Nicotinic environment affects the differentiation and functional maturation of monocytes derived dendritic cells (DCs)

Immunology Letters 95 (2004) 45–55

Nicotinic environment affects the differentiation and functional maturation of monocytes derived dendritic cells (DCs) Elisabeth Guinet, Keiichiro Yoshida, Mahyar Nouri-Shirazi∗ Department of Biomedical Sciences, Immunology Laboratory, Texas A&M University System Health Science Center, Baylor College of Dentistry, 3302 Gaston Avenue, Dallas, TX75246, USA Received 7 May 2004; received in revised form 5 June 2004; accepted 8 June 2004 Available online 1 July 2004

Abstract Differentiation of tissue monocytes into DCs is a critical phase in the development of a competent immune system. We show that in a nicotinic environment, while human monocytes differentiate into DCs (henceforth called nicDCs) with a typical morphology, they display unique phenotype and cytokine profile that adversely affect their function. Despite an increased capacity for receptor-dependent antigen uptake, nicDCs do not express CD1a and fail to fully up-regulate MHCs, molecules essential for their antigen-presenting function. Additionally, in response to bacterial antigen LPS, maturing nicDCs hardly express the chemotactic cytokine receptor 7 required for their entry into lymphatic vessels. Furthermore, in parallel with their differential expression of costimulatory molecules CD80 and CD86 and lack of IL-12, nicDCs display profoundly reduced Th1 promoting capacity. These findings thus indicate that nicotine impedes the development of cell-mediated immunity by skewing DC differentiation. These effects of nicotinic environment on DC differentiation may contribute to the increased risks of respiratory tract infection and various cancers in smokers. © 2004 Elsevier B.V. All rights reserved. Keywords: Dendritic cells; Nicotine; Differentiation; Th1

1. Introduction Cigarette smoke is a major health risk factor which significantly increases the incidence of cancers of various organs, as well as acute and chronic respiratory tract infections [1,2]. It has been reported that nicotine mimics the effects of cigarette smoke and alters a wide range of immunological functions including innate and adaptive immune responses [2]. Dendritic cells (DCs) are the key antigen-presenting cells that are pivotal in the initiation of a primary immune response [3]. Recently, our laboratory has shown that nicotine at dose reached in local tissues compromises competent DC functions [4].

Abbreviations: nicDC, DC generated in the presence of nicotine; DC-LAMP, DC-lysosome associated membrane glycoprotein; MFI, mean fluorescence intensity; SLC, secondary lymphoid-tissue chemokine; MIP-3␤, macrophage inflammatory protein ∗ Corresponding author. Tel.: +1 214 828 8138; fax: +1 214 828 8951. E-mail address: [email protected] (M. Nouri-Shirazi). 0165-2478/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2004.06.003

Bone marrow-derived circulating DC precursors, including monocytes, continuously exit the bloodstream and enter peripheral tissues where they eventually become resident cells [3]. During resolution of inflammation, endothelial tissues initiate differentiation of monocytes to DCs [5] that leave peripheral tissues by migrating to draining lymph nodes where they present antigens to T cells. Because in smokers, the generation of DCs takes place in a tissue environment composed of nicotine, we surmised that these cells possess properties that adversely influence the outcome of immune surveillance. In vitro, human monocytes can be directed to develop into potent immunostimulatory DCs when cultured in the presence of GM-CSF and IL-4 [6–9], cytokines that can be released in vivo by tissue mast cells [10]. To gain further insight into the distinctive package of effects which nicotine has on the DC system, we studied its effects on the in vitro differentiation of human monocytes into DCs, and their functional specialization with respect to antigen uptake, expression of migratory receptor, ability to secrete pro-inflammatory cytokines, and to modulate T-cell responses. We report the first in vitro

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results demonstrating that nicotinic environment skews the differentiation of monocytes into atypical DCs lacking parameters essential for the development of cell-mediated immunity.

2. Materials and methods 2.1. Media and reagents Complete culture media (CM): RPMI 1640, 1% l-glutamine, 1% penicillin/streptomycin, 50 ␮M 2-ME, 1% sodium-pyruvate, 1% non-essential aminoacids and heat-inactivated 10% FCS or human AB serum. Recombinant human cytokines: GM-CSF (Immunex, Seattle, WA), and IL-4 (R&D System, Minneapolis, MN). 7-Aminoactinomycin D (7-AAD), cycloheximide, (−)-nicotine and LPS were purchased from Sigma (St. Louis, MO). (−)-Nicotine was used at concentrations ranging from 0 to 200 ␮g/mL. FITC-dextran, CFSE and Lucifer yellow were purchased from Molecular Probes (Eugene, OR). 2.2. Generation of and immunophenotyping of DCs Immature monocyte-derived DCs were generated from peripheral blood adhering monocytes cultured in CM with GM-CSF (100 ng/mL) and IL-4 (10 ng/mL) as described previously [4,9]. Cultures were fed every 2 days with medium containing cytokines and nicotine (200 ␮g/mL). DC viability was quantitated by trypan blue staining and flow cytometry using FITC-labeled annexin V and propidium iodide (PI). Briefly, a total of 2 × 105 cells were incubated with 5 mL annexin V-FITC in binding buffer for 10 min, then washed and suspended in binding buffer before the addition of 5 mg/mL PI. On day 6 immature DCs and nicDCs were suspended in complete medium and then activated with LPS (1 ␮g/mL) for 48 h. DCs were stained with corresponding fluorochrome-labeled mAbs: CD1a (NA1/34), HLA-ABC (W6/32) (DAKO, Carpinteria, CA); CD14 (Tuk 4), CD32 (Fc␥R II, C1KM5) (CALTAG, Burlingame, CA); CD83 (HB15a, Beckman-Coulter, Brea, CA); HLA-DR (L243), CD80 (L307), CD86 (IT2.2), CD40 (5C3), CD36 (CB38), mannose receptor (19) (BD Biosciences, San Diego, CA); and analyzed by flow cytometry. Purified CCR7 (CCR7.6B3, BD Bioscience, San Diego, CA) staining was followed by biotinylated goat anti-mouse IgG F(ab )2 fragment conjugate (Molecular Probes, Eugene, OR), revealed by fluorochrome-labeled streptavidin (BD Biosciences, San Diego, CA) after neutralizing with mouse serum and analyzed by flow cytometry. 2.3. Confocal analysis DCs were allowed to adhere on polylysine-coated slides (Erie Scientific Company, Portsmouth, NH) for 45 min at

room temperature, fixed with 4% paraformaldehyde/PBS for 30 min, washed with 0.1% glycine/PBS, then permeabilized with 0.1% Triton/PBS. After blocking with 2 mg/mL BSA/PBS/goat serum, cells were labeled for 30 min at room temperature with purified mouse anti-human DC-LAMP (104.G4, Beckman-Coulter, Brea, CA) followed by biotinylated goat anti-mouse IgG F(ab )2 fragment conjugate, and revealed by Texas Red-labeled streptavidin (Sav-TR, BD Biosciences, San Diego, CA) after neutralizing with mouse serum. Cells were then stained with FITC-labeled anti-HLA-DR (L243, BD Biosciences, San Diego, CA) for 30 min. Slides were analyzed by confocal microscopy. 2.4. Antigen uptake by DCs Immature DCs or nicDCs were washed with PBS and suspended in CM containing FITC-dextran (0.5 mg/mL) or Lucifer yellow (1 mg/mL) (Molecular Probe, Eugene, OR). After 30 min of incubation at 37 or 4 ◦ C, as negative controls, cells were washed four times with cold PBS containing 1% FCS prior to analysis by flow cytometry. Induction of apoptotic cell death and labeling of apoptotic bodies were performed as described previously [4,11]. The labeled apoptotic cells were subsequently co-cultured with CFSE-labeled immature DCs or nicDCs at a 1:2 ratio. After 2 h at 37 ◦ C, cells were washed, and treated with 0.05% trypsin/0.02% EDTA for 5 min to disrupt cell–cell binding. Phagocytosis was quantified by flow cytometry as the percentage of double positive cells, CFSE+ 7-AAD+ . 2.5. T-cell purification and T-cell functional assays Purified na¨ıve T cells were obtained from Ficoll-separated PBMCs of healthy volunteers depleted of other cells using StemSepTM na¨ıve T cell enrichment kit (StemCell Technologies, Vancouver, Canada) containing CD14 (RMO52), CD16 (3G8), CD19 (J4.119), CD56 (NKH-1), HLA-DR (B8.12.2), glycophorin A (D2.10), CD45RO (UCHL1) monoclonal antibodies and magnetic colloid. The purity of the enriched population was > 98%. For proliferation assay, DCs and nicDCs were co-cultured (5 × 103 /well) in CM with 5% human AB serum with purified T cells (1 × 105 /well) in round-bottom 96 well plates (Falcon, Franklin Lakes, NJ). IL-12 (5 ng/mL) was added at the beginning of co-cultures. Proliferation was measured after 5 days by uptake of tritiated thymidine (1 ␮Ci/well for the last 16 h). For flow cytometry assay, DCs and nicDCs were co-cultured with purified allogeneic T cells at a ratio of 1:10 in CM containing 5% human AB serum. After 5 days, T cells were harvested and re-stimulated with fresh DCs for additional 40 h. Subsequently, aliquots of T cells from secondary co-cultures were stained with anti-CD4 (MT310, DAKO, Carpinteria, CA), anti-CD8 (Leu-2a), T-cell activation marker anti-CD25 (M-A251) (BD Biosciences, San Diego, CA) and analyzed by flow cytometry.

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2.6. Cytokine analysis

3. Results

Forty-eight hours following LPS stimulation, DCs or nicDCs culture supernatants were collected and the amounts of TNF-␣, IL-1␤, IL-10, and IL-12 were measured by ELISA (BD Biosciences, San Diego, CA). To assess T-cell cytokine production, culture supernatants from DC/T co-cultures were collected and amounts of IL-4 and IFN-␥ were measured by ELISA (BD Biosciences, San Diego, CA). For cytokine detection at the single cell level, after secondary co-cultures had been carried out, T cells were stimulated for 6 h with a leukocyte activation cocktail (BD Biosciences, San Diego, CA) before staining with anti-IFN-␥ (25723.11), anti-IL-4 (3010.211) and anti-CD3 (UCHT1) (BD Bioscience, San Diego, CA). The labeled cells were then analyzed by flow cytometry.

3.1. DC differentiation in a nicotinic environment

2.7. Statistical analysis Values are presented as mean ± S.D. Statistical significance of differences between nicDCs and controls was calculated using ANOVA test. A P < 0.05 was considered statistically significant.

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We first determined the extent to which the presence of nicotine in the microenvironment would affect monocyte differentiation into DCs by monitoring the acquisition of DC morphology and phenotype. When cultured in the presence of GM-CSF and IL-4 for 6 days, monocytes differentiated into large, non-adherent veiled cells with a typical immature DC morphology (Fig. 1A, left panel). Whereas addition of nicotine together with GM-CSF and IL-4 during DC differentiation did not affect the morphological development of generated DCs (henceforth called as nicDCs) (Fig. 1A, right panel), it skewed DCs towards the acquisition of an atypical phenotype characterized by selective absence of CD1a and CD80 and higher expression of CD86 and MHC class I and II molecules in a dose-dependent manner (Fig. 2 and Table 1). Identical results were obtained using nicotine hydrogen bitartrate (data not shown). It should be noted that nicDCs showed similar viability as compared to DCs even at the highest concentration of nicotine used in our studies (Fig. 1B).

Fig. 1. Effect of nicotine on DC differentiation. (A) Phase contrast photographs were taken directly from 6-day immature DCs generated in the absence (left image) or presence (right image) of 200 ␮g/mL nicotine. Numerous non-adherent DCs or nicDCs are visible. Original magnifications 10×. (B) Flow cytometry analysis of immature DCs and nicDCs stained with annexin V/PI. Annexin V+, annexin V+PI+, and PI+ staining represent early apoptotic, late apoptotic, and necrotic cells, respectively. One representative experiment of three is shown.

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Fig. 2. Dose-dependent effect of nicotine on differentiating DCs. Flow cytometry analysis of immature DCs generated in the absence or presence of different concentrations of nicotine. Open histograms represent the isotype control Abs. Filled black (DCs) and grey (nicDCs) histograms represent specific staining of the indicated cell-surface markers. One representative experiment of seven is shown.

3.2. Antigen uptake by nicDCs By and large, DCs are the first immune cells to encounter foreign antigens. Continuous sampling of the antigenic environment and delivery of processed antigens to na¨ıve T cells in draining lymph nodes are primary functions of tissue-resident DCs [3]. To evaluate the extent to which nicDCs fulfill these tasks, we first tested their ability to capture antigenic materials. nicDCs were allowed to internalize Lucifer yellow (macropinocytosis), FITC-dextran (receptor-mediated endocytosis), and 7-AAD-labeled apoptotic cells (receptor-mediated phagocytosis). Flow cytometry analyses revealed that while nicDCs

were less efficient in macropinocytosis, they augmented receptor-mediated antigen uptakes (Fig. 3A and B). The increased in receptor-dependent antigen uptake was correlated to higher expression of mannose (MFI: 331 versus 284) and Fc␥RII (20 versus 2) receptors on nicDCs compared to DCs. However, the expression of CD36 (107 versus 197), one of the molecules that mediate phagocytosis of apoptotic cells [11,12], was lessened (Fig. 3C). 3.3. The properties of mature nicDCs In response to maturation stimuli, DCs not only increase expression of costimulatory and antigen-presenting

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Table 1 Phenotype of immature and mature DC and nicDC CD1a DC Phenotype 534 355 143 213 278 135 207

CD14 nicDC

Mean 266 17 P-value 0.0005 Phenotype 179 233 146 207 177 78 291

DC

of immature 5 35 14 32 8 9 14

AB nicDC

DR

DC

nicDC

DC

nicDC

DC and nicDC (markers (immature)) 3 5 361 602 486 7 20 121 333 159 5 4 136 298 210 3 5 360 600 1336 16 9 111 249 228 2 1 65 150 608 9 8 285 467 486 6

7

0.7418

206

386

0.0474

54 0.0014

728

1162 0.0992

869

DC

CD86 # nicDC

520 246 322 1716 263 1432 522

16 23 11 13 16 26 23

0.3 7 6 3 0.1 4 9

717

18

4

502 0.4471

of mature DC and nicDC (markers (mature)) 5 14 474 1289 1062 32 7 429 1186 972 20 7 758 694 681 78 178 1637 1657 1153 5 23 692 875 743 31 67 396 822 609 44 80 709 1614 865

Mean 187 31 P-value <0.0001

CD80

0.0001

DC

CD40 nicDC

DC

CD83 nicDC

DC

nicDC

7 6 18 7 8 11 9

86 30 30 33 78 54 23

21 31 12 12 16 16 21

12 22 10 15 5 13 12

2 4 1 2 2 1 3

2 3 1 2 1 1 3

9

48

18

13

2

2

0.002

0.0977

0.5984

1747 748 832 1967 1405 897 1751

875 252 295 1688 560 448 881

141 130 69 221 142 74 205

16 34 23 62 16 37 71

383 260 242 637 339 135 648

132 73 56 173 115 55 193

60 52 27 92 53 23 117

25 26 17 35 20 16 72

17 26 12 47 30 10 49

5 4 3 15 13 9 17

1335

714

140

37

378

114

61

30

27

9

0.0391

0.0009

0.0052

0.0614

0.0158

Mean fluorescence intensity; (#) median fluorescence intensity; (immature) P < 0.05 was considered as statistically significant.

molecules, but also express CCR7 molecule, a chemokine receptor known to facilitate migration of activated DCs from the inflamed tissues to lymph nodes in response to SLC and MIP-3␤ [13–15]. Interestingly, while nicDCs reduced the expression of CD36 and mannose receptors, reminiscent of their response to maturational signal LPS (Fig. 3C), they hardly up-regulated the lymph node homing receptor, CCR7 (Fig. 4). High expression of MHC and costimulatory molecules and production of polarizing cytokines during DC maturation process are critical for optimal activation, proliferation and final differentiation of na¨ıve T cells to effector memory cells [16]. Therefore, we further assessed the characteristics of mature nicDCs. Except CD86 (MFI: 132 versus 129), mature nicDCs increased the expressions of MHC class I (1062 versus 602), class II (875 versus 520), CD80 (16 versus 0.3) and CD40 (25 versus 12) molecules compared to immature nicDCs (Figs 2 and 5A and Table 1). However, when compared with mature DCs, they showed lower levels of MHC class I (MFI: 1062 versus 1289) and class II (875 versus 1747), CD80 (16 versus 141), CD86 (132 versus 383), and CD40 (25 versus 60) expressions (Fig. 5A). Contrary to our expectation, a hallmark of DC maturation marker, CD83 [8], was not detected on these cells (Fig. 5A). Surprisingly, they restored high level of CD14 molecule, a monocyte and macrophage marker, which was down-regulated during differentiation (Fig. 5A). Confocal microscopy ex-

amination of mature nicDCs however, revealed the presence of DC-LAMP [17] confirming their DC characteristic (Fig. 5B). Additionally, we found major changes in the amount of cytokines produced by these cells in response to LPS. nicDCs produced to a greater extent the pro-inflammatory cytokines TNF-␣ (1.4-fold), IL-1␤ (3.3-fold), and IL-10 (2.2-fold) compared to control DCs (Fig. 6). More importantly, these cells produced essentially no IL-12 (up to 98% reduction), a key cytokine required for the development of effector memory Th1 cells [18] (Fig. 6). The same results were obtained when immature nicDCs were activated by soluble CD40L (data not shown). 3.4. Th-promoting capacity of nicDCs Next, we examined whether the aberrant expression of costimulatory molecules and lack of IL-12 production influence Th polarizing capacity of nicDCs. To this end, we co-cultured purified na¨ıve T cells with DCs. We found a two-fold reduction in the expansion of both CD4 and CD8 T cells, expressing T-cell activation marker CD25 when nicDCs were used as stimulators in primary co-cultures (Fig. 7A). This was correlated with the proliferative response of T cells co-cultured with nicDCs (Fig. 7B). In addition, fundamental difference was observed in the frequency of polarized T cells generated within actively proliferating cells in

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Fig. 3. nicDC antigen-capture properties. Day-6 immature DCs and nicDCs were cultured with 7-AAD labeled apoptotic cells (A); FITC-dextran (B, left panel) and Lucifer yellow (B, right panel). After indicated times, phagocytosis of apoptotic cells, FITC-dextran internalization and fluid phase uptake were analyzed by flow cytometry. (A) Contour plots represent the percentage of immature DCs and nicDCs that captured apoptotic cells. Filled black (DCs) and filled grey (nicDCs) histograms represent (B, left panel) endocytosis of FITC-dextran and (B, right panel) fluid phase uptake of Lucifer yellow at 37 ◦ C. Plain and dashed histograms represent staining at 4 ◦ C. (C) Filled black (DCs) and grey (nicDCs) histograms represent expressions of indicated cell-surface receptors/molecules on untreated or 48 h LPS-treated cells. Open histograms represent the isotype control Abs. One representative experiment of seven is shown.

response to nicDCs compared to DCs. Indeed, intracellular staining of these T cells rescued from primary co-cultures and restimulated with fresh DCs for IL-4 and IFN-␥ revealed a significant reduction (four-fold) in IFN-␥-producing with no obvious change in IL-4-producing T cells (Fig. 7C). Ac-

cordingly, we found a profound shortage (80% reduction, DC: 2248 versus nicDC: 472 pg/mL) in INF-␥ released by T cells originally co-cultured with nicDCs. Among the factors secreted by DCs, IL-12 biases the differentiation of CD4 T cells toward IFN-␥-producing Th1 cells [19,20]. However,

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Fig. 4. CCR7 expression by nicDCs. Day-6 immature DCs and nicDCs were left untreated or treated with LPS for 48 h. Filled black (DCs) and filled grey (nicDCs) histograms represent CCR7 expressions. Plain histograms represent the isotype control Abs. One representative experiment of five is shown.

we found that sole addition of exogenous IL-12 to primary co-cultures was not sufficient to restore Th1 polarizing capacity of nicDCs (Fig. 7A–C).

4. Discussion There are two sets of experiments suggesting that nicotine compromises smoker’s ability to respond to immunologically relevant insults by influencing DC system. We have recently demonstrated that exposure of competent DCs to nicotine impairs their immunostimulatory functions [4]. Here we show that nicotine also acts on DC precursors and adversely influences the immune system by changing the differentiation of human monocyte into atypical DCs. The concentration of nicotine used in this work was chosen according to previous studies [4,21–23]. It has been stated that the average cigarette contains approximately 10 mg of nicotine [24], and between 1 and 2 mg of nicotine are delivered to the lung when a cigarette is smoked [25]. In addition, it is reported that salivary concentration of nicotine reaches 1560 ␮g/mL in smokeless tobacco users [26]. One may infer that, after a cigarette has been smoked, the concentration of nicotine in local tissue reaches the level used in this work. While morphologically similar to DCs (Figs. 1A and 5B), immature and mature nicDCs were accompanied by a number of phenotypical and functional changes that are detrimental to their function as sentinels of the immune system. These cells reduced their constitutive macropinocy-

tosis that allows them to take up large volume of fluid containing soluble antigens [27] (Fig. 3B). However, immature nicDCs expressed higher levels of low affinity Fc␥RII and the mannose receptors, molecules that allow efficient capture of immune complexes and mannosylated antigens, respectively [27] (Fig. 3C). Accordingly, their overall capacity for receptor-dependent antigen uptakes was significantly increased (Fig. 3A and B). Importantly, in spite of this higher receptor-dependent antigen uptake, nicDCs might not ensure higher capacity for antigen presentation because these cells only moderately up-regulated MHC molecules, particularly class II (two-fold lower than control DCs) in response to maturational signal, LPS (Fig. 5A). Moreover, these cells did not express CD1a, non-classical Ag-presenting molecule involved in regulation of T-cell responses to microbial lipids and glycolipids-containing Ag (Figs. 2 and 5A and Table 1) [28]. Taken together, these observations indicate that nicotinic environment may diminish the quantity of antigen to be presented by nicDCs. Phagocytosis of apoptotic bodies occurs through receptors including CD36, and ␣v␤5 or ␣v␤3 [12,29]. Conflicting evidence exists for the importance of CD36 in phagocytosing apoptotic cells in vivo. On the one hand, blood monocytes from systemic lupus erythematosus patients demonstrate a decrease in CD36 levels paralleled by a deficiency in the phagocytosis of apoptotic cells. On the other hand, monocyte-derived macrophages from CD36-deficient patients show no defect in the phagocytosis of apoptotic neutrophils [30]. In our study, we found considerable increase (Fig. 3A) in phagocytosis of apoptotic bodies by

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Fig. 5. nicDC maturation in response to LPS. Day-6 immature DCs and nicDCs were left untreated or treated with LPS for 48 h. (A) Filled black (DCs) and grey (nicDCs) histograms represent specific staining of the indicated cell-surface markers. Open histograms represent the isotype control Abs. One representative experiment of seven is shown. (B) LPS-treated cells were fixed, permeabilized, labeled with anti-DC-LAMP (red) and anti-HLA-DR (green) mAbs and analyzed by confocal microscopy. One representative experiment of four is shown. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

nicDCs which was not attributed to CD36 level detected on these cells. Thus it remains to be determined whether alteration in the expression of other receptors such as ␣v␤5 or ␣v␤3 could explain the increased capacity of nicDCs to capture apoptotic bodies. Pathogen products such as LPS and the local production of TNF-␣ or IL-1 [31], all mediators of DC maturation, trigger

peripheral DC migration into the T-cell area of lymphoid organs where they activate na¨ıve T cells and probably also B cells and natural killer cells [3,32]. Importantly, while nicDCs produced more TNF-␣ and IL-1␤ in response to LPS (Fig. 6), they hardly up-regulated the lymph node homing receptor CCR7 (Fig. 4), suggesting that nicDCs may remain in inflamed tissues.

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Fig. 6. nicDC cytokines production in response to LPS. The culture supernatants from 48hr LPS-treated DCs (white bars) and nicDCs (grey bars) were harvested and cytokines production measured. Each symbol represents an independent experiment.

Inflammatory cytokines are also crucial for early responses to pathogens and the up-regulation of local host defenses. Among the cytokines secreted by DCs, IL-12 has been most extensively studied in both murine and human systems, because DC are a principle source of IL-12 in response to most pathogens and activated T cells through CD40L [33]; IL-12 bridges innate and specific immune responses by activating NK and T cells [18]; and IL-12 biases CD4 T cells toward Th1 differentiation [18]. We found a profound shortage of IL-12 (98% reduction) secreted by maturing nicDCs in response to LPS (Fig. 6). Interestingly, supplementation of IL-12 alone in primary co-cultures could not restore Th1 polarizing capacity of nicDCs (Fig. 7) suggesting the differential expression of costimulatory molecules and other possible mechanisms besides IL-12 preceded this defect. Up-regulation of costimulatory molecules ensures an efficient amplification of

signaling in na¨ıve T cells [16]. It is known that IL-10 produced by DC down-regulates the expression of MHC and costimulatory molecules, and the production of IL-12 by DCs [34–36]. From our data, it appeared that maturing nicDCs produced higher amount of IL-10 that might have moderated the up-regulation of MHC and costimulatory molecules on these cells in response to LPS (Fig. 5A and Table 1). The interaction of CTLA-4/CD28 on T cells and CD80/CD86 on DCs also plays a role in the regulation of Th1 versus Th2 cells development. In particular, B7.1/CD80 rather orients toward Th1 responses, whereas B7.2/CD86 ligation rather skews toward Th2 responses [37,38]. We observed that immature nicDCs express high level of CD86, while they lack CD80 (Fig. 2 and Table 1). Although, this higher CD86-to-CD80 ratio did not simply switch them towards Th2, it might have contributed to their compromised Th1 stimulatory mode. Contribution of the differential ex-

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Fig. 7. nicDC immunostimulatory properties. (A) Day-6 immature DCs and nicDCs were co-cultured with purified allogeneic na¨ıve T cells with or without IL-12. After 5 days, these T cells were subsequently re-challenged with competent DCs for additional 40 h. Flow cytometry analysis of T cells stained with corresponding mAbs. Gates indicate the frequency of actively proliferating CD4 and CD8 T cells expressing CD25. (B) Day-6 immature DCs and nicDCs were co-cultured with purified allogeneic na¨ıve T cells with or without IL-12. After 5 days of incubation, tritiated thymidine was added, and thymidine incorporation was measured 16 h later. T-cell proliferation is represented as the mean ± S.D. of (3 H) thymidine uptake from triplicate wells. (C) Intracellular cytokine analysis of IL-4 and IFN-␥-producing T cells following restimulation with competent DCs. The numbers in each gate indicate the respective frequency of T cells producing IL-4 and IFN-␥. One representative experiment of six is shown.

pression of costimulatory molecules and other factors in Th1 polarizing capacity of nicDCs is a subject of current investigation. The generation of memory T cells during the primary immune response is crucial for long-lasting protective immunity [39]. Our data finally indicate that nicDCs failed to

promote the development of memory Th1 cells. In fact, the frequency of IFN-␥-producing effector cells differentiated in response to nicDCs remained profoundly low even upon restimulation with fresh competent DCs (Fig. 7A and C). Altogether, this work presents a new evidence for the immunological alterations associated with nicotine, especially

E. Guinet et al. / Immunology Letters 95 (2004) 45–55

at the level of DC differentiation and development of effector memory Th1 cells. It may provide an explanation for the increased susceptibility of smokers to infection and cancer. In addition, this study is of importance to public health because it raises questions about the effectiveness and longevity of immunity elicited by vaccines in smokers as well as children exposed to environmental tobacco smoke.

Acknowledgements We thank Dr. David S. Carlson for his continuous support. We also thank Dr. Nasser Haghighat for performing statistical analysis of data. This work was supported by BCD-TAMUSHSC F0101 and BCD-TAMUSHSC Y2001-Y.

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