RESEARCH PAPER

IFN-c is essential for the inhibition of B16BL6 melanoma lungmetastasis in chronic alcohol drinking mice

Hui Zhang • Zhaohui Zhu • Jenifer M. McKinley •

Gary G. Meadows

Received: 18 November 2010 / Accepted: 30 December 2010 / Published online: 14 January 2011

� US Government 2011

Abstract We previously found that chronic alcohol

consumption (20% w/v in drinking water) that models the

level consumed by human alcoholics, when administered to

female C57BL/6 mice inhibits B16BL6 melanoma metas-

tasis to the lung; however, the mechanism is not known.

Chronic alcohol consumption increases IFN-c producing

NK, NKT, CD4?, and CD8? T cells. To examine the

impact of IFN-c on metastasis, we inoculated B16BL6

melanoma cells i.v. into control and chronic alcohol

drinking IFN-c knockout (KO) mice. Knockout of the ifn-cgene abrogated the anti-metastatic effects associated with

alcohol consumption. We examined metastasis in common

gamma-chain (cC) KO mice, which are deficient in NK,

NKT and CD8? T cells, and in Va14Ja281-/- KO mice,

which are deficient in invariant NKT (iNKT) cells, in order

to assess the importance of specific IFN-c producing cell

types to this effect. We found that the antimetastatic effect

of alcohol was still present in cC KO mice and also in cC

KO mice depleted of Gr-1? cells. Knockout of iNKT cells

reduced the degree but not the antimetastatic effect asso-

ciated with alcohol. These results indicate that the anti-

metastatic effect induced by chronic alcohol consumption

is IFN-c dependent and that multiple IFN-c producing cell

types contribute to this effect.

Keywords Alcohol � IFN-c � Knockout � Mice �Melanoma � Metastasis

Abbreviations

a-GalCer Alpha-galactosylceramide

CMF Phosphate buffered magnesium- and calcium-

free normal saline

cC KO Common gamma-chain knockout

FITC Fluorescein isothiocyanate

iNKT Invariant natural killer T cells

KO Knockout

NK Natural killer

PBL Peripheral blood leukocytes

PE Phycoerythrin

PerCP Peridinin chlorophyll protein

Introduction

There is strong epidemiological linking alcohol consump-

tion with an increased risk of developing cancer, including

but not limited to breast cancer, esophageal cancer, liver

cancer, and colorectal cancer [1–5]. It is now also becoming

clear that alcohol consumption also increases the risk of

developing melanoma skin cancer [6–10]. Since the inci-

dence of melanoma continues to increase every year, it is

important so study the impact of alcohol consumption on

this disease. While information linking alcohol consumption

to cancer risk continues to accumulate, it is not at all clear

what effect chronic alcohol consumption has on cancer

progression. Thus, it is not known to any great extent

whether the cancers associated with alcohol consumption

are more or less prone to metastasize.

Alcohol consumption is a well-known immunosuppres-

sant, and our previous research indicated that chronic high

alcohol consumption suppresses natural killer (NK) cell

cytolytic activity [11–16]. NK cells, which are the first line

of immune defense against tumors, play an initial role in

adaptive antitumor immune responses, and were found

several years ago to have an important role in the control of

H. Zhang � Z. Zhu � J. M. McKinley � G. G. Meadows (&)

Department of Pharmaceutical Sciences, College of Pharmacy,

Washington State University, Pullman, WA 99164-6534, USA

e-mail: meadows@wsu.edu

123

Clin Exp Metastasis (2011) 28:301–307

DOI 10.1007/s10585-011-9372-1

melanoma metastasis [17, 18]. Thus, we previously exam-

ined the hypothesis that alcohol consumption would

enhance metastasis of melanoma. Using the well charac-

terized B16BL6 melanoma model, which is highly invasive

and metastatic, we found to our surprise that 20% w/v

alcohol consumption inhibited both spontaneous metastasis

to the lung after intradermal inoculation into the pinna of the

ear as well as experimental metastasis after i.v. inoculation

via the lateral tail vein [19, 20]. The effect of alcohol on

B16BL6 melanoma metastasis is dose dependent [20] and

independent of perforin and granzymes related NK cell

cytolytic activity, as demonstrated in beige mice [21].

The mechanism underlying the inhibition of melanoma

metastasis associated with alcohol consumption is not

known. While multiple factors are likely involved, it is

known that IFN-c is a key player in controlling melanoma

metastasis [22, 23]. This cytokine, which can be produced

by a number of cells including but not limited to NK cells,

NKT cells, T cells, and macrophages, is also known to have

a significant impact on metastasis in other tumors [24–29].

We previously reported that chronic alcohol consumption

increases IFN-c producing CD3-NK1.1?, CD4? and CD8?

T cells [30, 31]. Herein we also report that chronic alcohol

consumption also increases IFN-c producing CD3?NK1.1?

(NKT) cells. This information provided a strong rationale to

examine the hypothesis that enhancement of IFN-c by

chronic alcohol consumption is responsible for the decrease

in B16BL6 melanoma metastasis to the lung. Herein, we

report that knockout of the ifn-c gene in mice abrogates the

antimetastatic effect induced by chronic alcohol consump-

tion, suggesting that the effect is IFN-c dependent.

Materials and methods

Animals and alcohol administration

Female C57BL6 mice at 6–7 weeks of age were purchased

from NIH Charles River Laboratories (Wilmington, MA)

and acclimated in the Wegner Hall Vivarium at Washington

State University for 1 week before using them in experi-

ments. These mice were used to determine the effect of

chronic alcohol administration on the percentage of IFN-cproducing NKT cells in the spleen. IFN-c knockout (KO)

mice (B6.129S7-Ifngtm1Ts/J) and breeding stock for cC KO

mice (B6.129S4-Il2rgtm1Wjl/J) were purchased from the

Jackson Laboratory (Bar Harbor, ME). Va14Ja281-/- NKT

KO mice were originally obtained through Dr. M. Taniguchi

at Chiba University (Chiba, Japan). The cC KO and the

iNKT KO mice were bred and housed in the Wegner Hall

Vivarium. Female KO mice from the three different strains

all on a C57BL/6 background were utilized in experiments

at 7–8 weeks of age. All mice were single housed in air filter

topped polystyrene cages. Experimental mice were ran-

domly divided into two groups. One group was provided

with sterilized double distilled, deionized water as a control

and the other another group was provided with 20% w/v

alcohol (Everclear, St. Louis, MO) as the sole drinking fluid.

Both groups had free access to Purina 5001 Rodent Labo-

ratory Chow. We previously validated this chronic model of

human alcoholism [32]. Mice given 20% w/v alcohol in the

drinking water consume[30% of their calories from alco-

hol, which is similar to the intake of human alcoholics. Mice

consumed alcohol for 3 months prior to using them in

experiments. Mice were then injected with melanoma, and

they continued to receive alcohol or water until the experi-

ments were terminated. The animal protocols used in this

research were approved by the Institutional Animal Care

and Use Committee at Washington State University.

Tumor cell culture, tumor inoculation and lung colony

counting

B16BL6 melanoma cells were cultured in Dulbecco’s

Modified Eagle’s Medium supplemented with 10% fetal

bovine serum, and 1% penicillin and streptomycin. Tumor

cells at 50–70% confluence were detached with 2 mM

ethylene glycol tetracetic acid and resuspended in phos-

phate buffered magnesium- and calcium-free normal saline

(CMF). Mice were inoculated with 5 9 104 melanoma

cells in 200 ll of CMF i.v. via the lateral tail vein and then

assessed for lung metastasis 3 weeks later. The lungs were

fixed at necropsy with Fekete’s solution and tumor colonies

on the 5 lobes of the lung were counted using a stereo

microscope.

Isolation and phenotyping of splenocytes and peripheral

blood leukocytes (PBL)

Splenocytes from C57BL/6 mice and PBL from cC KO

mice were isolated and stained with appropriately labeled

antibodies, and the cellular phenotype was determined by

flow cytometry according to previously published proce-

dures [33]. Cells were labeled with fluorescein isothiocy-

anate (FITC), phycoerythrin (PE), peridinin chlorophyll

protein (PerCP)-Cy5.5, or PE-Cy5.5 labeled anti-mouse

antibodies. Anti-CD11b (M1/70), anti-CD4 (L3T4), and

anti-CD8 (53-6.7) were purchased from BD Biosciences

Pharmingen (San Diego, CA). Anti-NK1.1 (PK136), anti-

Ly6G/Ly-6C (Gr-1) (RB6-8C5), and anti-CD3 (145-2C11)

were obtained from BioLegend (San Diego, CA).

Gr-1? cell depletion

Gr-1? cells were depleted by injection of 200 lg of purified

rat anti-mouse Gr-1 monoclonal antibody (Clone RB6-8C5,

302 Clin Exp Metastasis (2011) 28:301–307

123

National Cell Culture Center, Biovest International, Inc.,

Minneapolis, MN) in 200 ll of PBS via the lateral tail vein

24 h before and again 30 min before melanoma inoculation.

Control water and alcohol drinking mice were injected with

purified rat anti-IgG2b (BioLegend, San Diego, CA).

Intracellular staining of IFN-c

Splenocytes from cC KO mice were isolated according to

standard procedures and after activation and staining were

analyzed for IFN-c by flow cytometry as previously

described [33]. Splenocytes were activated with 80 ng/ml

alpha-galactosylceramide (a-GalCer) in RPMI1640 med-

ium containing 10% fetal bovine serum and GolgiStop for

5–6 h, fixed, and permeabilized by Cytofix/Cytoperm

Fixation/Permeabilization Solution Kit (BD Biosciences,

San Jose, CA) before staining for IFN-c. Cells were then

stained with anti-IFN-c (XMG1.2) antibody (BD Biosci-

ences Pharmingen, San Diego, CA) and analyzed by flow

cytometry [33].

Statistical analysis

Data were analyzed with the Microsoft Excel statistical

program or GraphPad Prism 5 (GraphPad Software Inc.).

The results are expressed as the mean ± SD. Differences

between groups were determined using two-tailed unpaired

Student’s t test. Values are considered different at P \ 0.05.

Results

The antimetastatic effects induced by chronic alcohol

consumption are abrogated in IFN-c KO mice

IFN-c plays a crucial role in controlling tumor metastasis

[24–27]. Chronic alcohol consumption significantly

increases the percentage of IFN-c producing splenic

CD3-NK1.1?, CD4? T, and CD8? T cells [30, 31]. Herein,

we determined that alcohol consumption also increased the

percentage of IFN-c producing cells in the CD3?NK1.1?

(NKT) splenic cell population (46.4 ± 2.3% (SD) in water

drinking mice compared to 55.1 ± 7.0% (SD) in alcohol

drinking mice (P = 0.015, n = 7)). Since the mean fluo-

rescence intensity of these cells was not different, this

indicates that it is the proportion of CD3?NK1.1? cells that

is increased by alcohol rather than the amount of IFN-c that

is produced by these cells. Together these data provided a

strong rationale to explore the hypothesis that the antimet-

astatic activity associated with alcohol consumption in the

B16BL6 melanoma tumor model resulted from elevated

IFN-c producing cells. The results in Fig. 1 indicate that

knocking out the ifn-c gene abrogates the antimetastatic

effects associated with alcohol consumption.

Depletion of NK, NKT, CD8? T cells, and B cells does

not abrogate the antimetastatic effects induced

by chronic alcohol consumption

We next sought to determine the cell type that played the

key role in producing IFN-c associated with inhibition of

B16BL6 melanoma metastasis. NK cells, NKT cells and

CD8? T cells are major producers of IFN-c; therefore, we

used a murine model for X-linked severe combined

immunodeficiency disease in humans that results in marked

deficiency or lack of NK and T cells. Previous studies in

male mice lacking cc expression (cC-/Y) indicated an

absence of NK and CD8? T cells in the spleen and greatly

diminished percentages of B cells [34]. Herein, we showed

similar results in the PBL from female cC KO (cC-/-)

mice (Fig. 2a–c). The results in Fig. 2d and e show that the

antimetastatic effect of alcohol consumption is not

impaired (Fig. 2d, e), and they suggest that NK, NKT, and

CD8? T cells are not critical to the antimetastatic effect.

Depletion of Gr-1? cells does not abrogate

the antimetastatic effects of alcohol consumption in cC

KO mice

Flow cytometric analysis revealed that CD11b? cells are a

dominant population in the PBL of cC KO mice (Fig. 3a).

These cells are a heterogeneous cell population comprised

of neutrophils, monocytes, and marcophages, and we found

that 40% of the PBL were CD11b?Gr-1? cells. These

CD11b?Gr-1? cells produce IFN-c upon activation [26].

Fig. 1 The effect of chronic alcohol consumption on experimental

lung metastasis of B16BL6 melanoma is abrogated in IFN-c KO

mice. Female IFN-c KO mice received alcohol for 3 months and then

were inoculated via tail vein with 5 9 104 B16BL6 melanoma cells.

Mice were euthanized 3 weeks after tumor inoculation. The figure

shows the distribution of lung colony numbers in which no

differences were observed between water and ETOH (alcohol)

drinking groups (P = 0.49). The bar indicates the mean number of

lung tumor colonies. Data are representative of two experiments with

similar results

Clin Exp Metastasis (2011) 28:301–307 303

123

The percentage of these cells increases significantly in the

blood of cC KO mice 2 days after i.v. B16BL6 inoculation

(Fig. 3b). Thus, the CD11b?Gr-1? cells could play a role

in the inhibition of B16BL6 lung colonization in the

alcohol drinking mice. To examine this hypothesis, anti-

Gr-1 monoclonal antibody was used to deplete the Gr-1?

cells. Figure 3c shows that the majority of the Gr-1? cells

were depleted after two injections of anti-Gr-1 antibody.

However, the depletion of Gr-1? cells did not abrogate the

antimetastatic effects induced by chronic alcohol con-

sumption in the cC KO mice (Fig. 3d). Liver tumor colo-

nies were observed in 10 out of 10 water drinking mice and

in 7 out of 10 alcohol consuming mice. The total number of

liver tumor colonies observed in the water drinking mice

was 42 compared to 23 in the alcohol consuming mice;

however, the difference between the two groups was not

significant (P = 0.2 by Mann–Whitney test).

Depletion of iNKT cells did not abrogate

the antimetastatic effects induced by chronic alcohol

consumption

Although cC KO mice are essentially devoid of

NK1.1?CD3? NKT cells in the blood (Fig. 2a), when

splenocytes from cC KO mice were stimulated with aGalCer

in vitro, the percentage of IFN-c producing cells were sig-

nificantly higher in the alcohol drinking than in the water

drinking mice (Fig. 4a, b). Additional phenotypic analysis

indicated that all of the IFN-c producing cells are Gr-1-CD

11b- CD3?. Gating on only the IFN-c producing cells, we

found that all cells are CD3?. Eleven percent of these cells are

CD3?NK1.1?. No CD3-NK1.1? cells were found in the

IFN-c producing cell population. The basis for the increase is

not clear; however, since aGalCer is the T-cell receptor ligand

of iNKT cells, this led us to further examine if iNKT cells play

a role in controlling B16BL6 lung metastasis in alcohol

drinking mice. The results in Fig. 4c indicate that iNKT KO

mice retain their sensitivity to the antimetastatic effect of

alcohol consumption, although the magnitude of the difference

between the lung colony numbers in the alcohol and water

drinking mice is not as great as in the cC KO mice (Fig. 2e).

Discussion

Little information exists regarding the effects of chronic

alcohol consumption on tumor progression and metastasis.

Using a mouse model of chronic alcohol consumption that

mimics intake in human alcoholics, we found that B16BL6

melanoma metastasis to the lung is greatly decreased

Fig. 2 Chronic alcohol consumption decreases lung metastasis of

B16BL6 melanoma in cC KO mice. Female cC KO mice received

alcohol for 3 months and then were inoculated via the tail vein with

5 9 104 B16BL6 melanoma cells. Mice were euthanized 3 weeks

after tumor inoculation. a Dot plot indicating the deficit of NK (upperleft quadrant) and NKT (upper right quadrant) in peripheral blood.

b Dot plot indicating the presence of CD8? T cells (upper leftquadrant) and CD4? T cells (lower right quadrant) in peripheral

blood. c Dot plot indicating the presence of CD19? B cells (upper leftquadrant) in the peripheral blood. d Representative example of

B16BL6 melanoma colonies on the lungs of mice given water or

ETOH (alcohol). e Distribution of the number of tumor colonies on

the lung of water and alcohol drinking mice. The bar indicates the

mean number of lung tumor colonies. Water drinking group different

from ETOH (alcohol) drinking group, P = 0.003

304 Clin Exp Metastasis (2011) 28:301–307

123

between 42 and 94% [19–21]. Our previous findings [19,

21] and the results of others [24, 25] indicate that NK cell

cytolytic activity is not the major mechanism underlying

control of melanoma metastasis. Until now the mechanisms

associated with this effect have not been elucidated. The

results of this investigation showed that IFN-c is essential

for the antimetastatic activity associated with alcohol

consumption. These results are consistent with other well-

documented reports that IFN-c is the crucial factor that

controls the metastasis of mesothelioma, breast cancer, as

well as melanoma [24–29]. A variety of IFN-c producing

cells were found to have a primary role in controlling

metastasis in these studies. IFN-c produced by tumor spe-

cific T cells was found to play a key role in control of

mesothelioma metastasis [28]. Phagocytic cells were found

to be the main source of IFN-c production that controls 4T1

breast cancer metastasis [26]. In this model, even small

amounts of endogenous of IFN-c produced by hematopoi-

etic cells within the tumor microenvironment had an

impact on metastasis [29].

Fig. 3 The antimetastatic effect

of chronic alcohol consumption

is sustained in CD11b?Gr-1?

depleted cC KO mice. a Dot

plot showing a high proportion

of CD11b?Gr-1? cells (graydots) in the PBL of water

drinking cC KO mice.

b Dynamic changes in the

percentage of CD11b?Gr-1?

cells in the PBL of cC KO mice

before (day 0) and after (day 1

and day 2) inoculation of

5 9 104 B16BL6 melanoma via

the lateral tail vein. * Different

from day 0, P \ 0.05. c Dot plot

showing depletion of

CD11b?Gr-1? cells in the PBL

of water drinking cC KO mice.

d Numbers of B16BL6

melanoma colonies on the lung

of water and ETOH (alcohol)

drinking cC KO mice after

depletion of Gr-1? cells. Water

drinking group different from

ETOH (alcohol) drinking group,

P = 0.03

Fig. 4 Chronic alcohol consumption increases aGalCer induced IFN-

c producing cells in the splenocytes from cC KO mice and inhibits

B16BL6 melanoma lung metastasis in iNKT KO mice. a Histogram

of the IFN-c producing splenocytes from water and ETOH (alcohol)

drinking cC KO mice. b Percentage of IFN-c producing cells in the

splenocytes of water drinking and ETOH (alcohol) drinking cC KO

mice after stimulation for 5–6 h with aGalCer in vitro. Water drinking

group different from ETOH (alcohol) drinking group, P = 0.04.

c Number of colonies in the lung of water drinking and alcohol

drinking iNKT mice. Water drinking group different from ETOH

(alcohol) drinking group, P = 0.007

Clin Exp Metastasis (2011) 28:301–307 305

123

In the present study, we were unable to identify one

specific cell type that was crucial to the production of IFN-

c and to the antimetastatic effect of alcohol consumption.

We found that knockout of NK, NKT, and CD8? T cells

that are major producers of IFN-c and also depletion of Gr-

1? cells that also can produce IFN-c did not abrogate the

antimetastatic effects of alcohol as expected. Even though

Cao et al. [34] found that IFN-c production was dramati-

cally reduced in cC KO mice, a very small amount was still

produced after stimulation of splenocytes with anti-CD3

and anti-CD28. Thus, it is possible that alcohol consump-

tion could facilitate production of IFN-c in residual cell

populations of the cC KO mice and that the additional

amount of IFN-c produced was sufficient to maintain the

antimetastatic effect in the alcohol drinking mice. This is

not an unreasonable possibility given the similar finding of

duPre et al. [29] in the 4T1 model of breast cancer. We did

not exclude a role for monocytes and macrophages in the

cC KO mice. It is also well known that alcohol consump-

tion increases the permeability of the gut to bacteria, which

can lead to an increase in the concentration of the bacterial

endotoxin, lipopolysaccharide, in the blood. This in turn

can activate monocytes and macrophages to produce IFN-c[35, 36]. Lipopolysaccharide also is known to induce tumor

regression as well as inhibit tumor growth in other exper-

imental tumor systems, an effect that is blocked by neu-

tralizing IFN-c [22].

We also found that treating splenocytes from cC KO

mice with aGalCer enhanced IFN-c production in alcohol

drinking mice compared to water drinking mice (Fig. 4b).

While we do not understand the underlying basis for this

finding, it is possible that alcohol facilitates production of

IFN-c in a unique population of iNKT cells that are not

dependant on the cC receptor for activity. This finding

provided additional rationale for examining the contribu-

tion of the specific role of iNKT cells to the antimetastatic

effect of alcohol. However, we found again that the anti-

metastatic effect of alcohol consumption was not abrogated

in the iNKT KO mice.

In summary, we showed that chronic alcohol con-

sumption facilitates the generation of IFN-c producing

cells and that IFN-c is essential to inhibition of murine

B16BL6 melanoma lung metastasis. Our findings support

those of duPre et al. [29] suggesting that only small

amounts of IFN-c are necessary to inhibit metastasis. In

addition, we found that no one IFN-c producing cell type

was the source of the antimetastatic effect in melanoma

associated with chronic alcohol consumption. The under-

lying mechanism associated with IFN-c is still unclear and

is the subject of ongoing research.

Acknowledgments The authors thank Dr. Danny Welch (Univer-

sity of Alabama, Birmingham, AL, USA) for reading the manuscript

and for his constructive comments. This work was supported by the

National Institute on Alcohol Abuse and Alcoholism grants R01A

A07293 and K05AA017149.

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