Alcohol & Alcoholism Vol. 38, No. 2, pp.

109–114, 2003
doi:10.1093/alcalc/agg049, available online at www.alcalc.oupjournals.org

NEUTROPHIL ACTIVATION BY MURINE RETROVIRAL INFECTION DURING

CHRONIC ETHANOL CONSUMPTION
YINHONG CHEN1,2, SAM MENDOZA2, GRACE DAVIS-GORMAN2, ZOË COHEN2, RAOUL GONZALES2,
HILLARY TUTTLE2, PAUL F. McDONAGH2 and RONALD R. WATSON1*
1
Divison of Health Prevention Science, College of Public Health and 2Cardiovascular and Thoracic Surgery and The Sarver Heart Center,
School of Medicine, University of Arizona, Tucson, AZ 85724, USA

(Received 8 August 2002; first review notified 11 October 2002; in revised form 29 October 2002; accepted 11 November 2002)

Abstract — Aims: Neutrophil adhesion molecule CD11b and reactive oxygen species (ROS) are neutrophil activation markers for
evaluating the functional activity of neutrophils. The aim of this study was to determine if neutrophils are activated in murine AIDS
and/or chronic ethanol consumption and if neutrophil CD11b expression and ROS production vary when progressive retrovirus infection
occurs. Methods: Four groups were studied: control, murine AIDS, ethanol and ethanol plus murine AIDS. Neutrophil activation was
assessed by CD11b expression and ROS production using flow cytometry. Results: We found that neutrophils lost their responsiveness
to fMLP due to retrovirus or ethanol exposure. In the murine AIDS group, neutrophil CD11b expression was up-regulated along with
a significant increase in ROS after 1 month of retroviral infection. After 2 months, neutrophil CD11b and ROS decreased. However,
neutrophil CD11b expression further increased after 3 months. In the ethanol consumption group, neutrophil CD11b expression was
down-regulated after 2 months, whereas ROS production increased in the first and third months. In the murine AIDS plus ethanol group,
there were significant increases in both ROS and CD11b expression during the 3-month observation period. Conclusions: These findings

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suggest that neutrophil function is impaired by LP-BM5 retrovirus infection and/or chronic ethanol consumption. The pattern of neutro-
phil CD11b expression and ROS production might help to predict the stage of murine AIDS. Ethanol may further compromise neutrophil
function in AIDS.

INTRODUCTION macrophages can further release abnormally large amounts

Neutrophils are the first line of defence against invading
foreign microorganisms. Impairment of neutrophil function
may contribute to the onset of certain life-threatening bacterial
and fungal infections in AIDS individuals (Ellis et al., 1988;
Murphy et al., 1988; Vecchiarelli et al., 1995). Neutrophil
CD11b expression and reactive oxygen species (ROS) prod-
uction are inflammatory responses to mechanical, bacterial
and viral injury. Under physiological conditions, circulating
neutrophils are in the resting state. CD11b, a component in the
CD11b/CD18 adhesion protein complex, is a sign of neutro-
phil activation by up-regulation of its expression. The presence
of this adhesion complex allows neutrophils to migrate from
the blood circulation into target tissue to perform their duty
(Cavanagh et al., 1998). ROS is another neutrophil activation
marker. Neutrophil-derived ROS may cause an intracellular
oxidant stress by retroviral infection. When neutrophils are
stimulated, the membrane-bound NADPH oxidase catalyses
the production of superoxide anions and hydrogen peroxide.
ROS production by neutrophils is also critical for microbial
killing. However, neutrophil activation is tightly regulated by
many factors. The underlying mechanism remains limited. It
may involve cytokine dysregulation, viral products and second-
ary infection in AIDS. Indeed, many investigators (Morse
et al., 1992; Akarid et al., 1995; Liang et al., 1997) described
the severity of cytokine dysregulation in murine AIDS. Increased
cytokine production by Th2 cells and infected macrophages is
a hallmark of murine AIDS. Production of IL-4, IL-6, IL-10
and TNF-α is dramatically elevated (Morse et al., 1992;
Akarid et al., 1995; Liang et al., 1997). Retrovirus-infected
*Author to whom correspondence should be addressed at: Division of Health
Prevention Science, College of Public Health, University of Arizona, P.O. Box
245155, Tucson, AZ 85724, USA.
of IL-1, TNF-α and platelet-activating factor (PAF) (Gelbard
et al., 1994; Westmoreland et al., 1996; Serradji et al., 2000).
It is well known that IL-1β, TNF-α and PAF are potent
neutrophil-activating factors. We therefore hypothesized that
neutrophil CD11b expression and ROS production are
elevated in murine AIDS.
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.
MacGregor’s 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-α. Ethanol consump-
tion 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 consump-
tion. 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.

109

© 2003 Medical Council on Alcohol

110 Y. CHEN et al.

MATERIALS AND METHODS
Reagents
The reagents and sources were as follows: LDS-751 and
2′,7′-dichlorofluorescein diacetate (DCFH-DA) were purchased
from Molecular Probes (Eugene, OR, USA); mouse fluorescein
isothiocyanate (FITC)-conjugated anti-CD11b mAb was
purchased from Pharmingen (San Diego, CA, USA); f-Met-
Leu-Phe (fMLP) was purchased from Sigma Chemical Co.
(St Louis, MO, USA). Phosphate-buffered saline (PBS) was
filtered using a 0.45 µm pore filter prior to all experiments.
Animals
Female C57BL/6N mice (National Cancer Institute) at 8–12
weeks of age and weighing about 20–22.5 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 20–22°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
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 inves-
tigation 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,

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leukaemia retrovirus infection, as done previously in our lab-
oratory (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 auto-
claved 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).
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 list–mode 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

Fig. 1. Representative FACS scans of a neutrophil subpopulation from a control sample.

Dot plot (A) gives representative side scatter (SSC), which is a measure of cell granularity, vs forward scatter (FSC), which is a measure of cell size,
from a blood sample. Dot plot (B) gives representative SSC and fluorescence intensity characteristics of the nucleated cells with positive LDS-751 stain.
The designated ellipse region in SSC vs FSC dot plot (A) is a lymphocyte population because lymphocytes occupy more than 70% of the leucocyte
region, have less granules, and are of a smaller size, compared to neutrophils and monocytes. Neutrophils contain more granules than monocytes
and lymphocytes. Thus, the designated sortrect region R1 in SSC vs LDS dot plot (B) is the neutrophil subpopulation. The designated R2
region contains lymphocyte and monocyte subpopulations.
 ETHANOL, RETROVIRUS AND NEUTROPHIL ACTIVATION 111

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 infec-
tion, 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 pro-
cessing was further analysed using WinMDI 2.8. Data were
expressed as total fluorescence intensity (TFI = mean channel
of fluorescence × % of positive events).
Haematology parameter
Blood cell counts were determined by an automated cell
neutrophil (%) and ANC at 2 months compared to the control
group (P < 0.05). However, after 3 months, neutrophil (%) and
ANC were dramatically increased.
Neutrophil CD11b expression and ROS production
In LP-BM5-induced murine AIDS, circulating neutrophil
CD11b (Fig. 2) increased, even in the first month of infection
(P < 0.05). In the following month of murine AIDS, neutrophil
CD11b expression went back to the control level. Three
months post retrovirus infection, CD11b expression reached a
new high point (P < 0.001). Neutrophil-derived ROS, another
marker for neutrophil activation and killing activity, increased
after 1 month of LP-BM5 infection (P < 0.001) (Fig. 3).
Thereafter, ROS production decreased. Increased neutrophil
ROS production was coupled with CD11b up-regulation in
1-month murine AIDS mice. After 2 months of LP-BM5
infection, neutrophil CD11b expression and ROS production
both fell back to unstimulated control levels. After 3 months
of infection, murine AIDS mice exhibited general malaise. At
this time, neutrophil CD11b expression dramatically increased,

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counter (Serono Diagnostics, Allentown, PA, USA; Model
9018 CP). Leucocyte differential counts were verified manually
on Wright stained blood smears.
Statistical analysis
All statistics were calculated using Prism Statistical soft-
ware (version 3.0). Comparisons between groups were made
using ANOVA with Newman–Keuls post hoc testing. Data are
presented as means ± SEM.
RESULTS
Total white blood cell (WBC) and neutrophil counts
The results of WBC and neutrophil counts are summarized
in Table 1. Although total WBC counts were not significantly
different among the groups, the per cent of neutrophil
[neutrophil (%)] and absolute neutrophil counts (ANC) were
significantly increased after 3 months of murine AIDS and
murine AIDS plus ethanol consumption compared to the
control group (P < 0.05). In the murine AIDS plus ethanol
consumption group, there was a significant decrease of
but ROS production remained consistent.
In the chronic ethanol consumption group, neutrophil CD11b
expression was down-regulated, especially after 2 months
of ethanol consumption (P < 0.05) (Fig. 2). Neutrophil ROS
production significantly increased after 1 month (P < 0.05),
returned to the control level after 2 months, and then increased
again after 3 months of ethanol consumption (P < 0.05) (Fig. 3).
After 1 month of ethanol consumption, an increased neutrophil
ROS production was not accompanied by CD11b up-regulation.
After 2 months, neutrophil CD11b was down-regulated,
but neutrophil ROS production did not change. After 3 months,
neutrophil ROS production was highly induced by chronic
ethanol consumption, but neutrophil CD11b expression was
not affected.
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 neutro-
phil 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

Table 1. Effect of murine AIDS and ethanol on haematology parameters

Total WBC count Neutrophil count

Group Month (103/µl) Neutrophils (%) (103/µl)

Control 0 3.10 ± 0.34 6.17 ± 1.50 0.19 ± 0.10

Murine AIDS 1 1.73 ± 0.26 11.98 ± 3.92 0.21 ± 0.11
2 2.68 ± 0.23 16.50 ± 2.19 0.44 ± 0.07
3 5.06 ± 1.18 26.83 ± 4.97* 1.36 ± 0.54*
Murine AIDS plus ethanol 1 2.95 ± 0.33 5.98 ± 1.23 0.17 ± 0.11
2 4.45 ± 0.44 2.00 ± 0.40** 0.09 ± 0.02**
3 5.59 ± 0.70 22.00 ± 6.44** 1.23 ± 0.50**
Ethanol 1 2.89 ± 0.25 5.73 ± 1.33 0.17 ± 0.13
2 1.97 ± 0.22 4.83 ± 0.87 0.10 ± 0.02
3 3.67 ± 0.41 11.76 ± 1.64 0.43 ± 0.09

Data are expressed as means ± SEM in eight mice for each group. The values at month zero represented the control level of white blood cell (WBC)
and neutrophil counts.
*P < 0.05, control vs 3 months of murine AIDS.
**P < 0.001, control vs 2 (decrease) or 3 months (increase) of murine AIDS plus ethanol consumption.
112 Y. CHEN et al.

Fig. 2. Effect of murine AIDS and ethanol on neutrophil CD11b expression.

Values were means ± SEM (bars) in eight mice for each group. The
values at month zero represented the control level of neutrophil CD11b
expression. Neutrophil CD11b expression significantly increased after
1 month of LP-BM5 infection and remained increased (*P < 0.05).
There was a decrease in neutrophil CD11b expression (#P < 0.05) after
2 months of ethanol consumption. After 3 months, CD11b expression

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reached a significantly high level (***P < 0.001) in murine AIDS, and
murine AIDS plus ethanol groups (**P < 0.01).

Fig. 3. Effect of murine AIDS and ethanol on neutrophil ROS production.

Values were means ± SEM (bars) in eight mice for each group. The
values at month zero represented the control level of neutrophil ROS
production. Neutrophil ROS production significantly increased
(***P < 0.001, **P < 0.001, *P < 0.05), respectively, 1 month after
LP-BM5 infection in either the presence or absence of ethanol consumption,
or ethanol consumption alone. After 2 months, ROS production in all
three different groups showed no significant change compared to the
control. However, there were significant increases of neutrophil ROS in the
murine AIDS plus ethanol (***P < 0.001), and ethanol (*P < 0.05) groups
after 3 months.
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
Fig. 4. CD11b expression from unstimulated and stimulated neutrophils
in all groups during the 3-month period.
(A) CD11b expression from unstimulated and stimulated neutrophils
during the different stages of murine AIDS. (B) CD11b expression from
unstimulated and stimulated neutrophils during the different stages of
murine AIDS in the presence of chronic ethanol consumption. (C) CD11b
expression from unstimulated and stimulated neutrophils during the
different stages of ethanol consumption. Black bars representing
unstimulated groups (blood incubated with FITC-CD11b antibody only).
White bars representing stimulated groups (blood incubated with FITC-
CD11b antibody and fMLP). Values are means ± SEM (bars) in eight mice
for each group. *P < 0.05, significant difference exhibited only in the control
between unstimulated to stimulated blood.
stimulate neutrophils for 15 min ex vivo. For neutrophil
CD11b expression (Fig. 4A–C), 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. 5A–C), similar results were found for
 ETHANOL, RETROVIRUS AND NEUTROPHIL ACTIVATION
Fig. 5. ROS expression from unstimulated and stimulated neutrophils in
all groups during the 3-month period.
(A) ROS expression from unstimulated and stimulated neutrophils
during the different stages of murine AIDS. (B) ROS expression from
unstimulated and stimulated neutrophils during the different stages of
murine AIDS in the presence of chronic ethanol consumption. (C) ROS
expression from unstimulated and stimulated neutrophils during the differ-
ent stages of ethanol consumption. Black bars representing unstimulated
groups (blood incubated with DCFH-DA only). White bars representing
stimulated groups (blood incubated with DCFH-DA and fMLP). Values
were means ± SEM (bars) in eight mice for each group. *P < 0.05,
significant difference exhibited in the control and the first month of
ethanol consumption groups between unstimulated to stimulated blood.
neutrophil CD11b expression. There was a significant increase
in neutrophil ROS production (P < 0.05) after being stimu-
lated 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.
113
DISCUSSION
Neutrophil CD11b expression plays a key role in mediating
firm adhesion of neutrophils to vascular endothelial cells prior
to transmigration into their target sites. Thereafter, activated
neutrophils release ROS that is toxic to microorganisms. In
this study, we applied fMLP ex vivo stimulation to determine
the ability of the neutrophil response to invading organisms.
We found that neutrophil CD11b expression and ROS
production were impaired in response to fMLP stimulation
during the different stages of LP-BM5 infection and/or chronic
ethanol consumption. This result suggests that neutrophil
function is diminished by AIDS and/or chronic ethanol con-
sumption. The neutrophil functional abnormalities may be due
to several factors, such as: (1) ethanol or its metabolites;
(2) viral particles from LB-BM5 infection; (3) cytokine dys-
regulation; and/or (4) neutrophil receptor desensitization.
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
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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-α 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 neutro-
phil 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 con-
tinually 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 kill-
ing, at this point ROS may trigger neutrophil apoptosis before
neutrophils reach the inflammatory site. This phenomenon
suggests that chronic ethanol consumption increases suscepti-
bility to infection (Jareo et al., 1995).
Cytokines released from infected cells are a hallmark of
LP-BM5 infection. Proinflammatory cytokines, such as TNF-α,
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
114 Y. CHEN et al.
(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.
Acknowledgements — This work was supported by NIH HL grants 63667 and
59794.
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