethanol consumption on immune competence of adult animals exposed to ethanol in utero.

Ethanol consumption results in significant changes in the immune system of experimental animals and humans. Previous work by ourselves and others has established that in utero exposure to ethanol results in alterations in the immune system of the offspring that persist into adult life. The present study was designed to determine if prenatal exposure to ethanol results in increased vulnerability to the immunosuppressive effects of ethanol consumption in adulthood. Male and female Sprague-Dawley offspring were selected in adulthood from prenatal ethanol (E), pair-fed (PF), and ad libitum-fed control (C) groups, and given either an ethanol-containing liquid diet or were pair-fed an isocaloric liquid diet without ethanol for 30 days. At the end of the 30-day feeding period, lymphocyte responses to the mitogens concanavalin A (Con A) and lipopolysaccharide, and to interleukin-2 (IL-2) were tested using in vitro assays. The results of this study support and extend previous data demonstrating long-term adverse effects of prenatal ethanol exposure on T-cell responses to mitogens, and provide further evidence that deficits seem to be more robust in male than in female offspring. Prenatal E males showed reduced T-lymphocyte proliferation to Con A and T-lymphoblast proliferation to IL-2, compared with their prenatal PF and C counterparts, regardless of whether they were exposed to the ethanol or the control diet in adulthood. In addition, T-lymphoblast proliferation to IL-2 was suppressed in prenatal E, compared with prenatal C, females exposed to control diet in adulthood. This is the first report of a deficit in T-cell aspects of immunity in E females, although it appears that this deficit may have been partially mediated by nutritional effects. A second major finding in this study is that consumption of ethanol diet in adulthood in itself had significant immunosuppressive effects on T-cell responses in both males and females. However, contrary to our expectation, previous exposure to ethanol in utero did not exacerbate the changes in immune responsiveness that were observed after adult ethanol consumption.

Decreased production of reactive oxygen intermediates is an early event during in vitro apoptosis of rat thymocytes.

Thymocyte apoptosis is one of the best characterized experimental models of apoptosis that can be induced by a variety of stimuli such as glucocorticoids, ionizing radiation, antibodies, and toxins. Recently, it has been suggested that oxidative stress is a common mediator of apoptosis. However, little is known about the production and possible function of reactive oxygen intermediates (ROI) in thymocytes. We used a highly sensitive flow cytometric assay with the hydrogen peroxide-sensitive dye, 2',7'-dichlorofluorescin diacetate (DCFH-DA), to measure intracellular ROI production in rat thymocytes, to study its primary sources, and to compare ROI levels in normal and apoptotic thymocytes. Apoptosis was induced by incubating the cells in the presence or absence of dexamethasone (Dex) at 37 degrees C in vitro. Normal thymocytes spontaneously produced significant amounts of ROI. Catalase or superoxide dismutase did not affect this intracellular fluorescence, presumably due to their failure to penetrate into the cells. However, N-acetyl-L-cysteine significantly attenuated the fluorescence in a dose-dependent manner. Significant inhibition of the intracellular fluorescence was also observed by addition of N-nitro-L-arginine methyl ester (L-NAME), that could not be reversed by L-arginine. The addition of N-nitro-D-arginine methyl ester (D-NAME) also caused considerable inhibition. This indicates that the inhibition by L-NAME or D-NAME is due to a direct scavenging effect, and nitric oxide production is not likely to be involved. In contrast to neutrophils and macrophages whose superoxide anions are released from membrane-bound NADPH oxidase, the production of ROI in thymocytes is likely to originate mainly from mitochondria, as indicated by the inhibitory effect of the addition of rotenone or antimycin A. The addition of lymphocyte simulators phytohemagglutinin (PHA), concanavalin A (Con A), or phorbol 12-myristate 13-acetate (PMA) enhanced intracellular fluorescence of thymocytes. This increase was abrogated by addition of rotenone or antimycin A. The ROI production was decreased with time after incubation of the thymocytes for 1, 3, and 6 h in vitro. The appearance of apoptosis of thymocytes in vitro, as indicated by DNA content of cells by flow cytometry and DNA ladder formation in agarose gel electrophoresis, was delayed, as compared to the time course of the decreased ROI production. The addition of Dex to the culture medium accelerated both of these processes. The results suggest that a decreased spontaneous production of ROI in thymocytes precedes the spontaneous in vitro apoptosis and Dex exaggerates these changes.

Different pathways of in vitro ethanol-induced apoptosis in thymocytes and splenic T and B lymphocytes.

To investigate the intracellular pathways leading to ETOH-induced apoptosis, thymocytes and splenic T and B cells were cultured 16 h with or without ETOH and different stimuli, and apoptotic cell death was determined. At concentrations of 0.4%-2% in culture, ETOH induced apoptosis in all three types of cells, but it had a more profound effect on thymocytes and B cells as compared with its effect on T cells. In thymocytes, ETOH-induced apoptosis was abrogated by chelation of extracellular calcium with EGTA, and inhibition of protein synthesis with CHX, or of PKC with H7 but not of PKA with HA 1004. ETOH potentiated the apoptosis of thymocytes induced with the calcium ionophore A23187 and suboptimal doses of PMA, but it had negligible effect on dAMP- and PGE2-induced apoptosis of thymocytes. In contrast to findings in thymocytes, the ETOH-induced apoptosis of T and B cells was almost completely abrogated by PMA, but not by H7 or CHX. In spleen cells, calcium chelation with EGTA triggered apoptosis. ETOH significantly inhibited EGTA-induced apoptosis of B cells but had little effect on EGTA-induced apoptosis of T cells. IL-4 reduced the ETOH-induced apoptosis of B and T cells, but it was not effective in the prevention of apoptosis of thymocytes. Inhibition of the calcium-dependent neutral protease calpain I did not rescue cells from apoptosis. Moreover, treatment with CI-I potentiated ETOH-induced apoptosis in T cells. These results suggest that both thymocytes and splenic T and B cells have relevant apoptotic pathways that can be induced by ETOH, but the mechanisms of ETOH-induced apoptosis differ in these cells.

Ethanol-induced depletion of lymphocytes from the mesenteric lymph nodes of C57B1/6 mice is associated with RNA but not DNA degradation.

The effects of ethanol (EtOH) consumption by adult female C57B1/6 mice on lymphocyte populations of the mesenteric lymph nodes (MLNs) were determined by feeding mice with the Lieber-DeCarli liquid diet by a pair-feeding paradigm. Histological analysis of the MLNs of EtOH-fed mice showed a progressive loss of lymphocytes from the medullary regions at 3, 5, and 7 days after initiation of the EtOH diet. The stromal cells in the medullary region also demonstrated a progressive alteration in stellate morphological features at times corresponding to those of loss of lymphocytes from this region. Microscopic evaluation of the follicle regions of MLNs obtained from mice fed an EtOH-containing diet showed no appreciable alterations in morphological characteristics. The number of tingible body macrophages in the germinal centers of the follicles, however, was increased after 3 days of EtOH diet feeding and declined progressively after this time. Flow cytometric analysis of isolated lymphocytes showed a depletion of both T and B cell populations from the MLNs. In contrast to B cells, however, T cells were depleted through 7 days of EtOH diet feeding. Total RNA isolated from the MLNs of mice consuming the EtOH-containing diet was progressively degraded. No degradation of DNA was observed. These study results establish that continuous consumption of dietary EtOH adversely affects the cellularity of MLN, resulting in a progressive loss of lymphocytes that is associated with degradation of total RNA.

Tumor necrosis factor and the pathogenesis of Pichinde virus infection in guinea pigs.

Pichinde virus (PIC) is a reticuloendothelial arenavirus of the New World tropics. A guinea pig passage-adapted strain of this virus (adPIC) is uniformly lethal for inbred guinea pigs, while the related, prototype strain (PIC3739) has attenuated virulence. The abilities of adPIC and PIC3739 to induce tumor necrosis factor (TNF) in vivo and in cultured macrophages were compared. Infection with adPIC, but not PIC3739, was associated with detectable serum TNF that peaked in week 2 of infection. Tumor necrosis factor was found in the spleens of adPIC- and PIC3739-infected animals in week 1 of infection; TNF alpha mRNA levels in spleens and livers of adPIC infected animals increased and remained high throughout infection, whereas PIC3739-infected organs showed down regulation of TNF alpha mRNA late in infection. Peritoneal macrophages explanted from adPIC-infected animals showed enhanced lipopolysaccharide-inducible TNF production. Altered regulation of TNF production may play a role in the pathogenesis of guinea pig arenavirus disease.

Effects of ethanol on cellular immunity to facultative intracellular bacteria.

Alcohol abuse has been associated with an increase in infectious diseases caused by pathogenic and opportunistic microorganisms. Study results obtained from this laboratory and other laboratories have shown that consumption of large amounts of ethanol is associated with numerous changes in the immune system. The purpose of this article is to report findings obtained from this laboratory, as well as review those obtained from other laboratories, from experiments designed to evaluate the effects of ethanol on various components of antimicrobial host-defense mechanisms. The effects of ethanol on various aspects of immunity obtained with the use of in vivo and in vitro model systems are reviewed as they pertain to antimicrobial defenses. All current data would support the suggestion that ethanol affects both the development of an antigen-specific immune response and the effector mechanisms of the cellular immune response. Findings obtained from animal models show that ethanol prevents the formation of granulomas in infected tissues, perhaps by inhibiting the response of macrophages to T-cell cytokines. Data obtained from this laboratory also support the suggestion that the inability of the immune system to control the intracellular growth of microorganisms results in an exaggerated inflammatory response that is responsible for at least a part of the tissue damage.

Pathological and virological features of arenavirus disease in guinea pigs. Comparison of two Pichinde virus strains.

A guinea pig passage-adapted strain of the arena-virus Pichinde (adPIC) is highly virulent in inbred guinea pigs, whereas the related strain PIC3739 is attenuated. Both viruses were macrophage tropic and infected peritoneal, splenic, liver, and alveolar macrophages during experimental Pichinde virus infection. Infection with the virulent strain was associated with unlimited viral replication in the face of exaggerated delayed-type hypersensitivity response, manifested by the macrophage disappearance reaction. Histopathological lesions unique to adPIC-infected guinea pigs included intestinal villus blunting with mucosal infiltration by pyknotic debris-laden macrophages and apoptosis of crypt epithelial cells. Splenic red pulp necrosis was also significantly associated with adPIC infection but not PIC3739 infection. These findings may provide clues to the pathogenesis of a group of poorly understood human viral hemorrhagic fevers.

Effects of ethanol consumption and withdrawal on B cell subpopulations in murine bone marrow.

We designed studies to examine the effects of ethanol consumption and withdrawal on the numbers of pre-B and B cells in murine bone marrow. Flow cytometric analysis of B220 and surface IgM expression on bone marrow cells revealed that consumption of ethanol by mice for 7 days led to a significant reduction in pre-B cells. The number of mature B cells in the bone marrow of these animals, however, did not differ from that of control mice. In contrast, examination of bone marrow obtained from mice at various times after withdrawal from ethanol showed significantly fewer numbers of mature B cells and an even greater loss of pre-B cells. This effect was seen for relatively long periods after withdrawal. These study findings are interpreted to suggest that ethanol consumption results in changes in the pre-B cell population in murine bone marrow. It also appears that withdrawal from ethanol results in more profound changes in the mature B cell population of the bone marrow than those that occur during ethanol consumption.

Increased susceptibility of experimental animals to infectious organisms as a consequence of ethanol consumption.

It has been shown by a number of studies that chronic alcohol abuse is associated with an increased incidence of infections. The mechanism of this increased susceptibility to infectious agents is multifactorial and certainly includes abuse of other drugs, smoking, malnutrition and other factors. Recently, it has become apparent that consumption of alcohol by human beings and experimental animals is associated with changes in the immune system. The increased incidence of tuberculosis and opportunistic bacterial and fungal infections described in alcoholics has led to the suggestion that alcohol affects the cell-mediated arm of the immune response. The present report will review the data that shows the effects of alcohol on the immune system. We will also review the data from our laboratory and other laboratories that show the effects of alcohol on host defense mechanisms to infectious agents. Where possible, mechanisms of increased susceptibility will be discussed or postulated.

The role of tumor necrosis factor in host defense against scrub typhus rickettsiae. I. Inhibition of growth of Rickettsia tsutsugamushi, Karp strain, in cultured murine embryonic cells and macrophages by recombinant tumor necrosis factor-alpha.

Recombinant murine tumor necrosis factor-alpha (TNF-alpha) inhibited intracellular growth of Rickettsia tsutsugamushi, Karp strain, in the mouse embryo cell line C3H/10T1/2 clone 8 at doses of 100 to 10 U/ml. The growth inhibitory effect of TNF-alpha was also evident when peritoneal exudate macrophages or bone marrow-derived macrophages were used as the host cell for rickettsial growth. Interferon-gamma (IFN-gamma), at doses up to 1,000 U/ml, did not affect the growth of this strain of rickettsiae in the mouse embryo cell line but, as expected, profoundly inhibited rickettsial growth in peritoneal exudate macrophages and bone marrow-derived macrophages. The effect of TNF-alpha on rickettsial growth in the mouse embryo cell line was not reproducibly enhanced by IFN-gamma. Treatment of the cell line with TNF-alpha delayed rickettsial cytopathic effects, but the rickettsiae ultimately grew to high numbers in the cells and caused cell death. These findings show that, at least in our system, R. tsutsugamushi is resistant to IFN-gamma-mediated antirickettsial effects in cells other than macrophages. The results of this study support the suggestion that TNF-alpha may inhibit rickettsial growth in cells other than macrophages.

The role of tumor necrosis factor in host defense against scrub typhus rickettsiae. II. Differential induction of tumor necrosis factor-alpha production by Rickettsia tsutsugamushi and Rickettsia conorii.

The present study was undertaken to investigate the ability of members of two different groups of Rickettsia to stimulate macrophages or immune lymphocytes to produce TNF. It was found that R. conorii, a spotted fever group rickettsia, readily induced murine peritoneal macrophages or the macrophage-like cell line P388D1 to produce relatively high levels of TNF. The interaction of macrophages with viable organisms or heat-killed organisms resulted in TNF production. In contrast, viable or killed R. tsutsugamushi did not stimulate the production of detectable TNF even though viable organisms grew to high numbers in both cell types. It was found that the appropriate immune spleen cells stimulated with heat-killed R. tsutsugamushi or R. conorii produced TNF, and TNF activity was found in the sera of immune mice after injection with rickettsial antigen. Infection of naive mice with viable R. tsutsugamushi resulted in high TNF levels in ascites, but TNF was not found in ascites obtained from infected athymic (nu/nu) mice. These data support the suggestion that spotted fever group rickettsiae, such as R. conorii, possess components perhaps on the surface that interact with macrophages to induce TNF production and this component is lacking in R. tsutsugamushi. Antigens of R. tsutsugamushi and R. conorii will stimulate immune cells to produce TNF activity. These data are compatible with the suggestion that the TH-1 subset of T cells is predominant in immunity to R. tsutsugamushi.

The influence of age on blood alcohol levels and ethanol-associated immunosuppression in a murine model of ethanol consumption.

Several study findings indicate that with ethanol ingestion a number of changes occur in the immune system. We studied the effects of ethanol consumption on mice at various ages. We used a murine model in which young (age 6-8 weeks), middle-aged (age 12 months), and old (age 24 months) male C57Bl/6 mice were pair-fed either a Leiber-DeCarli liquid diet containing 7% (v/v) ethanol or an isocaloric control diet. Consumption of ethanol diet for 8 days resulted in high blood alcohol levels in young and old mice; low levels were observed in middle-aged mice. Middle-aged mice consumed more ethanol than did either young or old mice and had the lowest percent body weight loss of all three age groups. Proliferation of spleen lymphocytes to T-cell stimuli (concanavalin A and alloantigens) in both young and old mice fed ethanol was diminished. T-cell function was unchanged in middle-aged mice consuming an ethanol diet when compared with that observed in age-matched mice pair-fed control diet. No effect of ethanol on proliferation to lipopolysaccharide was noted in any group. Proliferative response of T cells to soluble anti-CD3 monoclonal antibody was also decreased in middle-aged and old pair-fed control mice when compared with young control mice. The proliferative response to soluble anti-CD3 in all three age groups of mice fed ethanol, however, was not significantly affected by ethanol consumption.(ABSTRACT TRUNCATED AT 250 WORDS)

Ethanol ingestion increases susceptibility of mice to Listeria monocytogenes.

Excessive consumption of alcohol is associated with an increase in the frequency and severity of infectious diseases. Ethanol adversely affects specific and nonspecific aspects of the immune response. We used a murine model to determine whether ethanol ingestion impairs host mechanisms of resistance to Listeria monocytogenes. Naive mice and mice immune to L. monocytogenes were pair-fed either a Leiber-DeCarli liquid diet containing 7% (v/v) ethanol or an isocaloric control diet for 7 days. Then, nonimmune mice were given a sublethal dose of L. monocytogenes and studied 2 and 5 days after infection, and immune mice were challenged with a lethal dose of L. monocytogenes and studied 5 days after infection. Multifocal liver abscesses developed in nonimmune ethanol-treated and control mice 2 days after infection. Bacterial colony counts in the spleens were similar between the two groups; however, counts in the livers were slightly higher in ethanol-treated mice as compared with those in control mice. Five days after infection the nonimmune ethanol-treated mice had large necrotizing liver granulomas and organ bacterial colony counts 100 to 1000 times higher than those in control mice. Immune ethanol-treated mice had large areas of liver necrosis and inflammation containing numerous Gram-positive bacilli, whereas immune control mice had small, well-formed granulomas and much less necrosis. Organ bacterial colony counts were about 100 times higher in immune ethanol-treated mice as compared with those in immune control mice. Liver enzyme levels and mortality were significantly higher in ethanol-treated immune and nonimmune mice as compared with those in immune and nonimmune control mice. Data support the suggestion that ethanol consumption impairs the development and expression of T cell-mediated immunity of mice to L. monocytogenes, resulting in increased susceptibility to infection with this organism.