The pathogenesis of sepsis is characterized by an overwhelming systemic inflammatory response that induces the development of lethal multiple organ failure. Lipopolysaccharide (LPS), a major cell wall component of gram-negative bacteria, is known to activate monocytes and macrophages, thereby causing life-threatening shock (1). The stimulation by LPS induces the activation of nuclear factor (NF)-κB (2) and p38 mitogen-activated protein kinase (p38MAPK) (3), thus leading to the production of a variety of inflammatory proteins, such as cytokines, chemokines, and costimulatory molecules, such as B7, that are essential for the activation of T cells (4). The central components of LPS receptor have already been identified. CD14, a membrane protein on monocytes/macrophages, plays a critical role in the activation by LPS (5). Toll-like receptor (TLR)-4 is identified as the signal transducer for LPS (6). The LPS responses require CD14 and the serum protein LPS-binding proteins to present LPS to TLR-4 (7). The activation and proliferation of T cells by LPS requires direct cell-to-cell contact (8, 9). We previously reported that LPS induced tumor necrosis factor (TNF)-α production in peripheral blood mononuclear cells (PBMC), but it had no effect on the production of interleukin (IL)-18, IL-12, and interferon (IFN)-γ (10). Moreover, LPS at 1 ng/mL upregulated the expression of intercellular adhesion molecule (ICAM)-1, B7.1, B7.2, and CD40 on monocytes (10, 11). Antibodies (Abs) against ICAM-1, B7.1, and CD40 abolished the LPS-initiated TNF-α production in PBMC, thus suggesting that the engagement of adhesion molecules is involved in LPS-induced TNF-α production (10, 11). Recombinant IL-10 potently suppresses LPS-induced production of TNF-α, and pretreatment with IL-10 inhibits TNF-α activity and prevents LPS-induced mortality in mice (12). Therefore, inhibition of TNF-α levels and augmentation of IL-10 levels can be a quite effective way for treating or preventing septic shock.
The vagus nerve system is reported to downregulate inflammation in vivo by decreasing the release of TNF-α by endotoxin-stimulated macrophages (13). This anti-inflammatory effect is mediated by the interaction between acetylcholine, the principal neurotransmitter of the vagus nerve, and cholinergic nicotinic acetylcholine receptors on macrophages. Nicotine activates the nicotinic acetylcholine receptors (nAChRs) that belong to a family of ionotropic receptors which consist of 5 transmembrane subunits that make up the ion channels. The nAChRs are widely distributed throughout the central and peripheral nervous system, and they are also involved in the signal transmission at the skeletal neuromuscular junction, in the autonomic ganglia, and in the brain. In addition, nonneuronal cells, such as monocytes, also express nAChRs (14). In humans, 16 different subunits (α1-7, α9-10, β1-4, δ, ε, and γ) have been identified which form a large number of homopentameric and heteropentameric receptors with distinct structural and pharmacological properties (15, 16). The treatment of human or murine monocytes with nicotine, an immunomodulatory component, has been shown to significantly inhibit the production of IL-6, TNF-α, and IFN-γ in response to LPS stimulation (17-19). The stimulation of nAChR α7 subunit (α7-nAChR) on monocytes is involved in the inhibition of the TNF-α production (19). However, the effect of nicotine on immune cells is still not completely characterized and thus remains controversial.
A major product of cyclooxygenase (COX)-initiated arachidonic acid metabolism, prostaglandin E2 (PGE2), is the main prostaglandin produced in macrophages and monocytes (20, 21). PGE2 has high affinity for prostanoid EP1, EP2, EP3, and EP4 receptors, and the prostanoid EP2 and EP4 receptors are coupled to Gs and mediate increases in adenosine 3′, 5′-cyclic monophosphate (cAMP) and the activation of protein kinase A (PKA) (22). Recently, we found that PGE2 inhibited IL-18 production in human monocytes treated with LPS via prostanoid EP2 and EP4 receptors (23). Nicotine is reported to induce the expression of COX-2 and the synthesis of one of its major products, PGE2, in macrophages through α7-nAChR stimulation (24).
In the present study, the effects of nicotine on the expression of adhesion molecule and LPS-receptor complex were determined, and we examined the involvement of α7-nAChR in the nicotine actions.
MATERIALS AND METHODS
Reagents and drugs
Lipopolysaccharide from Escherichia coli (L8274, serotype 026:B6) was purchased from Sigma Chemical (St. Louis, Mo), and purified water produced by Millipore (Millipore Japan, Tokyo, Japan) was used as the solvent solution for it. Nicotine (1-methyl-2-[3-pyridyl]pyrrolidine), α-bungarotoxin mecamylamine, and H-89 were purchased from Sigma Chemical. NS398 and indomethacin were from Cayman Chemical (Ann Arbor, Mich). SN50 and SB203580 were purchased from Calbiochem (San Diego, Calif). For the flow cytometric analysis, fluorescein isothiocyanate (FITC)-conjugated mouse IgG1 monoclonal antibody (mAb) against ICAM-1/CD54 and Phycoerythrin (PE) conjugated anti-CD14 mAb were purchased from DAKO (Glostrup, Denmark). FITC-conjugated mouse IgG1 mAb against B7.1 was purchased from Immunotech (Marseille, France), FITC-conjugated mouse IgG1 mAb against B7.2 and CD40 were purchased from Pharmingen (San Diego, Calif). FITC-conjugated anti-TLR-4 Ab was purchased from MBL (Nagoya, Japan). Finally, an FITC-conjugated IgG1 class-matched control was obtained from Sigma Chemical.
Isolation of peripheral blood mononuclear cells and monocytes
Normal human PBMC were obtained from 10 healthy volunteers after acquiring institutional review board (IRB) approval (Okayama Univ. IRB No.106). Samples of 20 to 50 mL of peripheral blood were withdrawn from a forearm vein, after which the PBMCs were prepared, and monocytes isolated from PBMC were separated by counterflow centrifugal elutriation as previously described (10, 11). The PBMC and monocytes were then suspended at a final concentration of 1 × 106 cells/mL, and they were then washed 3 times in RPMI 1640 medium (Nissui Co., Ltd., Tokyo, Japan) supplemented with 10% (vol/vol) heat-inactivated fetal calf serum, 20 μg/mL kanamycin, and 100 μg/mL streptomycin and penicillin (Sigma Chemical). The endotoxin concentration of the medium described above was measured using an Endospecy (Seikagaku Kogyo, Tokyo, Japan) kit, and it was found to be lower than the detection limit of 0.06 EU/mL.
Double-labeling flow cytometric analysis for adhesion molecule expression
Changes in the expression of human leukocyte antigens CD14, TLR-4, ICAM-1, B7.1, B7.2, and CD40 on monocytes were examined by flow cytometry using Abs against respective antigens. Cultured cells at 5 × 105 cells/mL were prepared for the flow cytometric analysis as previously described (10, 11) and analyzed with a fluorescence-activated cell sorter Calibur (BD Biosciences, San Jose, Calif). The data were processed using the CELL QUEST program, and it was expressed as the relative fluorescent intensities against class-matched control.
Enzyme-linked immunosorbent assays
Peripheral blood mononuclear cells at 1 × 106 cells/mL used for analyzing TNF-α, IL-10, and PGE2 production were incubated for 24 h at 37°C in a 5% CO2/air mixture under different conditions. After culturing, PGE2 (Cayman Chemical), TNF-α, and IL-10 (R&D Systems, Minneapolis, Minn) in the cultured media were measured using enzyme-linked immunosorbent assay (ELISA) kits. The detection limit of the ELISA for TNF-α, IL-10, and PGE2 was 10 pg/mL.
Statistical significance was evaluated using analysis of variance followed by Dunnet test. The results were expressed as the means + SEM of triplicate findings from 5 donors. A probability value of less than 0.05 was considered to indicate statistical significance.
The effects of nicotine on CD14 and toll-like receptor 4 expression in human monocytes
The effects of nicotine at concentrations ranging from 0.1 to 100 μmol/L on the expression of CD14 and TLR-4 in the presence and absence of LPS were determined by flow cytometry after 24 h incubation of PBMC (Fig. 1). Lipopolysaccharide at 1 ng/mL reduced the expression of CD14 on monocytes, but not that of TLR-4. Nicotine inhibited the CD14 and TLR-4 expression in the presence and absence of LPS. The 50% inhibitory concentration (IC50) values for the inhibitory effect of nicotine on the expression of CD14 and TLR-4 in the presence of LPS were estimated to be 0.8 and 1 μmol/L, respectively.
The effects of nicotine on the production of tumor necrosis factor-α and interleukin 10 in human peripheral blood mononuclear cells
The effects of nicotine at concentrations ranging from 0.1 to 100 μmol/L on the production of TNF-α in the incubation media from PBMC were examined in the presence or absence of 1 ng/mL LPS (Fig. 2). LPS at 1 ng/mL enhanced the production of TNF-α, but had no respective effect on IL-10 production. Nicotine concentration-dependently inhibited the production of TNF-α in the presence of LPS, whereas it had no effect in the absence of LPS. Nicotine had no effect on the production of IL-10 in the presence or absence of LPS. The IC50 value for the inhibitory effect of nicotine on the TNF-α production was 1 μmol/L.
The effects of nicotine on the expression of intercellular adhesion molecule 1, B7.1, B7.2, and CD40 in human monocytes
Peripheral blood mononuclear cells at 1 × 106 cells/mL were treated with nicotine at concentrations ranging from 0.1 to 100 μmol/L in the presence or absence of 1 ng/mL LPS. After 24 h of incubation, the expression of ICAM-1, B7.1, and CD40 was determined by double-labeling flow cytometry (Fig. 3). Nicotine inhibited the LPS-enhanced ICAM-1, B7.1, and CD40 expression in a concentration-dependent manner (Fig. 3), but it had no effect on the expression of CD40L (data not shown). The IC50 values for the inhibitory effect of nicotine on the expression of ICAM-1, B7.1, and CD40 induced by LPS were estimated to be 0.8, 1.0, and 1.2 μmol/L, respectively. Nicotine had no effect on any type of adhesion molecule expression in the absence of LPS (data not shown).
The effects of nicotinic acetylcholine receptor α7 subunit antagonists on the nicotine-induced inhibition of CD14 and toll-like receptor 4 expression
To determine the involvement of α7-nAChR in the nicotine actions, we examined the effects of a nonselective α7-nAChR antagonist, mecamylamine, and a selective α7-nAChR antagonist, α-bungarotoxin, at concentrations ranging from 10 pmol/L to 10 nmol/L on CD14 and TLR-4 expression on monocytes in the presence of LPS at 1 ng/mL and nicotine at 100 μmol/L (Fig. 4). Mecamylamine and α-bungarotoxin reversed the effect of nicotine on CD14 and TLR-4 expression in the presence or absence of LPS. In the absence of LPS and nicotine, the α7-nAChR antagonists had no effect on the expression of CD14 and TLR-4 on monocytes (data not shown).
The effects of nicotinic acetylcholine receptor α7 subunit antagonists on the nicotine-induced inhibition of tumor necrosis factor-α production
Mecamylamine and α-bungarotoxin reversed the effect of nicotine on the production of TNF-α in the presence of LPS (Fig. 5). In the absence of LPS and nicotine, the α7-nAChR antagonists had no effect on the production of TNF-α (data not shown).
The effects of nicotinic acetylcholine receptor α7 subunit antagonists on the nicotine-induced inhibition of intercellular adhesion molecule 1, B7.1, and CD40 expression
Mecamylamine and α-bungarotoxin abolished the effect of nicotine on the expression of ICAM-1, B7.1, and CD40 in the presence of LPS (Fig. 6). In the absence of LPS and nicotine, the α7-nAChR antagonists had no effect on the expression of ICAM-1, B7.1, or CD40 (data not shown).
The involvement of nuclear factor-κB and p38 mitogen-activated protein kinase activation in the lipopolysaccharide-induced intercellular adhesion molecule 1, B7.1, and CD40 expression and tumor necrosis factor α production
We examined the involvement of the NF-κB (Fig. 7A) and p38MAPK activation (Fig. 7B) in the LPS-induced ICAM-1, B7.1, and CD40 expression on monocytes and TNF-α production. An NF-κB activation inhibitor, SN50, and a p38MAPK inhibitor, SB203580, reduced the LPS-enhanced expression of ICAM-1, B7.1, and CD40 and TNF-α production, not IL-10. In the absence of LPS, SN50, and SB203580 had no effect on TNF-α production (data not shown).
The effects of nicotine on the production of PGE2 in monocytes
As shown in Figure 8A, nicotine induced PGE2 production in the presence or absence of LPS, but was greater in its presence. A nonselective α7-nAChR antagonist, mecamylamine, and a selective α7-nAChR antagonist, α-bungarotoxin, prevented nicotine-stimulated PGE2 production in the presence or absence of LPS (Figs. 8,B and C). A nonselective COX-2 inhibitor, indomethacin, and a selective COX-2 inhibitor, NS398, again reduced nicotine-initiated PGE2 production in the presence or absence of LPS (Figs. 8,D and E). Without nicotine, neither α7-nAChR antagonists nor the COX-2 inhibitors had any effects on PGE2 production (data not shown).
The activation of afferent vagus nerve fibers by endotoxin is reported to stimulate hypothalamic-pituitary-adrenal anti-inflammatory responses that lead to anti-inflammatory signals through the efferent vagus nerve, which has been termed the cholinergic anti-inflammatory pathway (25). Excessive inflammation and TNF synthesis is known to cause morbidity and mortality in diverse human diseases, including endotoxemia, sepsis, rheumatoid arthritis, and inflammatory bowel disease. As shown in Figure 2, nicotine inhibited the LPS-induced TNF-α production in PBMC. The purpose of the present study was to examine the mechanism of nicotine actions in combination with LPS. The LPS actions on monocytes were reported to depend on the stimulation of CD14/TLR-4 complex (5-7). LPS suppressed the expression of CD14, but not TLR-4, on monocytes (Fig. 1). Soluble CD14 is reported to shed from monocytes after stimulation of LPS (27). However, little is known about the mechanism of CD14 shedding, and further study should be continued. In the present study, we found that nicotine inhibited the expression of CD14 and TLR-4 on monocytes (Fig. 1). Therefore, the regulation of CD14 and TLR-4 expression might result in the nicotine-induced modulation of the TNF-α production. We have indicated that the cell-to-cell interaction between monocytes and T-cells through the engagement of adhesion molecules is involved in the LPS-induced production of TNF-α (10, 11). As shown in Figure 3, nicotine reversed the LPS-enhanced expression of ICAM-1, B7.1 and CD40 on monocytes, thus suggesting that the suppression of the TNF-α production might depend on that of the adhesion molecule expression.
The α7-nAChR is required for the nicotine inhibition of macrophage TNF release (19). In the present study, we also found that nicotine inhibited the LPS-enhanced production of TNF-α in PBMC via α7-nAChR (Fig. 5). As shown in Figure 4, nicotine inhibited the expression of CD14 and TLR-4 on monocytes through the stimulation of α7-nAChR in the presence or absence of LPS. In addition, the stimulation of α7-nAChR suppressed the expression of ICAM-1, B7.1, and CD40 on monocytes in the presence of LPS (Fig. 6). As a result, the stimulation of α7-nAChR might regulate the immune response in the monocytes treated with LPS.
It is found that nicotine inhibits the LPS-induced activation of a transcription factor, NF-κ in monocytes, thus leading to the modulation of the TNF-α production (27). The nicotine action on NF-κB is reported to be caused by the stimulation of α7-nAChR (28). The LPS-induced activation of NF-κB is reported to result in the upregulation of ICAM-1, B7.1, and CD40 expression (29-31). LPS is known to activate monocytes by signaling through members of p38MAPK, thereby inducing transcription of TNF-α . As shown in Figure 7, we found that the NF-κB activation inhibitor, SN50, and the p38MAPK inhibitor, SB203580, suppressed the expression of ICAM-1, B7.1, and CD40 on monocytes and the production of TNF-α. Therefore, the effect of nicotine on ICAM-1, B7.1, and CD40 expression and the production of TNF-α might depend on the NF-κB and p38MAPK pathway.
Prostaglandin E2 primes naive human T cells and enhances their production of anti-inflammatory cytokines, while inhibiting their synthesis of proinflammatory cytokines (21, 22). Other examinations showed that nicotine induces the expression of COX-2 and the synthesis of one of its major products, PGE2, is released in whole blood, macrophages, and microglia through α7-nAChR stimulation (24, 32). In the present study, we also found that the stimulation of α7-nAChR resulted in the production of PGE2 in the incubation media from monocytes (Fig. 8), but not PGE1, PGD2, PGF2, PGI2, PGJ2, or thromboxane (data not shown). We recently reported that PGE2 had no effect on the expression of CD14 and TLR-4 and the LPS-enhanced expression of ICAM-1, B7.1, CD40, CD14, and TLR-4 and production of TNF-α in PBMC (11). Accordingly, the effects of nicotine on the expression of ICAM-1, B7.1, CD40, CD14, and TLR-4 and production of TNF-α were not reversed by a nonselective and a selective COX-2 inhibitor, indomethacin and NS398, respectively, and a PKA inhibitor, H89 (data not shown). IL-10 is known as an important regulator of TNF-α, which is induced by PGE2 (33), however nicotine (Fig. 2) and PGE2 (data not shown) had no effect on the production of IL-10 (34). These results suggested that the nicotine action might not depend on the production of PGE2. In conclusion, we found that nicotine inhibited the expression of CD14 and TLR-4 via α7-nAChR and NF-κB, thus leading to a suppression of both the production of TNF-α and the expression of ICAM-1, B7.1, and CD40 on monocytes. The α7-nAChR is a good target for new drug development, and further study on α7-nAChR regarding this is thus called for.
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