Cancer Prevention Research Center and the Department of Pharmaceutical Sciences, College of Pharmacy, Washington State University, Pullman, Washington 99164-6510
Received November 22, 2002; accepted January 23, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: NFB; AP-1; NK cells; IL-2; ethanol; perforin; granzyme A; granzyme B.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It is notable that EtOH exposure influences mRNA expression of a variety of genes and the activity of transcription factors. For instance, acute EtOH treatment in vitro inhibits LPS-stimulated mRNA expression of TNF-, IL-1ß, and IL-6, as well as NF-
B activity in monocytes and Küpffer cells (Mandrekar et al., 1997
; Szabo et al., 1995
). Transcription factors such as NF-
B, AP-1, C/EBP, and STAT, which are associated with the inhibition of mRNA expression of a variety of genes, are modulated in these cells (Chen et al., 1999
; Fox et al., 1996
; Mandrekar et al., 1999
; Zeldin et al., 1996
). No information is available regarding the effect of EtOH consumption in vivo on mRNA expression and transcription factor levels in NK cells after stimulation with IL-2.
In this paper, we further investigate whether decreased protein levels of perforin, granzyme A, and granzyme B by EtOH consumption in vivo are related to decreased gene expression and binding activity of NF-B, AP-1 in response to IL-2 stimulation in enriched murine NK cells. Our data show that EtOH consumption inhibits mRNA expression of perforin, granzyme A, and granzyme B, and the activity of NF-
B and AP-1. These data strongly suggest that EtOH consumption inhibits IL-2induced cytolytic activity of NK cells in part through interfering with the transactivation of genes integrally involved in the cytolytic process. This conclusion is supported by additional findings that the PDTC and TPCK, through inhibition of binding activity of NF-
B and AP-1 in EMSA, and through inhibition of perforin, granzyme A, and granzyme B mRNA expressions, block IL-2induced NK cytolytic activity.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mice, diet, and alcohol administration.
Five- to six-week-old specific pathogen-free female C57BL/6 mice were obtained from Charles River Laboratory (Wilmington, MA). The vivarium is accredited by the American Association for Accreditation of Laboratory Animal Care, and the animal protocol was approved by the Institutional University Animal Care Committee at Washington State University. Mice were acclimated to individual housing in polycarbonate cages for an additional week before administration of EtOH. The single housing paradigm was used so that adequate ethanol and food consumption could be monitored and to be consistent with previous studies. Collecting the nutritional parameters is important since nutritional status can modulate immune function. Single housing can be stressful; however, we addressed this issue previously and found that neither alcohol intake nor the housing paradigm elevated plasma glucocorticoid levels (Sipp et al., 1993).
Mice were fed water or 20% w/v ethanol as the sole fluid source for 4 weeks and also given pelleted rodent chow (Purina 5001) ad libitum. Ethanol and food consumption were monitored throughout the studies. All the monitored parameters were within the ranges of previous studies in our laboratory, where we clearly showed that 20% w/v ethanol inhibited inherent NK activity, IL-2 stimulated NK activity, and lymphokine-activated killer activity (Blank et al., 1991, 1992
; Gallucci et al., 1994
; Meadows et al., 1993
, 1989
). There were no differences in the total caloric intake or in overall body weight between water-drinking mice and alcohol-consuming mice at the time of the assay. EtOH-consuming mice did not exhibit any overt signs of toxicity or malnutrition. Mice were routinely analyzed for the common murine pathogens and found to be negative for Sendai virus, pneumonia virus, Mycoplasma pulmonis, minute virus, hepatitis virus, Reo virus, cytomegalovirus, and Theilers encephalolmyelitis virus. At the end of the EtOH-feeding period, mice were euthanized and their spleens were removed.
NK cell enrichment.
Enriched NK cells from spleens were used in all experiments. NK cells were enriched by negative selection from mixed splenocytes from 14 to 15 mice as previously described and modified herein (Gallucci and Meadows, 1995). Briefly, spleen cell suspensions were produced by forcing the spleen through a wire-mesh screen. Red blood cells were eliminated from the spleen mixture by hypotonic lysis or Lympholyte M (Accurate Chemical and Scientific Corp., Westbury, NY) gradient centrifugation separation. The splenocytes were resuspended in PBS buffer containing 0.1% BSA, and exposed to biotinylated IgG antibodies, including anti-CD8, anti-CD4, anti-CD19, anti-
TcR (GL3), antimacrophage (F4/80), antiMHC class II (I-Ab), antierythroid cell (TER-119), and antigranulocyte (Gr-1) at 4°C for 15 min. Unlabeled anti-FcR
(2.4G2) was added to block Fc receptor on the NK cell surface. The spleen cell suspension was washed twice with PBS and then treated with streptavidin microbeads (10:1 dilution) for 15 min at 4°C. The NK cells were then enriched by a MACS or auto-MACS cell separator system (Miltenyi Biotech, Auburn, CA), according to the manufacturers instructions. Purity was evaluated by flow cytometry and based on the percentage of NK 1.1+ cells. Total B cells, T cells, and macrophages were reduced to <1% of the eluted cells. Total enrichment in NK cells varied between 65 and 75% and viability ranged between 90 and 95%.
Cell culture.
The enriched NK cells from water-drinking and ethanol-drinking mice were divided into unstimulated and IL-2stimulated groups, respectively. The IL-2treated groups were stimulated with 1000 U/ml IL-2 in complete RPMI 1640 medium supplemented with 10% fetal bovine serum and cultured in a humidified incubator at 37°C and 5%CO2 for 4 h or 20 h indicated below.
Electrophoretic mobility shift assay.
Nuclear extracts were prepared as described previously (Zhou et al., 1999). From each extract, 6 µg nuclear protein was incubated with 32P-labeled NF
B (5'-AGT TGA GGG GAC TTT CCC AGG C-3') and AP-1 (5'-CGC TTG ATG AGT CAG CCG GAA-3') oligonucleotides (Promega, Madison, WI), respectively, in 10 µl of binding buffer (50 mM TrisHCl, pH 7.5, 20% glycerol, 5-mM MgCl2, 2.5-mM EDTA, 2.5 mM dithiothreitol (DTT), 250-mM NaCl and 0.25-mg/ml poly (dI-dC)] for 15 min at room temperature before loading onto 6% polyacryamide gels. For the supershift assays, the nuclear protein reaction mixture was incubated with antibodies against p50 and p65 for 30 min at room temperature. For the competition assays, 50-fold excess of unlabeled NF-
B and AP-1 oligonucleotides were used to challenge binding of labeled NF-
B and AP-1 oligonucleotides. The binding mixtures were run in 1x Tris borate buffer. The gel was then dried and exposed to autoradiographic film overnight at -70°C.
Semi-quantitative RT-PCR and real-time PCR.
Enriched NK cells were obtained from the pooled splenocytes of 7 to 8 water-drinking or EtOH-drinking mice. The pooled NK cells from both water-drinking and EtOH-drinking animals were treated in the presence or absence of 1000 IU/ml IL-2 for 20 h. Untreated NK cells were subjected to RNA isolation immediately after enrichment. Total RNA was extracted from the pooled splenic NK cells using a Qiagen RNeasy Mini kit. The reverse transcriptase (RT) step was conducted with 0.5 µg of total RNA per sample in a total volume of 20 ml, using the random primer and Superscrip preamplification system. Thirty-ng cDNA samples of RT were subjected to PCR amplification using synthetic oligonucleotide primers. The primers for the perforin cDNA are 5'-AGC CCC TGC ACA CAT TAC TG-3' / 5'-CCG GGG ATT GTT ATT GTT CC-3', for granzyme A are 5'-ATT CCT GAA GGA GGC TGT GAA-3' / 5'-GCA GGA GTC CTT TCC ACC AC-3', and for granzyme B are 5'-GCC CAC AAC ATC AAA GAA CAG-3' / 5'-AAC CAG CCA CAT AGC ACA CAT-3' (Makrigiannis et al., 1997). These primers generate 349 bp, 560 bp, and 213 bp PCR products, respectively. Primers for cyclophilin A are 5'-ATT TGG CTA TAA GGG TTC CTC-3' / 5'-ACG CTC CAT GGC TTC CAC AAT-3'. The amplified 291-bp PCR product of cyclophilin A was used as an internal control for quantification. PCR amplifications were performed using 0.25 µl Taq DNA polymerase in 25-µl reaction assemblies. To determine the linear region for the cycles of perforin, granzyme A, and granzyme B PCR signals, the PCR amplifications were calculated from densitometric measurement of the ethidium bromide-stained agarose gels and plotted on a logarithmic scale against the cycle number. The samples were amplified using 25 cycles for perforin, 26 cycles for granzyme A, 23 cycles for granzyme B, and 24 cycles for cyclophilin A at 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min 30 sec. These amplification cycles were predetermined to be in linear range relative to cycle number and band density. PCR products were analyzed by electrophoresis on a 1.5% agarose gel and visualized by staining with ethidium bromide.
The effect of EtOH consumption on the mRNA levels of perforin, granzyme A, and granzyme B in NK cells was further investigated by real-time PCR. This was performed on an iCycler iQ Multi-Color Detection System (Bio-Rad Laboratories, CA). Fifteen ng cDNA templates of RT were used for PCR amplification of perforin, granzyme A, granzyme B, and cyclophilin A, with 1 pmol of corresponding primer pairs as described above. The PCR reaction mixture (25 µl) also contained 60 U/ml platinum Taq DNA polymerase, 40 mM Trisx HCl, pH 8.4, 100 mM KCl, 6 mM MgCl2, 400 µM dNTP, 800 µM dUTP, 40 U/ml UDG, and 2.5 µl of SYBR green (1:10,000 dilution). The samples were amplified in duplicate at 50°C for 3 min and 95°C for 2 min followed by 40 cycles at 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min 30 sec. Cyclophilin was amplified as a quantification control. The fluorescence of SYBR green was measured at the end of each cycle using the comparative threshold cycle (Ct) method (User Bulletin 2 ABI PRISM 7700 sequence detection system, PE Application Biosystems).
Densitometric analyses of the data presented in Figures 1 and 2
and Table 1
were performed using NIH Image, V.1.54 for one-dimensional gels (Scion Corp., Frederick, MD). The relative difference in the intensity between two bands was expressed as fold induction or fold difference with respect to the appropriate control. The relative quantification between IL-2treated and untreated groups presented in Table 1
was determined following an arithmetic formula:
|
|
|
![]() |
for each group of data. Ct is a value of each reaction of each gene.
Cell viability assay.
The potential cytotoxicity of PDTC and TPCK was assessed by using the method of Mosmann (1983). NK cells were treated with each compound individually for 12 h, and then the medium was removed. One µl of RPMI 1642 (without phenol red) was added to each well followed by 100 ml of MTT prepared at 5 mg/ml. Cells were incubated for 3 h at 37°C. The blue formazan dye generated from MTT was solubilized with 1 ml of isopropanol in 0.04 M HCl. The resulting absorbance was measured at 570 nm.
Cytolytic assay (51Cr release assay).
Enriched splenic NK cells were stimulated with 1000 U/ml IL-2 in the presence or absence of 100 nM PDTC and 20 µM TPCK for 12 h and then analyzed in a standard 4-h 51Cr release assay as previously described (Gallucci et al., 1994). All assays and controls were performed in triplicate and the results were replicated in three different experiments. Cytolytic activity was calculated as the mean percentage of lysis of samples. These values were also converted to lytic units according to the method of Pross et al. (1981)
.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
EMSA was used to determine the potential inhibitory effect of EtOH consumption on the NF-B and AP-1 activity. In Figure 1A
, the data show that EtOH consumption inhibits IL-2induced NF-
B binding activity in enriched NK cells as compared to the water-drinking controls. The relative reduction in the induction of NF-
B in response to IL-2 was 3.6-fold. NF-
B was not detectable in untreated NK cells. Figure 1B
shows that AP-1 binding activity also is not constitutively present in NK cells irrespective of EtOH-consumption. IL-2induced AP-1 binding activity is reduced by 4.8-fold in the NK cells from EtOH-consuming mice as compared to water-drinking mice.
EtOH inhibits IL-2induced perforin, granzyme A, and granzyme B mRNA expression.
To investigate whether the inhibition of EtOH consumption is associated with the mRNA levels of perforin, granzyme A, and granzyme B, semi-quantitative RT-PCR and real time PCR experiments were conducted. The mRNA for perforin, granzyme A, and granzyme B is constitutively expressed at a low level in the untreated NK cells from both water- and EtOH-drinking groups, and there is no difference in expression between the groups. Treatment of murine NK cells with IL-2 leads to augmentation in the mRNA expression of perforin, granzyme A, and granzyme B in both water-drinking and EtOH-drinking groups (see Figs. 2A2C
).
However, the degree of increase was lower in the EtOH-consuming mice relative to water-drinking mice. Stimulation of perforin expression in water-drinking mice by IL-2 was increased by 3-fold as compared to a 1.5-fold in NK cells from EtOH-drinking groups (Fig. 2A). Granzyme A expression was increased 3.3-fold in water-consuming mice and 2.1-fold in EtOH-drinking mice (Fig. 2B
). Granzyme B expression was stimulated 5.8-fold by IL-2 in water-drinking mice and 4.2-fold in EtOH-drinking groups (Fig. 2C
).
The results from real-time PCR analysis in Table 1 show similar changes in perforin, granzyme A and granzyme B mRNA expression as compared to the changes measured by semiquantitative PCR analysis. A consistent finding from both analyses is that EtOH has the greatest inhibitory effect on perforin expression, which is essential for the cytolytic activity of NK cells (Kägi et al., 1994
; Simon et al., 1997
).
Inhibitors of NF-B and AP-1 reduce mRNA levels of perforin, granzyme A, granzyme B, and block IL-induced NK cytolytic activity.
PDTC and TPCK were used to address the roles of NF-B and AP-1 in the IL-2induced mRNA expression of perforin, granzyme A, and granzyme B, and NK cell cytolytic activity. It is known that PDTC prevents phosphorylation of I
B
as well as its subsequent degradation, thus inhibiting NF
B (Meyer et al., 1993
; Schreck et al., 1992
; Traenckner et al., 1994
). TPCK prevents the degradation of I
B proteins (Henkel et al., 1993
; Mellits et al., 1993
). Both agents have been applied in numerous cell types to study NF-
B-dependent events. In addition, PDTC is known to inhibit AP-1 as well as NF
B (Blazquez et al., 1997
; Li et al., 1997
).
Nuclear binding activity of NFB, as examined by EMSA, is shown in Figure 3
. The data in Figure 3A
show that there is a constitutive, faster-migrating binding band in both untreated and IL-2treated NK cells. Stimulation of NK cells with IL-2 resulted in an additional, slower-migrating, inducible binding band. In Figure 3A
, 100 nM PDTC and 20 µM TPCK completely abolished the IL-2induced slower-migrating band, whereas the faster-migrating bands were unaffected. Competition assay showed that 50-fold cold NF
B oligomer completely blocks the IL-2induced slower-migrating binding bands; while 50-fold AP-1 oligomer was not able to affect this binding band (see Fig. 3B
). This suggests that the IL-2induced band is specific for NF
B binding band. The supershift assay data in Figure 3B
show that anti-p50 antibody further retarded the mobility of the slower-migrating band. Although, anti-p65 antibody did not result in any supershift band of NF
B, the slower-migrating binding band was greatly reduced. These results indicate that the IL-2induced slower-migrating binding band is composed of p50 and p65 subunits. Anti-p50 and anti-p65 antibodies did not affect the constitutive, faster-migrating band.
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Transcription factors, which bind to cis regulatory elements, play critical roles in the regulation of de novo mRNA synthesis (Grimm and Bauerle, 1993). A variety of regulatory elements are present in these regions, including NF-
B, AP-1, SP1, Ets, and c-AMP response element binding protein (CREB), and these elements are found in the 5' untranslated regions of murine perforin, granzyme A and granzyme B genes (Babichuk et al., 1996
; Ebnet et al., 1992
; Koizumi et al., 1993
; Youn et al., 1991
). The 5'-flanking region of the murine perforin gene is homologous to the analogous region of the human gene (Lichtenheld et al., 1989
). Perforin gene expression is regulated by IL-2Rß signals through two enhancers that reside 15 kb (Zhang et al., 1999
) and 1 kb upstream of the promoter (Sica et al., 1997
; Yu et al., 1999
; Zhang et al., 1999
). STAT5 transcription factors are also essential for NK cell development (Moriggle et al., 1999
) and STAT5 binding sites are present in both upstream promoters and enhancers of perforin (Yu et al., 1999
; Zhang et al., 1999
). STAT5 is activated by the well-defined JAK/STAT pathway emerging from the IL-2R (Beadling et al., 1994
) and also through IL-15R (Lin et al., 1995
; Nelson and Willerford, 1998
). Recently we showed that IL-2R signaling also activates an NF-
B signaling pathway in NK cells, and that this pathway is involved in the control of perforin expression in NK cells (Zhou et al., 2002
). Thus, the inhibition of NF
B binding activity in NK cells related to EtOH consumption is highly likely to be correlated to the decrease in perforin mRNA expression. Interestingly, EtOH consumption does not affect expression of STAT5 protein in splenic NK cells from C57BL/6 mice (unpublished results).
Less is known about the specific regulation of granzyme expression in NK cells and the role of NF-B and AP-1 in regulation of these genes. Mutational studies of murine granzyme B promoter show that it is regulated by AP-1, cyclic AMP-responsive element (CRE), Ikaros and core-binding factor (CBF/PEBP2) in cytotoxic T cells (Babichuk et al., 1997
). The fact that EtOH decreases AP-1 binding activity in murine NK cells suggests an important role for this transactivation factor in NK functioning and is a fertile area for future research.
PDTC and TPCK have been widely used to block the NF-B pathway directly and indirectly (Henkel et al., 1993
; Li et al., 1995
; Schreck et al., 1992
; Xie, QW, et al., 1994
; Zhou et al., 1999
); however, as described below, these inhibitors also block AP-1. Both compounds suppress cytolytic activity in human NK cells (Blazquez et al., 1997
), and we confirmed this in murine NK cells. In addition, both compounds dramatically decreased perforin and granzyme-A expression and, to a lesser extent, granzyme-B expression. Thus, there is a strong association between the cytolytic activity, and NF-
B and AP-1 binding activity in regulation of perforin, and granzyme mRNA expression in response to IL-2 stimulation in NK cells. It is likely that decreased NF-
B and AP-1 binding activity and decreased mRNA and protein expression of perforin, granzyme A, and granzyme B only partly explain the decrease in IL-2stimulated NK cell activity, since we previously showed that target-induced release of granzymes is also affected by EtOH consumption (Spitzer et al., 1999
).
PDTC and TPCK, although they inhibited various functions associated with NK cytolytic activity, do so in a nonspecific manner. PDTC inhibits NF-B and AP-1 activity induced by PMA and TPA in human NK cells and JB6 mouse promotion-sensitive cells (Blazquez et al., 1997
; Li et al., 1997
). It has no effect on TNF-
-induced AP-1 activity in JB6 mouse promotion-sensitive cells (Li et al., 1997
), and in some cell types it increases AP-1 activity (Blazquez et al., 1997
). However, we found in this study that both inhibitors suppress IL-2induced NF-
B and AP-1 activity in murine NK cells. Thus, it is possible that they influence NK cytolytic activity and/or gene expression not only through inhibition of NF-
B and AP-1 activity but also through other unidentified pathways.
Because NF-B and AP-1 are not constitutively active in resting NK cells, it is unlikely that these transactivation factors influence the cytolytic activity of resting NK cells. The most likely explanation for the influence of EtOH on the baseline cytolytic activity is decreased activity of granzyme A and inhibition of target-induced release of granzyme A (Spitzer et al., 1999
). It is also known that EtOH consumption inhibits the cytolytic activity of NK after stimulation with IL-2 or poly I:C. This inhibition in cytolytic activity is associated with altered protein expression of perforin, granzyme A, and granzyme B by EtOH (Collier et al., 2000
; Spitzer et al., 1999
). The inhibition of NK cytolytic activity is not due to the altered expression of the IL-2 receptor (Gallucci et al., 1996
, 1994
). Previously, we also showed that expression of perforin, granzyme A, and granzyme B proteins is lower in EtOH-consuming mice as compared to water-drinking mice after stimulation of enriched NK cells in vivo with IL-2. We now show that the decrease in protein expression is associated with decreased mRNA expression and with decreased nuclear binding activity of NF-
B and AP-1. Further support for a role of NF-
B and AP-1 in regulation of cytolytic activity is the finding that PDTC and TPCK inhibit NF-
B and AP-1 binding activity and expression of perforin, granzyme A, and granzyme B as well as IL-2induced NK cell cytolytic activity. Taken together, these data indicate that NF-
B and AP-1 are involved the control of NK cytolytic activity and suggest that the inhibition of NK cytolytic activity by EtOH consumption in part results from decreased activity of these transactivation factors.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
NOTES |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Babichuk, C. K., Duggan, B. L., and Bleackley, R. C. (1996). In vivo regulation of murine granzyme B gene transcription in activated primary T cells. J. Biol. Chem. 271, 1648516493.
Beadling, C., Guschin, D., Witthuhn, B. A., Ziemiecki, A., Ihle, J. N., Kerr, I. M., and Cantrell, D. A. (1994). Activation of JAK kinases and STAT proteins by interleukin-2 and interferon , but not the T-cell antigen receptor in human T lymphocytes. EMBO J. 13, 56055615.[Abstract]
Blank, S. E., Duncan, D. A., and Meadows, G. G. (1991). Suppression of natural killer-cell activity by ethanol consumption and food restriction. Alcohol Clin. Exp. Res. 15, 1622.[ISI][Medline]
Blank, S. E., Johansson, J.-O., Origines, M. M., and Meadows, G. G. (1992). Modulation of NK cell activity by moderate intensity endurance training and chronic ethanol consumption. J. Appl. Physiol. 72, 814.
Blank, S. E., Pfister, L. J., Gallucci, R. M., and Meadows, G. G. (1993). Ethanol-induced changes in peripheral blood and splenic NK cells. Alcohol Clin. Exp. Res. 17, 561565.[ISI][Medline]
Blazquez, M. V., Luque, I., Collantes, E., Aranda, E., Solana, R., Pena, J., and Munoz, E. (1997). Cellular redox status influences both cytotoxic and NF-B activation in natural killer cells. Immunology 90, 455460.[CrossRef][ISI][Medline]
Chen, J., Kunos, G., and Gao, B. (1999). Ethanol rapidly inhibits IL-6-activated STAT3 and C/EBP mRNA expression in freshly isolated rat hepatocytes. FEBS Lett. 457, 162168.[CrossRef][ISI][Medline]
Collier, S. D., and Pruett, S. B. (2000). Mechanisms of suppression of poly I:C-induced activation of NK cells by ethanol. Alcohol 21, 8795.[CrossRef][ISI][Medline]
DeBlaker-Hohe, D. F., Yamauchi, A., Yu, C. R., Horvath-Arcidiacono, J. A., and Bloom, E. T. (1995). IL-12 synergizes with IL-2 to induce lymphokine-activated cytotoxicity and perforin and granzyme gene expression in fresh human NK cells. Cell Immunol. 165, 3343.[CrossRef][ISI][Medline]
Ebnet, K., Kramer, M. D., and Simon, M. M. (1992). Organization of the gene encoding the mouse T-cell-specific serine proteinase "granzyme A". Genomics 13, 502508.[ISI][Medline]
Ericsson, C. D., Kohl, S., Pickering, M. D., Davis, J., Glass, G. S., and Faillace, L. A. (1980). Mechanisms of host defense in well nourished patients with chronic alcoholism. Alcohol Clin. Exp. Res. 4, 261265.[ISI][Medline]
Fox, E. S., Cantrell, C. H., and Leingang, K. A. (1996). Inhibition of the Kupffer cell inflammatory response by acute ethanol: NF- B activation and subsequent cytokine production. Biochem. Biophys. Res. Commun. 225, 134140.[CrossRef][ISI][Medline]
Gallucci, R. M., and Meadows, G. G. (1995). Ethanol consumption reduces the cytolytic activity of lymphokine-activated killer cells. Alcohol Clin. Exp. Res. 19, 402409.[ISI][Medline]
Gallucci, R. M., and Meadows, G. G. (1996). Ethanol consumption suppresses the IL-2induced proliferation of NK cells. Toxicol. Appl. Pharmacol. 138, 9097.[CrossRef][ISI][Medline]
Gallucci, R. M., Pfister, L. J., and Meadows, G. G. (1994). The effects of ethanol consumption on enriched natural killer cells from C57BL/6 mice. Alcohol Clin. Exp. Res. 18, 625631.[ISI][Medline]
Grimm, S., and Baeuerle, A. (1993). The inducible transcription factor NF-B: Structure-function relationship of its protein subunits. Biochem. J. 290, 297308.[ISI][Medline]
Henkel, T., Machleldt, T. A. I., Krönke, M., Ben-Neriah, Y., and Baeuerle, P. A. (1993). Rapid proteolysis of IB-
is necessary for activation of transcription factor NF-
B. Nature 365, 182185.[CrossRef][ISI][Medline]
Herberman, R. B., Reynolds, C. W., and Ortaldo, J. R. (1986). Mechanism of cytotoxicity by natural killer (NK) cells. Annu. Rev. Immunol. 4, 651680.[CrossRef][ISI][Medline]
Kägi, D., Ledermann, B., Bürki, K., Seiler, P., Odermatt, B., Olsen, K. J., Podack, E. R., Zinkernagel, R. M., and Hengartner, H. (1994). Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin-deficient mice. Nature 369, 3137.[CrossRef][ISI][Medline]
Koizumi, H., Horta, M. F., Youn, B.-S., Fu, K.-C., Kwon, B. S., Young, J. D.-E., and Liu, C.-C. (1993). Identification of a killer-cell-specific regulatory element of the mouse perforin gene: An Ets-binding site-homologous motif that interacts with Ets-related proteins. Mol. Cell Biol. 13, 66906701.[Abstract]
Kornbluth, J., and Hoover, R. C. (1988). Changes in gene expression associated with IFN-ß and IL-2induced augmentation of human natural killer cell function. J. Immunol. 141, 32343240.
Li, C. C., Dai, R. M., and Longo, D. L. (1995). Inactivation of NF-B inhibitor I
B
: ubiquitin-dependent proteolysis and its degradation product. Biochem Biophys Res Commun. 215, 292301.[CrossRef][ISI][Medline]
Li, J. J., Westergaard, C., Ghosh, P., and Coburn, N. H. (1997). Inhibitors of both nuclear factor-kappa ß and activator protein-1 activation block the neoplastic transformation response. Cancer Res. 57, 35693576.[Abstract]
Lichtenheld, M. G., and Podack, E. R. (1989). Structure of the human perforin gene. A simple gene organization with interesting potential regulatory sequences. J. Immunol. 143, 42674274.
Lin, J. X., Migone, T. S., Tsang, M., Friedmann, M., Weatherbee, J. A., Zhou, L., Yamauchi, A., Bloom, E. T., Mietz, J., John, S., and Leonard, W. J. (1995). The role of shared receptor motifs and common Stat proteins in the regulation of cytokine pleiotropy and redundancy by IL-2, IL-4, IL-7, IL-13, and IL15. Immunity 2, 331339.[ISI][Medline]
Lu, P., Garcia-Sanz, J. A., Lichtenheld, M. G., and Podack, E. R. (1992). Perforin expression in human peripheral blood mononuclear cells: Definition of an IL-2independent pathway of perforin induction in CD8+ T cells. J. Immunol. 148, 33543360.
MacGregor, R. R. (1986). Alcohol and immune defense. J. Amer. Med. Assn. 256, 14741479.[CrossRef][ISI][Medline]
Makrigiannis, A. P., and Hoskin, D. W. (1997). Inhibition of CTL induction by rapamycin: IL-2 rescues granzyme B and perforin expression but only partially restores cytotoxic activity. J. Immunol. 159, 47004707.[Abstract]
Mandrekar, P., Catalano, D., and Szabo, G. (1997). Alcohol-induced regulation of nuclear regulatory factor-B in human monocytes. Alcohol Clin. Exp. Res. 21, 988994.[ISI][Medline]
Mandrekar, P., Catalano, D., and Szabo, G. (1999). Inhibition of lipopolysaccharide-mediated NF-B activation by ethanol in human monocytes. Int. Immunol. 11, 17811790.
Martinez-Martinez, S., Gomez del Arco, P., Armesilla, A. L., Aramburu, J., Luo, C., Rao, A., and Redondo, J. M. (1997). Blockade of T-cell activation by dithiocarbamates involves novel mechanisms of inhibition of nuclear factor of activated T cells. Mol. Cell. Biol. 17, 64376447.[Abstract]
Meadows, G. G., and Blank, S. E. (1993). Modulation of natural killer-cell activity by alcohol. In Alcohol, Immunity, and Cancer (R. Yirmiya and A. N. Taylor, Eds.), pp. 5585. CRC Press, Boca Raton.
Meadows, G. G., Blank, S. E., and Duncan, D. D. (1989). Influence of ethanol consumption on natural killer-cell activity in mice. Alcohol Clin. Exp. Res. 13, 476479.[ISI][Medline]
Mellits, K. H., Hay, R. T., and Goodbourn, S. (1993). Proteolytic degradation of MAD3 (I kappa B alpha) and enhanced processing of the NF-B precursor p105 are obligatory steps in the activation of NF-
B. Nucleic Acids Res. 21, 50595066.[Abstract]
Meyer, M., Schreck, R., and Baeuerle, P. A. (1993). H2O2 and antioxidants have opposite effects on activation of NF-B and AP-1 in intact cells: AP-1 as secondary antioxidant-responsive factor. EMBO J. 12, 20052015.[Abstract]
Moriggle, R., Topham, D. J., Teglund, S., Sexl, V., McKay, C., Wang, D., Hoffmeyer, A., van Deursen, J., Sangster, M. Y., Bunting, K. D., et al. (1999). Stat5 is required for IL-2induced cell cycle progression of peripheral T cells. Immunity 10, 249259.[ISI][Medline]
Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Meth. 65, 5563.[CrossRef][ISI][Medline]
Nelson, B. H., and Willerford, D. M. (1998). Biology of the interleukin-2 receptor. Adv. Immunol. 70, 181.[ISI][Medline]
Pross, H. F., Baines, M. G., Rubin, P., Shragge, P., and Patterson, M. S. (1981). Spontaneous human lymphocyte-mediated cytotoxicity against tumor target cells: IX. The quantitation of natural killer cell activity. J. Clin. Immunol. 1, 5163.[ISI][Medline]
Saxena, Q. B., Mezey, E., and Adler, W. H. (1980). Regulation of natural killer activity in vivo: II. The effect of alcohol consumption on human peripheral blood natural killer activity. Int. J. Cancer 26, 413417.[ISI][Medline]
Schreck, R., Meier, B., Männel, D. N., Dröge, W., and Baeuerle, P. A. (1992). Dicthiocarbamates as potent inhibitors of nuclear factor B activation in intact cells. J. Exp. Med. 175, 11811194.[Abstract]
Seitz, H. K., Poschl, G., and Simanowski (1998). Alcohol and cancer. Recent Dev. Alcohol. 14, 6795.[Medline]
Sica, A., Dorman, L., Viggiano, V., Cippitelli, M., Ghosh, P., Rice, N., and Young, H. A. (1997). Interaction of NF-B and NFAT with the interferon-
promoter. J. Biol. Chem. 272, 3041230420.
Simon, M. M., Hausmann, M., Tran, T., Ebnet, K., Tschopp, J., ThaHla, R., and Müllbacher, A. (1997). In vitro- and ex vivo-derived cytolytic leukocytes from granzyme A x B double knockout mice are defective in granule-mediated apoptosis but not lysis of target cells. J. Exp. Med. 186, 17811786.
Sipp, T. L., Blank, S. E., Lee, E. G., and Meadows, G. G. (1993). Plasma corticosterone response to chronic ethanol consumption and exercise stress. Proc. Soc. Exp. Biol. Med. 204, 184190.[Abstract]
Spitzer, J. H., and Meadows, G. G. (1999). Modulation of perforin, granzyme A, and granzyme B in murine natural killer (NK), IL-2 stimulated NK, and lymphokine-activated killer cells by alcohol consumption. Cell Immunol. 194, 205212.[CrossRef][ISI][Medline]
Szabo, G. (1999). Consequences of alcohol consumption on host defense. Alcohol 34, 830841.
Szabo, G., Mandrekar, P., and Catalano, D. (1995). Inhibition of superantigen-induced T-cell proliferation and monocyte IL-1 ß, TNF-, and IL-6 production by acute ethanol treatment. J. Leuk. Biol. 58, 342350.[Abstract]
Traenckner, E. B., Wilk, S., and Baeuerle, P. A. (1994). A proteasome inhibitor prevents activation of NF-B and stabilizes a newly phosphorylated form of I
ß-d that is still bound to NF-
B. EMBO J. 13, 54335441.[Abstract]
Wu, W. J., Wolcott, R. M., and Pruett, S. B. (1994). Ethanol decreases the number and activity of splenic natural killer cells in a mouse model for binge drinking. J. Pharmacol. Exp. Ther. 271, 722729.[Abstract]
Xie, Q. W., Kashiwabara, Y., and Nathan, C. (1994). Role of transcription factor NF-B/Rel in induction of nitric oxide synthase. J. Biol. Chem. 269, 47054708.
Youn, B.-S., Liu, C.-C., Kim, K.-K., Young, J. D.-E., Kwon, M. H., and Kwon, B. S. (1991). Structure of the mouse pore-forming protein (perforin) gene: Analysis of transcription initiation site, 5' flanking sequence, and alternative splicing of 5' untranslated regions. J. Exp. Med. 173, 813822.[Abstract]
Yu, C.-R., Ortaldo, J. R., Curiel, R. E., Young, H. A., Anderson, S. K., and Gosselin, P. (1999). Role of a STAT binding site in the regulation of the human perforin promotor. J. Immunol. 162, 27852790.
Zeldin, G., Yang, S. Q., Yin, M., Zhi, H., Rai, R., and Diehl, A. M. (1996). Alcohol and cytokine-inducible transcription factors. Alcohol Clin. Exp. Res. 20, 16391645.[ISI][Medline]
Zhang, J., Scordi, I., Smyth, M. J., and Lichtenheld, M. G. (1999). Interleukin-2 receptor signaling regulates the perforin gene through signal transducer and activator of transcription (Stat) 5 activation of two enhancers. J. Exp. Med. 190, 12971307.
Zhou, J., Struthers, A. D., and Lyles, G. A. (1999). Differential effects of some cell signaling inhibitors upon nitric oxide synthase expression and nuclear factor-B activation induced by lipopolysaccharide in rat aortic smooth muscle cells. Pharmacol. Res. 39, 365373.
Zhou, J., Zhang, J., Lichtenheld, M. G., and Meadows, G. G. (2002). A role for NF-B activation in perforin expression of NK cells upon IL-2 receptor signaling. J. Immunol. 169, 13191325.