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The Journal of Immunology, 2002, 169: 2915-2924.
Copyright © 2002 by The American Association of Immunologists

Effects of Nicotine Exposure on T Cell Development in Fetal Thymus Organ Culture: Arrest of T Cell Maturation1

Aaron J. Middlebrook*, Cherie Martina{dagger}, Yung Chang{dagger}, Ronald J. Lukas{ddagger} and Dominick DeLuca2,*

* Department of Microbiology and Immunology, University of Arizona College of Medicine, Tucson, AZ 85274; {dagger} Department of Microbiology, Arizona State University, Tempe, AZ 85287; and {ddagger} Division of Neurobiology, Barrow Neurological Institute, Phoenix, AZ 85013


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
There is evidence for both physiological functions of the natural neurotransmitter, acetylcholine, and pharmacological actions of the plant alkaloid, nicotine, on the development and function of the immune system. The effects of continuous exposure to nicotine over a 12-day course of fetal thymus organ culture (FTOC) were studied, and thymocytes were analyzed by flow cytometry. In the presence of very low concentrations of nicotine many more immature T cells (defined by low or negative TCR expression) and fewer mature T cells (intermediate or high expression of TCR) were produced. In addition, the numbers of cells expressing CD69 and, to a lesser extent, CD95 (Fas) were increased. These effects took place when fetal thymus lobes from younger (13–14 days gestation) pups were used for FTOC. If FTOC were set up using tissue from older (15–16 days gestation pups), nicotine had little effect, suggesting that it may act only on immature T cell precursors. Consistent with an increase in immature cells, the expression of recombinase-activating genes was found to be elevated. Nicotine effects were partially blocked by the simultaneous addition of the nicotinic antagonist d-tubocurarine. Furthermore, d-tubocurarine alone blocked the development of both immature and mature murine thymocytes, suggesting the presence of an endogenous ligand that may engage nicotinic acetylcholine receptors on developing thymocytes and influence the course of normal thymic ontogeny.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Nicotine has been reported to affect both humoral and cell-mediated branches of the immune system (1, 2, 3) and to produce an altered immune response that is characterized by a decrease in inflammation, a decreased Ab response, and a reduction in T cell-receptor-mediated signaling (4). These effects probably stem from the direct impact that nicotine has on T lymphocytes. It has been reported that treatment of T lymphocytes with nicotine provides signals that mimic TCR-mediated cell activation signals, thus leading to partial activation of T cells, resulting in anergy (5, 6, 7). Nicotine exposure has also been associated with the induction of regulatory T cells (8). These studies suggest that nicotine is a potent immunopharmacological agent with regard to T cell behavior in the periphery. Aside from its direct effect on peripheral T cells, nicotine may also influence T cell development, given the presence of nicotinic acetylcholine receptors (nAChR)3 in thymus (9). However, nAChR engagement within the thymus and the subsequent impact on T cell development remain unknown.

Using fetal thymus organ culture (FTOC) as an in vitro model for T cell maturation, we investigated the influence that nicotine may have on murine T cell development. During the development of the thymus, T cells initially express either CD4 or CD8 without CD3 (immature single positive (SP)) before they become double-positive (DP) CD3- cells. When TCR genes rearrange, a low level of this receptor is expressed on the cell surface, linked to CD3 (immature DP) (10). After positive and/or negative selection of these immature DP T cells based on the ability of their TCR to bind to self peptide and MHC molecules (11), DP T cells transiently express high levels of CD3-associated TCR (mature DP T cells) (12). They then rapidly differentiate into mature CD4 or CD8 T cells, which express high levels of TCR-associated CD3 molecules (12). These functional T cells are exported into the periphery.

Our data suggest that nicotine may be a potent regulator of T cell development, in that FTOC incubated with very low concentrations of nicotine have an apparent blockage in T cell development at the transition between the DP to mature SP stage of T cell development. This event is accompanied by an increase in CD69 expression, indicating that activation of the cells has occurred. In addition, there is an increase in CD95 induction, suggesting that activation-induced cell death may occur. Nicotine exposure also results in an increase in recombinase-activating gene (RAG) gene activity, presumably due to the induction of a negative selection signal on the immature T cells and/or a reduction of mature T cells that are no longer recombination active. Finally, the nAChR antagonist d-tubocurarine can itself inhibit the development of T cells. This latter result suggests that an endogenous ligand for nAChR may normally play a role in the regulation of T cell production.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

C57BL/6 (B6) mice were purchased from the National Cancer Institute (Frederick, MD). Timed pregnant females were used. The fetuses were removed from pregnant females at the indicated time points. Developmental assessment of these mice was based on vaginal plug date (plug date = day 0) and on their characteristics, as reported previously (13). Fetuses from pregnant mice that displayed any disease conditions were not used.

Fetal thymus organ culture

Thymus lobes were dissected from 13–16 days gestation fetal mice and were placed on the surface of Millipore (25 µm thick, 0.45-µm pore size; Millipore, San Francisco, CA) filters, which were supported on blocks of surgical Gelfoam (Upjohn, Kalamazoo, MI) in 10 x 35-mm plastic petri dishes with 3 ml medium. Organ culture medium consisted of DMEM (4.5 g/l D-glucose; JRH, Lenexa, KS) supplemented with 20% FBS (HyClone Laboratories, Logan, UT), streptomycin (100 µg/ml), penicillin (250 mg/ml), gentamicin (10 µg/ml), nonessential amino acids (0.1 mM), sodium pyruvate (1 mM), 2-ME (2 x 10-5 M), and 3.4 g/l sodium bicarbonate. The cultures were grown in a humidified incubator in 5% CO2 at 37°C. Cells were harvested as previously described (15). Briefly, the thymus lobes were placed into a solution of collagenase (from Clostridium histolyticum, type V, clostridiopeptidase A; Sigma-Aldrich, St. Louis, MO) 0.4 mg/ml in 0.2 M phosphate buffer with 0.2 mg/ml EDTA. The tissue was incubated at 37°C for 30 min. The lobes were then dispersed into a single-cell suspension by gentle aspiration with a Pasteur pipette. This treatment disaggregates most of the lymphoid cells from the tissue. However, many lymphoid cells remain, and to obtain these as well as the non-lymphoid stromal cells of the cultures, the fragments of thymus tissue were retreated with a solution of 0.12–0.25% trypsin (type II crude from porcine pancreas; Sigma-Aldrich) in the same EDTA/phosphate buffer as the collagenase for an additional 15–30 min at 37°C. After washing once in HBSS plus 5% FBS to prevent further enzyme action, cell viability in both collagenase- and trypsin-extracted samples was determined by 1% trypan blue exclusion. Viability was always >95%. The results are expressed as the total cells recovered x 104 per lobe and reflect the combined pool of cells recovered from both collagenase and trypsin treatments. Unless otherwise noted, FTOC was conducted for 12 days.

Reagents

FITC- and PE-conjugated hamster isotype controls, PE-conjugated anti-mouse CD3{epsilon} and CD95, CD45RA, FITC-conjugated anti-mouse CD8, and Tri-Color (TC) anti-mouse CD4 were purchased from Caltag Laboratories (South San Francisco, CA), and PE-conjugated anti-mouse CD8 was purchased from BD PharMingen (San Diego, CA). Bo-dipy-conjugated {alpha}-bungarotoxin was purchased from Molecular Probes (Eugene, OR). Nicotine (purchased from Sigma-Aldrich), was dissolved in PBS and brought to physiological pH (7.4) with 1 M NaOH. It was diluted to the indicated concentrations with standard organ culture medium (described above) before being added to cultures.

Flow cytometric (FC) analysis

Cell suspensions were stained with mAbs directly conjugated with TC (CD4), FITC (CD8), PE (CD8), PE (CD3{epsilon}), or Bo-dipy ({alpha}-bungarotoxin). The Abs were used at a concentration of 1 µg/106 cells, and {alpha}-bungarotoxin was used at a concentration of 175 nM. After staining, cells were fixed in 1% paraformaldehyde before FC analysis. Three-color FC analysis was performed using a FACScan (BDIS, San Jose, CA) equipped with photomultiplier tubes and optical fibers as recommended by the manufacturer. FITC, PE, and TC were excited by a 488-nm argon laser. Fluorescence data were collected using 3-decade logarithmic amplification on 10,000 viable lymphoid cells as determined by forward and 90° light scatter intensity to exclude stromal and other non-lymphoid elements (see Fig. 2Go for lymphocyte gate). Data were collected with CellQuest (Santa Rosa, CA) and were analyzed using FlowJO (TreeStar, San Carlos, CA) software.



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  		FIGURE 2. Effects of nicotine treatment on T cell phenotypes. FTOC of 13–14 day gestation C57BL/6 thymi were treated over 12 days of culture with increasing doses of nicotine and analyzed by three-color flow cytometry. Untreated control (A) and 10-18 M nicotine-treated (B) samples are shown. Boxes shown from left to right depict forward vs side scatter, CD8 FITC vs CD4 TC of the lymphocyte gate, and CD3 PE histograms of each indicated quadrant (SP8, DP, DN, SP4). The gates on the histograms show four relevant divisions: N, negative expression; D, dull intensity; I, intermediate intensity; and B, bright intensity. These gates were then used to define immature (N+D) and mature (I+B) cells. One representative experiment of six is shown.

 
Statistical analysis

There is some variability in total cell production across FTOC preparations (i.e., across experiments). However, total cell production from replicate samples within an experiment are in much closer agreement (~20–25%), and proportions or features of nicotine effects did not change markedly across experiments. However, because of the wide variation in cell production between some organ cultures set up at different times, the values were normalized to a percentage of the untreated control cultures. Statistical analyses (mean and SEM) were then performed on these values for comparison purposes. Paired Student’s t tests were performed on all data shown. The p values are not listed; however, determinations that achieved significance at the 90% confidence level (p <= 0.1) and those that achieved significance at the 95% confidence level (p <= 0.05) are indicated.

RNA preparation and RT-PCR for RAG gene analysis

RNA was prepared from cultured FTOC thymocytes using a CsCl ultracentrifugation method (16). RNA was reverse transcribed into cDNA using random oligonucleotides. The cDNA was serially diluted and amplified for RAG-2 and {beta}2-microglobulin ({beta}2m) genes using the oligonucleotides described previously (16). Amplification of the {beta}2m message served as an internal control for input cDNA. These PCR products were analyzed by Southern blot. A probe for RAG-2 was prepared by PCR amplification of RAG-2 constructs (provided by D. G. Schatz, Yale University, New Haven, CT) (14). The probe for {beta}2m was a gel-purified PCR product made with primers specific for {beta}2m cDNA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of nAChRs on the surface of murine thymocytes produced by FTOC

It has been demonstrated that thymocytes express nAChR subunits (9). We determined whether these receptors are present on the cells that are derived from FTOC. Accordingly, 14 day gestation fetal thymus lobes from C57BL/6 mice were organ-cultured for 6 days (producing mainly CD4+/8+ DP cells; Fig. 1GoA) or 12 days (producing mainly CD4+ or CD8+ SP cells; Fig. 1GoB). Cells produced by murine FTOC at 6 days of culture consisted of mainly immature DP cortical T cells, while at 12 days of culture more mature SP medullary T cells were produced (15) (Fig. 1Go). {alpha}-Bungarotoxin (which binds specifically to nAChR subtypes containing {alpha}1 or {alpha}7 subunits) staining from both 6- and 12-day FTOC at 175 nM is shown. Cells could not be stained with 25 or 125 nM {alpha}-bungarotoxin. The mature cells derived from 12-day FTOC stained more brightly (Fig. 1GoB) than the relatively immature cells produced by 6-day FTOC (Fig. 1GoA). This was true for all phenotypes of T cells, CD8+, DP, DN, and CD4+. The amount of labeled {alpha}-bungarotoxin required to detect binding was nearly 10-fold greater than that used to stain nervous tissue (17). Even with this higher level of {alpha}-bungarotoxin, the staining was also relatively weak, with only a few of the cells in the 12-day FTOC classified as intermediate staining (that is, in the intensity range >102–103). It appears that expression, at least of {alpha}-bungarotoxin binding nAChR, gradually increases along with T cell progression from the DP stage to the SP stage. The staining of {alpha}-bungarotoxin among all phenotypes of T cells, CD8+, DP, DN, and CD4+ in both 6- and 12-day cultures was also specifically inhibited by preincubation with d-tubocurarine (table at bottom of Fig. 1Go).



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  		FIGURE 1. {alpha}-Bungarotoxin staining of CD4/CD8-labeled cells. Cells from a 12-day FTOC derived from 13–14 day gestation thymi were stained with Abs specific for CD4 (TC-labeled) and CD8 (PE-labeled) and then with {alpha}-bungarotoxin (1.75 nM; Bo-dipy-labeled) and analyzed by three-color flow cytometry. T cell phenotypes were defined by CD4/8 staining as shown. The intensity of {alpha}-bungarotoxin staining is shown for each of the CD4/8-defined subpopulations of T cells as a separate log fluorescence intensity histogram divided into negative (N), dull (D), intermediate (I), and bright (B) staining by vertical lines (roughly the same scale used to evaluate TCR expression). All numbers represent percentages of cells in each intensity category. The cells were also preincubated with 10-4 M d-tubocurarine for 45 min at 4 C° before {alpha}-bungarotoxin staining. The table at the bottom of this figure shows the results of this experiment. The staining was determined to be specific, as d-tubocurarine significantly inhibited positive staining.

 
Effect of exogenous nicotine addition on the development of T cells in murine FTOC

Our studies focused on the ability of nicotine to affect 12-day FTOC made with 13–14 day gestation B6 fetal mouse thymus tissue. Initially, we studied higher concentrations of nicotine (as high as 10-2 M, which was found to be toxic and lead to low cell recovery and low viability), but we found that as we continued to titer out the drug, nicotine could still affect T cell development. Once it was established that nicotine concentrations >10-4 M were toxic and those <10-18 M produced no significant effect, the experimental range was narrowed to 10-18–10-4 M for all subsequent experiments. Compared with the untreated control, the percentage of lymphocytes in the 10-18 M nicotine-treated culture was not greatly changed (56.9 vs 51.1%; Fig. 2Go). The frequency of DP cells was considerably decreased, and that of SP cells was increased. However, the frequency of mature T cells, shown in Fig. 2Go as having intermediate or bright staining for PE-labeled anti-CD3, was decreased. For example, mature T cells were reduced from 20.5% of the CD8+ SP population to only 1.8% by treatment with 10-18 M nicotine (Fig. 2GoB). Concomitantly, the frequency of immature T cells, shown in Fig. 2Go as having negative or low staining for anti-CD3, was increased from 79.5 to 98.2%.

Total immature vs mature cell recovery was then analyzed at several concentrations of nicotine (shown as a ratio of the untreated control; Fig. 3Go). Mature CD8+ cell maturation was impaired in all concentrations except 10-14 M (Fig. 3GoA). Mature CD4+ cell maturation appeared to be less sensitive than CD8+ cells, in that the degree of inhibition was lower, but the dose-response curve for nicotine was similar (Fig. 3GoD). The numbers of immature CD8+ or CD4+ cells increased dramatically in low dose nicotine culture, and both these cell types also increased to a lesser degree after 10-8–10-6 M nicotine treatment. Similarly, immature DP cells exhibited significant increases at the low (10-16–10-18 M nicotine) dose range and less impressive increases at the high (10-8 M nicotine) dose range. In an interesting contrast to the mature SP T cells, mature DP T cells increased in a pattern similar to that of immature DP (Fig. 3GoB). This latter observation suggests that the effect of nicotine may occur at the DP to SP transition during T cell development or after the cells have committed to the CD4 or CD8 lineage.



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  		FIGURE 3. Profiles for mature and immature CD4 and CD8 cells from nicotine-treated FTOC made with tissue from 13–14 day gestation C57BL/6 pups. FTOCs were treated with the indicated concentrations of nicotine. The cells recovered were counted, and the cells were stained with Abs specific for CD4, CD8, and CD3. The stained cells were analyzed using three-color flow cytometry, and the percentage of gated lymphoid cells was determined for each of the three markers. The data are expressed as the total number of the indicated cell type produced per thymus lobe (x104). Values were then normalized to the untreated control cultures. Statistical analyses (mean and SEM) were performed on these values for comparison purposes. The p values were calculated using paired Student’s t test. Determinations that achieved significance at the 90% confidence level (p <= 0.1) are indicated with one asterisk, and those that achieved significance at the 95% confidence level (p <= 0.05) are indicated with two asterisks. The production of immature T cells (CD3- or CD3low; {diamondsuit}) and that of mature T cells (CD3int or CD3brite; {square}) are shown. Range of cell numbers (per lobe): immature: CD8, 8–18 x 104; DP, 25–36 x 104; DN, 7–16 x 104; CD4, 2–11 x 104; mature: CD8, 2–5 x 104; DP, 2–3 x 104; DN, 6–8 x 104; CD4, 5–7 x 104 (n = 6).

 
A low dose peak and a high dose peak (10-4–10-8 M nicotine) pattern was found when total numbers of CD8, CD4, DP, and DN T cell subpopulations in FTOC were analyzed (Fig. 4Go). The increase in cell numbers seemed to be distributed among all cell phenotypes. The increases in absolute numbers of SP CD4 cells, SP CD8 cells, and DN cells in cultures treated with low dose nicotine (10-18 M) were paralleled with a small increase in the percentages of these cell types at the expense of a decrease in the percentage of DP cells. Across the nicotine dose profile the proportion of CD8+ cells was slightly elevated, the proportion of DN cells was slightly elevated, the percentage of DP cells was slightly depressed at low nicotine concentrations, and the percentage of CD4+ cells was first increased slightly at low nicotine concentrations, then declined slightly with increasing nicotine dose.



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  		FIGURE 4. Quantitation of T cell subsets following nicotine treatment of FTOC. Thirteen to 14 day gestation (dg) C57BL/6 FTOCs were treated with the indicated concentrations of nicotine or were left untreated. The cells recovered were counted and analyzed for the total recovery of CD4+, CD8+, DP, and DN T cells. The data are shown as the number of cells recovered per thymus lobe (x104; {diamondsuit}) and as a percentage of the total lymphocytes ({square}). Values were then normalized to the percentage of the untreated control cultures. Statistical analyses (mean and SEM) were performed on these values for comparison purposes. Determinations that achieved significance at the 90% confidence level (p <= 0.1) are indicated with one asterisk, and those that achieved significance at the 95% confidence level (p <= 0.05) are indicated with two asterisks. Range of cell numbers (per lobe): CD8, 12–21 x 104; DP, 26–38 x 104; DN, 14–25 x 104; CD4, 8–19 x 104 (n = 6).

 
We noticed a difference in the way T cells reacted to nicotine depending on the age of the lobes used. When older tissue (15–16 days gestation) was used as the source of 12-day FTOC compared with 13–14 days gestation tissue, control cultures produced ~2-fold more CD8+ cells per thymus lobe (12.8 x 104 (±1.9 SEM) vs 22 x 104 (±5.6 SEM)), ~2-fold more CD4+ cells (10 x 104 (±1.3 SEM) vs 24.3 x 104 (±5.1 SEM)), and about equal numbers of DN cells, but ~3-fold fewer DP cells (27 x 104 (±3.1 SEM) vs 8 x 104 (±4.6 SEM)). These data are consistent with earlier work showing the production of mature T cells by cultures derived from older fetal thymus lobes grown in FTOC for the same period of time (15). Moreover, the effects of nicotine were less dramatic in FTOC derived from 15–16 day (Fig. 5Go) compared with 13–14 day (Fig. 4Go) gestation tissue. The nicotine effect on cell numbers was generally absent (flat dose-response profile) for CD8+ cells (Fig. 5GoA); in fact, treatment with a low dose nicotine of 13–14 day gestation-derived FTOC gave numbers of CD8+ cells similar to those in control 15–16 day gestation-derived FTOC thymus (21.2 x 104 (±4.4 SEM) vs 22 x 104 (±5.6 SEM) produced by untreated 15–16 day tissue)). The nicotine effect on cell numbers was also generally absent for DP cells, DN cells, and CD4+ cells (Fig. 5Go, B–D). The highest concentration of nicotine (10-2 M) killed all T cell phenotypes in FTOC derived from either older or younger tissues, and after the studies shown in Fig. 5Go, this concentration was subsequently dropped from the titration experiments. The inherent difference in the way more mature FTOC reacts to exogenous nicotine compared with less mature cultures may be due to the fact that the most delicate and easily influenced cell types in the FTOC are immature T cells (DP and immature SP). As mentioned above, this population makes up a large portion of the total cell number in the 13- to 14-day-derived FTOC, but is significantly reduced in the more mature 15- to 16-day-derived FTOC.



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  		FIGURE 5. Effects of nicotine treatment on T cell production in FTOC derived from older fetal thymi. The total cell recovery of CD4+, CD8+, DP, and DN T cells in murine 12-day C57BL/6 FTOC derived from 15–16 day gestation thymi and treated with increasing doses of nicotine is shown as the number of cells recovered per thymus lobe (x104; {diamondsuit}) and as a percentage of the total lymphocytes ({square}). Values were then normalized to the percentage of the untreated control cultures. Statistical analyses (mean and SEM) were performed on these values for comparison purposes. Determinations that achieved significance at the 90% confidence level (p <= 0.1) are indicated with one asterisk, and those that achieved significance at the 95% confidence level (p <= 0.05) are indicated with two asterisks. Range of cell numbers (per lobe): CD8, 3–29 x 104; DP, 2–20 x 104; DN, 2–28 x 104; CD4, 2–31 x 104 (n = 3).

 
Induction of CD69 expression in FTOC treated with nicotine

To determine how far these expanded populations of T cells in FTOC from younger thymi had matured in terms of thymic education and selection, we examined the expression of CD69. This early activation marker has been shown to be expressed just following the MHC-dependent phase of positive selection (18). Its expression is believed to be the final stage of DP development preceding CD4+ or CD8+ lineage commitment, yet T cells remain CD69+ as they continue the maturation process (19). We found that the numbers of T cells of every phenotype expressing CD69 increased in FTOC treated with both low and high doses of nicotine roughly in proportion to increases in total cell numbers (compare Fig. 6Go to Fig. 4Go), although the magnitude of the low dose nicotine effect on CD69 expression for CD4 SP was modest. Thus, the types of T cells that are increased after nicotine exposure have CD69 on their surface, suggesting that nicotine may have delivered a positive selection signal to these cells.



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  		FIGURE 6. Effects of nicotine exposure on CD69 expression in FTOC. Thirteen to 14 day gestation (dg) C57BL/6 FTOCs were treated with the indicated concentrations of nicotine or were left untreated. The cells recovered were counted and were stained with Abs specific for CD4, CD8, and CD69. The stained cells were analyzed using three-color flow cytometry, and the percentage of gated lymphoid cells was determined for each of the three markers. The data are shown as the number of cells recovered per thymus lobe (x104) that were CD69+ ({diamondsuit}) and as the percentage of each lymphocyte subpopulation that was CD69+ ({square}). Values were then normalized to the percentage of the untreated control cultures. Statistical analyses (mean and SEM) were performed on these values for comparison purposes. Determinations that achieved significance at the 90% confidence level (p <= 0.1) are indicated with one asterisk, and those that achieved significance at the 95% confidence level (p <= 0.05) are indicated with two asterisks. Range of cell numbers that were CD69+ (per lobe): CD8, 3–7 x 104; DP, 1–2 x 104; DN, 9–16 x 104; CD4, 2–5 x 104 (n = 6).

 
Induction of CD95 (Fas) expression in FTOC treated with nicotine

We also found that the production of CD95+ T cells of all phenotypes (DN, CD8 SP, CD4 SP, DP) was increased when nicotine was added (Fig. 7Go), clearly in proportion to increases in total cell numbers (see Fig. 4Go). Interestingly, however, the increase in CD95+ cells seen at the lower doses of nicotine for DN, DP, and CD8 SP was not found for CD4 SP. This result parallels the very modest effect of this dose of nicotine on the expression of CD69 on these cells. Overall, these data suggest that nicotine seems to drive the activation, selection, and expansion of T cells. There also appears to be a sustained expression of molecules involved in programmed cell death among the expanded populations of cells.



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  		FIGURE 7. Effects of nicotine treatment on CD95 expression in FTOC. Thirteen to 14 day gestation (dg) C57BL/6 FTOCs were treated with the indicated concentrations of nicotine or were left untreated. The cells recovered were counted and were stained with Abs specific for CD4, CD8, and CD95. The stained cells were analyzed using three-color flow cytometry, and the percentage of gated lymphoid cells was determined for each of the three markers. The data are shown as the number of cells recovered per thymus lobe (x104) that were CD95+ ({diamondsuit}) and as the percentage of each lymphocyte subpopulation that was CD95+ ({square}). Values were then normalized to the percentage of the untreated control cultures. Statistical analyses (mean and SEM) were performed on these values for comparison purposes. Determinations that achieved significance at the 90% confidence level (p <= 0.1) are indicated with one asterisk, and those that achieved significance at the 95% confidence level (p <= 0.05) are indicated with two asterisks. Range of cell numbers that were CD95+ (per lobe): CD8, 7–12 x 104; DP, 23–33 x 104; DN, 12–22 x 104; CD4, 6–14 x 104 (n = 6).

 
Increase in the number of immature T cells by low levels of nicotine and reversal of these effects by d-tubocurarine

Next we attempted to determine whether the effects of exposure to nicotine on the production of immature T cells vs mature T cells could be reversed by the coadministration of d-tubocurarine, a competitive inhibitor of nicotine binding by nAChR. The enhancing effects of low dose nicotine on the production of immature T cells were indeed reversed as the numbers of these cells were returned to control levels (Fig. 8Go). Mature T cell inhibition by low levels of nicotine was also generally returned close to that of the control by d-tubocurarine treatment, although the differences among the control, nicotine-treated, and nicotine- plus d-tubocurarine treated FTOC were not statistically different from one another due to the low and variable numbers of these cells produced by the cultures. Interestingly, the presence of d-tubocurarine alone also affected the ability of thymocytes to mature; d-tubocurarine reduced the numbers of both immature and mature T cells among all phenotypes. These latter results suggest that there is an endogenous nicotine-like ligand that is responsible for regulating the development of T cells via a d-tubocurarine-sensitive mechanism.



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  		FIGURE 8. Effects of d-tubocurarine treatment alone or in combination with nicotine on T cell profiles in FTOC. Samples were treated with 10-18 M nicotine, d-tubocurarine, or both or were left untreated. Cultures that received both nicotine and its inhibitor were pretreated with d-tubocurarine (10-6 M in all cases) for 45 min in FTOC before the addition of nicotine. These cells were then analyzed by three-color flow cytometry. Data are plotted as the numbers of immature ({blacksquare}) and mature ({square}) cells per thymus lobe (x104). Values were normalized to a percentage of the untreated control cultures. Statistical analyses (mean and SEM) were performed on these values for comparison purposes. Determinations that achieved significance at the 90% confidence level (p <= 0.1) are indicated with one asterisk, and those that achieved significance at the 95% confidence level (p <= 0.05) are indicated with two asterisks. Range of cell numbers (per lobe): immature: CD8, 3–15 x 104; DP, 8–36 x 104; DN, 4–12 x 104; CD4, 2–10 x 104; mature: CD8, 1–4 x 104; DP, 0.5–2 x 104; DN, 2–6 x 104; CD4, 2–9 x 104 (n = 4).

 
Induction of RAG genes in FTOC treated with nicotine

RAG-1 and RAG-2 are involved in the rearrangements of B cell and TCR genes. During T cell development these genes are expressed in DN and immature DP cells and are required for the production of the TCR. RAG-1 and RAG-2 RNA were assessed by RT-PCR. We found that RAG expression varied with nicotine concentration and showed increases across almost all nicotine concentrations tested. Similar to the changes observed in immature T cell recovery (compare Fig. 3Go to Fig. 9Go), RAG expression was most dramatic at the low dose (10-16 M). Because these results were presented already normalized for input RNA levels, they indicate an induction of RAG expression per cell or an increased number of T cells that express these genes, such as DN and immature DP cells.



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  		FIGURE 9. Effects of nicotine exposure on RAG expression in FTOC. Thirteen to 14 day gestation (dg) C57BL/6 FTOC were treated with the indicated concentrations of nicotine or were left untreated for the 12 days of organ culture. The cells were counted, and their concentrations were adjusted to the same total number of cells before being processed for RT-PCR. Using RAG-1-, RAG-2-, and {beta}2m-specific probes, RNA levels were determined. The data are expressed as the ratio of PCR products for RAG-1 or RAG-2 vs {beta}2m, which is expressed in all cells (n = 2).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The salient findings of this study are 1) nAChR, capable of binding {alpha}-bungarotoxin, were expressed on T cells that develop in organ culture, and the expression of these receptors increased as the cells mature; 2) exposure of T cells to exogenous nicotine across a wide range of doses in FTOC derived from 13–14 day gestation mouse pups resulted in an increase in the production of immature T cells 12 days later with a concomitant decrease in mature T cell production; 3) more mature T cell precursors in 12-day FTOC derived from older mouse pups (15–16 days gestation) were resistant to the effects of exogenous nicotine noted for 13–14 day gestation FTOC; 4) immature T cells increased by the appropriate concentrations of exogenous nicotine bear activation markers CD69 and CD95 associated with both positive and negative selection; 5) inhibitors of nAChR can reverse the effects of nicotine as well as prevent full development of both immature and mature T cells when added alone to FTOC; 6) the pattern of increased production of immature T cells at the appropriate doses of nicotine shows some similarity to that of RAG gene expression, suggesting that the affected T cells are attempting secondary rearrangements of their TCR.

It is well established that once T cell precursors enter the thymus, they differentiate along a well-defined pathway, which can be delineated based on the expression of CD44, CD25, c-Kit, CD3, CD4, CD8, and TCR (20). Before cells express CD3, CD4, and CD8, they exist as a triple negative (TN) phenotype, and they differentiate successively into TN CD44+ CD25-, TN CD44+ CD25+, TN CD44- CD25+, and TN CD44- CD25- (21). Then the cells express either CD4 or CD8 without CD3 (immature SP) (22, 23) before they become DP CD3- cells. When TCR genes rearrange, a low level of this receptor is expressed on the cell surface, linked to CD3 (immature DP). This receptor is used for positive selection based on the avidity of the TCR for self peptides presented on thymic stromal cells bound to MHC proteins. Those cells with a TCR that recognizes the MHC-peptide complexes with low to moderate avidity are positively selected, and those with very high avidity (that would cause autoimmunity) are negatively selected. Cells that do not produce a TCR that can recognize self- peptide/MHC, which are the vast majority of thymocytes, die via programmed cell death. The cells that remain after these processes of selection are mature CD4-CD8+ SP or CD4+CD8- SP mature T cells with a high level of TCR-associated CD3 (22, 23, 24, 25, 26, 27). These detailed pathways have also been determined for mouse thymocytes and hold true for cells produced in our FTOC (15).

Our work suggests that this pathway is somehow regulated through nAChR engagement. This regulation could be manifested by two possible mechanisms. In the first, the development of T cells is blocked by nAChR engagement at the DP stage, with an increase in the production of these cells, both immature and mature, as well as their immediate precursors, the immature SP cells. The blockade of DP cells results in a decrease in their products, the mature SP cells. The alternative explanation is that the production of immature T cells is increased by direct signaling through the nAChR, and the production of mature T cells is decreased by this signal. In the former model, nAChR signals increase immature T cell production, while in the latter model, the loss of mature SP T cells may be the result of increased negative selection or the deletion of these cells by virtue of the fact that they express higher levels of the {alpha}7-nAChR subtype, which binds {alpha}-bungarotoxin (Fig. 1Go) and has been associated with non-neuronally derived cell types such as epithelial and endothelial cells (28, 29).

However, other nAChR subtypes mediating the effects of nicotine may be higher or lower in SP cells. More importantly, FTOC produced from older mouse tissue yielding large numbers of SP cells showed resistance to the effects of exogenous nicotine, consistent with a decline in functional nAChR expression in those cells. Therefore, the idea that nicotine signaling of mature SP cells could lead to their selective loss in the experiments using tissue from younger pups is not supported by these data. The selective loss of mature SP cells by nAChR engagement also does not readily explain the net increase in the production of immature T cells (CD3- SP or CD3-/low/high DP) seen in nicotine-treated FTOC derived from young mouse pups.

Our data, therefore, favor the first interpretation, namely that nAChR engagement blocked T cell development at the DP stage. It has been shown that that IL-7 added to chimeric FTOC can enhance the production of immature T cells while preventing the development of mature T cells (30). We have recently confirmed this observation and shown that anti-IL-7, added at the time when mature T cells are being produced, can enhance the production of these cells (31). The subcapsular region of the FTOC was found to stain for the presence of IL-7, while the medullary region, where mature T cells would be found, was devoid of IL-7. Thus, there is precedent for signaling of early T cells to not only support the growth and development of these cells, but also to block the development of more mature T cells.

Our d-tubocurarinine data also suggest that nicotine exposure alters the positive selection of immature T cells by mimicking the activity of an endogenous thymic ligand for nAChR. One of our hypotheses is that exogenous nicotine interacts with conventional (or novel?) nAChR subtypes, causing the opening of receptor ion channels, leading to subtle changes in intracellular levels of calcium ions (32, 33). Normal maturation of thymocytes involves TCR ligation, which itself induces calcium ion influx (34, 35). In this fashion exogenous nicotine may mimic TCR signals via ionotropic means and effectively change the way T cells normally mature. This signal could sustain developing thymocytes as they undergo the thymic selection process, resulting in the expansion of immature thymocytes (Fig. 3Go) that otherwise would have been lost due to their lack of a TCR specific for self-peptide/self-MHC. Conversely, the lack of signals, as may occur though blockade of endogenous ligands in the presence of the nicotinic antagonist d-tubocurarine, might inhibit normal development of both mature and immature T cells. This idea may explain why d-tubocurarine treatment of FTOC exposed to low levels of nicotine still produced lower levels of immature and mature DP cells (Fig. 8Go), in that an excess of the antagonist could block T cell production in addition to reversing the effects of nicotine. Signals generated via nAChR upon exogenous nicotine administration appear to alter positive selection such that CD69-positive T cells, which are normally produced in the murine FTOC system, were increased (Fig. 6Go). The expansion of these populations may be due to the direct activation by nicotine or the prevention of their progression down the T cell differentiative pathway caused by a block in positive selection. The activation signal delivered by nicotine may be sufficient to cause the T cells to down-regulate their TCR, perhaps by interfering with the recycling of the CD3/TCR complex after ligation by MHC-peptide complexes (36). This result would be manifested as a conversion of mature CD3-expressing SP T cells that had previously been selected via their TCR’s interaction with self-peptide/MHC into CD3low-expressing cells that would appear as immature cells by our criteria. However, this mechanism would not explain the large increase in the total number of immature CD3low-expressing cells over control values that we found, unless the original CD3high-expressing SP cells had also been driven to proliferate by nicotine. Most T cells in the thymus do not bear TCR specific for the Ags presented there, and the number of mature TCRhigh DP cells was actually increased by nicotine treatment. Therefore, we favor the concept that T cell development is impacted by nicotine at the DP stage, without induction of TCR down-modulation in mature TCRhigh SP T cells.

However, since positive selection must also be accompanied by sustained signaling through the TCR, a signal that T cells will not receive, since the vast majority of them do not have a TCR specific for self-MHC and self-peptide required for normal positive selection, these cells may eventually begin to undergo programmed cell death. Thus, Fas, which is associated with apoptosis of DP T cells (37), appears on the immature cells that accumulate in FTOC treated with exogenous nicotine. Other workers (38) have reported a similar connection between nicotine and the induction of apoptosis in maturing thymocytes.

The increased expression of Fas may be associated with an increase in RAG activity seen after exposure to nicotine doses that also increased the production of immature T cells (Fig. 9Go). Petrie et al. (12) demonstrated that T cells receiving TCR activation signals strong enough to elicit negative selection attempt to produce another, less avid, TCR by rearranging additional TCR {alpha}-chain genes. These additional TCR rearrangements result in reactivation of RAG. T cells derived from the cord blood of infants born to mothers exposed to passive tobacco smoke also have increased RAG gene expression (39). We hypothesize that since positive selection is altered in T cell precursors exposed to nicotine, the T cells may attempt to further rearrange their TCR to affect a normal positive selection signal, causing an increase in RAG activity and Fas expression. Thus, the increase in RAG expression can be caused by an elevated expression per recombination-active cell and/or an increased fraction of RAG-expressing cells in total thymocytes. The higher levels of RAG expression in individual cells may reflect the attempt by these potentially tolerized T cells to undertake receptor editing. Alternatively, an elevated number of RAG-expressing cells among total thymocytes is an indicator of the immature status of the T cell population, i.e., the relative increase in DN and DP cells, which is consistent with our observation in T cell recovery, shown in Figs. 3Go and 4Go.

Both these scenarios are consistent with our finding of enhanced production of immature T cells that are recombination active along with a reduction in mature SP T cells. The fact that nicotine treatment also increased the production of DP T cells bearing high levels of TCR (Fig. 3Go) would suggest that the control point of T cell development impacted by nicotine is the transition between DP and SP T cells. However, we also found decreases in the percentages of DP T cells produced in these cultures and increases in the production of DN T cells, which are precursors of the other cell types. This result, along with the fact that d-tubocuarinine treatment inhibited the production of all T cells in FTOC suggest that nAChR engagement may affect early precursors of T cells before the DP stage of development as well.

The low dose range of nicotine used in our work (10-16–10-18 M) is much lower than the range of concentrations of nicotine found in the plasma of smokers (2–4 x 10-7M), where we found some effects of nicotine (2). The higher dose of nicotine (10-2 M), which inhibited the development of T cells entirely, is substantially greater than plasma levels. However, concentrations of nicotine that acutely activate the function of known and characterized nAChR subtypes half-maximally are in the 10-4–10-6 M range, and maximal effects of acetylcholine occur at 10-3 M, approximately the concentration of acetylcholine attained transiently at neuronal synapses (40, 41, 42). Thus, some of the effects observed in this study of nicotine action occur at concentrations compatible with actions through known nAChR subtypes, which function as nicotinic agonist-gated Na+ and/or Ca2+ channels mediating cation influx into cells. However, the low dose effects observed at 10-16–10-18 M suggest that novel nAChR subtypes might exist. These observations have given rise to current attempts to identify these potentially novel nAChRs. Direct measurement of the affinities of FTOC-derived nAChRs will require the production of cloned cells expressing these receptors exclusively. Alternatively, conventional nAChR subtypes in FTOC may mediate novel signaling cascades much more sensitive to nicotine activation than opening of nAChR channels, especially among DP T cells, which are known to be exquisitely sensitive to nearly any signaling event.

Because the effects of nicotine in FTOC occur at wide range of concentrations, it is also evident that multiple nAChR subtypes must be involved. Immature FTOC-derived thymocytes express {alpha}3, {alpha}5, {alpha}7, {beta}2, and {beta}4 subunit genes as message, while mature thymocytes express {alpha}2, {alpha}5, and {alpha}7 subunits. Thymic stromal cells express {alpha}2, {alpha}3, {alpha}4, {alpha}7, and {beta}4 subunits. Other subunits, such as {alpha}2 and {alpha}4, are expressed at marginal levels on immature T cells, and {alpha}4 is expressed at marginal levels on thymic stromal cells. Moreover, levels of expression of these subunits are developmentally regulated through fetal and into early postnatal life (43). For example, {alpha}3, {alpha}7, and {beta}4 subunit genes appear to be expressed at the highest level in scid/scid FTOC at 15 days gestation, the stage when immature TCR- T cells, which appear to be sensitive in the present study, would be expected to be produced. The differences in subunit expression between thymocytes and stromal cells in conjunction with developmental regulation may help explain why nicotine is able to exert effects at both a low (10-16–10-18 M) and a high (10-6–10-10 M) dose range.

The lowest concentrations of nicotine required to influence T cell development are smaller than those of other agents known to be active in causing alterations in T cell survival, such as vasoactive peptide (10-14 M) for inhibition of Ag-induced apoptosis (44), nominal peptide itself (10-10 M) in the activation of mature CD8+ T cells in TCR transgenic spleen cells, or the same peptide in the selection of mature CD8 SP T cells in FTOC (10-5 M) (45). However, in all these systems the assay involved mature T cells, not the immature cells that we examined in our studies. Developing thymocytes, especially the DP population, are known to be exquisitely sensitive to signals such as peptide and cortisone. Nevertheless, the sensitivity of FTOC-derived T cells to nicotine was unexpected and led to an extensive analysis to determine the lowest level of nicotine that could reproducibly affect T cell development. It is conceivable that the very low concentrations of nicotine found to be effective in our studies are actually concentrated by stromal cells in the thymus for presentation to the T cells, or the amounts we added supplemented the endogenous nicotinic ligand(s) already made by the thymus to cause the effects. Interestingly, mature CD3+ cells derived from FTOC using young (13–14 days gestation) pups or SP T cells from FTOC using older (15–16 days gestation) pups were relatively resistant to nicotine. These results suggest that sensitivity to nicotine signaling is also developmentally regulated.

Our d-tubocurarine blocking data suggest a role for nAChR engagement in normal T cell development and are in line with the observation that cholinergic input to the thymus seems to regulate thymocyte maturation. Transections of the right vagus nerve produce a decrease in the number of lymphocytes released from the thymus into the venous circulation (46). This effect disappears after sectioning of the recurrent laryngeal nerve. Vagal stimulation produces a transient increase in the number of lymphocytes released from the thymus, an effect that also disappears after section of the recurrent nerve. The effects of vagotomy are mimicked by nicotine-blocking agents, which also suppress the effects of vagal stimulation. Selectivity for nicotinic cholinergic signaling is suggested, because muscarinic cholinergic agents are ineffective in mimicking the effects of vagotomy. A recent development along this line of research is the identification of lynx1, an endogenous peptide that has been shown to be a potent modulator of nicotinic receptor function with a high degree of structural and genetic homology to the Ly-6 family of immune-associated Ags (47). However, it has only been detected in brain to date.

Taken together, our observations implicate a definitive role for a possibly unknown nAChR ligand in normal thymic ontogeny and implicate a direct interaction between the nervous system and the immune system, shared resources between the immune and nervous systems, or both. Our results extend those of other workers (29) who have found nAChRs expressed on nonexcitable cells and have suggested that these receptors may modulate cell proliferation and differentiation in response to locally produced acetylcholine. Most interesting of all, perhaps, is the extremely low concentrations of nAChR ligand needed to exert notable effects on the process of immune development. In light of this finding, the wide use of nicotine-containing products in modern society may have more impact on immune system development than previously thought. Indeed, we have preliminary evidence that exposure to nicotine during pregnancy profoundly alters the ability of human cord blood-derived T cell precursors to develop in organ cultures.


   Acknowledgments
 
We thank Jennifer Michaels, Ty Lebsack, and Donald Shaw for their excellent technical assistance.


   Footnotes
 
1 This work was supported by Grants 9910 and 5001 from the Arizona Disease Control Research Commission (to D.D.). Back

2 Address correspondence and reprint requests to Dr. Dominick DeLuca, Department of Microbiology and Immunology, University of Arizona College of Medicine, Life Sciences North, Room 648, 1501 North Campbell Avenue, Tucson, AZ 85724. E-mail address: deluca{at}u.arizona.edu Back

3 Abbreviations used in this paper: nAChR, nicotinic acetylcholine receptor; {beta}2m, {beta}2-microglobulin; DN, double negative (CD8-CD4-); DP, double positive (CD8+CD4+); FC, flow cytometric; FTOC, fetal thymus organ culture; RAG, recombinase-activating gene; SP, single positive (CD4+ or CD8+); TC, Tri-Color; TN, triple negative (CD8-CD4-CD3-). Back

Received for publication December 11, 2001. Accepted for publication June 25, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Johnson, J. D., D. P. Houchens, W. M. Kluwe, D. K. Craig, G. L. Fisher. 1990. Effects of mainstream and environmental tobacco smoke on the immune system in animals and humans: a review. Crit. Rev. Toxicol. 20:369.[Medline]
  2. Geng, Y., S. M. Savage, L. J. Johnson, J. Seagrave, M. L. Sopori. 1995. Effects of nicotine on the immune response. I. Chronic exposure to nicotine impairs antigen receptor-mediated signal transduction in lymphocytes. Toxicol. Appl. Pharmacol. 135:268.[Medline]
  3. Kalra, R., S. P. Singh, S. M. Savage, G. L. Finch, M. L. Sopori. 2000. Effects of cigarette smoke on immune response: chronic exposure to cigarette smoke impairs antigen-mediated signaling in T cells and depletes IP3-sensitive Ca2+ stores. J. Pharmacol. Exp. Ther. 293:166.[Abstract/Free Full Text]
  4. Sopori, M. L., W. Kozak, S. M. Savage, Y. Geng, D. Soszynski, M. J. Kluger, E. K. Perryman, G. E. Snow. 1998. Effect of nicotine on the immune system: possible regulation of immune responses by central and peripheral mechanisms. Psychoneuroendocrinology 23:189.[Medline]
  5. Evavold, B. D., J. Sloan-Lancaster, P. M. Allen. 1993. Tickling the TCR: selective T-cell functions stimulated by altered peptide ligands. Immunol. Today 14:602.[Medline]
  6. Sloan-Lancaster, J., P. M. Allen. 1995. Significance of T-cell stimulation by altered peptide ligands in T cell biology. Curr. Opin. Immunol. 7:103.[Medline]
  7. Geng, Y., S. M. Savage, S. Razanai-Boroujerdi, M. L. Sopori. 1996. Effects of nicotine on the immune response. II. Chronic nicotine treatment induces T cell anergy. J. Immunol. 156:2384.[Abstract]
  8. Menard, L., M. Rola-Pleszcynski. 1987. Nicotine induces T-suppressor cells: Modulation by the nicotinic antagonist d-tubocurarine and myasthenic serum. Clin. Immunol. Immunopathol. 44:107.[Medline]
  9. Talib, S., T. B. Okarma, J. S. Lebkowski. 1993. Differential expression of human nicotinic acetylcholine receptor alpha subunit variants in muscle and non-muscle tissues. Nucleic Acids Res. 21:233.[Abstract/Free Full Text]
  10. Kearse, K. P., Y. Takahama, J. A. Punt, S. O. Sharrow, A. Singer. 1995. Early molecular events induced by T cell receptor (TCR) signaling in immature CD4+ CD8+ thymocytes: increased synthesis of TCR-{alpha} protein is an early response to TCR signaling that compensates for TCR-{alpha} instability, improves TCR assembly, and parallels other indicators of positive selection. J. Exp. Med. 181:193.[Abstract/Free Full Text]
  11. Ashton-Rickardt, P. G., A. Bandeira, J. R. Delaney, L. Van Kaer, H. P. Pircher, R. M. Zinkernagel, S. Tonegawa. 1994. Evidence for a differential avidity model of T cell selection in the thymus. Cell 76:651.[Medline]
  12. Petrie, H. T., F. Livak, D. G. Schatz, A. Strasser, I. N. Crispe, K. Shortman. 1993. Multiple rearrangements in T cell receptor {alpha} chain genes maximize the production of useful thymocytes. J. Exp. Med. 178:615.[Abstract/Free Full Text]
  13. Rugh, R.. 1968. The Mouse: Its Reproduction and Development Burgess, Minneapolis.
  14. Schatz, D. G., M. A. Oettinger, D. Baltimore. 1989. The V(D)J recombination activating gene, RAG-1. Cell 59:1035.[Medline]
  15. DeLuca, D., J. A. Bluestone, L. D. Shultz, S. O. Sharrow, Y. Tatsumi. 1995. Programmed differentiation of murine thymocytes during fetal thymus organ culture. J. Immunol. Methods 178:13.[Medline]
  16. Chang, Y., G. C. Bosma, M. J. Bosma. 1995. Development of B cells in scid mice with immunoglobulin transgenes: implications for the control of V(D)J recombination. Immunity 2:607.[Medline]
  17. Wheeler, S. V., S. D. Jane, K. M. Cross, J. E. Chad, R. C. Foreman. 1994. Membrane clustering and bungarotoxin binding by the nicotinic acetylcholine receptor: role of the {beta} subunit. J Neurochem 63:1891.[Medline]
  18. Hare, K. J., E. J. Jenkinson, G. Anderson. 1999. CD69 expression discriminates MHC-dependent and -independent stages of thymocyte positive selection. J. Immunol. 162:3978.[Abstract/Free Full Text]
  19. Aguila, H. L., K. Akashi, J. Domen, K. L. Gandy, E. Lagasse, R. E. Mebius, S. J. Morrison, J. Shizuru, S. Strober, N. Uchida, et al 1997. From stem cells to lymphocytes: biology and transplantation. Immunol. Rev. 157:13.[Medline]
  20. Godfrey, D. I., A. Zlotnik. 1993. Control points in early T-cell development. Immunol. Today 14:547.[Medline]
  21. van Ewijk, W., G. Hollander, C. Terhorst, B. Wang. 2000. Stepwise development of thymic microenvironments in vivo is regulated by thymocyte subsets. Development 127:1583.[Abstract]
  22. Guidos, C. J., I. L. Weissman, B. Adkins. 1989. Intrathymic maturation of murine T lymphocytes from CD8+ precursors. Proc. Natl. Acad. Sci. USA 86:7542.[Abstract/Free Full Text]
  23. Guidos, C. J., I. L. Weissman, B. Adkins. 1989. Developmental potential of CD4-8- thymocytes: peripheral progeny include mature CD4-8- T cells bearing {alpha}{beta} T cell receptor. J. Immunol. 142:3773.[Abstract]
  24. Mathieson, B. J., B. J. Fowlkes. 1984. Cell surface antigen expression on thymocytes: development and phenotypic differentiation of intrathymic subsets. Immunol. Rev. 82:141.[Medline]
  25. Scollay, R., P. Bartlett, K. Shortman. 1984. T cell development in the adult murine thymus: changes in the expression of the surface antigens Ly2, L3T4 and B2A2 during development from early precursor cells to emigrants. Immunol. Rev. 82:79.[Medline]
  26. Scollay, R., J. Smith, V. Stauffer. 1986. Dynamics of early T cells: prothymocyte migration and proliferation in the adult mouse thymus. Immunol. Rev. 91:129.[Medline]
  27. Guidos, C. J., J. S. Danska, C. G. Fathman, I. L. Weissman. 1990. T cell receptor-mediated negative selection of autoreactive T lymphocyte precursors occurs after commitment to the CD4 or CD8 lineages. J. Exp. Med. 172:835.[Abstract/Free Full Text]
  28. Wang, Y., E. F. Pereira, A. D. Maus, N. S. Ostlie, D. Navaneetham, S. Lei, E. X. Albuquerque, B. M. Conti-Fine. 2001. Human bronchial epithelial and endothelial cells express {alpha}7 nicotinic acetylcholine receptors. Mol. Pharmacol. 60:1201.[Abstract/Free Full Text]
  29. Conti-Fine, B. M., D. Navaneetham, S. Lei, A. D. Maus. 2000. Neuronal nicotinic receptors in non-neuronal cells: new mediators of tobacco toxicity?. Eur. J. Pharmacol. 393:279.[Medline]
  30. Plum, J., M. De Smedt, G. Leclercq. 1993. Exogenous IL-7 promotes the growth of CD3-CD4-CD8-CD44+CD25+/- precursor cells and blocks the differentiation pathway of TCR-{alpha}{beta} cells in fetal thymus organ culture. J. Immunol. 150:2706.[Abstract]
  31. DeLuca, D., D. R. Clark. 2002. Interleukin-7 negatively regulates the development of mature T cells in fetal thymus organ cultures. Dev. Comp. Immunol. 26:365.[Medline]
  32. Eisenhour, C. M., E. Puchacz, R. J. Lucas. 1993. Calcium ion influx in clonal cell lines expressing nicotinic acetylcholine receptors. Soc. Neurosci. Abstr. 19:1533.
  33. McGehee, D. S., L. W. Role. 1995. Physiological diversity of nicotinic acetylcholine receptors expressed by vertebrate neurons. Annu. Rev. Physiol. 57:521.[Medline]
  34. Noble, A., J. P. Truman, B. Vyas, M. Vukmanovic-Stejic, W. J. Hirst, D. M. Kemeny. 2000. The balance of protein kinase C and calcium signaling directs T cell subset development. J. Immunol. 164:1807.[Abstract/Free Full Text]
  35. Andjelic, S., N. Jain, J. Nikolic-Zugic. 1993. Immature thymocytes become sensitive to calcium-mediated apoptosis with the onset of CD8, CD4, and the T cell receptor expression: a role for bcl-2?. J. Exp. Med. 178:1745.[Abstract/Free Full Text]
  36. Liu, H., M. Rhodes, D. L. Wiest, D. A. Vignali. 2000. On the dynamics of TCR:CD3 complex cell surface expression and downmodulation. Immunity 13:665.[Medline]
  37. Nishimura, Y., A. Ishii, Y. Kobayashi, Y. Yamasaki, S. Yonehara. 1995. Expression and function of mouse Fas antigen on immature and mature T cells. J. Immunol. 154:4395.[Abstract]
  38. Rinner, I., T. Kukulansky, P. Felsner, E. Skreiner, A. Globerson, M. Kasai, K. Hirokawa, W. Korsatko, K. Schauenstein. 1994. Cholinergic stimulation modulates apoptosis and differentiation of murine thymocytes via a nicotinic effect on thymic epithelium. Biochim. Biophys. Acta 203:1057.
  39. Finette, B. A., J. P. O’Neill, P. M. Vacek, R. J. Albertini. 1998. Gene mutations with characteristic deletions in cord blood T lymphocytes associated with passive maternal exposure to tobacco smoke. Nat. Med. 4:1144.[Medline]
  40. Lukas, R. J.. 1989. Pharmacological distinctions between functional nicotinic acetylcholine receptors on the PC12 rat pheochromocytoma and the TE671 human medulloblastoma. J. Pharmacol. Exp. Ther. 251:175.[Abstract/Free Full Text]
  41. Lukas, R. J.. 1995. Diversity and patterns of regulation of nicotinic receptor subtypes. Ann. NY Acad. Sci. 757:153.[Abstract]
  42. Fenster, C. P., M. F. Rains, B. Noerager, M. W. Quick, R. A. Lester. 1997. Influence of subunit composition on desensitization of neuronal acetylcholine receptors at low concentrations of nicotine. J. Neurosci. 17:5747.[Abstract/Free Full Text]
  43. Kuo, Y., L. Lucero, J. Michaels, D. DeLuca, and R. J. Lukas. 2001. Differential expression of nicotinice acetylcholine receptor subunits in fetal and neonatal mouse thymus. J. Neuroimmunol. In press.
  44. Delgado, M., E. Garrido, C. Martinez, J. Leceta, R. P. Gomariz. 1996. Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptides (PACAP27) and PACAP38) protect CD4+CD8+ thymocytes from glucocorticoid-induced apoptosis. Blood 87:5152.[Abstract/Free Full Text]
  45. Sebzda, E., V. A. Wallace, J. Mayer, R. S. Yeung, T. W. Mak, P. S. Ohashi. 1994. Positive and negative thymocyte selection induced by different concentrations of a single peptide. Science 263:1615.[Abstract/Free Full Text]
  46. Antonica, A., F. Magni, L. Mearini, N. Paolocci. 1994. Vagal control of lymphocyte release from rat thymus. J. Auton. Nerv. Syst. 48:187.[Medline]
  47. Miwa, J. M., I. Ibanez-Tallon, G. W. Crabtree, R. Sanchez, A. Sali, L. W. Role, N. Heintz. 1999. Lynx1, an endogenous toxin-like modulator of nicotinic acetylcholine receptors in the mammalian CNS. Neuron 23:105.[Medline]



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