Cytokine fingerprinting of chemical allergens: species comparisons and statistical analyses
Introduction
Occupational respiratory allergy to chemicals or proteins is an important worldwide health problem (Hendrick, 2001). Among those chemicals known to cause allergic sensitization of the respiratory tract are acid anhydrides (including trimellitic anhydride; TMA), isocyanates (such as toluene diisocyanate; TDI, diphenylmethane diisocyanate and hexamethylene diisocyanate), certain reactive dyes and some platinum salts (Chan-Yeung and Malo, 1993). Chemical-induced respiratory allergy represents a major toxicological challenge since there are no well-validated or widely accepted methods yet available for the prospective identification of such materials (Kimber, and Wilks, 1995, Kimber, Bernstein, Karol, Robinson, Sarlo, and Selgrade, 1996). The development of appropriate methods for hazard identification has been constrained largely by continuing uncertainty regarding the immunobiological mechanisms of respiratory sensitization to chemicals (Kimber et al., 1998). Whereas immediate-type respiratory responses and asthma to proteins are generally acknowledged to be dependent on IgE-mediated mechanisms, there is no such consensus regarding chemical respiratory hypersensitivity (Cullinan, 1998, Mapp, Boschetto, Miotto, De Rosa and Fabbri, 1999). For all known chemical respiratory allergens, specific IgE antibody has been detected in at least some symptomatic individuals. However, particularly in the case of isocyanate-induced asthma, these individuals are often in the minority, with only between 5 and 20% of symptomatic patients displaying measurable specific IgE antibody (Tee, Cullinan, Welch, Burge, and Newman Taylor, 1998, Tarlo, 1999). A contributing factor to the failure to detect IgE antibody in some cases may be the interval between last exposure to isocyanate and serum sampling, given that it has been demonstrated recently that IgE measurements are optimal when serum was sampled within 30 days of last exposure to isocyanate (Tee et al., 1998). There is a much stronger relationship between specific IgE antibody and disease status for other respiratory allergens, especially some of the acid anhydrides (Baur, and Czuppon, 1995, Yokota, Yamaguchi, Takeshita, and Morimoto, 1998).
Although there is some doubt regarding a universal association of IgE antibody and occupational chemical respiratory allergy, there is a more general consensus that, in common with protein allergens, chemicals with the potential to cause sensitization of the respiratory tract induce in humans the activation of T cell subpopulations with a polarized type 2 phenotype (Kimber, and Dearman, 1997, Mapp, Boschetto, Miotto, De Rosa and Fabbri, 1999). It has been demonstrated that both CD4 T helper (Th) cells and CD8 T cytotoxic (Tc) cells differentiate into two main populations, designated Th1 and Th2, and Tc1 and Tc2 (Mosmann and Sad, 1996). Type 2 cells produce cytokines that are required for the induction and maintenance of IgE antibody responses and which facilitate the development and expression of allergic sensitization (reviewed in Kimber and Dearman, 1997). The development in humans of asthma to high molecular weight allergens is commonly associated with T lymphocytes of a type 2 phenotype (Durham et al., 1992, Robinson, Hamid, Bentley, Ying, Kay, and Durham, 1993). In addition, for patients with isocyanate-induced asthma, there is evidence that similar patterns of T lymphocyte infiltration and eosinophil recruitment occur (Bentley et al., 1992) and Tc2-type cells producing interleukin (IL)-5 have been detected in individuals with asthma to TDI (Del Prete et al., 1993, Maestrelli et al., 1994).
It has been shown also that topical exposure of mice to chemicals that are known or suspected to cause respiratory allergy and occupational asthma in humans (including glutaraldehyde and certain acid anhydrides, platinum salts and diisocyanates) provokes the development of selective type 2 cytokine expression profiles by activated lymph node cells (LNC) (Dearman, and Kimber, 1999, Dearman, and Kimber, 2001, Hayashi, Higashi, Kato, and Kaneko, 2001, Vandebriel et al., 2000). These analyses have focused primarily on the differential expression by allergen-activated draining LNC of a limited number of cytokines; the type 2 cytokines interleukin (IL)-4 and IL-10 and the type 1 cell product interferon γ (IFN-γ). Thus, epicutaneous application of the respiratory allergen TMA over a 13-day period stimulates the preferential development of type 2 cells with expression of high levels of IL-10 and mitogen-inducible IL-4, but relatively low amounts of the type 1 cytokine IFN-γ. The converse selective type 1 cytokine profile is elicited by treatment with chemicals such as 2,4-dinitrochlorobenzene (DNCB), which induce contact allergy but which apparently lack the potential to cause sensitization of the respiratory tract (Dearman, and Kimber, 1999, Dearman, and Kimber, 2001, Vandebriel et al., 2000, Hayashi, Higashi, Kato, and Kaneko, 2001). More recently it has been demonstrated that TMA provokes the vigorous secretion of the additional type 2 cytokines IL-5 and IL-13 by activated LNC (Dearman and Kimber, 2001). In apparent conflict with these data, it has been reported that contact allergens stimulate the secretion of type 2 cytokines (Ulrich et al., 2001). In these experiments a different sensitization protocol was employed and all cytokines were measured after polyclonal restimulation in vitro with anti-CD3 antibody. Furthermore, direct comparisons between the ability of DNCB and TMA to elicit type 1 and type 2 profiles, respectively, are not possible since concentrations of DNCB were used which resulted in considerably lower levels of immunogenicity. However, the observation that DNCB and other contact allergens provoked detectable type 2 cytokines is not inconsistent with the fact that using the experimental design described herein, spontaneous expression of type 2 cytokines is also detectable following treatment with DNCB, albeit at much lower levels than that stimulated by TMA. The preferential development of a Th2-type cytokine secretion profile will favor the elicitation of immediate type hypersensitivity reactions independently of the induction of IgE antibody by the Th2 cell product IL-4 (Azuma et al., 1987, Finkelman et al., 1988). IL-5 plays a central role in the differentiation of eosinophils and in the accumulation of these cells at sites of allergic inflammation (Iwamoto, and Takatsu, 1995, Foster, Hogan, Ramsay, Matthaei, and Young, 1996), whereas IL-4 and IL-10 are mast cell growth factors (Smith, and Rennick, 1986, Thompson-Snipes, Dhar, Bond, Mosmann, Moore, and Rennick, 1991). IL-13 is emerging as an important mediator of the respiratory allergic response, with administration of this recombinant cytokine alone sufficient to induce many aspects of the asthmatic phenotype, including airway hyperresponsiveness, mucus hypersecretion and eosinophilia (Grunig et al., 1998, Wills-Karp et al., 1998).
The studies described above, in which it has been shown that chemical contact and respiratory allergens stimulate the preferential activation of type 1 and type 2 T cell subpopulations, respectively, have been performed using mice exclusively (Dearman, and Kimber, 1999, Dearman, and Kimber, 2001, Vandebriel et al., 2000, Hayashi, Higashi, Kato, and Kaneko, 2001). The purpose of the present investigations was to explore the ability of chemical allergens to provoke divergent immune responses in an alternative species, the Brown Norway (BN) rat. To this end, the patterns of cytokine secretion induced in BALB/c strain mice and BN strain rats following topical exposure to the reference chemical respiratory allergen TMA have been analyzed and compared with those stimulated by concurrent exposure to the reference contact allergen DNCB. Expression of the type 2 cytokines IL-4, IL-10 and IL-13 and the type 1 cell product IFN-γ has been examined in both species. The ability of allergen-stimulated LNC to secrete IL-12 has been examined also. This cytokine, a product primarily of various antigen-processing and presenting cells, including dendritic cells (Trinchieri, 1994, Heufler et al., 1996), plays a pivotal role in the development of differentiated type 1 T lymphocytes (Morris et al., 1994, Trinchieri, 1994, Heufler et al., 1996).
Section snippets
Animals
Young adult (6–12 weeks old) female Brown Norway strain rats (Harlan Seralab, Oxon, UK) or adult (8–12 weeks old) female BALB/c strain mice (Animal Breeding Unit, Alderley Park) were used throughout these studies. Rats were randomized on the basis of body weight and then acclimatized for at least 5 days prior to the start of the studies. Animals were housed two to five per cage on flushing metal racks with materials provided for environmental enrichment (paper and cardboard nesting materials
Results
Initial experiments were conducted to identify concentrations of DNCB and TMA which stimulated similar levels of immune activation (for each species) in the absence of local or systemic toxicity. Immunogenicity was assessed as a function of increased cell numbers and proliferative responses induced in the lymph node draining the site of topical treatment (Fig. 1). Topical exposure of BALB/c strain mice to 1% DNCB or to 10% TMA caused significant immune activation, with lymph node cellularity
Discussion
BALB/c strain mice and BN strain rats were exposed topically to concentrations of the reference contact allergen DNCB and the reference respiratory sensitizer TMA of equivalent immunogenicity. Under these exposure conditions, divergent cytokine secretion profiles consistent with the preferential activation of type 1 and type 2 T cell subpopulations were elicited. Thus, in both species, DNCB-activated LNC elaborated relatively high levels of IFN-γ and IL-12, but little of the type 2 cytokines
Acknowledgements
This work was supported in part by grants from the UK Health and Safety Executive (HSE) and the Long-range Research Initiative (LRI) of the European Chemical Industry Council (CEFIC).
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