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Proceedings of the Society for Experimental Biology and Medicine 224:141-146 (2000)
© 2000 Society for Experimental Biology and Medicine

Original Article

Differential Effects of Nicotine and Aging on Splenocyte Proliferation and the Production of Th1- Versus Th2-Type Cytokines

Nora Hallquist*, Amal Hakki*, Lynn Wecker{dagger}, Herman Friedman* and Susan Pross*,1


* Department of Medical Microbiology and Immunology, and
{dagger} Department of Pharmacology and Therapeutics, University of South Florida College of Medicine, Tampa, Florida 33612


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Nicotine has a multitude of biological actions in the central and peripheral nervous systems where nicotinic acetylcholine receptors are found. Nicotinic acetylcholine receptors have also been identified on immune cells, but the effects of nicotine on immune responses are not well characterized. These studies tested the hypotheses that nicotine has an effect on both T-lymphocyte proliferation and the production of cytokines by activated T cells, processes that are necessary for effective T-cell–mediated immune responses. In addition, the effects of nicotine on these immune responses in aging animals and the effects of nicotine exposure prior to immunostimulation were investigated. Murine splenocytes were exposed to nicotine and stimulated with concanavalin A (ConA). The highest concentration of nicotine (128 µg/ml) significantly depressed proliferation of T cells both when nicotine and ConA were added concurrently and when nicotine was added 3 hr prior to ConA. Nicotine, added concurrently with ConA at concentrations between 0.25 and 64 µg/ml, significantly inhibited the production of IL-10 by splenocytes from young adult mice, whereas the inhibition of production of IL-10 by splenocytes from old mice was significantly inhibited, but the response was more variable, depending on the nicotine concentration. In contrast, the production of IFN-{gamma} by splenocytes from either young adult or old mice was not affected when nicotine (0.016–64 µg/ml) was added concurrently with ConA. Pre-exposure to 1 µg/ml of nicotine for 3 hr significantly enhanced the production of IFN-{gamma} by splenocytes from young adult mice, whereas pre-exposure to 0.016 µg/ml of nicotine tended to but did not significantly enhance IFN-{gamma} production. Nicotine is now being used as an over-the-counter drug by people who differ in age and general immunocompetence. Therefore, the effects of nicotine on immune responses, independent from the effects of the other chemicals found in tobacco, need to be investigated.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Nicotine, a small organic alkaloid synthesized by tobacco plants, is the addictive component of tobacco products (1, 2). The use of nicotine-containing tobacco products has been associated with immunomodulation (3) and an increase in specific diseases, such as respiratory tract infections (4), chronic airway disease (5), asthma in children exposed to second-hand smoke (6), allergies (7), and lung and other cancers (8, 9). Although the specific role of nicotine in tobacco-related diseases is not clear, nicotine alone has been shown to alter immune responses by decreasing inflammation, decreasing the antibody-forming cell response of splenocytes, decreasing T-cell-receptor–mediated signaling (10), and decreasing proliferation of peripheral blood mononuclear cells (11). Nicotine is an agonist at nicotinic acetylcholine receptors, which are found not only in the central and peripheral nervous systems but also on other cells throughout the body, including immune cells (12, 13). Thus, nicotine could enhance or interfere with immune responses in a receptor-mediated manner.

The use of nicotine-containing alternative products, such as gums and patches, as aids to stop smoking is rising. In addition, it has been suggested that nicotine and nicotine agonists can be developed to treat Alzheimer's and Parkinson's diseases (14), two disorders predominantly associated with aging. Physiological compromises of the immune system occur with aging, subsequently advancing disease processes in the elderly. For example, poor T-cell responses to mitogen stimulation and altered cytokine profiles occur in the elderly. Specifically, increased IL-3, IL-4, and IL-6 production and decreased IL-2 production have been documented in older individuals (15, 16). It has also been reported that the proliferation of T cells decreases with age (17). It follows that T-cell immunity is altered in aged animals contributing to diminished responses to pathogens as well as uncontrolled cellular growth in cancer (18). In addition, tissue repair processes diminish with age. Older people who smoke are more susceptible to vascular disease (19) and lung diseases including lung cancer (20) than age-matched nonsmokers. However, little is known about the actions of nicotine on the immune system of aged individuals, especially those who may already have a diminished immunologic potential.

Results of these experiments indicated that nicotine decreased proliferation of mitogen-activated murine spleen cells. In ConA-stimulated splenocytes from young adult and old mice, nicotine added concurrently with Con A altered the production of IL-10, a defining Th2 cytokine, whereas it had little detectable effect on the production of IFN-{gamma}, indicating no consequence of nicotine exposure to a Th1 cytokine response. In contrast, pre-exposure of splenocytes to 1 µg/ml of nicotine significantly increased the production of IFN-{gamma}.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice.
Female BALB/c mice (Jackson Labs, Bar Harbor, ME), 8–9 weeks old (young adult mice) or 18–24 months of age (old mice), were used for these studies. They were housed in IACUC-approved animal facilities, fully accredited by the American Association for Accreditation of Laboratory Animal Care. Mice were sacrificed by asphyxiation with carbon dioxide.

Splenocytes.
Spleens were removed, and single cell suspensions were prepared with a Stomacher 80 Lab Blender (Tekmar, Cincinnati, OH) in Hank's Balanced Salt Solution (Sigma Chemical Co., St. Louis, MO). Red blood cells were lyzed in a solution containing 155 mM ammonium chloride, 10 mM potassuim bicarbonate, and 100 µM EDTA (Sigma Chemical Co.). The cells were washed and cultured in RPMI 1640 medium supplemented with 10% FCS, 100 units penicillin/ml, 100 µg streptomycin/ml, 2 mM glutamine, and 0.5 µM ß-mercaptoethanol (Sigma Chemical Co.). Cell viability exceeded 95% by trypan blue exclusion.

Nicotine Preparation and Exposure.
Nicotine hydrogen bitartrate (Sigma Chemical Co., St. Louis, MO) was stored in a dessicator jar and shielded from light. Nicotine was prepared fresh for each experiment. The timing of cellular exposure was either: i) cells concurrently exposed to nicotine and mitogen; or ii) cells pre-exposed to nicotine 3 hr prior to mitogen. The chosen nicotine concentrations were 0, 0.016, 0.031, 0.063, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, and 128 µg nicotine/ml. The lower concentrations of nicotine (< 1 µg/ml) can be found in the blood of light to heavy smokers (21, 22), and nicotine at higher concentrations (1–10 µg/g) can be found deposited in body tissues (23, 24).

Mitogenic Stimulation of Immune Cells.
Whole splenocyte populations were stimulated with concanavalin A (ConA) (Sigma Chemical Co.), a plant lectin that preferentially stimulates T cells by binding to carbohydrate residues in glycoproteins specifically involved in T-cell activation (25).

Lymphocyte Proliferation.
To assess whether nicotine impacted cellular proliferation, 1 x 106 splenocytes/ml were stimulated with 5 µg/ml of ConA and incubated in 96-well flat bottom plates (Corning Costar Corp., Cambridge, MA) with or without nicotine for 72 hr. The cells were pulsed for the last 18 hr with 2 µCi 3H-thymidine/ml (Amersham Pharmacia Biotech Inc., Arlington Heights, IL) and harvested on glass fiber filters. Proliferation was determined by measuring the incorporation of radioactivity in a liquid scintillation counter.

Cytokine Production.
To determine whether nicotine altered the production of cytokines typically produced by Th1 or Th2 cells, 2.5 x 106 splenocytes/ml were incubated in 24-well tissue culture plates (Corning Costar Corp.) with 5 µg/ml of ConA, with or without nicotine, for 48 or 72 hr. Supernatants were collected and tested by ELISA for the production of IL-10 or IFN-{gamma}. Medium-bind 96-well Costar EIA plates were coated overnight at 4°C with 10 µg IL-10/ml or 4 µg IFN-{gamma}/ml of antimurine cytokine antibodies (PharMingen, San Diego, CA). After the plates were blocked with bovine serum albumin (BSA; Sigma Chemical Co.), either serially diluted standard cytokines (PharMingen) or supernatants from cultures were added to wells in triplicate and incubated for 1 hr. Four µg/ml for IL-10 or 2 µg/ml for IFN-{gamma} of biotinylated antimurine cytokine antibodies (PharMingen) were added and incubated for 1 hr. Streptavidin-horseradish peroxidase, 1:1000 dilution, (PharMingen) was added and incubated for 30 min followed by 50 µl of 3,3',5,5',- tetramethylbenzidine (TMB) liquid substrate system (Sigma Chemical Co.). The enzymatic reaction was stopped with 1 N sulfuric acid, and the color produced was detected at 450 nm on an Emax Microplate Reader (Molecular Devices, Menlo Park, CA).

Statistical Analyses.
Data from identical triplicate wells were averaged and analyzed as one experimental n. The data were analyzed by Mann-Whitney rank sum test, Kruskal-Wallis one-way analysis of variance, or one-way or two-way analysis of variance, and followed by Student Newman Keuls multiple comparisons test where appropriate (n = 3–11 mice). Specific tests are listed in each figure legend. Analyses were considered significantly different at P < 0.05 using SigmaStat: Statistical Software for Working Scientists (Jandel Scientific, San Rafael, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Lymphocyte Proliferation.
To determine whether nicotine affected lymphocyte proliferation, splenocytes from young mice were exposed to various concentrations of nicotine and concurrently stimulated with ConA. Exposure of splenocytes to the highest concentration of nicotine (128 µg/ml) significantly reduced the ability of the cells to incorporate 3H-thymidine (Fig. 1)Go. Lower concentrations of nicotine had no effect. Similar results were seen when splenocytes were exposed to nicotine for 3 hr prior to stimulation with ConA (Fig. 1)Go. Trypan blue exclusion studies confirmed cell viability even at the higher nicotine concentrations.



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  		Figure 1.   Effect of nicotine on splenocyte proliferation. Splenocytes from young adult mice were immunostimulated with ConA. Nicotine was added concurrently with ConA (0 hr pre) or 3 hr prior to ConA (3 hr pre), and the samples were incubated for 72 hr. Proliferation was measured by 3H-thymidine incorporation and calculated as a percentage of control within treatment group. One-way analysis of variance followed by Student Newman Keuls multiple comparisons test showed that nicotine decreased lymphocyte proliferation at the highest concentrations of exposure (128 µg nicotine/ml): P < 0.001, n 0 hr pre = 8; P < 0.001, n 3 hr pre = 4. Two-way analysis of variance showed that the effect of nicotine was not dependent on timing of nicotine exposure: P = 0.455.

 
Cytokine Production.
The effect of nicotine and aging on cytokine production was evaluated by exposing splenocytes from young adult or old mice to nicotine in vitro for 48 hr. In the unexposed (no nicotine) immunostimulated controls, splenocytes from young adult mice produced a significantly greater amount of IL-10 (3129 + 382 pg/ml) than splenocytes from old mice (1626 + 161 pg/ml) (P = 0.034) indicating that aging was associated with a 50% decrease in IL-10 production. Upon nicotine exposure, IL-10 production was significantly inhibited as compared with unexposed immunostimulated controls at the higher concentrations of nicotine tested in young adult mice (0.25–64 µg/ml) and at various concentrations of nicotine tested in old mice (0.25, 0.5, 16, and 32 µg/ml) (Fig. 2)Go. In contrast, in the unexposed (no nicotine) immunostimulated controls, a significant 3-fold increase in the amount of IFN-{gamma} was produced by splenocytes from old mice (17642 + 5028 pg/ml) as compared with splenocytes from young adult mice (5196 + 927 pg/ml) (P = 0.006). Upon nicotine exposure, the production of IFN-{gamma} by splenocytes from young adult or old mice was not altered (Fig. 3)Go.



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  		Figure 2.   (a) Splenocytes from young adult mice and (b) splenocytes from old mice. Effect of nicotine on IL-10 production by splenocytes. Splenocytes from young adult or old mice were immunostimulated with ConA and concurrently exposed to nicotine for 48 hr. IL-10 production was measured by ELISA and calculated as a percentage of age-matched unexposed (no nicotine) controls. Kruskal-Wallis one-way analysis of variance followed by Student Newman Keuls multiple comparisons test showed that nicotine significantly decreased IL-10 production by splenocytes from young adult mice at the higher concentrations of nicotine tested (0.25–2 and 8–64): P = 0.003, n = 5–10; and from old mice at various concentrations of nicotine tested (0.25, 0.5, 16, and 32 µg nicotine/ml): P = 0.002, n = 3–4. Two-way analysis of variance using concentrations of nicotine between 0.25 and 64 µg/ml showed that the effect of nicotine was not significantly different in mice of different ages: P = 0.157, n = 3–10.

 


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  		Figure 3.   (a) Splenocytes from young adult mice and (b) splenocytes from old mice. Effect of nicotine on IFN-{gamma} production by splenocytes. Splenocytes from young adult or old mice were immunostimulated with ConA and concurrently exposed to nicotine for 48 hr. IFN-{gamma} production was measured by ELISA and calculated as a percentage of age-matched unexposed (no nicotine) controls. Kruskal-Wallis one-way analysis of variance showed that nicotine did not alter IFN-{gamma} production by splenocytes from young adult mice: P = 0.611, n = 7–11, or old mice: P = 0.291, n = 4–5. Two-way analysis of variance using concentrations of nicotine between 0.25 and 64 µg/ml showed that the effect of nicotine on IFN-{gamma} production was significantly different in mice of different ages: P < 0.001, n = 4–11.

 
To determine whether exposure to nicotine prior to mitogen altered the production of cytokines by splenocytes, spleen cells were exposed to nicotine for 3 hr prior to stimulation with ConA and then incubated for 72 hr. Concentrations of 0.016 and 1 µg/ml of nicotine were chosen because these concentrations of nicotine can be found in the blood of light or heavy smokers (22). At 1 µg/ml of nicotine, the production of IFN-{gamma} was significantly increased when nicotine was added 3 hr prior to ConA. This significant increase was seen both when comparing pre-exposed immunostimulated to concurrently exposed immunostimulated splenocytes from young adult mice and when comparing pre-exposed immunostimulated to unexposed (no nicotine) immunostimulated controls from young adult mice (Fig. 4)Go. The production of IFN-{gamma} by young adult mice was not significantly affected at 0.016 µg/ml of nicotine although it tended to increase when nicotine was added 3 hr prior to ConA, similar to preincubation with 1 µg/ml of nictoine (Fig. 4)Go. After 72 hr, the amount of IL-10 was severely decreased as compared with 48 hr possibly due to re-uptake or breakdown of IL-10 by the splenocytes or other experimental error. Thus, the IL-10 data concerning preincubation with nicotine was not analyzed.



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  		Figure 4.   Effect of pre-exposure to 1 and 0.063 µg/ml of nicotine on IFN-{gamma} production by splenocytes from young adult mice. Splenocytes were immunostimulated with ConA. Nicotine was added concurrently with ConA (0 hr pre) or 3 hr prior to ConA (3 hr pre), and the samples were incubated for 72 hr. IFN-{gamma} production was measured by ELISA and calculated as a percentage of age-matched unexposed (no nicotine) controls. In unexposed (no nicotine) immunostimulated controls, the concentration of IFN-{gamma} was 3408 + 496 pg/ml. Mann-Whitney rank sum tests compared 0 hr preincubation with 3 hr preincubation (^) or compared unexposed (no nicotine) 100% immunostimulated controls (horizontal line) with 3 hr preincubation (*). At 1 µg nicotine/ml, the production of IFN-{gamma} at 3 hr pre was significantly enhanced compared with 0 hr pre (^) (P = 0.036, n 0 hr pre = 4, n 3 hr pre = 7). At 0.063 µg nicotine/ml, the production of IFN-{gamma} at 3 hr pre was not significantly enhanced compared with 0 hr pre (^) (P = 0.264, n 0 hr pre = 4, n 3 hr pre = 7). Similarily, when compared with the unexposed (no nicotine) 100% immunostimulated controls (horizontal line), the production of IFN-{gamma} by splenocytes preincubated with 1 µg/ml of nicotine was enhanced: (*) (P = 0.010, n 3 hr pre = 7).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Nicotine is available over the counter to people of various ages and states of general health. Also, nicotine or nicotine agonists may become available for the treatment of certain brain disorders, especially those associated with the elderly. The objectives of these studies were to determine whether nicotine affects T-lymphocyte proliferation or the production of cytokines by activated T cells. Also investigated were the effects of nicotine on these immune responses in splenocytes from older mice and in splenocytes that were exposed to nicotine prior to immunostimulation.

It was demonstrated that at the highest concentrations of nicotine tested, proliferation of mitogen-stimulated splenocytes was inhibited when cells were exposed to nicotine either concurrently or prior to mitogen. Since it is necessary for lymphocyte populations to increase in size to mount an effective immune response, loss of cell division due to nicotine exposure would hinder the immune response of the host. However, 128 µg nicotine/ml is 100 times greater than concentrations of nicotine that can found in the blood plasma. Although nicotine concentrations of 5–10 times greater than plasma levels can be deposited in tissues including immune tissues (23), these physiological concentrations of nicotine did not affect lymphocyte proliferation. Nicotine affected concurrently stimulated and pre-exposed splenocytes in the same manner, suggesting that the effect of nicotine is independent of the timing of nicotine exposure with regard to immune cell proliferation.

The cytokines measured represent those responsible for regulating Th2 (antibody-mediated) and Th1 (cell-mediated) immune responses. IL-10 activates Th2 responses and inhibits Th1 responses, whereas IFN-{gamma} activates Th1 responses and inhibits Th2 responses (26). In the unexposed (no nicotine) immunostimulated controls, the production of IL-10 by splenocytes from old mice was decreased whereas the production of IFN-{gamma} by splenocytes from old mice was increased. The findings that T cells from older mice produce less IL-10 and more IFN-{gamma} are supported in the literature. Published data depicted decreased production of IL-10 by immunostimulated memory T cells from old mice (27) and increased production of IFN-{gamma} by immunostimulated helper T cells from old mice (28), though contrasting results have been published (29). Beyond this background of already altered cytokine production by older animals, nicotine had its own effects. These investigations demonstrated that production of IL-10 by splenocytes from both young adult and old mice was inhibited upon exposure to higher concentrations of nicotine, suggesting a decrease in Th2 immune responses at least in heavy smokers. Smoking has been shown to alter cytokine profiles and decrease inflammation of the bowel in people with ulcerative colitis, a disease associated with an overactive Th2 immune response (30, 31). Discontinuing the use of tobacco or nicotine products increased intestinal damage, suggesting that nicotine suppressed the overactive immune response (32). Furthermore, when transdermal nicotine was given to healthy male nonsmokers, peripheral blood mononuclear cells from these volunteers produced less IL-10, whereas the production of IFN-{gamma} and TNF-{alpha} was unchanged (33). Thus, nicotine in vivo, independently from smoking, inhibited this Th2 immune cell function similar to the findings reported here in vitro. Nicotine may not be as effective at alleviating the symptoms of ulcerative colitis in older individuals since IL-10 production was inhibited at various concentrations of nicotine used in these experiments, including concentrations of nicotine that can be found in the tissues of smokers. Physiological responses by older individuals tend to be variable, especially within the human population where both genetic differences and environmental differences contribute to the physiological responses. However, splenocytes from old mice that were not exposed to nicotine produced less IL-10; therefore, even a small decrease of this Th2 response by nicotine may be biologically relevant in elderly individuals.

In contrast to ulcerative colitis, smoking intensifies the inflammatory symptoms in the gastrointestinal tract of Crohn's patients, possibly due to increased production of the Th1 cytokine, IFN-{gamma} (34). The results reported here showed that nicotine did not alter the production of IFN-{gamma} after mitogen stimulation in vitro, presumably allowing a fully active Th1 response to occur in both young adult and old mice. These results are supported by other investigators who found that the production of both IL-4 and IFN-{gamma} by activated human peripheral blood mononuclear cells was not altered by nicotine exposure (35). Even though nicotine did not affect IFN-{gamma} production, aging itself did have an impact. Splenocytes from older mice produced more IFN-{gamma} than splenocytes from younger mice, confirming results that have been published elsewhere (28). Altered immune responses, including altered cytokine profiles from aged animals, and varying modulations of Th-1 versus Th-2 cytokines are well documented (15, 16, 28).

Pre-exposure to nicotine was employed as an attempt to simulate chronic exposure to nicotine, as would occur in users of tobacco or nicotine products. However, even in the blood of heavy smokers, nicotine concentrations transiently increase and decrease allowing for resensitization of the nicotinic acetylcholine receptors. In this system, the nicotinic acetylcholine receptors would desensitize rapidly and not respond to the nicotine. This situation could possibly exist in nicotine patch users, but only locally and at very low concentrations of nicotine. It is interesting that the production of IFN-{gamma} was significantly elevated when spleen cells were pre-exposed to nicotine. Although IFN-{gamma} was not elevated when mitogen and nicotine were added concurrently, elevated IFN-{gamma} due to pre-exposure to nicotine may have biological implications for drug developers who have recently attempted to use nicotine or nicotine agonists to alleviate symptoms of diseases such as Alzheimer's or Parkinson's (32). The possible mechanisms of the effects of nicotine on these and other Th1- and Th2-mediated diseases need to be investigated to determine whether nicotine has consistent effects on T-helper cells independent of the disease.

The effects of nicotine on immune cell functions could occur via either nonreceptor- or receptor-mediated pathways. The nicotinic acetylcholine receptor protein has been found on both intact lymphocytes and lymphocyte membranes (36, 37), and the mRNA of the {alpha}2–7 and ß2–4 subunits of the nicotinic acetylcholine receptor has been found in human peripheral blood mononuclear cells (12, 38). However, the functional role of nicotinic acetylcholine receptors in the mechanisms of nicotine-induced modulation of immune cells has not been clarified. The discoveries of nicotinic acetylcholine receptors on immune cells give mechanistic support to the hypothesis that nicotine alters immune cell functions via its receptor. A typical step in the signal transduction pathway of nicotine is altered intracellular calcium concentrations. Nicotine exposure resulted in a downregulation of intracellular calcium after immunostimulation of T and B cells when cells were exposed to nicotine in vivo (39) and an upregulation of intracellular calcium after immunostimulation of human peripheral blood cells or leukemic cell lines when cells were exposed to nicotine in vitro (40), supporting a role for nicotine in altered T-cell signal transduction. Therefore, it is possible that nicotine affected Th1 and Th2 immune cell functions by binding to its receptor directly and altering the transduction of the stimulation signal supplied by the mitogen. Future work in this laboratory will pursue the mechanisms of altered immune cell responses to nicotine.


   Footnotes
 
This research was supported by the National Institutes of Health training grant # 2T32-DA07245–10.

1 To whom requests for reprints should be addressed at the Department of Medical Microbiology and Immunology, University of South Florida College of Medicine, MDC-10, 12901 Bruce B. Downs Blvd., Tampa, FL 33612. E-mail: spross{at}com1.med.usf.edu Back


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication September 20, 1999. Accepted for publication February 10, 2000.




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