Toxicology Letters 136 (2002) 121 /132 www.elsevier.com/locate/toxlet
Temporal changes in cytokine gene expression profiles induced in mice by trimellitic anhydride Catherine J. Betts a,*, Rebecca J. Dearman a, Brian F. Flanagan b, Ian Kimber a a
b
Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire SK10 4TJ, UK Department of Immunology, Duncan Building, University of Liverpool, Daulby Street, Liverpool L69 3GA, UK Received 16 May 2002; received in revised form 25 July 2002; accepted 25 July 2002
Abstract Prolonged (13 day) topical exposure of BALB/c strain mice to the chemical respiratory allergen trimellitic anhydride (TMA) induces a selective T helper (Th) 2 profile of cytokine secretion in cells isolated from the draining lymph node. The ability of chemical respiratory allergens to elicit preferential type 2 immune responses is a distinguishing characteristic and provides the theoretical basis for cytokine fingerprinting, a novel approach to hazard identification. This study aimed to further characterize the cytokine expression profile induced by TMA, and to investigate the kinetics of cytokine production at both the protein and mRNA level by comparison of acute (3 day) and chronic (13 day) exposure regimes. Acute exposure resulted in the expression of high levels of mRNA for both Th1- and Th2-type cytokines, including interleukins 4, 10, 15 (IL-4, IL-10, IL-15) and interferon g (IFN-g), and the inflammatory cytokine IL-6, as determined by ribonuclease protection assay (RPA). However, following chronic exposure marked downregulation of message for IL-6 and IFN-g was observed along with concomitant up-regulation of IL-4 and IL-10 expression. These cytokine mRNA profiles were broadly paralleled at the protein level. There was also a marked increase with time of mRNA for the Th2 cytokine IL-9, a cytokine not associated previously with chemical allergy. These data show that as the immune response to TMA develops, the cytokine gene expression profile of allergenactivated lymph node cells evolves from a mixed Th1/Th2 phenotype to a more polarized Th2 profile. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Chemical respiratory allergy; Trimellitic anhydride; Th subsets; Interleukin 9
1. Introduction Abbreviations: AOO, acetone:olive oil; con A, concanavalin A; ELISA, enzyme-linked immunosorbant assay; FCS, foetal calf serum; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IFN, interferon; IL, interleukin; LNC, lymph node cells; PBS, phosphate buffered saline; RPA, ribonuclease protection assay; TMA, trimellitic anhydride. * Corresponding author. Tel.: /44-1625-514047; fax: /441625-590996 E-mail address:
[email protected] (C.J. Betts).
A variety of chemicals has been shown to cause sensitization of the respiratory tract, and occupational asthma remains an important health issue (Ross et al., 1995; Hendrick, 2001). An intriguing question, that has attracted much attention, is the reason why only some chemical allergens cause respiratory sensitization, whereas others (the ma-
0378-4274/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 4 2 7 4 ( 0 2 ) 0 0 2 8 9 - 8
122
C.J. Betts et al. / Toxicology Letters 136 (2002) 121 /132
jority) are associated exclusively with skin sensitization and allergic contact dermatitis (Kimber, 1995). Although there is some debate about a universal mandatory requirement for IgE antibody in the pathogenesis of chemical respiratory allergy (Cullinan, 1998; Kimber et al., 1998), there now exists a substantial body of evidence that selective development of type 2 immune responses is an important feature (Kimber and Dearman, 1997). In mice it has been shown that whereas skin sensitizers induce preferential type 1 immune responses, chemical respiratory allergens stimulate the development of selective type 2 responses (Kimber and Dearman, 1997). As judged by the distribution of antibody isotypes and/or cytokine secretion patterns, it has been demonstrated previously that known chemical respiratory sensitizers, including various acid anhydrides, diisocyanates, platinum salts, glutaraldehyde and cyanuric chloride, all provoke in mice the evolution of type 2 immune responses (Dearman and Kimber, 1992, 2001; Vandebriel et al., 2000). The majority of analyses to date have focused on the differential expression of a limited number of cytokines; primarily, interleukins 4 and 10 (IL-4 and IL-10) and interferon g (IFN-g) and this approach forms the basis of cytokine fingerprinting for the identification of chemical respiratory allergens. With the purpose of refining further cytokine fingerprinting, the objectives of the investigations reported here were to examine in greater detail and in a more holistic fashion the patterns of cytokine expression induced in mice by exposure to a reference chemical respiratory allergen, trimellitic anhydride (TMA) (Zeiss et al., 1977; Topping et al., 1986). To this end we have here measured, using a ribonuclease protection assay (RPA), TMA-induced changes in the expression of mRNA for IL-4, IL-10, IFN-g and a number of additional cytokines; IL-2, IL-5, IL-6, IL-9, IL-13 and IL-15. In parallel investigations, the secretion of some of these same cytokines by cultured lymph node cells (LNC) was measured by enzyme-linked immunosorbent assays (ELISA). Finally, it has been found previously that following treatment of mice with chemical respiratory allergens preferential type 2 immune responses with respect to differential IL-4 and IFN-g expres-
sion evolve with time from first exposure (Dearman et al., 1994; Moussavi et al., 1998). We have chosen here, therefore, to measure patterns of cytokine expression following both acute and more chronic exposure of mice to TMA.
2. Materials and methods 2.1. Animals Young adult (6 /12 weeks old) female BALB/c strain mice (Harlan Seralab, Oxon, UK) were used throughout these studies. Mice were housed in metal cages and food (SDS PCD pelleted diet; Special Diets Services Ltd., UK) and water were available ad libitum. The ambient temperature was maintained at 219/2 8C and relative humidity was 559/10% with a 12 h light /dark cycle. All experiments were conducted under the provisions of the Animals (Scientific Procedures) Act, 1986. 2.2. Chemicals TMA (97% pure) was obtained from the Aldrich Chemical Co. (Gillingham, Dorset, UK) and dissolved in 4:1 acetone:olive oil (AOO) immediately prior to application. 2.3. Sensitization of mice for cytokine production Groups of mice (n /10) either received 50 ml of 10% TMA bilaterally on each shaved flank (for chronic exposure) on days 0 and 5 or were untreated (acute exposure). After a further 5 days (day 10), all animals received 25 ml of 10% TMA on the dorsum of both ears daily for 3 consecutive days. A further control group remained untreated (naı¨ve) throughout the duration of the experiment. This concentration of TMA was chosen based on previous experience demonstrating that it was of marked immunogenicity and capable of polarizing the cytokine response at the protein level following a chronic exposure regime (Dearman and Kimber, 2001).
C.J. Betts et al. / Toxicology Letters 136 (2002) 121 /132
2.4. Preparation of cells from the draining lymph nodes Thirteen (chronic) or 3 (acute) days after first exposure, the draining auricular lymph nodes were excised and pooled for each experimental group. A single cell suspension was prepared under aseptic conditions by mechanical disaggregation through sterile 200-mesh steel gauze. LNC were cultured in RPMI-1640 growth medium (GibcoBRL, Renfrewshire, UK) supplemented with 25 mmol/l HEPES, 400 mg/ml streptomycin, 400 mg/ml ampicillin (Sigma Chemical Co., St. Louis, MA, USA) and 10% heat-inactivated foetal calf serum (FCS) (GibcoBRL). Cells (1 ml) at 107/ml were seeded into 24-well tissue culture plates and maintained at 37 8C in a humidified atmosphere of 5% CO2 in air. Culture was terminated after 120 h, the supernatants collected, centrifuged at 100 /g for 5 min and stored at /70 8C until analysis. Alternatively, immediately after isolation aliquots of cells were centrifuged at 1500 rpm, the pellet resuspended in TRIZOL (GibcoBRL) at 1 ml per 3 /107 cells and the samples stored at /70 8C until total RNA extraction. 2.5. Cytokine determinations The cytokine content of culture supernatants derived from LNC following a 120 h culture period was determined by sandwich ELISA. All assays used plastic microtitre plates from Nunc (Copenhagen, Denmark) and the optical density at 450 nm was measured using an automated reader (Multiskan, Flow Laboratories, Irvine, Ayrshire, UK). Capture and detection antibody combinations for each assay were as follows: 2.5.1. IFN-g Capture antibody: 1 mg/ml hamster monoclonal anti -mouse IFN-g (R &D Systems Europe, Abingdon, Oxon, UK). Standard: Recombinant murine IFN-g (R&D Systems Europe). Detection: 0.1 mg/ml biotinylated goat anti mouse IFN-g (R&D Systems Europe). Lower limit of detection: 78 pg/ml.
123
2.5.2. IL-5 and IL-10 Capture, standard and detection reagents purchased as complete set (OptEIA ELISA kit, Pharmingen, San Diego, CA, USA). Lower limits of detection: 19.5 (IL-5) and 156 pg/ml (IL-10). 2.5.3. IL-6 Capture antibody 2.5 mg/ml rat monoclonal anti -mouse IL-6 ( R & D Systems Europe). Standard: Recombinant murine IL-6 (R&D Systems Europe). Detection antibody: 8 mg/ml goat anti -mouse IL-6 (R&D Systems Europe). Lower limit of detection: 156 pg/ml. 2.5.4. IL-12p40 Capture antibody: 4 mg/ml rat monoclonal anti mouse IL-12 (Clone C15.6; R&D Systems Europe). Standard: Recombinant murine IL-12 (R&D Systems Europe). Detection antibody: 0.1 mg/ml biotinylated rat anti -mouse IL-12 (R&D Systems Europe). Lower limit of detection: 78 pg/ml. 2.5.5. IL-13 Capture antibody: 10 mg/ml monoclonal anti mouse IL-13 (R&D Systems Europe). Standard: Recombinant murine IL-13 (R&D Systems Europe). Detection antibody: 0.4 mg/ml biotinylated rat anti -mouse IL-13 (R&D Systems Europe). Lower limit of detection: 156 pg/ml. 2.6. Preparation of total RNA for ribonuclease protection assay analysis Total RNA was extracted from cell pellets resuspended in TRIZOL (GibcoBRL) as per the manufacturer’s instructions. Briefly, to each ml of cells in TRIZOL, 200 ml of chloroform were added and the tubes agitated by hand. The aqueous and organic phases were separated by centrifugation at 14,000 /g, 4 8C for 15 min and the upper layer was removed to a fresh 1.5 ml tube. Following RNA precipitation with an equal volume of isopropanol at /20 8C overnight, the RNA was
124
C.J. Betts et al. / Toxicology Letters 136 (2002) 121 /132
recovered by centrifugation at 14,000 /g , 15 min at 4 8C. The RNA pellet was washed with 70% ethanol in DEPC /H2O and then centrifuged as above. Finally, the RNA pellets were air-dried and resuspended in 50 ml of DEPC /H2O. Yield was established by spectrophotometer readings at 260 nm using a Genequant RNA/DNA calculator (Pharmacia Biotech, St. Albans, UK) and protein contamination monitored by analysis of the optical density 260/280 nm ratio. No samples presented ratios indicating unacceptable protein contamination. TRIZOL reagent routinely delivers high quality RNA, the integrity of which was confirmed by 1% agarose gel electrophoresis in the presence of ethidium bromide.
2.7. RNAse protection assay T7 RNA polymerase was used to synthesize 32P radiolabelled antisense RNA probes, using RiboQuant cDNA template mCK-1 (containing IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-15, IFN-g, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and L32) according to the manufacturer’s instructions (Pharmingen). Probe solutions were hybridized to 20 mg of total RNA at 56 8C overnight followed by digestion with RNAse A and T1 (Pharmingen). Samples were then treated with proteinase K to remove RNAses, phenol extracted and precipitated in ethanol. Each sample was loaded onto a 6% polyacrylamide urea gel, run at 38 mA in 0.5 / TBE (Tris /borate EDTA buffer) alongside the undigested probe as size marker. Gels were exposed to autoradiography film (KODAK XAR-5) at /70 8C with intensifying screens. Densitometric analysis was carried out using the ‘Image’ programme (National Institutes of Health, USA).
2.8. Statistics The statistical significance of differences between groups with respect to relative levels of cytokine mRNA expression or cytokine protein production was assessed by two-sided Student’s ttest.
3. Results 3.1. Cytokine mRNA profiles following exposure to TMA In the current experiments, we have compared cytokine mRNA expression ex vivo and protein production patterns induced in mice by either acute or chronic topical exposure to TMA. Four independent experiments were performed; the composite polyacrylamide gel from RPA analysis is displayed in Fig. 1. Quantitative measurements were made by densitometer and levels of cytokine mRNA relative to GAPDH mRNA expression for each of the four individual experiments are shown in Fig. 2 with mean and SE of relative cytokine expression and the statistical significance of changes in cytokine levels with time displayed in Table 1. LNC derived from naı¨ve control animals expressed little or no detectable type 2 cytokines, and only low levels of the type 1 cytokines IL-2, IL-15 and IFN-g, under conditions where relatively high levels of expression of the housekeeping genes L32 and GAPDH was observed (Fig. 1). In contrast, treatment with allergen caused marked cytokine mRNA production. Acute exposure to TMA resulted in high levels of expression of IL-6 and IFN-g with somewhat lower, and similar, levels of transcripts detected for each of IL-2, IL-4, IL-10, IL-13 and IL-15. Following more prolonged application of TMA, however, a more polarized (Th2-type) response was observed, with down-regulation of IFN-g and IL-6, and concomitant up-regulation of IL-4, IL-9 and IL-10 (Figs. 1 and 2). Examination of the changes in gene expression relative to GAPDH mRNA levels revealed considerable consistency in the kinetic changes in the cytokine mRNA expression (Fig. 2 and Table 1). In each experiment, mRNA levels for IL-2, IL-13 and IL-15 did not change significantly with time, although there were consistent trends between experiments, with IL-2 and IL-13 tending to increase following chronic exposure, whereas somewhat higher levels of mRNA for IL15 were expressed after acute exposure. IL-5 mRNA was not detectable at any time point in any of the four experiments. IL-6 and IFN-g were the two cytokines which were the most highly
C.J. Betts et al. / Toxicology Letters 136 (2002) 121 /132
125
Fig. 1. Cytokine mRNA expression profiles following topical application of TMA. Mice were exposed topically to 10% TMA in AOO vehicle over a 3 (acute, A) or a 13 day (chronic, C) period. Draining auricular lymph nodes were excised from TMA-treated animals (a) and naive untreated controls (b), a single cell suspension prepared and RNA extracted from cell pellets using TRIZOL. Levels of cytokine mRNA and housekeeping genes were measured by multiprobe RPA. A representative autoradiograph of four independent experiments is shown. Undigested probe samples were run to provide the size markers.
expressed following acute (3 day) exposure to TMA, whereas after chronic (13 day) treatment, IL-4 and IL-10 were the most highly expressed genes. Thus, after acute exposure to TMA, IL-6 and IFN-g mRNA levels (relative to GAPDH expression) were on average 40.4 and 47.7%, respectively, decreasing to 16.8 and 26.2%, respectively, following more prolonged treatment. Whereas IL-4 and IL-10 mRNA production (relative to GAPDH levels) increased from 13.8 and 12.0% after acute treatment to 32.7 and 29.7%, respectively, 13 days after initiation of exposure. Interestingly, a statistically significant up-regulation with time of mRNA for IL-9 was also observed, with mRNA levels relative to
GAPDH expression increasing from an average of 9.4 to 23.4%. 3.2. Cytokine protein secretion profiles following exposure to TMA In parallel experiments, cells isolated from mice following acute or chronic exposure to TMA were cultured for 120 h and cytokine secretion profiles measured by ELISA. Supernatants were analyzed for IL-4, IL-5, IL-6, IL-10, IL-12 (p40), IL-13 and IFN-g expression (Fig. 3). There was no detectable spontaneous IL-4 production by cultured draining LNC derived from mice treated acutely or chronically with TMA (data not shown). As reported previously (Dearman and Kimber, 2001; Dearman
126
C.J. Betts et al. / Toxicology Letters 136 (2002) 121 /132
Fig. 2. Relative cytokine mRNA expression profiles induced following topical treatment with TMA. Mice were exposed topically to 10% TMA in AOO vehicle over a 3 (I) or a 13 day (j) period. Draining auricular lymph nodes were excised from TMA-treated animals, a single cell suspension prepared and RNA extracted from cell pellets using TRIZOL. Levels of cytokine mRNA and housekeeping genes were measured by multiprobe RPA and quantitated by densitometric analysis. Results are expressed as relative levels of cytokine mRNA compared with GAPDH control transcripts for each of four independent experiments (a, b, c, d).
et al., 1994, 1996) allergen-activated LNC require restimulation in vitro with the T-cell mitogen concanavalin A (con A) in order for IL-4 secretion to be detectable. LNC derived from naı¨ve or from vehicle-treated animals, however, failed to elaborate detectable IL-4 in the presence or absence of mitogen (Dearman and Kimber, 2001; Dearman et al., 1994, 1996). Allergen-stimulated LNC isolated from animals following acute exposure to TMA did, however, express relatively high levels of IFNg, IL-6, IL-12 and IL-13 and detectable levels of
IL-5 and IL-10; a pattern of cytokine secretion consistent with an undifferentiated T helper phenotype. After more prolonged (13 day) exposure, IL-6 and IFN-g production was significantly down-regulated (P B/0.01 and 0.05, respectively) whereas IL-12 and IL-13 levels did not signifciantly change with time, although reduced levels of cytokine in each of the four experiments were recorded following chronic exposure. In contrast, IL-5 and IL-10 secretion were consistently upregulated (although only the changes in IL-10
C.J. Betts et al. / Toxicology Letters 136 (2002) 121 /132
127
Table 1 Cytokine mRNA expression in draining LNC isolated following acute or chronic exposure to TMA Cytokine
Acute exposure
Chronic exposure
Statistical significance (P value)
IL-4 IL-5 IL-10 IL-13 IL-15 IL-9 IL-2 IL-6 IFN-g
13.8 ND 12.0 13.7 16.1 9.4 16.5 40.4 47.7
32.7 ND 29.7 17.1 10.6 23.4 20.4 16.8 26.2
(1.52)
B/0.01 (**)
(3.95) (2.56) (1.23) (3.57) (1.88) (3.28) (4.53)
0.014 0.142 0.072 0.018 0.282 0.022 0.036
(1.91) (3.25) (2.90) (2.20) (2.47) (2.73) (6.85) (6.76)
(*) (NS) (NS) (*) (NS) (*) (*)
Mice were exposed topically to 10% TMA in AOO. Three (acute) or 13 (chronic) days after initiation of exposure, a single cell suspension of auricular lymph nodes was prepared and total RNA isolated using TRIZOL. Relative levels of cytokine mRNA expression in comparison with GAPDH control transcript levels were analyzed by multiprobe RPA. Data are shown as mean values with SE in parentheses of four independent experiments. ND, not detectable. Statistically significant temporal changes in cytokine expression were assessed by two-sided Student’s t- test and are illustrated by * (P B/0.05), ** (P B/0.01) and NS, not statistically significant.
concentration reached statistical significance) resulting in a more Th2-type cytokine secretion profile.
4. Discussion We have examined, at both the protein and the mRNA level, the kinetics of cytokine responses induced in the draining lymph node following topical exposure of BALB/c strain mice to the respiratory sensitizing acid anhydride TMA. Changes in cytokine gene expression have been profiled using a multi-probe RPA, a sensitive and specific method for the detection and quantitation of several mRNA species simultaneously within a single sample of total RNA (Gilman, 1993). This technique allows comparative analysis of the expression of different genes concurrently within the same sample and the incorporation of housekeeping gene transcripts facilitates comparisons of mRNA levels between samples. Unlike the technique of reverse transcription-polymerase chain reaction (RT-PCR), there is no amplification step in RPA analyses, ensuring a more quantitative assessment of cytokine gene expression levels, although there is a concomitant decrease in sensitivity. However, the RPA is more sensitive than is Northern blotting and requires consider-
ably less material for the analysis of an equivalent panel of cytokines (Gilman, 1993). RPA analyses performed with RNA derived from four independent experiments revealed that this technique is indeed sensitive enough to detect cytokine expression by allergen-activated LNC, with very little inter-experimental variation observed. However, cells derived from the quiescent lymph nodes of naı¨ve animals, despite displaying high levels of message for the two housekeeping genes, expressed only very low levels of mRNA for most of the cytokines examined, with IL-15, IL-2 and IFN-g transcripts present just above the limit of detection. This pattern of cytokine expression is what might be expected from a population of cells where the majority is unlikely to have encountered allergen and is consistent with data reported previously for murine lymphoid tissue, with freshly isolated auricular LNC from vehicle-treated mice expressing no detectable mRNA for the same cytokines (Manetz et al., 2001). In each experiment, acute exposure to TMA resulted in a mixed cytokine profile, with the expression of mRNA for both Th1- (IL-2, IL-15 and IFN-g) and Th2-types (IL-4, IL-9, IL-10 and IL-13) cytokines, with particularly high levels of IFN-g and the inflammatory cytokine IL-6. Following chronic exposure, IFN-g and IL-6 transcripts were markedly down-regulated, with a
128
C.J. Betts et al. / Toxicology Letters 136 (2002) 121 /132
Fig. 3.
C.J. Betts et al. / Toxicology Letters 136 (2002) 121 /132
129
Fig. 3. (Continued) Relative cytokine protein secretion profiles induced following topical treatment with TMA. Mice were exposed topically to 10% TMA in AOO vehicle over a 3 day (I) or a 13 day (j) period. Draining auricular lymph nodes were excised from TMA-treated animals, a single cell suspension prepared and cultured for 120 h at 107 cells/ml. Supernatants were analyzed for cytokines by ELISA. Individual cytokine secretion profiles (a, IFN-g, IL-6, IL-12 and IL-13; b, IL-5 and IL-10) for each of four independent experiments are shown with mean and SE. Statistically significant temporal changes in cytokine expression were assessed by two-sided student’s t- test and are illustrated by *(P B/0.05) and **(P B/0.01).
concomitant increase observed in the expression of Th2-type cytokines IL-4, IL-9 and IL-10. In contrast, IL-2, IL-13 and IL-15 gene expression did not change significantly with time and allergen exposure. These patterns of expression were largely paralleled by cytokine protein secretion profiles. Thus, LNC isolated 3 days after initiation of exposure to TMA secreted significantly more IFNg and IL-6 than did those stimulated over 13 days, whereas the converse was true of the Th2-type cytokine IL-10. Interestingly, IL-13 expression (at
both the mRNA and the protein level) did not change significantly with time, suggesting that early high level production of this cytokine may be one of the factors responsible for polarization of the response to TMA. There was considerably more inter-experimental variation in the absolute levels of cytokine protein secretion than there was in the levels of mRNA detected, however, in each case the pattern of cytokine production remained the same. Interestingly, although levels of IL-5 transcripts were not detectable using RPA (indi-
130
C.J. Betts et al. / Toxicology Letters 136 (2002) 121 /132
cating the relatively low sensitivity of this technique), TMA-activated LNC expressed measurable levels of IL-5 protein, albeit at lower levels (50 / 600 pg/ml) compared with the other Th2 cytokines IL-10 and IL-13 (250 /1250 and 2000 /10,000 pg/ ml, respectively). It should be noted, however, that the biological activity of these cytokines was not assessed and therefore the low level (with respect to absolute amount of cytokine protein present) expression of IL-5 is likely to be biologically significant. Measurement of IL-5 secretion was also indicative of a polarization toward Th2-type responses with time; in every experiment higher levels of this cytokine were detected following chronic compared with acute treatment with TMA. As reported previously (Dearman and Kimber, 2001; Dearman et al., 1994, 1996), it was not possible to detect the spontaneous secretion of IL-4 by TMA-activated LNC. IL-4 protein expression by allergen-activated LNC is detectable only following secondary stimulation in culture with the T-cell mitogen con A. Mitogen stimulation per se does not stimulate IL-4 expression by murine LNC. Thus, only LNC activated in vivo by previous topical exposure to respiratory allergens such as TMA, and not to contact sensitizers or cells derived from vehicle-treated or naı¨ve mice, can be stimulated by con A to produce this cytokine vigorously (Dearman and Kimber, 2001; Dearman et al., 1994, 1996). In the current experiments, we have confirmed that IL-4 is strongly represented at the message level in TMA-activated murine LNC and that there is an up-regulation of mRNA transcripts for this cytokine as the immune response develops with time into a more polarized Th2-type phenotype. These data are in agreement with previously published experiments in which it was demonstrated that chronic exposure to TMA resulted in marked expression of transcripts for IL-4 (measured by RPA) compared with treatment with the contact allergen 2,4-dinitrochlorobenzene (DNCB) or with vehicle alone (Plitnick et al., 2002). The inability to detect IL-4 protein in culture supernatants may be a result of the reutilization of this cytokine by the cells in culture in autocrine fashion. Indeed, it has been demonstrated previously that the sensitivity of detection of IL-4 protein expression by LNC
derived from parasite (Trichuris muris )-infected mice could be enhanced by blocking uptake of this cytokine with anti -IL-4 receptor antibody (Bancroft et al., 1998). Interestingly, a marked up-regulation with time in IL-9 mRNA expression by TMA-stimulated cells has been identified. This cytokine was initially described as a murine T-cell growth factor produced by activated T cells (Uttenhove et al., 1988) which also plays a role in the development of mast cells (Hultner et al., 1990). More recently, IL-9 has been defined as a Th2-type cytokine and it has been suggested to play a critical role in many aspects of the development of the pathophysiology of asthma in experimental animals, including eosinophilic inflammation, bronchial hyperresponsiveness, elevated IgE levels and increased mucus secretion (Temann et al., 1998; Soussi-Gounni et al., 2001). Furthermore, increased expression of IL-9 and corresponding receptor has been described in allergic individuals compared with those with non-allergic lung disease or normal controls (Shimara et al., 1999). The observation that chronic exposure to TMA is associated with enhanced expression of IL-9 mRNA further confirms the Th2-phenotype of these cells. Together with expression of IL-5 and IL-13, the potential involvement of IL-9 in respiratory allergy provides for a non-IgE mediated effector mechanism which may contribute to the inflammatory and immune responses induced by this chemical allergen. These data show that with repeated topical exposure to TMA and with time, a polarized type 2 phenotype of cytokine mRNA and protein production develops. This type 2 cytokine phenotype (high levels of IL-4, IL-5, IL-9, IL-10 and IL13) evolved with time from an undifferentiated or mixed T helper profile, consistent with the ability of TMA to provoke immediate type hypersensitivity responses (Zeiss et al., 1977; Topping et al., 1986). It remains to be determined whether other chemical respiratory allergens induce similarly robust and distinctive patterns of cytokine gene expression. Other investigators have shown that another respiratory allergen, toluene diisocyanate, does indeed stimulate a Th2-type phenotype of cytokine mRNA expression compared with the contact allergen 2,4-dinitrofluorobenzene which
C.J. Betts et al. / Toxicology Letters 136 (2002) 121 /132
provoked the converse Th1-type profile (Manetz et al., 2001). In this case, the induction of these divergent cytokine transcript profiles required restimulation of LNC for 24 h with the T-cell mitogen con A (Manetz et al., 2001). Furthermore, in another independent investigation it has been demonstrated previously using this technique that chronic topical exposure of mice to TMA does indeed provoke a Th2-type pattern of cytokine mRNA, however, exposure to DNCB failed to stimulate the converse Th1-type profile in the absence of mitogenic restimulation (Plitnick et al., 2002). Plitnick et al. (2002) also investigated the influence of dose on cytokine mRNA profiles induced by TMA and observed that a Th2-type phenotype was stimulated independent of dose. In our investigations, acute exposure to TMA induced marked IFN-g mRNA expression and although this was down-regulated following chronic exposure, transcripts for this cytokine were still detectable. These data together suggest that the identification of contact allergens by the measurement of cytokine expression at the message level using RPA may not be sufficiently sensitive to identify contact allergens. However, in the context of characterization of chemical respiratory and contact allergens, cytokine profiling at the level of protein secretion using some of the same cytokines by allergen-activated LNC has been demonstrated to be reliable for the identification and classification of both respiratory and contact sensitizers (Dearman and Kimber, 2001). Further experience with a wider range of allergens is required to determine whether analysis of cytokine profiles by RPA is a suitable replacement for the identification of respiratory chemical allergens.
Acknowledgements This work was supported in part by a grant from the Health and Safety Executive, UK and EC Concerted Action Grant BMH4-CT98-3951.
131
References Bancroft, A.J., McKenzie, A.N.J., Grencis, R.K., 1998. A critical role for IL-13 in resistance to intestinal nematode infection. J. Immunol. 160, 3453 /3461. Cullinan, P., 1998. Occupational asthma, IgE and IgG. Clin. Exp. Allergy 28, 668 /670. Dearman, R.J., Kimber, I., 2001. Cytokine fingerprinting and hazard assessment of chemical respiratory allergy. J. Appl. Toxicol. 21, 153 /163. Dearman, R.J., Kimber, I., 1992. Divergent immune responses to respiratory and contact chemical allergens: antibody elicited by phthalic anhydride and oxazolone. Clin. Exp. Allergy 22, 241 /250. Dearman, R.J., Moussavi, A., Kemeny, D.M., Kimber, I., 1996. Contribution of CD4 and CD8 T lymphocyte subsets to the cytokine secretion patterns induced in mice during sensitization to contact and respiratory allergens. Immunology 89, 502 /510. Dearman, R.J., Ramdin, L.S.P., Basketter, D.A., Kimber, I., 1994. Inducible interleukin-4-secreting cells provoked in mice during chemical sensitization. Immunology 8, 551 / 557. Gilman, M., 1993. Ribonuclease protection assay. In: Ausubel, F.M., et al. (Ed.), Current Protocols in Molecular Biology, vol. 1. John Wiley and Sons , New York, pp. 4.7.1 /4.7.8. Hendrick, D.J., 2001. The world wide problem of occupational asthma. Clin. Exp. Allergy 31, 1 /4. Hultner, L., Druez, C., Moeller, J., Uttenhove, E., Schmitt, E., Rude, E., Dormer, P., Van Snick, J., 1990. Mast cell growth enhancing activity (MEA) is structurally related and functionally identical to the novel mouse T-cell growth factor P40/TCGFIII (interleukin 9). Eur. J. Immunol. 20, 1413 / 1416. Kimber, I., Dearman, R.J., 1997. Cell and molecular biology of chemical allergy. Clin. Rev. Allergy Immunol. 15, 145 /168. Kimber, I., Warbrick, E.V., Dearman, R.J., 1998. Chemical respiratory allergy, IgE and the relevance of predictive test methods: a commentary. Hum. Exp. Toxicol. 17, 537 /540. Kimber, I., 1995. Contact and respiratory sensitization by chemical allergens: uneasy relationships. Am. J. Contact. Dermat. 6, 34 /39. Manetz, T.R., Pettit, D.A., Meade, B.J., 2001. The determination of draining lymph node cell cytokine mRNA levels in BALB/c mice following dermal sodium lauryl sulphate, dinitrofluorobenzene, and toluene diisocyanate exposure. Toxicol. Appl. Pharmacol. 171, 174 /183. Moussavi, A., Dearman, R.J., Kimber, I., Kemeny, D.M., 1998. Cytokine production by CD4 and CD8 T cells in mice following primary exposure to chemical allergens: evidence for functional differentiation of T lymphocytes in vivo. Int. Arch. Allergy Immunol. 116, 116 /123. Plitnick, L.M., Loveless, S.E., Ladics, G.S., Holsapple, M.P., Selgrade, M.J., Sailstad, D.M., Smialowicz, R.J., 2002. Cytokine profiling for chemical sensitizers: application of the ribonuclease protection assay and effect of dose. Toxicol. Appl. Pharmacol. 179, 145 /154.
132
C.J. Betts et al. / Toxicology Letters 136 (2002) 121 /132
Ross, D.J., Sallie, B.A., McDonald, J.C., 1995. SWORD ’94: surveillance of work-related and occupational respiratory disease in the UK. Occup. Med. 45, 175 /179. Shimara, A., Christodoulopoulos, P., Soussi-Gounni, A., Olivenstein, R., Nakamura, Y., Levitt, R.C., Nicolaides, N.C., Holroyd, K.J., Tsciopoulos, A., Lafitte, J.-J., Wallaert, B., Hamid, Q.A., 1999. IL-9 and its receptor in allergic and nonallergic lung disease: increased expression in asthma. J. Allergy Clin. Immunol. 104, 108 /115. Soussi-Gounni, A., Kontolemos, M., Hamid, Q., 2001. Role of IL-9 in the pathophysiology of allergic diseases. J. Allergy Clin. Immunol. 107, 575 /582. Temann, A., Geba, G.P., Rankin, J.A., Flavell, R.A., 1998. Expression of interleukin 9 in the lungs of transgenic mice causes airways inflammation, mast cell hyperplasia, and bronchial hyperresponsiveness. J. Exp. Med. 188, 1307 / 1320. Topping, M.D., Venables, K.M., Luczynska, C.M., Howe, W., Newman Taylor, A.J., 1986. Specificity of the human IgE
response to inhaled acid anhydrides. J. Allergy Clin. Immunol. 77, 834 /842. Uttenhove, C., Simpson, R.J., Van Snick, J., 1988. Functional and structural characteristics of P40, a mouse glycoprotein with T-cell growth factor activity. Proc. Natl. Acad. Sci. USA 85, 6934 /6938. Vandebriel, R.J., De Jong, W.H., Spiekstra, S.W., Van Dijk, M., Fluitman, A., Garssen, J., Van Loveren, H., 2000. Assessment of preferential T-helper 1 or T-helper 2 induction by low molecular weight compounds using the local lymph node assay in conjunction with RT-PCR and ELISA for interferon-g and interleukin-4. Toxicol. Appl. Pharmacol. 162, 77 /85. Zeiss, C.R., Patterson, R., Pruzansky, J.J., Miller, M., Rosenberg, M., Levitz, D., 1977. Trimellitic anhydride-induced airway syndrome: clinical and immunological studies. J. Allergy Clin. Immunol. 60, 96 /103.
