Manuscript Ver. III

8/8/2019 Manuscript Ver. III

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Phagocytosis is one component of innate immunity, vital in providing the first line of

defense against infection from foreign pathogens. In contrast to acute alcohol

intoxication wherein immunosuppressant effects remain transient, medical literature has

long established that chronic alcohol consumption has long term effects, weakening the

body’s immune system, resulting in increased susceptibility to infections. It is therefore

the purpose of this study to investigate the consequence of different concentrations of

alcohol in the phagocytic process of male albino mice (Mus musculus), as well as to

establish a correlation between alcohol concentrations with degree of anti-phagocytic

effect, through the chronic administration of high (70%) and low (6%) ethanol

concentrations and a subsequent Staphylococcus aureus challenge. Unpaired t-tests

performed on results show significant reduction in phagocytosis occurring between the

70% ethanol treatment group compared and the controls, as well as between the 70% and

6% ethanol treatment groups. No significant difference was seen between the 6% ethanol

treatment group and the controls. This study has revealed that chronic ethanol

administration causes impaired inflammatory and phagocytic functions in male albino

mice against Staphylococcus aureus infection. However, significant impairment of

phagocytic mechanisms are only observed in mice exposed to high ethanol

concentrations, this effect being almost negligible with low ethanol concentrations,

suggesting a minimum threshold ethanol concentration wherein significantly impaired

phagocytic functions may be seen. Further information can be obtained in this respect by

increasing the number of equally graded concentrations of ethanol administered, in order

to identify the aforementioned minimum threshold concentration.

Keywords: Phagocytosis; chronic; ethanol; Staphylococcus aureus, male albino mice

(Mus musculus)

Introduction

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Overview of Innate Immunity

The innate immune system is an essential component of immunity, designed to provide

the first line of defense against infection from foreign pathogenic microorganisms.

Innate immunity is achieved by several factors, one of which is the mechanism of

phagocytosis, which comprise polymorphonuclear leukocytes (PMNs); the neutrophils

and macrophages. These cells also mediate the inflammatory response through the

localization of the invading microorganisms at its site of infection and cytokine

production.

Phagocytosis

Phagocytosis is vital mechanism comprising a series of events, utilized in the

neutralization, regulated engulfment, intracellular destruction and subsequent clearance

from the circulation or body fluids of invading pathogens and apoptotic cellular debris.

Recognition and binding of particles is mediated by specific cell-surface receptors

located on the circulating inflammatory leukocytes. This is followed by engulfment of

the pathogen complex, which requires the formation of an actin-rich membrane assembly,

pseudopod extension and fusion of the phagosome, forming a mature phagolysosome 1.

Intracellular compartmentation of pathogen destruction is maintained in the

phagolysosome, protecting the host from conditions required to achieve microbial

digestion. Such conditions involve the production of reactive oxygen species (ROS),

cytokines, the generation of an acidic pH of less than pH 5.5 within the cell 2, and

presence of hydrolytic enzymes and specific antimicrobial pore-forming peptides through

fusion with lysosomes, the combination of which, facilitate the destruction of pathogens.

The entire process of phagocytosis is further facilitated by complement, a group of

proteins and proteolytic peptides that are involved in regulating both the innate and

acquired immune response. In the case of the innate immune response against invasion

of foreign pathogens, the alternative and lectin pathways of complement fixation come

into play, while the classical pathway is reserved for cell-mediated immunity, wherein

antigen-antibody interactions are required for complement activation (Fig 1) 3.

3



Figure 1. Pathways for complement activation. Lifted from Foster, 2005 3.

Complement activation promotes opsonization through the release of small peptide

molecules such as C3a and C5a, common to all pathways, which are recognized by cell

surface receptors in phagocytes, thus serving for the recruitment and recognition of these

cells to the site of infection. It should also be noted that phagocytes also exhibit cell

surface receptors able to recognize the effector regions of antibodies attached to the

bacterial surface 4.

Acquired immunity is subsequently activated through antigenic processing and

presentation of pathogenic fragments by macrophages and dendritic cells, stimulating

components of cell-mediated immunity5,6

.

Pathogens evolve to escape the deleterious effect of phagocytosis by professional

phagocytes. Avoiding phagocytosis, killing phagocytes or surviving inside them are the

actions of pathogens for them to continue existing. Bacterial pathogens are also using

induction of phagocytic entry into non-specific phagocytic cells, such as epithelial cells,

as a strategy of survival and multiplication 7.

Staphylococcus aureus

Staphylococcus aureus is a microorganism commonly found in the moist squamous

lining epithelium of the anterior nares, either permanently or transiently present in

4



approximately 80% of the population 8. It is frequently found to be the causative agent in

infections involving the skin, creating abscesses, or more serious invasive infections that

may lead to sepsis 9.

Invasion of S. aureus is facilitated by cell surface proteins which make possible its

adherence host tissues and cells, including red blood cell membrane proteins and the

presence of a polysaccharide coat 10,11,12. Tissue damage results from the secretion of lytic

enzymes such as proteases, hyaluronidases, lipases and nucleases, as well as various

other toxins targeting cell membranes 13,14.

As with most infectious pathogens, following penetration of external barriers such as the

skin and mucosal surfaces, the first line of defense against S. aureus infections are

neutrophils and macrophages. As described in the previous section, neutrophils and

macrophages migrate to the site of infection, recognize and bind invading S. aureus

facilitated by host antibodies and complement.

Similarly to other microorganisms, S. aureus have developed mechanisms to circumvent

the host innate immune system. This is achieved through secretion or _expression of

polysaccharides and proteins that prevent antibody recognition and therefore

opsonization and complement activation. Further, S. aureus has also been shown to

survive in the phagosomal environment, cause neutrophil destruction and display

resistance to antimicrobial substances and lysosomal enzymes. S. aureus also suppresses

both the innate and cell-mediated immunity through the secretion of immunomodulatory

proteins and the _expression of superantigens. Lastly, the evolution of antibiotic resistant

strains to such antibiotics as meticillin and vancomycin, which were previously generally

effective further enhances the virulence of this microorganism 15,16.

Chronic alcohol exposure

In contrast to acute alcohol intake, whose dampening effects on the immune and

inflammatory response have been shown to be transient, chronic alcohol intake may have

more long term effects 17,18. In fact, acute consumption of moderate amounts of alcohol

has been shown to have beneficial effects, as demonstrated by a prospective cohort study

by Watzl and colleagues. This study revealed that moderate wine consumption is

associated with a decreased risk of common colds. The study clearly showed that acute

consumption of a moderate amount of red wine and of a 12% ethanol solution had no

short-term effect on immune functions of men. Also an acute intake of wine constituents

5



such as polyphenol-rich beverages (dealcoholized red wine and red grape juice) did not

manifest any decrease in the host defense mechanism of the subjects 19.

As such, chronic ethanol consumption has been associated with impaired immune

responses, which result in increased susceptibility to infectious diseases, observed in both

human patients and experimental animals. Studies have reported an approximately

doubly increased incidence of pulmonary infections such as pneumonia and tuberculosis

in alcoholics compared to nondrinkers or light drinkers. In addition, the alcoholic group

has been found to respond less effectively to therapy 20.

Susceptibility in not merely limited to pulmonary infections, but include increased risk of

developing head, neck and upper gastrointestinal cancers, as well as bacterial peritonitis

and spontaneous bacteremia, which is apparent particularly in those with alcoholic

cirrhosis. More recently, studies have demonstrated decreased resistance and poorer

prognosis to human immunodeficiency virus (HIV) infection with ethanol exposure 21,22,23.

Mechanisms proposed for the anti-phagocytic effect of alcohol

While the lifestyle of alcoholics are believed to contribute the their exposure to infectious

microorganisms, it has become increasingly apparent that alcohol negatively influences

the immune system, in particular the mechanism of phagocytosis of pathogenic

microorganisms, with increasing evidence from human and animal studies from in vivo as

well as in vitro experiments. The antimicrobial activity of mononuclear phagocytes and

neutrophils falls into two categories, oxygen-dependent and independent mechanisms.

Toxic free radicals such as superoxide anion, singlet oxygen, hydroxyl radical and

hydrogen peroxide are potent antimicrobial agents that are oxygen dependent. In

contrast, lysosomal enzymes act independently of oxygen.

Hepatic effects

It has been proposed that acute or prolonged consumption of alcohol has been shown to

depress the microbicidal activities of phagocytes, which may be attributed at least in part

to the downregulation of the release of oxygen derived radicals 24. This theory is

supported by Bautista and colleagues, whose study involved the feeding of ethanol to

male Wistar rats for 32 weeks. Results showed that E. coli phagocytosis and superoxide

anion production by Kupffer cells were downregulated in the ethanol-fed group 25.

6



On the contrary, several studies propose that alcohol is able to achieve this through the

production of ROS, which is believed to be involved in the impairment of macrophage

function, as well as decreasing cell viability 26,27,28. The ingestion of alcohol produces

oxidative stress generating free radicals of oxygen and ethanol. These free radicals have

a molecular reactive ability and, therefore, play an important role in the development of

the injury, which often appears in the liver and in other organs and tissues. This

mechanism is supported by a study done by Colome and colleagues, where

administration of 50 mM ethanol showed a statistically significant increase in oxidative

stress in the cells of all three phagocytic cell types (lymphocytes 9.19%, monocytes 32%

and granulocytes 36%) 24.

Chronic ethanol intake has been shown to affect the production of ROS in phagocytes

which contribute to the development of alcoholic liver disease (ALD). A study by

Parlesack and colleagues observed ROS release and phagocytosis of neutrophils and

monocytes following administration of endotoxin and 22 or 44 mM ethanol in 60 patients

with varying severity of ALD and 28 healthy controls. Results showed that neutrophils

from patients with severe ALD produce more ROS than the healthy controls. Further,

this production was found to correlate with severity of disease. It also appears that ROS

formation was reduced in the healthy controls in a dose-dependent manner, a response

which was absent in patients with ALD. It is believed that this enhanced ROS production

contributes to decreased immunity 29.

Additional evidence for the impaired phagocytic mechanism in the liver upon ethanol

administration is provided by McVicker and colleagues. Within the liver, the

asialoglycoprotein receptor (ASGP-R) has been shown to be involved in the phagocytosis

of apoptotic hepatocytes, as well as altered cellular endocytic events after ethanol

administration. The study showed the capacity of ASGP-R to phagocytose apoptotic

cells in relationship to the damaging events that occur with alcohol consumption. The

results of this assay indicated that the phagocytosis of apoptotic cells was decreased

significantly (30% to 42%, P <.05) 30.

Pulmonary effects

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The effect of ethanol in relation to oxidative stress on the pulmonary immune response

has also been studied. Zhang and colleagues determined the activity of PMNs and

alveolar macrophages (AMs) in rats intraperitoneally administered with 20% ethanol at a

dose of 5.5 g of ethanol/kg and subsequently challenged with intratracheal endotoxin

(300 ug/kg in 0.5 ml saline). Results showed that ethanol ingestion suppressed

phagocytosis by circulating PMNs with a concurrent reduction in hydrogen peroxide

production by AMs. Thus, alveolar macrophage immune function is impaired by alcohol

abuse, rendering patients susceptible to pneumonia 31. This study supports the initial

proposal that the mechanism employed by alcohol in the depression of the anti-microbial

actions of phagocytes is through the downregulation of ROS.

A recent study by Joshi and colleagues supplied evidence that the mechanism of ethanol-

induced inhibition of phagocytosis extends to the molecular level. Their study

demonstrated a decrease in granulocyte-macrophage colony-stimulating factor (GM-

CSF) receptor gene _expression, responsible for the maturation of alveolar macrophages,

as well as _expression and nuclear binding of PU.1, the transcription factor that activates

GM-CSF-dependent macrophage functions. These effects were observed in rats that

ingested ethanol for six weeks 32. Additionally, a study done by Schleifer and colleagues,

which compared alcohol dependent patients with no medical disorders to persons free

from substance abuse and medical disorders, showed altered granulocyte function in the

alcohol-dependent sample, with a consequent reduction in phagocytic activity in the

alcohol-dependent males 33.

Central Nervous System effects

Data on the effects of chronic ethanol ingestion in the central nervous system is also

provided by Aroor and Baker, who demonstrated the inhibition of phagocytosis of E.coli

by microglia, presenting further evidence of the various mechanisms involved and the

widespread systemic effects of ethanol ingestion on the immune system 19.

Additional evidence

Mandel and colleagues have shown the significance of humoral and cellular immunity in

host defense against the Lyme disease spirochete, B. burgdorferi, and against Listeria

monogenes 34. Related studies done by Mendelhall and colleagues noted that ethanol

interferes with the ability of rats to mount optimal humoral and cellular immune

8



responses against borrelial antigens. Such ethanol-induced immunosuppression closely

resembles short-term immune system impairment, where there is increased bacterial

burden in Listeria-challenged rats fed with intoxicating amounts of ethanol. This is in

close agreement with the reported results showing that ethanol ingestion lowers resistance

in Listeria-infected mice. Increased bacterial colonization of the liver have been

observed in rats consuming ethanol and infected with a related intracellular organism,

Mycobacterium bovis 35.

A study by Pavia and colleagues observed the effects of ethanol ingestion on the

antimicrobial immunity of rats both in vivo and in vitro. Nonimmune Long-Evans rats

were given a short-course treatment of excessive amounts of alcohol per gram. Spleen

cell suspensions were exposed to two bacterial pathogens, Listeria monocytogenes and

Borrelia burgdorferi, in vitro. Subsequent determination of their ability to mount a

defense against such pathogens was resolved by counting the cultured CFU ( Listeria) or

upon microscopic examination ( Borrelia). Results showed that the spleen cells from the

ethanol-treated rats killed fewer bacteria than the pair-fed controls. The in vivo

experiment involved the intraperitoneal infection of Listeria to ethanol-treated and

control rats. Systemic infection was then assessed based on the number of organisms

present in their livers and spleens. Elevated bacterial CFU counts were observed in both

organs of the ethanol-treated rats two days following listerial challenge. These results

support the concept that acute ethanol ingestion may impair host defense mechanisms,

especially those expressed at the cellular level 36.

Here, chronic administration of high and low concentrations of ethanol with a subsequent

Staphylococcus aureus challenge were used to investigate the consequence of different

concentrations of ethanol in the phagocytic process of male albino mice (Mus musculus).

As several studies have identified the phagocytic mechanism as one of the factors that

chronic ethanol consumption interferes with, it is hypothesized that subjects treated with

ethanol would exhibit impaired phagocytic processes when challenged with S. aureus.

Furthermore, the degree of suppression of phagocytosis exhibited by the subjects is

hypothesized to correlate with the percent of ethanol ingested by the mice.

The results of this study can contribute to further understanding of the effects of varying

concentrations of ethanol on the phagocytic mechanism in male albino mice, and is

clinically significant in that the results obtained could potentially be correlated to that of

different alcoholic drinks, and thus extrapolated to the human population.

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Objectives

Based on the establishment of the anti-inflammatory and anti-phagocytic effects of ethanol by previous investigations, the purpose of this study is to compare the percentage

of male albino mice (Mus musculus) that will exhibit an anti-phagocytic effect under

varying concentrations of ethanol, and to evaluate if there is a significant difference

among treatment groups.

Materials and Methods

Research Design

This study was designed to evaluate the anti-phagocytic effect of varying concentrations

of ethanol using male albino mice (Mus musculus) as test animals. In this experiment,

70% and 6% ethanol concentrations as treatment interventions were used to represent

different alcoholic drinks, as well as distilled water in the control group. The manner of

administration was through force feeding of the assigned treatments for each group using

gavage tubes. Treatments were administered once daily for a period of seven days. On

the final day of administration, each mouse was inoculated intraperitoneally with

standardized Staphylococcus aureus solution. Mice were sacrificed an hour following

inoculation. Smears of peritoneal blood were made and examined for the presence of

phagocytes with ingested bacteria. The presence of ingested bacteria within a phagocyte

suggests a positive result for normal phagocytosis, otherwise, an impaired or absence of

phagocytic activity of the cell.

Preparation of Test Animals

Fifty six male albino mice were procured from the Bureau of Food and Drugs (BFAD,

Filinvest, Alabang). The test animals were of the ICR strain (Institute of Cancer

Research), weighed approximately 18-19 g each, and were 4-5 weeks old. These mice

were randomly distributed to three groups with 18 mice in the control group, which was

administered with distilled water and 19 mice each in the 70% and 6% ethanol treatment

groups. The mice were kept in factions of 3 in wire mesh cages and fed with mice pellets

and distilled water, ad libitum. Distilled water, ethanol and mice pellets were provided

10



for by the Department of Physiology, University of Santo Tomas (Manila, Philippines).

The mice were housed in an appropriate and well-ventilated indoor facility (Manila,

Philippines).

Treatment procedure

Each mouse was force fed once daily for a period of seven days with 0.5 ml of the

assigned treatment. Force feeding was done using gavage tubes and administration was

performed at the same time of day throughout the course of the study (Fig 2).

Figure 2. Administration of assigned treatment through force feeding.

Experiment Proper

On the seventh and final day of the trial, mice were inoculated intraperitoneally with 1 ml

standardized S. aureus broth culture an hour following administration of each groups’

respective assigned treatment (Fig 3). S. aureus broth cultures were obtained from St.Vincent’s Clinical Lab (Bulacan, Philippines) which were standardized using the

McFarland standard.

Figure 3. Intraperitoneal inoculation of S. aureus.

The mice were sacrificed by cervical dislocation one hour after inoculation. The

abdomen was cut and the peritoneal cavity was exposed. Impression smears of the

11



peritoneal blood of each mouse were made by pressing a glass slide to the exposed

peritoneal cavity (Fig 4). The smears were air-dried and labeled. Fixation and staining

were done by immersing the slides in reagent-filled couplin jars. Fixation of the smears

with absolute methanol was done for one minute, followed by exposure to undiluted

Wright’s stain solutions (Rapi Stain, Crescent Diagnostics) for another minute. Water

was used to flush the stain from the slide, then allowed to air-dry.

Figure 4. Impression smear of the peritoneal cavity.

Histological Examination

Each slide was examined under oil immersion with 1000x magnification using a bright

field microscope (Olympus) for the presence of phagocytes containing ingested bacteria.

The entire slide was scored using the crenellation method of counting cells positive for

phagocytosis. The presence of ingested bacteria within a phagocyte was considered a

positive indicator of normal phagocytic activity while the absence of ingested bacteria in

the phagocyte indicated an impaired or the absence of phagocytic activity of the cell.

Statistical Analysis

The number of phagocytes with ingested bacteria per slide was summarized in each

treatment groups. The means of the control and treatment groups were compared against

each other using unpaired student’s t-tests using SPSS ver.14 statistical software. The

unpaired t-test can be used to compare groups with an unequal number of samples and is

appropriate for this type of data. The t-tests were done at a 95% confidence interval with

a p-value of 0.05 or less considered determinate of a statistically significant difference.

Results

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Please note that preliminary results have been included in this initial draft of the

manuscript as scoring of duplicate slides has yet to be completed. It is anticipated that

final results will be available by 13 February 2006.

A total of 46 slides were examined to determine the number of macrophages that were

positive for phagocytic activity: 14 for the distilled water control group, 19 for the 6%

ethanol group and 13 for the 70% ethanol group.

Figure 5. Phagocyte in the process of bacterial ingestion. Peritoneal smear from thedistilled water control group (1000x magnification).

Figure 6. Phagocyte in the process of bacterial ingestion. Peritoneal smear from the 6%

ethanol treatment group (1000x magnification).

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Figure 7. Phagocyte in the process of bacterial ingestion. Peritoneal smear from the 70%ethanol treatment group (1000x magnification).

The distilled water control group had an average of 3.45 active macrophages per slide.

There was a single outlier in the distilled water group that was eliminated using the

interquartile range method. This single outlier was disregarded in the computation for the

mean. The 6% ethanol treatment group had an average of 2.60 active macrophages per

slide. The 70% ethanol group had an average of only 0.409 active macrophages per slide

(Fig 8). Refer to Figures 9-11 for the frequency distribution of each group.

Figure 8. Tabulation of Number of Phagocytes with Ingested Bacteria (positive

for Phagocytosis) per Slide for Each Group

No. of (+) Phagocytes per

slide

No. of slides

70% EtOH

(n = 22; Ave. =

0.409)

6% EtOH

(n = 30; Ave. =

2.60)

distilled H2O

(n = 33; Ave. =

3.45 )

0 13 - 2

1 9 9 12 - 10 7

3 - 3 8

4 - 4 7

5 - 2 5

6 - 1 1

7 - - -

8 - 1 2

9 - - -

10 - - -

11 - - -12 - - -

13 - - -

14 - - 1*

Total 22 30 34

*outlier, not factored in t-test and mean computation

14



0

1

2

3

4

5

6

7

8

9

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

No. of (+) phagocytes per slide

N o . o f

s l i d e s

Figure 9. Frequency distribution for the distilled water/ control group

0

2

4

6

8

10

12

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15


N o . o f s l i d e s

Figure 10. Frequency distribution for the 6% ethanol treatment group

0

2

4

6

8

10

12

14

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15


N o . o f s l i d e s

Figure 11. Frequency distribution for the 70% ethanol treatment group

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There was an insignificant and negligible difference between the 6% ethanol treatment

group and the distilled water control group as indicated by a p-value of 0.063. The single

outlier in the distilled water group was again disregarded in the unpaired t-test

computation. In contrast, there was a significant difference between the distilled water

control group and the 70% ethanol treatment group with a p-value of less than 0.0001 at a

95% confidence interval. Likewise, there is also a significant difference between the 6%

and 70% ethanol groups with a p-value of less than 0.0001 (Fig 12).

Figure 12. Comparisons of P-values of the Unpaired T-test

Groups Compared p-value (psig < 0.05)

70% EtOH and distilled H2O <0.0001

70% EtOH and 6% EtOH <0.00016% EtOH and distilled H2O 0.063

Discussion

It can be deduced from the results of this study that chronic administration of high

ethanol concentrations leads to impaired phagocytosis, but this effect is not seen with low

ethanol concentrations.

In contrast to other studies which have employed administration of interventions for

prolonged time periods to investigate chronic ethanol exposure, this study has used a

relatively short period (seven days) in order to evaluate chronic ethanol-induced

alterations in phagocytic function over a period wherein subject survival is

uncompromised. Based on a pilot study performed prior to the experiment proper (results

not shown), the aforementioned treatment schedule and administered ethanolconcentrations were determined to be the most appropriate to investigate the anti-

phagocytic effect of different concentrations of ethanol on male albino mice.

17 of the 18 mice in the control group, 15 of the 19 mice in the 6% ethanol treatment

group and 11 of the 19 mice in the 70% ethanol treatment group remained viable at the

conclusion of the treatment period. All four deaths in the 6% ethanol treatment group

were recognized to be from aspiration. Of the eight deaths in the 70% ethanol treatment

group, six were attributed to aspiration as well, while two were unexplained. The one

mouse in the control group had managed to escape while in the process of cervical

dislocation, and thus was considered to be an unavoidable circumstance.

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Upon observation that deaths occurred only in the ethanol treatment groups and not in the

control group, as well as the struggle of the mice during ethanol administration, which

was also not seen in the control groups, it is thus believed that ethanol is the cause of

these effects, as all other variables remained constant. Struggle during force feeding of

ethanol was attributed to initial irritation of administration of a foreign substance with an

unpalatable flavor. However, deaths occurred within 24 hours of the previous

administration, and therefore have been due to the failure of these mice to recover from

the initial stress of ethanol consumption.

Significant differences seen between the control and in those mice treated with 6% and

70% ethanol were as predicted, with several studies having established the anti-

inflammatory and anti-phagocytic effects of ethanol consumption. These investigations

have proposed various mechanisms to explain these observed outcomes, among which

predominates the increased exposure to reactive oxygen species (ROS) secondary to

ethanol consumption, as discussed in previous sections.

Joharapurkar and colleagues have stated that free radicals are the underlying cause for the

decreased immunity. Their study demonstrated that chronic ethanol treatment decreases

reduced glutathione (GSH), and as the latter has an important role in activating T cells

and macrophages, its reduction results in impaired phagocytosis and cell-mediated

immunity. It was further stated that intake of vitamin C and E, which helps the body

overcome oxidative stress, prevented the influence of ethanol on GSH levels.

Furthermore, the production of nitric oxide by macrophages, which normally results in

the activation of T cells, is downregulated along with NF-kappa B, in the sustained

presence of free radicals 37.

Gauthier and colleagues provided further evidence in support of the above study,

demonstrating impaired alveolar macrophage phagocytic function following ethanol

exposure. A rise in apoptosis and diminished cell viability was said to be due to

increased lipid peroxidation caused by oxidative stress. However, while their

investigation also concluded that this decrease in phagocytic processes was a function of

the reduction of GSH availability in the presence of oxidative stress due to ethanol

exposure, their study contrasts to that of Joharapurkar, wherein their test subjects, being

fetal guinea pigs, were already deficient in GSH prior to ethanol exposure. Nevertheless,

observations were similar to that of adults, albeit with augmented effects, wherein

decreased GSH:GSSH (oxidized glutathione) ratios were seen in subjects exposed to

ethanol as compared to controls of the same gestational age. GSH and vitamin E

supplementation was shown to improve alveolar macrophage development, thus

17



enhancing phagocytic functions 38. Consequently, both these studies conclude that the

mechanism of ethanol-induced reduction in phagocytic activity is through the reduction

of GSH availability, postulated to be secondary to an increase in oxidative stress, which

is essential to macrophage development.

According to a more recent study by Zima and colleagues (2005), the increased

production of reactive oxygen species (ROS) in the mitochondria and cytochrome P-450

2E1 (CYP2E1) impairs antioxidant defenses. Ethanol also activates Kupffer cells to

release ROS, reactive nitrogen species (RNS) and cytokines. These ROS and free

radicals in return, induce apoptosis, produces direct hepatocellular damage, and

stimulates collagen deposition by hepatic stellate cells. Lipid peroxidation products and

acetaldehyde-malondialdehyde adducts also contribute to hepatic inflammation 39. In

addition to Kupffer cells, ethanol also stimulates hepatocytes and sinusoidal cells to

produce nitric oxide 40.

In addition, Bautista and Spitzer introduced the involvement of endotoxins. Chronic

consumption of ethanol enhanced gut permeability, which increases endotoxin influx in

the circulation. This will cause influx of lipopolysaccharide or a reduction in the

clearance of endotoxin by Kupffer cells. The increase in endotoxin will activate hepatic

macrophage to produce TNF, IL-1, and superoxide anions. TNF and IL-1 activate

mononuclear phagocytes and PMNs to undergo respiratory burst and release of free

radicals. Bautista and Spitzer also demonstrated that these cytokines enhance the

expression of adhesion molecules and increase chemotaxis, promoting PMNs to migrate

to the liver 41.

On the other hand, while most studies state that ethanol represses the immune system

through ROS production, there are studies that observed otherwise. One such study by

Morio and colleagues oppose the above study by Bautista and Spitzer, observing a

decrease in chemotaxis towards the complement fragment C5a in alveolar macrophages

in rats consuming ethanol for 9-12 weeks. A decrease in superoxide anion, nitric oxide

and NO synthase was also noted, along with a lower cell adhesion molecule _expression

in hepatic macrophages, thus opposing other studies that have demonstrated dramatic

increases in ROS production by phagocytes 42.

Carvalho and colleagues, have shown that ethanol inhibits the production of

inflammatory cytokines in chronically ethanol-fed mice 43. As evident in the slides

examined, the slides obtained from the distilled water control group showed a gram

positive cocci in the cytoplasm of the monocytes while those slides taken from ethanol

18



fed group revealed no bacteria in the cytoplasm. The absence of cocci within the

phagocytes observed in the ethanol fed group could indicate that the phagocytic activity

had been impaired whereas the presence of ingested bacteria within the phagocytes in the

distilled water control group could signify presence of phagocytic activity.

These studies combined support the data obtained from this investigation, that high

ethanol concentrations indeed reduces the phagocytic activity of the host cell against

bacterial infection, in this instance Staphylococcus aureus.

Studies demonstrating no significant reduction in phagocytic activity upon ethanol

exposure should also be considered. Kvietys and colleagues propose that the

administration of ethanol actually enhances various neutrophil functions such as

adherence, chemotaxis and degranulation. Their study involved the measurement of

neutrophil adherence to venules and extravasation into the interstitium following ethanol

application on the surface of cat mesentery. Results showed that ethanol is

proinflammatory at concentrations achieved in the mucosal interstitium during acute

alcohol intoxication. The ethanol-induced leukocyte adherence and extravasation is

dependent on the expression of adhesive glycoproteins. Leukocyte-endothelial cell

interactions initiated by ethanol were not affected by inflammatory mediators such as

platelet-activating factor (PAF) and leukotriene B4 (LTB4) 44.

Another study showed that alcohol does not have any effect on the immune system.

Healthy men consumed 12% ethanol over a period of two weeks. They did not observe

any significant effects on neutrophil and monocyte phagocytosis. Further, they observed

that monocyte phagocytosis via Fc-receptor increased directly with the dose of ethanol.

Lymphocyte apoptosis and lytic activity of NK cells were not affected as well. TNF-á,

TGF-â, and cytokine (IL-2, IL-4) production were also not significantly affected by the

treatment. Thus, there was no observed effect on TH2-lymphocytes 45.

There appears to be threshold concentration of alcohol required before the immune

system is affected, as observed by Watzl and colleagues. For instance, 440 mM of

ethanol was observed to significantly decrease TNF-á production by monocytes and an

ethanol concentration of 25 mM reduced TNF-á mRNA levels. Binge drinking up to 3.ll

of beer also reduced IL-2 production45

.

Such an effect was in fact seen in our investigation, where no significant difference was

observed between the 6% ethanol treatment group and the control group. This result

contrasts with the expectation that despite the relatively low dose of administered ethanol

19



(6%), similar anti-phagocytic manifestations would be seen, albeit to a lesser degree than

that of the 70% ethanol group. These observations suggest a minimum threshold ethanol

concentration wherein significantly impaired phagocytic functions may be seen.

The following investigation has reported significantly impaired phagocytic and

bactericidal function mediated by elevated ethanol concentrations against S. aureus

infections in male albino mice. Based on the literature, this effect appears to be

widespread, involving various mechanisms affecting phagocytosis, as well as influencing

numerous organ systems. Conversely, these effects were not observed in mice

administered with low ethanol concentrations, suggesting a minimum threshold level

wherein significant changes in phagocytic function can be seen. Data obtained from this

investigation may contribute to the increasing evidence of the involvement of ethanol as a

risk factor for the increased incidence of opportunistic infections in alcoholics. However,

caution must be exercised on this point as to the limitations of this study in terms of mice

being used as test subjects and the relatively short experimental duration.

Conclusions

In accordance with previous investigations, this study has revealed that ethanol

administration causes an impairment of the inflammatory and phagocytic functions of

male albino mice against Staphylococcus aureus infection. However, significant

impairment of phagocytic mechanisms are only observed in mice exposed to a high

(70%) ethanol concentration, while an almost negligible effect on the immunity of mice

when treated with a low (6%) ethanol concentration compared to untreated (distilled

water) controls is seen, suggesting a minimum threshold ethanol concentration wherein

significantly impaired phagocytic functions may be noted.

Future Recommendations

This investigation can be further improved by increasing the number of equally graded

concentrations of ethanol administered. This serves the purpose of gaining a more

accurate comparison of the anti-phagocytic activity of ethanol, while also identifying the

minimum threshold concentration wherein significantly impaired phagocytic mechanisms

can be observed. Furthermore, the use of other test animals such as rats or hamsters

could provide a more comprehensive and wider-range of information, since their greater

20



total body weight than mice and hence may have a higher tolerance for ethanol intake.

Alternatively, other routes of ethanol administration such as through inhalation or

intraperitoneal administration should be considered as these procedures would avoid the

gag reflex the mice exhibited during treatment administration as well as ensuring the

complete and direct absorption of the entire volume of solution administered. Finally, a

prolonged treatment period is suggested as this would most likely be more representative

of true chronic ethanol consumption.

Acknowledgements

We thank our supervisors Dr. Llarena and Dr. Sibulo for their advice and support, as well

as the Department of Physiology, University of Santo Tomas for their assistance in

procuring materials and use of their facilities during the course of this work.

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