1 Divison of Health Prevention Science, College of Public Health and
2 Cardiovascular and Thoracic Surgery and The Sarver Heart Center, School of Medicine, University of Arizona, Tucson, AZ 85724, USA
Received 8 August 2002; in revised form 29 October 2002; accepted 11 November 2002
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ABSTRACT |
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INTRODUCTION |
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Because cytokines involved in neutrophil activation are mainly secreted by immune cells, such as T and B cells, and macrophages, the degree of cell destruction by retroviral infection in the different stages of murine AIDS may influence cytokine levels. Eventually, it will reflect on neutrophil activities, such as neutrophil CD11b adhesion molecule expression and ROS production. Therefore, levels of neutrophil CD11b expression and ROS production may become useful prognostic markers to monitor the progressive stages of AIDS.
MacGregors studies indicated that ethanol intoxication has profound effects on neutrophil kinetics (Gluckman and MacGregor, 1978; MacGregor et al., 1978
, 1988
). Of most significance is the inhibition of neutrophil mobilization to sites of inflammation. Most of these effects are likely to be mediated by the mechanism of inhibited neutrophil adhesion molecular expression through cytokine dysregulation. Indeed, Arbabi et al.(1999)
found that acute ethanol intoxication inhibits the production of IL-8 and TNF-
. Ethanol consumption could directly increase production of ROS in neutrophils by ethanol dehydrogense, microsomal oxidation systems and catalase. However, few investigators used both neutrophil CD11b expression and ROS production for neutrophil kinetic and/or functional studies after chronic ethanol consumption. Chronic ethanol consumption in AIDS patients is common. Thus, 14% of HIV-infected patients misuse alcohol (Welch, 2000
). Retrovirus and ethanol may interact in a complex manner on neutrophils. Therefore, we investigated both factors on neutrophil CD11b expression and ROS production.
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MATERIALS AND METHODS |
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Animals
Female C57BL/6N mice (National Cancer Institute) at 812 weeks of age and weighing about 2022.5 g were randomly assigned to four different groups: control, murine AIDS, ethanol and murine AIDS plus ethanol. Mice were housed in transparent plastic cages with a stainless wire lid in a room at 2022°C with constant humidity and a 12 h:12 h light:dark cycle (lights on at 07.00). Murine AIDS was induced by LP-BM5 murine leukaemia retrovirus infection, as done previously in our laboratory (Wang et al., 1995; Liang et al., 1997
). Infection leads to the rapid induction of clinical symptomatology that is similar to human AIDS. In the first week, 10% (v/v) ethanol in autoclaved tap water was made available to the chronic ethanol-fed mice in a 300 ml plastic bottle with a stopper. The ethanol concentration was increased in increments of 10% at 1-week intervals, from an initial 10% to a final concentration of 20% (v/v) and kept at 20% (v/v) for the rest of the treatment period. Mice were active at night; therefore, we took blood samples at night (after lights were turned off for 3.5 h) to estimate blood-ethanol concentration (Sigma Diagnostics Alcohol Kit). The average blood-ethanol concentration was 66.9 mg/dl (0.0669%). The non-ethanol fed mice were given the same diet, except that the water bottles contained only water. No weight loss was found at the end of the experimental period between the non-ethanol and ethanol-fed mice.
Neutrophil CD11b expression and ROS production
Flow cytometry was used to identify neutrophils and their activity in whole blood. Freshly drawn, citrated whole blood was mixed with the vital nucleic acid stain, LDS-751 (1 µg/ml). Labelling leucocytes with LDS-751 allowed for the investigation of leucocyte characteristics in whole blood and thus avoided leucocyte isolation procedures, which are known to cause artificial leucocyte activation (Macey et al., 1992). This mixture of whole blood and LDS-751 was used for all subsequent leucocyte experiments. Neutrophil CD11b and ROS measurements were performed by incubating saturating concentrations of FITC-labelled mouse CD11b antibody, and 80 mM DCFH-DA with the whole blood/LDS-751 mixture. Samples were incubated in a 37°C water bath for 15 min, and then diluted with 0.5 ml of cold PBS. During FACS (fluorescence-activated cell sorter) scanning (Becton Dickinson, FACScan Clinical Flow Cytometer), a 488 nm argon laser light was used for excitation, and fluorescence emission was detected as forward scatter (FSC), which is a measure of cell size, and side scatter (SSC), which is a measure of cell granularity. In addition, a threshold fluorescence was set on the LDS-751 signal that allowed listmode data collection on leucocytes in whole blood without interference from erythrocytes. Thus, neutrophil subpopulations can be separated on the basis of their dot plots pattern on FSC, SSC and the fluorochrome intensity of LDS-751 (red) in the FL3 channel (Fig. 1
) (detector FL3 is for red fluorochrome). The green fluorescence intensity due to bound FITC-labelled CD11b antibody was monitored in the FL1 channel (detector FL1 is for green fluorochrome). For measurement of ROS, this method (Himmelfarb et al., 1992
) used the properties of DCFH-DA, which rapidly diffused across the cell membrane and was then trapped within the cell by a deacetylation reaction. In the presence of hydrogen peroxide, this compound was oxidized to DCF, which is highly fluorescent in the FL1 channel. To investigate the capacity of neutrophils to up-regulate the CD11b adhesion molecule expression and ROS generation in response to bacterial infection, we used fMLP (10-6 M final concentration), a bacterial peptide, to stimulate neutrophils for 15 min ex vivo before analysis by flow cytometry. The data from the FACS processing was further analysed using WinMDI 2.8. Data were expressed as total fluorescence intensity (TFI = mean channel of fluorescence x % of positive events).
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Statistical analysis
All statistics were calculated using Prism Statistical software (version 3.0). Comparisons between groups were made using ANOVA with NewmanKeuls post hoc testing. Data are presented as means ± SEM.
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RESULTS |
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In the murine AIDS with chronic ethanol consumption group, no significant difference in neutrophil CD11b expression (Fig. 2) was observed in the first or second month. However, a significant increase in neutrophil CD11b expression occurred (P < 0.01) during the 3-month observation period. The neutrophil ROS production (Fig. 3
) had a complex pattern. It increased in the first month (P < 0.001), then tended to decrease in the second month and then increased (P < 0.001) again after 3 months. In the first month, neutrophils induced a higher level of ROS without CD11b up-regulation. In the second month, no significant difference of neutrophil CD11b expression and ROS production was observed. Thereafter, an increased neutrophil CD11b expression was parallel to ROS production in the third month of retroviral infection plus ethanol consumption.
fMLP stimulated neutrophil CD11b expression and ROS production
To determine the capability of the neutrophil response to bacterial infection, we used fMLP, a bacterial peptide, to stimulate neutrophils for 15 min ex vivo. For neutrophil CD11b expression (Fig. 4AC), fMLP stimulation caused a significant increase (P < 0.05) that was exhibited in the control group only. No significant differences were found in all other groups in the presence or absence of fMLP. For neutrophil ROS production (Fig. 5AC
), similar results were found for neutrophil CD11b expression. There was a significant increase in neutrophil ROS production (P < 0.05) after being stimulated by fMLP in the control group and the early first month of ethanol consumption. However, no significant difference in neutrophil ROS production was found after stimulation by fMLP in the different stages of murine AIDS, murine AIDS plus ethanol consumption, and chronic ethanol consumption alone.
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DISCUSSION |
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Ethanol intoxication has been observed to decrease the adherence of neutrophils to endothelial cells (Gluckman and MacGregor, 1978; MacGregor et al., 1978
). Our results further support this observation because neutrophil CD11b adhesion molecules decrease after moderate ethanol consumption. The impairment of neutrophil CD11b expression may be related to decreased proinflammatory cytokine production, such as TNF-
and IL-8 (Arbabi et al., 1999
), which are potent neutrophil stimulators. It is well known that ethanol induces oxidative stress in many cell types (Brown et al., 2001
; Sun and Sun, 2001
). Neutrophil ROS may be induced directly by the microsomal ethanol-oxidizing systems or indirectly by inflammatory cytokines. In our study, an initial peak of neutrophil ROS production may be dominated by ethanol-oxidizing systems. After 2 months of ethanol consumption, a reduced level of neutrophil ROS with down-regulated neutrophil CD11b suggests that decreased proinflammatory cytokines override the effect of ethanol-oxidizing systems. As mice continually drink, an increase of neutrophil ROS production with normal neutrophil CD11b expression suggests that neutrophils are tolerant to the response of proinflammatory cytokines; however, ethanol-oxidizing systems in neutrophils may be not affected. Although ROS is a necessary agent for bacterial killing, at this point ROS may trigger neutrophil apoptosis before neutrophils reach the inflammatory site. This phenomenon suggests that chronic ethanol consumption increases susceptibility to infection (Jareo et al., 1995
).
Cytokines released from infected cells are a hallmark of LP-BM5 infection. Proinflammatory cytokines, such as TNF-, IL-1, IL-6, and PAF, increase in AIDS (Gelbard et al., 1994
; Akarid et al., 1995
; Liang et al., 1997
). They are all potent neutrophil activators. In our study, we found that increased neutrophil ROS production was accompanied by neutrophil CD11b up-regulation in the first month of LP-BM5 infection, even though the neutrophil response to fMLP was diminished. At this time, up-regulation of neutrophil CD11b and ROS may be due to cytokine stimulation. After 2 months of LP-BM5 infection, both neutrophil CD11b expression and ROS production returned to the baseline level. This decrease may be due to: (1) a decrease in cytokine production; and/or (2) neutrophil receptor desensitization. A decreased cytokine level may be related to a massive destruction of cytokine-producing cells due to rapid viral replication. Neutrophil desensitization could be also related to cytokine dysregulation (Clark-Lewis et al., 1991
). Thus, normal CD11b expression and ROS production do not suggest the improvement of disease in that stage. In the later stages of LP-BM5 infection, neutrophil CD11b and ROS were further up-regulated. The extremely high ROS in circulating neutrophils may induce apoptosis before neutrophils reach the target sites. The pattern of neutrophil CD11b expression and ROS production may help to predict the different stages of murine AIDS.
Ethanol may further compromise neutrophil function in AIDS (Prakash et al., 1998). We do find that circulating neutrophils are highly activated, especially neutrophil ROS production, which is extremely elevated during the 3-month observation period. These results suggest that neutrophils may be more susceptible to apoptosis in murine AIDS mice with chronic exposure to ethanol. This may contribute to exacerbating the progression of murine AIDS.
In summary, neutrophil CD11b expression and ROS production are compromised by LP-BM5 retrovirus infection and/or chronic ethanol consumption. Neutrophil response to bacterial infection is impaired. Neutrophil CD11b expression and ROS production may become promising prognostic markers to predict the progression of murine AIDS. Ethanol may further compromise neutrophil function in AIDS.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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REFERENCES |
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Arbabi, S., Garcia, I., Bauer, G. J. and Maier, R. V. (1999) Alcohol (ethanol) inhibits IL-8 and TNF: role of the p38 pathway. Journal of Immunology 162, 74417445.
Brown, L. A., Harris, F. L. and Guidot, D. M. (2001) Chronic ethanol ingestion potentiates TNF-alpha-mediated oxidative stress and apoptosis in rat type II cells. American Journal of Physiology Lung Cellular and Molecular Physiology 281, L377L386.
Cavanagh, S. P., Gough, M. J. and Homer-Vanniasinkam, S. (1998) The role of the neutrophil in ischemia-reperfusion injury: potential therapeutic interventions. Cardiovascular Surgery 6, 112118.[CrossRef][ISI][Medline]
Clark-Lewis, I., Schumacher, C., Baggiolini, M. and Moser, B. (1991) Structureactivity relationships of interleukin-8 determined using chemically synthesized analogs. Critical role of NH2-terminal residues and evidence for uncoupling of PMN chemotaxis, exocytosis, and receptor binding activities. Journal of Biological Chemistry 266, 2312823134.
Ellis, M., Gupta, S., Galant, S., Hakim, S., VandeVen, C., Toy, C. and Cairo, M. S. (1988) Impaired neutrophil function in patients with AIDS or AIDS-related complex: a comprehensive evaluation. Journal of Infectious Diseases 158, 12681276.[ISI][Medline]
Gelbard, H. A., Nottet, H. S., Swindells, S., Jett, M., Dzenko, K. A., Genis, P., White, R., Wang, L., Choi, Y. B. and Zhang, D. (1994) Platelet-activating factor: a candidate human immunodeficiency virus type 1-induced neurotoxin. Journal of Virology 68, 46284635.[Abstract]
Gluckman, S. J. and MacGregor, R. R. (1978) Effect of acute alcohol intoxication on granulocyte mobilization and kinetics. Blood 52, 551559.[Abstract]
Himmelfarb, J., Hakim, R. M., Holbrook, D. G., Leeber, D. A. and Ault, K. A. (1992) Detection of granulocyte reactive oxygen species formation in whole blood using flow cytometry. Cytometry 13, 8389.[ISI][Medline]
Jareo, P. W., Preheim, L. C. Lister, P. D. and Gentry, M. J. (1995) The effect of ingestion on killing of Streptococcus pneumoniae, Staphylococcus aureus and Staphylococcus epidermidis by rat neutrophils. Alcohol and Alcoholism 30, 311318.[Abstract]
Liang, B., Zhang, Z., Araghiniknam, M., Eskelson, C. and Watson, R. R. (1997) Prevention of retrovirus-induced aberrant cytokine secretion, excessive lipid peroxidation, and tissue vitamin E deficiency by T cell receptor peptide treatments in C57BL/6 mice. Experimental Biology and Medicine 214, 8794.[Abstract]
Macey, M. G., Jiang, X. P., Veys, P., McCarthy, D. and Newland, A. C. (1992) Expression of functional antigens on neutrophils. Effects of preparation. Journal of Immunological Methods 149, 3742.[CrossRef][ISI][Medline]
MacGregor, R. R., Gluckman, S. J. and Senior, J. R. (1978) Granulocyte function and levels of immunoglobulins and complement in patients admitted for withdrawal from alcohol. Journal of Infectious Diseases 138, 747753.[ISI][Medline]
MacGregor, R. R., Safford, M. and Shalit, M. (1988) Effect of ethanol on functions required for the delivery of neutrophils to sites of inflammation. Journal of Infectious Diseases 157, 682689.[ISI][Medline]
Morse, H. C., 3rd, Chattopadhyay, S. K., Makino, M., Fredrickson, T. N., Hugin, A. W. and Hartley, J. W. (1992) Retrovirus-induced immunodeficiency in the mouse: MAIDS as a model for AIDS. AIDS 6, 607621.[ISI][Medline]
Murphy, P. M., Lane, H. C., Fauci, A. S. and Gallin, J. I. (1988) Impairment of neutrophil bactericidal capacity in patients with AIDS. Journal of Infectious Diseases 158, 627630.[ISI][Medline]
Prakash, O., Zhang, P., Xie, M., Ali, M., Zhou, P., Coleman, R., Stoltz, D. A., Bagby, G. J., Shellito, J. E. and Nelson, S. (1998) The human immunodeficiency virus type I Tat protein potentiates ethanol-induced PMN functional impairment in transgenic mice. Alcoholism: Clinical and Experimental Research 22, 20432049.[ISI][Medline]
Serradji, N., Bensaid, O., Martin, M., Kan, E., Dereuddre-Bosquet, N., Redeuilh, C., Huet, J., Heymans, F., Lamouri, A., Clayette, P., Dong, C. Z., Dormont, D. and Godfroid, J. J. (2000) Structureactivity relationships in platelet-activating factor (PAF). From PAF antagonism to inhibition of HIV-1 replication. Journal of Medicinal Chemistry 43, 21492154.[CrossRef][ISI][Medline]
Sun, A. Y. and Sun, G. Y. (2001) Ethanol and oxidative mechanisms in the brain. Journal of Biomedical Science 8, 3743.[CrossRef][ISI][Medline]
Vecchiarelli, A., Monari, C., Baldelli, F., Pietrella, D., Retini, C., Tascini, C., Francisci, D. and Bistoni, F. (1995) Beneficial effect of recombinant human granulocyte colony-stimulating factor on fungicidal activity of polymorphonuclear leukocytes from patients with AIDS. Journal of Infectious Diseases 171, 14481454.[ISI][Medline]
Wang, Y., Huang, D. S., Wood, S. and Watson, R. R. (1995) Modulation of immune function and cytokine production by various levels of vitamin E supplementation during murine AIDS. Immunopharmacology 29, 225233.[CrossRef][ISI][Medline]
Welch, K. J. (2000) Correlates of alcohol and/or drug use among HIV-infected individuals. AIDS Patient Care and STDs 14, 317323.[CrossRef][ISI][Medline]
Westmoreland, S. V., Kolson, D. and Gonzalez-Scarano, F. (1996) Toxicity of TNF alpha and platelet activating factor for human NT2N neurons: a tissue culture model for human immunodeficiency virus dementia. Journal of Neurovirology 2, 118126.[ISI][Medline]