Effects of water-soluble cigarette smoke extracts upon ... - Springer Link

© Birkhäuser Verlag, Basel, 2003 Inflamm. res. 52 (2003) 461– 469 1023-3830/03/110461-09 DOI 10.1007/s00011-003-1202-8

Inflammation Research

Effects of water-soluble cigarette smoke extracts upon the release of b -hexosaminidase from RBL-2H3 basophilic leukaemia cells in response to substance P, compound 48/80, concanavalin A and antigen stimulation C. J. Fowler, M. Sandberg and G. Tiger Department of Pharmacology and Clinical Neuroscience, Umeå University, SE-901 87 Umeå, Sweden, Fax: ++ 46 90 7852752, e-mail: [email protected] Received 6 December 2002; returned for revision 1 April 2003; accepted by I. Ahnfelt-Rønne 5 June 2003

Abstract. Objective and Design: To determine whether water-soluble constituents of cigarette smoke affect mast cell function using an in vitro model, RBL-2H3 basophilic leukaemia cells. Materials and methods: RBL-2H3 cells were induced to degranulate in response to compound 48/80 and substance P, as assessed by monitoring the release of the granular enzyme b-hexosaminidase, by treatment for 7 days with 20 mM quercetin. Responses to concanavalin A and antigen were determined by measuring the b-hexosaminidase release from cells cultured on fibronectin-coated plates. Results: The b-hexosaminidase release response to compound 48/80 induced by quercetin treatment was accompanied by a release of lactate dehydrogenase, suggesting that degranulation is not the only process triggered by compound 48/80 under these conditions. Quercetin treatment reduced the b-hexosaminidase release response to concanavalin A. Precoating of the culture wells with rat fibronectin enhanced the b-hexosaminidase response to calcimycin, but not to concanavalin A. Under these conditions, concanavalin A did not induce a release of lactate dehydrogenase. The responses to c48/80, substance P, calcimycin, concanavalin A and antigen (after IgE pretreatment) were reduced by treatment with cigarette smoke solution obtained from standard and low-tar cigarettes (IR3 and IR5F). The effect of cigarette smoke solution from IR5F cigarettes upon the b-hexosaminidase release elicited by compound 48/80 (in quercetin-treated cells) and by concanavalin A (in cells cultured on fibronectin-coated wells) could be prevented by N-acetyl-L-cysteine, but not with either hemoglobin, a-tocopherol, catalase or palmitoylethanolamide. N-acetyl-L-cysteine also reduced the effect of cigarette smoke solution upon the degranulation response to antigen. Conclusions: Under the conditions used, oxidants present in cigarette smoke solution from IR5F cigarettes reduce the Correspondence to: C. J. Fowler

ability of RBL-2H3 cells to degranulate in response to both immunological and non-immunological stimuli. Key words: Cigarettes – Mast cell degranulation – Substance P – Quercetin – Concanavalin A – Palmitoylethanolamide – Compound 48/80 – Antigen

Introduction Mast cells are multifunctional immune cells and have been implicated in immediate hypersensitivity and inflammatory reactions [1]. Activation of mast cells leads to their degranulation and release of a number of inflammatory mediators including histamine, interleukin-4 and tumour necrosis factor-a (TNFa). Recent findings implicating mast cells and mast-cell derived cytokines in the development of airway hyper-responsiveness [2, 3] are examples of the renewed interest in establishing the contribution made by mast cells to the development of lung diseases [4]. Chronic obstructive pulmonary disease affects 15–20% of all smokers [5]. There is good evidence that inflammatory reactions occur in the lungs of smokers, and several studies have suggested that mast cells may be involved in smokinginduced lung disease. Kalenderian et al. [6] found that the levels of the mast cell mediators histamine and tryptase were considerably elevated in bronchoalveolar lavage fluids from smokers. The authors concluded that these data indicated ‘a greater propensity for mast cell-mediated injury in the smoker’. Subsequent studies have borne out this conclusion [7, 8]. In 1992, Thomas et al. [9] reported that a water soluble extract of cigarette smoke (both gas and particulate phases) dose-dependently increased the release of histamine and tryptase from a canine mastocytoma cell line. Thus, histamine release increased from a control level of 3.5 ± 0.7% to a maximum of 53 ± 12% in response to the extract, a

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response that was not due to cell toxic effects of the extract. The histamine release in response to the calcium ionophore calcimycin (A23187) was not affected by the extract, suggesting that a calcium-independent mechanism was involved. The cigarette smoke extract did not induce de novo synthesis of prostaglandin D2, and in fact reduced the synthesis produced by incubation with either the A23187 or arachidonic acid. Thomas et al. [9] concluded that ‘examination of the effect of cigarette smoke on isolated mast cells … will lead to a greater understanding of the pathogenesis of tobacco-related lung disease’. However, the mechanism(s) by which cigarette smoke affect mast cell function were not investigated. In addition, it has been argued that the response of mast cells to stimuli is highly dependent upon their microenvironment. Mast cells in peripheral tissue are surrounded by other cells (such as fibroblasts) and extracellular matrix proteins (such as fibronectin) and receive signals via cell surface adhesion molecules [10]. In this respect, mast cell lines bind to fibronectin, and the bound cells show an increased sensitivity to stimulation [11]. In consequence, in the present study, we have investigated the effects of cigarette smoke solution upon the responsiveness of rat basophilic leukemia 2H3 (RBL-2H3) cells either cultured on fibronectin-treated plates and/or treated with quercetin, a flavonoid structurally related to sodium cromoglycate. These cells normally only degranulate in response to immunogenic stimuli, but can be induced to respond to nonimmunogenic stimuli such as compound 48/80 and substance P after treatment with quercetin [12]. The ability of antioxidant agents and palmitoylethanolamide, a compound known to have antiinflammatory properties (see [13]) and to affect mast cell function in vivo [14], to prevent the effects of the cigarette smoke solution have also been investigated.

Materials and methods Materials Reference cigarettes IR3 and IR5F were purchased from The Tobacco and Health Research program at the University of Kentucky, KY, USA. Quercetin dihydrate, rat fibronectin, substance P, concanavalin A [Con A], compound 48/80, hemoglobin (pig), mannitol, N-acetyl-L-cysteine, catalase (from bovine liver; 51,000 U/mg protein), a-tocopherol, phenylmethylsulfonyl fluoride, anti DNP IgE (monoclonal anti-dinitrophenyl), DNP-HSA (albumin, human dinitrophenyl) and calcimycin were purchased from the Sigma Chemical Co., St. Louis, MO, USA. Palmitoylethanolamide was obtained from the Cayman Chemical Co., Ann Arbor, MI, USA. Quercetin was dissolved in 60 mM propylene glycol and then diluted to the appropriate concentration in RPMI 1640 medium (without L-glutamine, with phenol red). Palmitoylethanolamide and a-tocopherol were dissolved in ethanol. Substance P stock solutions were made in 10 mM acetic acid, due to its poor stability in distilled water. A23187, Con A and compound 48/80 were dissolved in water. The stimulating agents were then diluted to the appropriate concentration in RPMI 1640 medium (without L-glutamine, without phenol red). Culture medium, sera and supplements were obtained from Invitrogen, Sweden.

Cell culture RBL-2H3 cells (rat basophilic leukaemia cells) were purchased from the American Type Culture Collection, MD, USA and used over a pas-

C. J. Fowler, M. Sandberg and G. Tiger

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sage range of 14–41. The RBL-2H3 cells were cultured in 75 cm2 tissue culturing flasks (T75) and incubated at 37°C in an atmosphere with high humidity and a CO2 gas content of 5 %. RBL-2H3 cells were cultured in Eagle’s Minimal Essential Medium with Earl´s salts and L-glutamine. The medium was supplemented with 15% fetal bovine serum (FBS) and 1% PEST (100 U/ml penicillin + 100 mg/ml streptomycin). Medium was changed three times a week and the cells were harvested and set into new culture flasks once a week.

Quercetin sensitisation of RBL-2H3 cells RBL-2H3 cells were harvested by trypsinization and centrifuged at 1100 rpm for five minutes. They were resuspended in RPMI 1640 medium (without L-glutamine, with phenol red) supplemented with 10 % FBS, 1% PEST and 2 mM L-glutamine. The cell density of the resuspended cells was determined using a Bürkner chamber. The RPMI 1640 medium was supplemented with 20 mM quercetin and the cells seeded in 24-well plates at a density of 0.13 ¥ 106 cells/ml. The cells were grown under these conditions for 7 days with one change of medium during this period, unless otherwise stated.

Fibronectin coating of wells The wells were treated essentially as described by Paulusson et al. [15]. Briefly, culture wells were incubated with 200 ml of rat fibronectin in PBS (10 mg/ml) at 37°C for appproximately 5 h in a cell incubator. The plates were then washed with PBS containing 2% BSA. RBL-2H3 cells were then applied as described above. For the experiments investigating the antigen sensitivity, medium was changed on Day 6 and new medium containing DNP IgE (30 ng/ml) was added. The cells were then incubated overnight.

Preparation of the cigarette smoke solution (CSS) CSS from two IR3 or IR5F reference cigarettes (per day) was prepared essentially as described by Roth et al. [16]. Puffs (35 ml) of smoke were drawn into a 60 ml plastic syringe. The smoke was then bubbled through a 0.80 mm ¥ 80 mm gauge into 8 ml of PBS (154 mM NaCl, 1.5 mM KH2PO4, 2.7 mM Na2HPO4, pH 7.2) during a period of about one min. Eight puffs of smoke were drawn from each cigarette. The slightly acidic smoke solution was filtered through a filter paper and titrated with a 0.1 M NaOH solution to pH 7.2. The CSS was used within half an h of preparation.

b-hexosaminidase assay Mast cell degranulation was detected measuring the release of the granular enzyme b-hexosaminidase by a method first described by Smith et al. [17]. Medium was removed, replaced by new RPMI 1640 and incubated (pre-incubation) for 30 min at 37 °C. The cell monolayers were washed twice with PBS before RPMI 1640 medium (without L-glutamine, without phenol red) was added. The cells were incubated with the test compounds or antigen for another fifteen minutes at 37 °C. Aliquots (50 ml) of the overlying medium were collected from each well and pipetted into a 96-well plate. Substrate, p-nitrophenyl Nacetyl-b-D-glucosaminide (50 ml of a 2 mM solution diluted in 0.2 M citrate buffer, pH 4.5), was added to each well. The samples were then incubated with the substrate for two hours at 37 °C. All incubations were undertaken at this temperature because all the reactions are sensitive to changes in temperature. Medium and chemicals were also kept at 37 °C during the experiment to avoid undesired impact on the experimental result caused by changes in temperature. After two h, reactions were stopped by adding 150 ml of 1M Tris-buffer pH 9.0 and the

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absorbances were measured in a ThermoMax microplate reader at 405 nm. The total b-hexosaminidase content of the cells was determined in parallel wells where the cells were dissolved in 0.01 % Triton X-100. The background release from unstimulated cells was also determined. This background release was found to vary somewhat between experimental series (from ~ 5 % – ~ 12 % of total release). Whether this reflects different levels of b-hexosaminidase release induced by cell damage during the assay procedures is unclear. However, it can be noted that the highest levels were seen in the first of the experimental series reported here. Release of cytoplasmic lactate dehydrogenase was determined in an analagous manner using a commercially available kit (Roche Molecular Biochemicals, Mannheim, Germany).

Statistical treatment of data In order to negate for the variation in responsiveness of the cells between experiments, significance was assessed using one way ANOVA for repeated measures with post-hoc Bonferroni-Dunn tests using the Statview computer programme (SAS institute Inc., Cory, NC, USA).

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Results Effects of cigarette smoke solution (CSS) upon the responsiveness of RBL-2H3 cells to non-immunogenic stimuli RBL-2H3 cells were induced to respond to non-immunogenic stimuli (compound 48/80, substance P) by prolonged treatment with quercetin. Initial experiments indicated that in our hands concentrations of quercetin of 30 and particularly of 100 mM were deleterious to RBL-2H3 cell proliferation. However, at a concentration of 20 mM, quercetin treatment induced a responsiveness of the cells to compound 48/80 (Fig. 1, Panel A) and substance P (data not shown) as measured by the release of the granular enzyme b-hexosaminidase. Preincubation of the quercetin-treated cells with EDTA (10 mM) followed by washing the cells with PBS greatly reduced the effect of compound 48/80 upon b-hexosaminidase release (Fig. 2, Panel D). In contrast, preincubation of the cells with palmitoylethanolamide (3 – 100 mM) was without effect on the response to

Fig. 1. Panels A and B. Effects of culturing conditions upon the b-hexosaminidase response to A, compound 48/80; B, calcimycin. RBL2H3 cells were cultured for 7 days on either uncoated or fibronectin-coated plates in the absence or presence of 20 mM quercetin, as indicated. Data are means ± s.e.m., n = 4 (Panel ) or 9 (Panel B). * P < 0.05 vs. corresponding value in the absence of stimulant for the same culturing conditions. Panels C and D, effects of CSS treatment upon the b-hexosaminidase response to C, compound 48/80 and D, Substance P and calcimycin. RBL2H3 cells were cultured for 7 days on uncoated plates in the presence of 20 mM quercetin. Cells were subjected to a preincubation phase of 30 min followed by change of medium and incubation for 15 min with the stimulating agents. CSS (100 ml in PBS in the 400 ml assay) was present during the preincubation and/or incubation phases, as indicated in the figures. Data are means ± SEM, n = 4 (Panel C) or 5 (Panel D). *P < 0.05 vs. the corresponding control value for the same stimulant concentration. # Significant ANOVA value for the group, but no significant differences were found upon post-hoc testing.

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Fig. 2. b-Hexosaminidase (Panels A, C and D) and LDH (Panel B) release from quercetin-treated RBL-2H3 cells, cultured in uncoated wells, in response to compound 48/80 (15 mg/ml). The quercetin treatment was as is described in the legend to Fig. 1. Cells were preincubated with either PBS (unfilled columns) or CSS (70 ml in PBS + 30 ml PBS, filled columns) from IR5F cigarettes together with the compounds shown for 30 min prior to washing twice with PBS and incubation with compound 48/80. Abbreviations and concentrations: N-acetyl-L-cysteine (N-AC, 1 mg/ml), hemoglobin (Hb, 1 mg/ml), EtOH (ethanol vehicle), a-tocopherol (Vit E, 10 mM), palmitoylethanolamide (PEA, 100 mM), catalase (Cat, 10000 U/ml), EDTA (10 mM). Data are means ± SEM, n = 9 (Panels A and D), 6 (Panel B) or 5 (Panel C). *P < 0.05 for the comparisons shown. # P < 0.05 vs. the corresponding value in the absence of EDTA.

compound 48/80, even when palmitoylethanolamide breakdown was prevented by the concomitant addition of phenylmethylsulfonyl fluoride (10 mM) (data not shown). The quercetin treatment in addition increased the sensitivity of the cells to degranulation by the calcium ionophore calcimycin (Fig. 1, Panel B). Compound 48/80 (15 mg/ml), however, also produced a large release of the cytoplasmic enzyme lactate dehydrogenase (LDH) in the quercetin-treated cells (Fig. 2, Panel B). The use of fibronectin-coated plates rather than uncoated plates did not produce a further increase in the sensitivity of the cells to compound 48/80 (Fig. 1, Panel A), although the responsiveness of untreated RBL-2H3 cells to calcimycin was greater when they were cultured on fibronectin-coated plates than on uncoated plates (Fig. 1, Panel B). The effects of exposure of quercetin-treated RBL-2H3 cells to CSS from either standard (IR3) or low tar/nicotine (IR5F) cigarettes upon their responsiveness to non-immunogenic stimuli are shown in Fig. 1, Panels C and D. The CSS (100 ml in a 400 ml assay) was either preincubated with the cells for 30 min prior to addition of the degranulation and/or added concomitantly to the degranulating agents and thereby incubated with the cells for 15 min. After the preincuba-

tion phase, the cells were washed twice with PBS. The CSS from the two cigarettes both reduced the response to c48/80 (5 mg/ml) and to calcimycin (5 mM), whereas only the CSS from the IR5F cigarettes reduced the response to substance P (300 nM) with no large or consistent effects upon the basal release of b-hexosaminidase. In general, the presence of CSS during the preincubation phase was more important than its presence during the incubation phase (Fig. 1, Panel C). Further studies using the preincubation phase alone demonstrated a concentration-dependency for the effect of CSS upon the response to c48/80, with inhibition being found after exposure to 100 and 70 ml, but not to 10 – 30 ml (data not shown). The inhibitory effect of 70 ml CSS could be blocked when N-acetyl-L-cysteine was also present in the incubation phase, whereas hemoglobin, a-tocopherol, palmitoylethanolamide, catalase (Figs. 2, Panels A, C and D) and mannitol (1 mM) (data not shown) were without effect. Interestingly, the release of LDH in response to compound 48/80 was not affected by preincubation with 70 ml CSS (Fig. 2, Panel B).

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Fig. 3. Panel A, Effects of culturing conditions upon the b-hexosaminidase response to concanavalin A. RBL2H3 cells were cultured for 7 days on either uncoated or fibronectin-coated plates in the absence or presence of 20 mM quercetin, as indicated. Data are means ± SEM, n = 7. *P < 0.05 vs. corresponding value in the absence of stimulant for the same culturing conditions. Panel B, effects of CSS treatment upon the bhexosaminidase response to concanavalin A. RBL2H3 cells were cultured for 7 days on fibronectin-coated plates. Cells were subjected to a preincubation phase of 30 min followed by change of medium and incubation for 15 min with the stimulating agent. CSS (100 ml in the 400 ml assay) was present during the preincubation and/or incubation phases, as indicated in the figures. Data are means ± SEM, n = 5. * P < 0.05 vs. the corresponding control value for the same stimulant concentration. # Significant ANOVA value for the group, but no significant differences were found upon post-hoc testing.

Effects of CSS upon the responsiveness of RBL-2H3 cells to concanavalin A Concanavalin A is believed to produce degranulation by crosslinking Fc eRI receptors [18]. The compound produced a robust release of b-hexosaminidase when used at concentrations of 0.2 and 0.3 mg/ml, and a lower release at 1 mg/ml (Fig. 3, Panels A and B). The sensitivity to concanavalin A was similar when the cells were cultured on uncoated and fibronectin coated wells, but was lost for the quercetin-treated cells (Fig. 3, Panel A). For cells cultured on fibronectin coated wells, the degranulation response elicited by concanavalin A was not accompanied by a concomitant release of LDH (Fig. 4, Panel B). The effect of CSS from IR3 and IR5F cigarettes is shown in Fig. 3, Panel B. In these initial experiments, the relatively large data spread reduced the level of significance attained, although the tendency was for the CSS to reduce the release of b-hexosaminidase. However, in the series of experiments summarised in Fig. 4 (excluding the samples with ethanol controls to avoid possible confounding solvent effects), the

b-hexosaminidase release in response to 0.3 mg/ml concanavalin A (17 ± 2.2% of maximum attainable release) was signicantly reduced after preincubation with 70 ml CSS from IR5F cigarettes (8.3 ± 0.86% of maximum attainable release; P < 0.001, two-tailed paired t-test) (means ± s.e.m., N = 15; basal levels of b-hexosaminidase release measured in the same experiments were 5.3 ± 0.22%). The inhibitory effect of 70 ml CSS could be blocked when N-acetylcysteine was also present in the incubation phase, whereas hemoglobin, a-tocopherol, palmitoylethanolamide and catalase were without effect (Fig. 4, Panels A, C and D). The release of LDH either per se or following concanavalin A stimulation was not affected by preincubation with 70 ml CSS (Fig. 4, Panel B). Effects of CSS upon the responsiveness of RBL-2H3 cells to antigenic stimulation RBL-2H3 cells were sensitised by overnight incubation with DNP-IgE (30 ng/ml). Incubation of the cells with antigen

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Fig. 4. b-Hexosaminidase (Panels A, C and D) and LDH (Panel B) release from RBL-2H3 cells, cultured in fibronectin-coated wells, in response to concanavalin A (0.3 mg/ml). The fibronectin treatment was as is described in the legend to Fig. 3. Cells were preincubated with either PBS (unfilled columns) or CSS (70 ml in PBS + 30 ml PBS, filled columns) from IR5F cigarettes together with the compounds shown for 30 min prior to washing twice with PBS and incubation with concanavalin A. Abbreviations and concentrations of the antioxidants are given in the legend to Fig. 2. Data are means ± SEM, n = 9 (Panel A), 7 (Panels B and D) or 6 (Panel C). *P < 0.05 for the comparisons shown. # P < 0.05 vs. the corresponding value in the absence of N-acyl-L-cysteine.

(DNP-HSA, 10 ng/ml) produced a large release of b-hexosaminidase, that was reduced by EDTA and almost obliterated by 70 ml CSS from IR3 and IR5F cigarettes (Fig. 5). The inhibitory effect of CSS from IR5F cigarettes was almost blocked by N-acetylcysteine, when both compounds were present during the preincubation phase alone (Fig. 5A), whereas only a small, but significant, effect of this compound was seen when CSS and N-acetylcysteine were present during both preincubation and incubation phases (Fig. 5B). A broadly similar pattern was seen for CSS from the IR3 cigarettes, although in the preincubation phase alone experiments, the effect of N-acetylcysteine did not reach significance. Discussion In the present study, the effects of CSS upon the release of the preformed granular enzyme b-hexosaminidase has been investigated in RBL 2H3 cells, a mastocyte tumour cell line with good homology to rat mucosal mast cells [19]. RBL2H3 cells bind to fibronectin-coated surfaces, and the binding enhances the histamine release response to both antigen and calcimycin stimulation [11]. In our hands, the b-hex-

osaminidase response to calcimycin stimulation was enhanced whereas the response to concanavalin A was essentially unchanged. The finding of a robust response to concanavalin A in these cells is worthy of comment, since earlier studies suggested that RBL-2H3 cells did not degranulate in response to this lectin [20, 21]. However, a bell-shaped curve for concanavalin A stimulation of b-hexosaminidase, which has been reported for other mast cells [22], may provide an explanation for this discrepancy. Under normal conditions, RBL-2H3 cells do not respond to non-immunogenic stimulation other than calcium ionophores. However, in 1993, Trnovsky et al. [23] reported that prolonged treatment of RBL-2H3 cells with quercetin (a flavonoid structurally related to sodium cromoglycate) led to a change in phenotype, so that the cells appeared to shift towards a more mature phenotype. Senyshyn et al. [12] found that quercetin-treated RBL-2H3 cells acquire sensitivity to several agents (c48/80, substance P, VIP and somatostatin) without affecting the response to antigen stimulation. The acquired sensitivity, which was maximal after two days of treatment with 30 mM quercetin followed by medium change, was accompanied by an increased expression of Gai2 and Gai3, and was pertussis toxin-dependent [12, see also Ref. 24]. In our hands, this concentration of quercetin was cell toxic, but

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Fig. 5. b-Hexosaminidase release from RBL-2H3 cells, cultured in fibronectin-coated wells, in response to antigen (DNP-HSA, 10 ng/ml). Cells were cultured for 6 days in fibronectin-coated wells. Medium was changed to medium containing sensitizing antibody (anti-DNP IgE, 30 ng/ml) and the cells incubated overnight prior to assay. In Panel A, cells were preincubated with either either PBS or CSS (70 ml in PBS + 30 ml PBS) together with vehicle (medium), N-acetyl-L-cysteine (N-AC, 1 mg/ml), or EDTA (10 mM) in medium as appropriate prior to medium change and antigen stimulation during the 15 min incubation phase. In Panel B, the CSS, N-AC and EDTA were present during both preincubation and incubation phases. Data are means ± SEM, n = 5. * P < 0.05 vs. the corresponding value in the absence of CSS for the same N-AC/EDTA concentration. # P < 0.05 vs. the corresponding value in the absence of N-AC or EDTA.

a response to non-immunogenic stimuli could be attained following 7 days of treatment with 20 mM quercetin. However, this treatment reduced the sensitivity to stimulation by concanavalin A. Furthermore, the release of b-hexosaminidase from quercetin-treated cells in response to 15 mg/ml of compound 48/80 was accompanied by a large release of the cytoplasmic enzyme LDH, suggesting that degranulation is not the only process triggered by this concentration of compound 48/80 under the conditions used here. This differs from the finding that RBL-2H3 cells can be induced to respond to compound 48/80 (1–30 mg/ml) in a manner that is also partially reduced by EDTA, but with no concomitant release of LDH, after a two week co-culture of the cells with 3T3 fibroblast cells [25]. A reasonable conclusion from the present study is that the 3T3 co-culture method is a more appropriate way of inducing a responsiveness to nonimmunogenic stimuli. Nevertheless, the finding in the present study that the effects of CSS upon b-hexosaminidase release in response to compound 48/80 differ from the corresponding effects upon LDH release suggests that the two release processes are separate. The main goal of the present study was to investigate further the finding of Thomas et al. [9] that CSS affected mast cell responsiveness. These authors reported that CSS increased the release of pre-formed mediators (histamine and tryptase) from disaggregated mastocytoma cells, whereas the release of prostaglandin D2 was not affected. In contrast, the CSS reduced the prostaglandin D2 response to stimulation by calcimycin [9]. In our hands, CSS had no dramatic effect per se but reduced the release of the preformed granular protein b-hexosaminidase in response to stimulation by compound 48/80, calcimycin, substance P, concanavalin A and antigen. Thus, the effect of CSS upon the function of mast cell- like cell lines appears to be critically dependent upon the mast cell line and/or the assay conditions used.

According to information from the manufacturer of the cigarettes (The Tobacco and Health Research program at the Univestity of Kentucky), the nicotine levels in the smoke of the IR3 cigarretes (1.14 mg/cigarette with a 30 mm butt remaining) are considerably higher than the corresponding nicotine levels in the smoke of the filter-tipped IR5F cigarettes (0.16 mg/cigarette with a 35 mm butt remaining). The tar levels in the smoke are also considerably lower. Since the effects of both cigarette types on the b-hexosaminidase release response to stimulants were generally rather similar, it is likely that constituents other than nicotine are responsible for the inhibitory effect. This is consistent with the report of Thomas et al. [9] who found that nicotine tartrate had no effect upon either the basal release of histamine or upon calcimycin-induced prostaglandin D2 release from canine mastocytoma cells. In our view, most likely explanation for the inhibitory effect of CSS from IR5F cigarettes upon b-hexosaminidase release is that it is a result of oxidants present in the CSS. It is well establishd that cigarette smoke contains a number of free radicals and that smokers show signs of free radical attack [26]. With respect to identification of the oxidant responsible for the deleterious effects of CSS on mast cell function, there are a number of candidates from which to choose [26]. In this respect, Hoshino et al. [27] found that the cytotoxic effects of CSS on A549 cells (an alveolar type II cell-derived cell line) were abolished by the non-specific antioxidant N-acetylcysteine, whereas mannitol (hydroxyl radical scavenger), oxyhaemoglobin (nitric oxide scavenger), catalase (hydrogen peroxide scavenger) and uric acid (peroxynitrite scavenger) were only able partially to reduce the effects at the concentrations used. In contrast, mannitol was ineffective in preventing CSS-induced protein oxidation, whereas glutathione was partially effective and ascorbic acid

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was completely effective [28]. For the effects found in the present study, the only antioxidant which effectively blocked the actions of CSS from IR5F cigarettes was N-acetylcysteine. In conclusion, the present study has demonstrated that the effects of CSS on mast cell function are rather dependent upon the mast cell line used, and that in RBL-2H3 cells the reduced responsiveness to degranulating stimuli, at least in the case of IR5F cigarettes, is presumably the result of the presence of oxidants in the smoke. It is of course difficult to extrapolate data from cultured cells in vitro to the situation in vivo. However, the present results would suggest that a sensitisation of mast cells to stimuli (as opposed to an increased density of mast cells) is not a likely explanation for the finding of increased levels of preformed mast cell mediators in the bronchalveolar fluid of smokers [6]. The findings, however, may have some bearing upon the observation that the incidence of an extrinsic allergic alveolitis seen in pigeon breeders is lower in smokers than in non-smokers. In this disorder, smokers have been shown to produce lower levels of IgG in the bronchoalveolar lavage fluid in response to inhaled pigeon antigens [29]. A reduced sensitivity of the mast cells to antigen stimulation as a result of exposure to cigarette smoke would also contribute to a reduced allergic response. A final note concerns the experiments with palmitoylethanolamide. This endogenous N-acyl ethanolamine has been reported to reduce the incidence of acute respiratory infections when given to soldiers [30], and to reduce the degranulation of mast cells in response to substance P in vivo [14]. An initial study using RBL-2H3 cells reported that palmitoylethanolamide could prevent degranulation in response to an immunogenic stimulus in vitro [31]. However, subsequent in vitro studies using either isolated mast cells or mast cell lines have not been able to demonstrate consistent effects of palmitoylethanolamide [32–34]. One explanation for this difference between in vivo and in vitro effects of palmitoylethanolamide was that the mast cells in the in vitro studies behaved differently than those in vivo due to the lack of interactions with other cells or cell components. The present finding that preincubation of RBL-2H3 cells cultured in fibronectin coated plates with palmitoylethanolamide, is without effect on the b-hexosaminidase release response to concanavalin A, would argue against this explanation and suggest instead that the effects of this compound in vivo are not the result of a direct action upon mast cells. Acknowledgements. We thank Ingrid Persson for excellent technical support. The authors are grateful to the Rådet för Medicinsk Tobaksforskning for financial support. In addition, the experiments dealing with the development of the RBL-2H3 model and the effects of palmitoylethanolamide upon RBL-2H3 function were supported by the Swedish Medical Research Foundation (Grant no. 12158), the Swedish Asthma- and Allergy Association’s Research Foundation, the Swedish Psoriasis Association, and Konung Gustav V’s and Drottning Victorias Foundation.


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