Restoration of ethanol-compromised Th1 responses by sodium orthovanadate
Serge Ostrovidov1,
Laurence M. Howard1,
Masato Ikeda1,
Akiko Ikeda1 and
Carl Waltenbaugh1
1 Department of MicrobiologyImmunology, Northwestern University School of Medicine, 303 East Chicago Avenue, Chicago, IL 60611, USA
Correspondence to: C. Waltenbaugh; E-mail: waltenbaugh{at}northwestern.edu
Transmitting editor: D. R. Green
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Abstract
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Alcohol consumption often diminishes antigen-specific cell-mediated immunity. In alcohol-consuming mice IFN-
and delayed-type hypersensitivity (DTH) responses are blunted, although antigen-specific T cell proliferation and IL-2 responses are largely unaffected, suggesting that alcohol differentially affects signal transduction pathways. In the present report we explore the use of the phosphatase inhibitor, Na3VO4 to restore IFN-
secretion in the presence of ethanol both in vivo and in vitro. We show that Na3VO4 restores IFN-
in vitro and antigen-specific DTH in vivo to the levels seen in alcohol non-consuming mice. Our data support the contention that ethanol, by up-regulating phosphotyrosine phosphatase, diminishes the IFN-
signal transduction pathway.
Keywords: alcohol, IFN-
, mouse, Na3VO4, protein tyrosine phosphatase
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Introduction
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When consumed in excess, alcohol alters normal immune function, evident in the increased frequency and severity of infections seen in alcoholic patients (1,2). Cell-mediated and humoral immune responses are affected, impairing delayed-type hypersensitivity (DTH) and elevating serum Ig levels (3,4). Likewise, in experimental animals, ethanol consumption for 710 days impairs cell-mediated immunity, while humoral immunity is enhanced (57). In humans and mice, two types of CD4+ T lymphocytes, termed Th1 and Th2, defined by cytokine secretion and immune function, are largely responsible for mediating cell-mediated (type 1) and humoral (type 2) immunity (8,9). In general, ethanol polarizes the immune response away from type 1 and toward type 2 (7,10), and is evident by the diminished production of the Th1-related cytokine IFN-
(11).
Antigen-presenting cells (APC) influence the development of type 1 and type 2 adaptive immune responses. Ethanol affects APC (e.g. dendritic cells, macrophages or B cells) function (12,13) by decreasing IL-12, a cytokine central to the skewing of T cell response toward Th1 function (14). Ethanol consumption by BALB/c APC donor mice decreases both IL-12 production and subsequent ovalbumin (OVA)-stimulated IFN-
production by purified T cells from
ß TCR transgenic mice (DO11.10) (15).
Alcohol consumption impairs IFN-
and IL-12 production, although other type 1 parameters such as antigen-specific T cell proliferation and IL-2 production are unaffected (7,15), indicating that alcohol may affect IL-2/proliferation and IL-12/IFN-
pathways differently. Events that ultimately lead to Th1 IFN-
production are complex, involving a number of signal transduction molecules [for review, see (16)]. Ethanol impairs inositol triphosphate (IP3) production, intracellular Ca2+ mobilization and subsequent cAMP production, and augments extracellular Ca2+ uptake (17,18). Ethanol may interfere with early cell surface-associated signal transduction phosphorylation events that, under normal circumstances, ultimately lead to the activation of phospholipase C (PLC)
1 (1719). The tyrosine phosphorylation pathway has been implicated in the regulation of IFN-
production (20). Here we show that Na3VO4, a phosphotyrosine phosphatase (PTP) inhibitor, restores IFN-
in vitro and antigen-specific DTH in vivo. Our data support the contention that ethanol up-regulates PTP activity, subsequently shortening the half-life of phosphorylated signal transduction molecules at the cell membrane and leads to attenuation of the strength or duration of TCR signals which results in reduced capacity for Th1 functional responses.
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Methods
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Animals
Female BALB/c mice were purchased from the Small Animal Production Unit, National Cancer Institute (Frederick, MD) and were held for a 2-week acclimation period prior to use. OVA-specific
ß TCR transgenic D011.10 mice (21), syngeneic with BALB/c mice, were bred and maintained in the Northwestern University vivarium. All mice were 810 weeks of age at the initiation of experiments. All animal protocols met with prior approval of the Northwestern University Animal Care and Use Committee.
Diets and feeding
Mice were fed a solid diet consisting of laboratory chow (TekLad 7022, NIH-07 diet; Harlan TekLad, Madison, WI) and water ad libitum, a liquid ethanol diet (LED) or pair-fed a liquid control diet (LCD), as previously described (7). Liquid diets that meet the American Institute of Nutrition (AIN) standards for rodents were compounded in our laboratory. Briefly, the laboratory-formulated diets contained sucrose (7.84 g/l; United Sugar, Minneapolis, MN), corn oil (1.28 g/l; Dyets, Bethlehem, PA; 401150), AIN mineral mixture 76 (893 mg/l; Dyets; 200000), AIN vitamin mixture 76A (255 mg/l; Dyets; 300050), DL-methionine (77 mg/l; Dyets; 402950), choline bitartrate (51 mg/l; Dyets; 400750), xanthine gum (200 mg/l; Dyets; 402525) and casein hydrolysate (5.1 g/l; US Biochemical, Cleveland, OH; 12855). BALB/c mice were fed diets containing ethanol at 45.8 g/l (30% ethanol-derived calories; LED30). The LCD isocalorically substituted sucrose for ethanol. All liquid diets contained 1 kcal/ml (4.184 kJ/ml), and provided 20% protein-, 11% fat (corn oil)- and 66% carbohydrate (sucrose and/or ethanol)-derived calories. Solid laboratory chow and water were removed from the groups fed liquid diets, and were replaced with the appropriate liquid diet. Some diets were supplemented by the daily addition of 104 M Na3VO4 (
6.5 mg Na3VO4/kg; Sigma; S-6508). Mice were pair-fed, i.e. caloric intake by the LED-fed mice determined the caloric intake for the LCD-fed mice for the subsequent day.
Assessment of DTH
Female BALB/c mice were fed LED30, pair-fed LCD or solid laboratory chow and water for the entirety of the experiments. On dietary day 4 the mice were primed s.c. with 100 µg F
G emulsified in complete Freunds adjuvant (H37Ra; Difco, Detroit, MI) as previously described (13). Six days after immunization, mice were anesthetized with sodium pentobarbital (60 mg/kg), baseline ear thickness was measured using a Mitutoyo dial thickness gauge (model 7326) (22) and 20 µg fowl
-globulin (F
G) in 10 µl PBS was injected intradermally into the dorsal surface of the ear using a syringe fitted with a 30-gauge needle. Antigen-induced ear swelling, determined 24 h later in a blind reading, is expressed in units of 104 inches (22).
Tissue culture
Single-cell suspensions of 5 x 105 viable spleen cells were cultured with 18 µM OVA, concanavalin A (Con A; 0.124 µg) or anti-CD3 mAb (
2 ng; hybridoma 145-2C11 supernatant) in 96-well round-bottom culture plates in DMEM:F12 medium (Hyclone, Logan, UT) supplemented with 50 µM ß2-mercaptoethanol, 3 µM glutamine and 1% Nutridoma (a serum substitute; Roche, Indianapolis, IN). At optimal times cell-free supernatants were harvested for determination of IL-2 (24 h) and IFN-
(72 h for OVA and 48 h for Con A or anti-CD3) levels. T cell proliferation was determined at optimal times (72 h for OVA and 48 h for Con A or anti-CD3) in triplicate cultures pulsed 24 h previously with 0.5 µCi/well [3H]thymidine.
Cytokine analysis
ELISA was used to determine cytokine concentrations in tissue culture supernatants. Briefly, wells of 96-well Maxisorp (Nunc, Rochester, NY) plates were coated with anti-IL-2 (Caltag, Burlingame, CA; RM9120) or anti-IFN-
(Caltag; RM9110) antibody overnight, then blocked with PBS containing 2% BSA. Standards, recombinant IL-2 (Preprotech, Rocky Hill, NJ; 212-12) or recombinant IFN-
(Preprotech; 31505) and diluted culture supernatants were incubated for 2 h at 37°C on the coated plates. The plates were washed and biotinylated anti-IL-2 (Caltag; RM90215) or anti-IFN-
(Caltag; RM90015) was added for 1 h at 37°C. The plates were washed and horseradish peroxidase-conjugated streptavidin (KPL Laboratories, Rockville, MD) was added. After washing, TMB substrate (BioFX Laboratories, Owings Mills, MD) was added. Chromogenic development was stopped by the addition of 0.18 M H2SO4. Plates were read in a SpectraMax190 plate reader (Molecular Devices, Palo Alto, CA) at 450 nm and SoftMax Pro (Molecular Devices) curve fitting software used to determine concentrations.
Single-cell cytokine secretion was visualized by a modification of ELISA, the ELISPOT assay. Briefly, sterile 96-well flat-bottom PVDF membrane plates (Unifilter 350; Whatman, Clifton, NJ; 7770-001) were coated with sterile anti-IFN-
(PharMingen; 554430) antibody diluted in PBS, incubated overnight at 4°C and then blocked for 2 h at 37°C with PBS containing 1% BSA. Single-cell suspensions of 250,000 viable, unimmunized BALB/c spleen cells were stimulated with anti-CD3. After 24 h culture, the plates were washed to remove cells and culture supernatants, and incubated with biotinylated anti-IFN-
(Caltag; RM90015) overnight at 4°C. After washing, alkaline phosphatase-labeled anti-biotin antibody (Vector, Burlingame, CA; SP-3020) was added to each well and incubated for 2 h at 22°C, and then washed extensively. Spots were developed by the addition of NBT/BCIP (BioFX; BCID-0100-01) chromogenic substrate to each well. Spot development was stopped after 520 min by washing with distilled water. Plates were air-dried, and then spots were read and enumerated on an automated reader (CTL Analyzers, Cleveland, OH).
Statistical analysis
Data are reported as the arithmetic mean ± SEM. Statistical significance was assessed by two-tailed Students t-test using JMP software from the SAS Institute (Cary, NC).
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Results
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Effect of alcohol on T cell proliferation and IFN-
production in vitro
Antigen-specific T cell proliferative and antigen-driven IL-2 responses are largely unaffected in lymphocyte cultures derived from ethanol-consuming mice (23), although IFN-
responses are severely impaired (15,23). Addition of 100 mM ethanol to cultures derived from normal, unimmunized DO11.10 mice does not affect the ability of these OVA-specific, TCR transgenic spleen cells to proliferate in response to antigen (Fig. 1A), although IFN-
production is impaired (Fig. 1D). A similar pattern of unimpaired proliferation, but diminished IFN-
production is seen following Con A (Fig. 1B and E) or anti-CD3 stimulation (Fig. 1C and F). Ethanol impairment of IFN-
production is dose dependent (Fig. 1F). These data show that alcohol differentially affects IFN-
- and IL-2-dependent T cell proliferative pathways in antigen- or mitogen-driven cultures.
Effects of Na3VO4 in vitro
The molecular mechanisms regulating IFN-
responses are not well characterized (20). PTP inhibitors, such as Na3VO4, have been reported to greatly enhance IFN-
production (20,24,25). Data in Fig. 2 show an inverse relationship between T cell proliferation and IFN-
secretion in cultures containing up to 80 µM Na3VO4, addition of vanadate in excess of 80 µM significantly impairs both proliferation and IFN-
secretion. No decrease in viability or cell number was seen for the concentrations of vanadate used (data not shown). Inhibition of tyrosine dephosphorylation of STAT-1 by Na3VO4 exacerbates the anti-proliferative effect of IFN-
(26) and may account for the decreased proliferation seen for cultures containing
40 µM vanadate.
We used ELISPOT to determine whether increased IFN-
production results from a greater number of cytokine-secreting cells or from an increase in the quantity of IFN-
produced by individual cells. Data in Fig. 3 show that within the first 24 h, cultures containing ethanol exhibit decreased numbers of IFN
ELISPOT. As little as 5 µM vanadate added to ethanol-containing cultures restores IFN-
ELISPOT numbers to that of untreated cultures. Addition of up to 20 µM vanadate increases the IFN-
ELISPOT number to comparable levels for both control and ethanol-containing cultures. Further increases in vanadate concentration cause no further increase in IFN-
ELISPOT number (not shown). Although not shown, there was no apparent increase in the mean diameter of IFN-
spots in treated cultures, suggesting that vanadate does not increase the amount of IFN-
secreted by individual cells.
Previously we showed both in vivo (15) and in vitro (23) that ethanol directly acts upon APC and not T cells. We used a co-culture system to determine whether vanadate would ameliorate the effect of ethanol (Fig. 4). Data show that 4 h ethanol (100 mM) pretreatment of purified DO11.10 T cells does not impair IFN-
production (cf. Fig. 4B with A), although this same ethanol treatment of APC is inhibitory (cf. Fig. 4C with A). Addition of Na3VO4 to co-cultures of ethanol-pretreated cells restores their capacity to produce IFN-
(Fig. 4D and E). Addition of Na3VO4 only during the 4-h pretreatment period does not restore IFN-
(Fig. 4F and G). IL-2 production and T cell proliferation are unaffected by ethanol or vanadate treatment. These data show that vanadate does not affect IL-2 or T cell proliferation and suggest that the effects of vanadate are only seen during antigen stimulation.
Effects of Na3VO4 in vivo
To determine whether vanadate would restore Th1 function, female BALB/c mice were fed LED30, pair-fed LCD or solid diet ± 100 µM Na3VO4 for 11 day. On dietary day 4, mice were immunized with F
G emulsified in complete Freunds adjuvant; 6 days later their ears were challenged with F
G and 24 h later antigen-specific DTH determined. Figure 5 shows that ethanol-associated impairment of DTH in antigen-primed mice is abrogated by the inclusion of vanadate in the diet. Dietary vanadate does not significantly increase the degree of DTH for solid diet-consuming immunized mice nor does supplementation of the diet with vanadate result in ear swelling in naive mice challenged with the eliciting antigen. Although not shown, the amount of vanadate used to supplement the diet is crucialtoo much and the mice die, too little and no effect is seen. We previously showed (13) that ethanol consumption just prior and through the time of immunization would impair development of DTH responses. We have also shown (23) that 68 days of ethanol consumption by mice is critical for the alteration of immune parameters such as IFN-
production, B cell numbers and increase in serum IgE responses. Data in Fig. 6 show that vanadate supplementation for the entire dietary period restores DTH response. However, addition of vanadate prior and through immunization did not reverse ethanols inhibition of DTH. In contrast, supplementation with vanadate during the last half of the dietary period and, more specifically, during the last 2 days or elicitation phase of the DTH response almost totally restored DTH to solid diet control levels.
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Discussion
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Prolonged, excessive alcohol consumption by humans decreases CD4-mediated cellular and increases humoral immune responses (2). Similarly, immunization of mice fed an ethanol-supplemented diet also show diminished antigen-specific CD4-mediated DTH responses (13,15). However, the mechanism by which this inhibition occurs is still unclear. In this paper, we address a number of questions as to the mechanism of how ethanol affects Th1 cell differentiation and in vivo type 1 responses, and propose a possible central process by which Th1 cell function is controlled in vivo.
Using both DTH and in vitro antigen-specific APCT cell co-culture techniques, we show that ethanol acts during the cognitive phase of the immune response when APC and T cells interact in the recognition of antigen (13,15). IL-2 production and subsequent T cell proliferation are not influenced by ethanol, but IFN-
production is diminished by ethanol addition at the start of culture. Previous studies support this influence of ethanol on IL-12 and IFN-
production, and the effector phase of a type 1 response (15,27). Collectively these data may be interpreted to suggest that early events in the induction of naive T cell responses (IL-2 production and T cell expansion/proliferation) are largely unaffected by alcohol, but their later functional differentiation towards type 1 pro-inflammatory cells (ability to secrete IFN-
and IL-12) is impaired.
T cell activation results from a complex balance of phosphorylation by protein tyrosine kinase (PTK) and dephosphorylation by PTP of signal transduction molecules (2830). Whether a T cell becomes activated or remains quiescent depends upon the relative balance of PTK to PTP function (29). Ethanol has a marked effect on signal transduction in T cells (1719). Phosphorylation and subsequent migration of PLC
1 to the cytoskeleton, downstream IP3 generation and intracellular calcium reactions are all inhibited by ethanol (1719,30) upon TCR engagement. To date there is little or no evidence beyond decreased PLC
1 activation showing reduced kinase activity associated with TCR activation. That we observed normal proliferation and IL-2 secretion by T cells under the influence of ethanol in this study, and others (15,23), suggests that TCR engagement and signal transduction is relatively normal, but that one or more elements may be modified. While ethanol inhibits intracellular calcium mobilization, for example, it augments extracellular calcium uptake during activation (19). We show here, that the effects of ethanol were exclusively on T cell IFN-
production (Figs 1 and 4). PTP strongly suppress IFN-
production (20) and orthovanadate, a PTP inhibitor (20,31), can reverse this effect. Thus, we addressed the possibility that diminished IFN-
secretion by T cells is due to reduced PLC
1 activation as a consequence of higher levels of PTP over PTK activity, induced by the presence of ethanol. Addition of Na3VO4 to ethanol-containing cultures restores their ability to produce IFN-
. Similarly in vivo, orthovanadate supplementation during the effector phase, alone, when ears of mice were challenged with antigen, almost fully restored DTH responses in ethanol-consuming mice.
One or more mechanisms may account for these observations. It is possible that Na3VO4 may compensate for the effects of ethanol using a completely separate mechanism to that observed by ethanol inhibition. Although unlikely, if this were the case, PLC
1 phosphorylation may not be observably increased in T cells cultured in the presence of ethanol supplemented with Na3VO4, but that IP3 or downstream intracellular calcium flux may return to normal levels. Second, Na3VO4 may increase PLC
1 phosphorylation, by decreasing PTP activity, while PTK activity may remain inhibited by ethanol, resulting in a modified equilibrium between PTP and PTK function. Equally possible is the converse, in which PTP activity, increased in the presence of ethanol, is inhibited by Na3VO4, resulting in PLC
1 phosphorylation. Similarly, NAD(P)H levels may also be affected by Na3VO4, which may provide greater kinase potential during the signal transduction cascade.
In mice fed alcohol for the duration of the experiment, normal antigen-specific DTH responses were observed when dosed with Na3VO4 just 2 days before antigenic challenge (Fig. 6). This contrasts with our earlier observations that alcohol administration prior to and during immunization, but not after immunization, inhibits DTH (13). We see several possible explanations for this disparity. First, our present data may be interpreted to suggest that Th cell differentiation, prior to administration of orthovanadate, resulted in normal Th1 cell differentiation. However, Th1 cells in alcohol-consuming individuals are blunted in IFN-
secretion. Orthovanadate, by inhibiting PTP activity, may increase Th1 effector function in a population of cells which were blunted during initial differentiation, in the presence of alcohol alone, by increasing their capacity for IFN-
-secretion in later rechallenge. Second, since ethanol also down-modulates specific APC functions as shown by diminished IL-12 production (15,27) and Na3VO4 has been shown to augment APC function (32), orthovanadate may also act either directly by increasing the capacity of APC to cross-talk with T cells or indirectly by increasing APC sensitivity to IFN-
produced by antigen-specific Th1 cells.
It has been proposed that T cell activation occurs only upon sufficient lipid raft aggregation (33). In resting cells, small lipid rafts contain limited numbers of associated molecules. PTP, such as CD45 (34) that are excluded from the rafts during T cell activation (35), can readily access and dephosphorylate proteins or ITAM within the raft. Upon TCR engagement, a dramatic capping of lipid rafts into larger aggregates occurs, with a concomitant exclusion from these aggregates of PTP enzymes (33). This facilitates PTK interactions and limits PTP activity. Lipid raft formation is dependent upon cholesterol (36,37) and ethanol reduces cholesterol in the cell membranes, increasing membrane fluidity (38,39). In this context, it is probable that a reduction in lipid raft aggregation in the membranes of alcohol fed mice, results in a greater capacity for PTP activity on smaller raft aggregates (SMAC), decreasing effective PTK signal transduction for optimal PLC
1 activation. Inhibition of PTP activity would therefore be redressed.
The present study extends our understanding of the action of alcohol on the immune response. We show that the addition of ethanol to T cell cultures selectively down-regulates IFN-
production by polyclonal- or APC-mediated antigen-specific stimulation without affecting IL-2 production or T cell proliferation. We further show that Na3VO4, a PTP inhibitor, can reverse the inhibitory effects of alcohol and restore IFN-
secretion to normal levels. We demonstrate the in vivo consequences of this effect, by showing that treatment with Na3VO4 of mice fed alcohol diets restores type 1 inflammatory T cell effector function to that of control levels. Clinical implications of these data, reflecting diminished type 1 effector responses and its augmentation by vanadate in alcohol-fed mice, imply that Th1 cell development may occur normally, but inhibition of type 1 effector function may be a central factor in the observed impaired immune response seen in alcoholics. Our data suggest that ethanol may increase PTP over PTK activity, resulting in the domination of dephosphorylation of PLC
1 over that of TCR-induced PTK activation of this enzyme.
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Acknowledgements
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Supported in part by National Institutes of Health grants AA08275 and AA10058
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Abbreviations
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AINAmerican Institute of Nutrition
APCantigen-presenting cell
Con Aconcanavalin A
DTHdelayed-type hypersensitivity
F
Gfowl
-globulin
IP3inositol triphosphate
LCDliquid control diet
LED30liquid ethanol diet containing 30% ethanol-derived calories
OVAovalbumin
PLCphospholipase C
PTKprotein tyrosine kinase
PTPphosphotyrosine phosphatase
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