Allergy and automobile pollution: experiments in animals

REVUE FRANCAISE D'ALLERGOLOGIE

ET DqMMUNOLOGIE CLINIQUE

Allergy and automobile pollution: experiments in animals T. SANDSTROM, A. BLOMBERG, R. HELLEDAY, B. RUDELL

SUMMARY

RESUME

This review has b e e n d i r e c t e d to d e s c r i b e t h e p r e s e n t state o f e x p e r i m e n t a l a n i m a l studies investigating t h e effects o f a u t o m o b i l e associated air p o l l u t i o n o n a l l e r g e n sensinzation a n d responsiveness. T h e r e is m o u n t i n g e v i d e n c e f r o m a large b o d y o f studies in a n i m a l s i n d i c a t i n g t h a t oxidative, as well as p a r t i c u l a t e air p o l l u t a n t s , m a y e n h a n c e a l l e r g e n responses. O z o n e a n d diesel e x h a u s t are t h e p o l l u t a n t c o m p o n e n t s for w h i c h t h e l i t e r a t u r e o n a n i m a l studies is m o s t conclusive. T h e e x p e r i e n c e s f r o m e x p e r i m e n t a l a n i m a l studies suggest t h e plausibility o f t h e i n f l u e n c e o f a m b i e n t air p o l l u t i o n t o g e t h e r with o t h e r factors, o n airway h e a l t h a n d allergy in h u m a n s .

Pollution a u t o m o b i l e et aUergie: exp6rience sur les animaux. - Cette revue entend d6crire l'6tat actuel des &udes exp6rimentales sur les effets de la pollution a6rienne par l'automobile sur la sensibilisation et la r6activit6 ~tl'allergbne. I1 est de mieux en mieux d6montr6 par un grand nombre d'&udes snr les animaux que les polluants a6riens tant chimiques que particulaires peuvent renforcer les r6ponses aux allerg6nes. L'ozone et les gaz d'6chappement sur les composants de la pollution pour lesquels la litt6rature sur les 6tudes animales est la plus concluante. Les conclusions des &udes exp6rimentales sur les animaux sugg~rent qu'il est vraisemblable que la pollution de Fair ambiant, associ6e ~t d'autres facteurs, influencent aussi chez l'homme les alt6rations allergiques des voies respiratoires.

KEY-WORDS' Air pollution. - Diesel exhaust. - Ozone. Nitrogen diojdde.

MOTS-CLI~S: Pollution adrienne. - Gaz d'6chappement. Ozone. - Dioxyde d'azote.

The increasing prevalence of asthma and allergy has g a i n e d i n c r e a s i n g interest d u r i n g the last decades. I n parallel, the adverse h e a l t h events associated with p o l l u t i o n have c o m e m u c h into focus. E p i d e m i o l o g i c a l a n d e x p e r i m e n t a l h u m a n studies have given f i r m s u p p o r t f o r the hypothesis t h a t a u t o m o b i l e associated air p o l l u t i o n affects l u n g f u n c t i o n a n d s y m p t o m s in individuals with a s t h m a a n d C O P D . T h e s e c i r c u m s t a n c e s have led to explorative studies in a n i m a l m o d e l s as r e g a r d s the toxicology o f air pollutants. An i n t e r e s t i n g

p o i n t in this r e s e a r c h field has b e e n to evaluate the air p o l l u t i o n - allergy i n t e r a c t i o n . T h e r e is m o u n t i n g e v i d e n c e t h a t p o l l u t a n t s such as o z o n e (O3), n i t r o g e n d i o x i d e (NOz), s u l p h u r d i o x i d e (SO2) a n d particulates have adverse effects o n i m m u n e c o m p e t e n t cells a n d airway responsiveness in a n i m a l s as r e c e n t l y reviewed [18]. This review has b e e n d i r e c t e d to u p d a t e the allergy a n d a u t o m o b i l e p o l l u t i o n i n t e r a c t i o n in animals a s s o c i a t e d with two m a j o r p o l l u t a n t s , n a m e l y o z o n e a n d diesel e x h a u s t particulates.

Department of respiratory medicine and allergy, University Hospital of Ume~t, UMEA and .Department of occupational and environmental medicine, LINKOPING (Sweden). Tir6s fi part : Dr T. Sandstr6m, Department of Respiratory Medicine and Allergy, University Hospital of Umefi, S-90185 UME~,. (Sweden).

SANDSTROM 1_., BLOMBERG A., HELLEDAY R, RUDELL B. Allergy and automobile pollutron: experiments rn animals Rev. fr. Allergol., 2000, 40 (1), 47-51.

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• T. S A N D S T R O M E T COLLABORATEURS /

OZONE

Ozone is the strongest oxidant agent in polluted air and is p r o d u c e d during photochemical processes in polluted air. Complementary to the body of h u m a n exposure studies, experimental animal studies have been performed, notably usually with quite high ozone concentrations. This has often been taken as a token of a low relevance to the h u m a n exposure situation, but this may not necessarily be so. It is clear that there are certain species differences in susceptibility. Still, it should be noted that, as described by Hatch et al. at the US EPA, physical characteristics including nose breathing, nasal surface and bronchial branching in small rodents result in need of higher exposure concentrations to deliver the same dose to airway surfaces than in humans. An interesting early Japanese study demonstrated that ozone in concentrations of 5-10 p p m caused an increased sensibilization to ovalbumin with an enhanced systemic anaphylaxis in guineapigs [9]. Later this group showed that pre-exposure to 2 p p m ozone enhanced the anaphylactic reaction after inhalation of an ovalbumin aerosol in sensitized guinea-pigs. Immunoglobulin production represents another and perhaps more relevant aspect of allergen response, as more closely connected to the immunization process. Osebold et al. p e r f o r m e d analyses in lung lavage fluid after 03 exposure in mice. Significantly increased levels of i m m u n o g l o b u l i n e s were demonstrated in airway lavages [12]. This increased the interest in the field, pulling more scientists into exploring allergen sensitization after air pollution exposure. Subsequently, ozone has been found to reduce the antigen threshold also in guinea pigs. Following exposure to 5 p p m ozone, less ovalbumin was n e e d e d to cause sensitization and a greater antibody production was induced, compared to air exposure [11]. Furthermore, Sumitomo et al., showed that exposure to 1 and 3 p p m ozone for two hours caused an increased response to ovalbumin after prior sensitization. Similar results were recently reported by Campos et al. [3]. Guinea pigs sensitized to ovalbumin for three weeks were exposed to 3 p p m ozone for one hour. Apart from the increased neutrophil content in BAL fluid in both sensitized and nonsensitized animals, guinea pigs exposed to ozone increased the i m m u n o b r o n c h o c o n s trictor response to both antigen and histamine inhalation. Lower 03 concentrations have more recently been explored. Osebold et al. intermittently expo-

sed mice to 0.5 and 0.8 p p m ozone during three days and sensitized the animals with aerosolized ovalbumin. By injecting antigen intravenously, it was evident that the ozone-exposed animals experienced far more p r o n o u n c e d systemic anaphylaxis than their air exposed controls. Inhalation of ozone has been reported to increase IgE and IgG containing cells in the lungs also in mice. Gershwin et al. [7] reported that exposure to 0.5 and 0.8 p p m 03 respectively, induced a 34-fold increase in IgE positive cells in mice receiving aerosolized antigen after prior sensitization. The increase in IgE cell counts correlated well to the enhanced anaphylactic sensitivity to intravenous challenge with ovalbumin [1]. Other excitatory effects on immunoglobuline production has been shown by Osebold et al. in a study which was not focused u p o n allergens sensitization. Specific pathogen free mice were exposed to 0.5 and 0.8 p p m 03 and a significant increase in IgA containing cells was shown in the b r o n c h u s associated lymphoid tissue together with increases in IgA, IgG1 and IgG2 concentrations in lung lavage. Interestingly the numbers of IgM and IgG containing cells did not increase [12]. Studies have also been carried out in rats, such as Norwegian brown rats which were sensitized to ovalbumin and then exposed to filtered air, 0.12 or 0.6 p p m ozone for four hours. Subsequently ovalbumin or saline was inhaled. The higher 03concentration in combination with inhalation of ovalbumin significantly increased lung resistance. Similar response was seen with 0.12 p p m ozone, though statistical significance was not reached. Both ozone and ovalbumin caused a bronchoalveolar neutrophilia and the combination caused a close to significant trend towards a synergistic increase [23]. The issue whether this response might be isolated to rodents, was addressed by Yanai et al. The Japanese group consequently exposed mongrel dogs to 3 p p m ozone and investigated its effect on sensitization of ascaris s u u m antigen. It was shown that ozone indeed caused an increased sensitization also in this species. Biagini et al. investigated ozone effects in a platinum asthma primate model. Cynomolgus monkeys were exposed to 1 p p m ozone and sensitized with platinum salt during a three month period. Combined ozone and platinum exposures significantly increased both platinum salt sensitivity and metacholine bronchial hyperreactivity considerably more than either exposure alone. Exposure to ozone also resulted in enhanced incidence of positive skin prick test. Rev. f r AlIergol., 2000, 40, 1

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/ A L L E R G Y AND A UFOMOBILE POLLUTION: EXPERIMENTS I N A N I M A L S •

Table I. - Diesel e x h a u s t a n d allergy responses. Author

Speczes

Allergen

Admmistratmn route

Exposure tzme

Change m endpomt (+_)

Bice et al, 1985 [2]

Rat Mice

SRBC after DE SRBC after DE

intra-tracheal intra-tracheal

6, 12, 18, 24 m o n t h s 6, 12, 18, 24 m o n t h s

Antibody f o r m i n g cells +

M u r a n a k a et al., 1986 [10]

Mice

DEP+ ovalbumin DEP + pollen

peritoneal

1-8 weeks

IgE +

Takafuji et al., 1987 [22]

Mice

DEP + ovalbumin

intra-nasal

1-13 weeks

IgE +

Mice

pyrene + ovalbumin DEP + ovalbumin pyrene + pollen

intra-peritoneal intra-peritoneal intra-peritoneal

1-11 weeks 1-11 weeks 1-11 weeks

IgE + IgE + IgE +

Suzuki et al., 1993 [19] Fujimaki et al., 1994 [4]

Mice

DEP + ovalbumin DEP + pollen

mtra-tracheal mtra-tracheal

1-7 weeks 1-7 weeks

IL-4 + IL-4 +

Fujimaki et al., 1995 [5]

Mice

DEP + ovalbumin

intra-nasal

3-9 weeks

IL-4+, IgE +

Takano et al., 1997 [21]

Mice

DEP+ ovalbumin

intra-tracheal

9 weeks

IL-4+, IL-5+, GM-CSF

Takano et al., 1998 [20]

Mice

DEP+ ovalbumin

intra-tracheal

9 weeks

IL-5+, GM-CSF

DIESEL EXHAUST

The main focus on automobile associated air pollution has gradually drifted from the oxidative gaseous air pollutants towards the particulate matter air pollution. Diesel exhaust being a major source of small, mainly submicron, particles in ambient air has drawn much attention. Major concern has also been given to the fact that diesel powered engines increase rapidly in Europe, and particularly in France. Fuel efficiency, long annual running, and long endurance of the diesel engines mean that they may considerably increase their contribution to ambient PM in the next millenium. There are a n u m b e r of certain characteristics of diesel exhaust particles that are of importance for their reactivity and biological effects. They are extremely small, the majority having a diameter less than 0.5 gm and a substantial amount is ten-hundred times smaller and, hence, easily respirable. The n u m b e r is commonly extremely high compared to the particle concentrations we know from larger inhalable particles (1-5 gm). These small insoluble particles mainly consist of a carbonaceous core with a large surface area relative to the mass, u p o n which many other components are adsorbed. Consequently, effects by diesel exhaust may not only be caused by gases and pure particles, but mainly due to components on the surfaces of the particles that may make them far more aggressive to tissues. Hydrocarbons, acids, aldehydes, and transition Rev. fr. Allergol , 2000, 40, 1

metals are examples of such components. O f major importance is the fact that this large numbers of small particles may penetrate into the airways and deposit easily. Furthermore, the small particles are able to penetrate through the epithelium deep into the airway tissue, and may also penetrate vascular walls and be transported by the blood to distal organs. O f importance for allergic immunization is the fact that these fine particles may, following the deposition in the lung, be translocated to the lung-associated lymph nodes. The lung-associated lymph nodes are important by receiving the lymphatic drainage from the lungs and are sites of proliferation of immune cells and antibody production. The particles are retained for a considerable amount of time in the lung lymph nodes, rather than following the lymph into the major circulation. A summary of animal studies on DEP- allergy interaction is given in Table I. Bice et al. [2] Have been exploring effects of diesel exhaust particles in various situations. This group exposed rats and mice to 350 - 7000 gg diesel exhaust particles/m 3 for 6-24 months. They found that the lung-associated lymph nodes accumulated diesel particles that had been cleared in the lung lymphatics, resulting in an increased cellularity in the lymph nodes. I m m u n e functions were only marginally affected after exposure to low levels of diesel exhaust, but tended to be more elevated in animals exposed to medium or high levels. The authors speculated that particu-

50 late effects on macrophages as well as damage to the airway epithelium together with effects on lymphoid cells could be of importance [8]. In Japan Muranaka et al. [6] showed, in mice immunized with intraperitoneal injection of ovalbumin, that diesel exhaust particulates mixed t o g e t h e r with ovalbumin or pollen antigens caused a higher production of IgE antibodies, compared to immunization with antigens alone [10]. The authors continued by examining how inoculation with diesel exhaust particles intranasally would affect the response to allergen. Mice were immunized intranasally, at three week interval, with ovalbumin alone or mixed with diesel exhaust particles. Even a small dose such as l m g of the particles administered at a 3-week interval caused an increased immunization. The interval between the immunizations did n o t clearly affect the response as also one week interval caused relatively similar effects [22]. More recently, another Japanese group studied BALB/c mice [4]. These mice were instilled intratracheally with diesel exhaust particles mixed with ovalbumin or Japanese cedar pollen three times at a three week interval. After the last instillation the proliferative response and lymphokine-producing activity of mediastinal lymph node cells were examined i n vitro. In mice injected with diesel exhaust particles (DEP) plus ovalbumin the proliferative responses were enhanced 4-17 times compared to the controls. DEP also increased the IL4-producing activity by ovalbumin, and similarly IgE antibody production was higher after the combined exposure. DEP also increased the IL-4 levels following Japanese cedar pollen stimulation. The intratracheal instillation of diesel exhaust particles consequently appeared to affect the IgE responses, probably via local T-cell activation, induced by enhanced IL-4 production. This interesting study, with its mechanistic approach, definitely pointed out directions for an enhanced understanding of the interaction between pollutants and the development of allergic responses [5]. Consequently, the same group continued by evaluating whether similar responses would occur after nasal instillation. DEP was again mixed with ovalbumin and instilled up to three times. Cervical lymph node cells were cultured in vitro and the proliferative response to ovalbumin was significantly higher in animals, which have been instilled with DEP together with ovalbumin, in comparison with ovalbumin alone. Ovalbumin stimulated IL-4 production in the lymph node cells was highest when ovalbumin had been combined with the DEE In contrast, stimulation with oval-

• T. SANDSTROMET COLLABORATEURS /

bumin alone increased interferon- 7 more than if DEP was added. Consequently, the authors concluded that intranasal instillation of the diesel exhaust particles and antigen in mice modulated the in vitro antigen-stimulated cytokine production together with an increased IgE production. Intratracheal DEP instillation has more recently been shown to affect a wider range of asthma and allergy associated components. Epithelial increases in eosinophils, lymphocytes, goblet cells and IgG and IgE were seen together with increased IL-4, IL-5 and GM-CSF mRNA and protein production [20]. Adjuvant activity of pyrene in diesel exhaust on allergen sensitization in mice has only recently b e e n investigated [19]. Pyrene was selected among the chemical c o m p o u n d s in diesel exhaust particles as being of particular interest. The mice were immunized intraperitoneally six times at two week intervals with ovalbumin alone or in combination with pyrene or diesel exhaust particulates. Both pyrene and diesel exhaust particles clearly enhanced the IgE antibody production. Sensitization was also done with Japanese cedar pollen allergen with pyrene and again a significant e n h a n c e m e n t of IgE antibody production compared with allergen alone was shown. Metal content in particles has been hypothesised to have a central role in immune responses to particulates. The metal content is especially rich in residual oil fly ash, but also important in DE PM due to wear and tear in the engine. Gavett et al. sensitized mice intraperitoneally with ovalbumin and p e r f o r m e d challenges two weeks later with ovalbumin aerosol. Six hours after the ovalbumin challenge they were intratracheally instilled with 3 mg residual oil fly ash and ambientair particulate matters from US cities. Ovalbumin and the particulates generated an eosinophil increase in bronchoalveolar lavage fluid. Ovalbumin challenge together with residual oilily ash, was associated with a synergistic increase in eosinophil numbers. Ambient particulates from major US cities also caused synergistic effects together with ovalbumin, even though less pronounced. In contrast protein, LDH, glucosaminidase and albumin, as markers of lung injury, were not potentiated by the allergen challenge. Air reactivity was more p r o n o u n c e d after exposure to particulates and fly ash than following corresponding saline challenge [6]. Data on metals and immunotoxicological effects from residual oil fly ash have become available following intense activity at the US EPA. Data are likely to be soon available in the biomedical literature also for diesel exhaust. Rev. fr. Allergol.,

2000,40, 1

/ ALLERGY AND AUTOMOBILE POLLUTION: EXPERIMENTS IN ANIMALS •

CONCLUSIONS

A large n u m b e r of studies that have suggested that oxidative air pollutants as well as particulate pollutants such as diesel exhaust may enhance the sensibilization as well as respiratory and systemic responses to allergen exposures. This has found support from experimental studies in humans and is likely to gain an increased attention and

51

appreciation. The contribution of automobile exhaust pollution in general, and from diesel engines in particular, to adverse health effects is in major focus in the c u r r e n t E u r o p e a n Commission Framework 5 activity. The increasing research activity in the immediate coming years will lay the foundation for the EC and the national regulatory bodies to develop new strategies to reduce adverse health effects from air pollution.

REFERENCES 1. Biagini R.E., Moorman w.J., Lewis T.R., Bernstein I.L - Ozone enhancement of platinum asthma m a primate model. Am. Rev. Respzr Dzs, 1986, 134, 719-725. 2. Bice D.E., MauderlyJ.L., Jones R.K., McClellan R O. - Effects of inhaled diesel exhaust on immune responses after lung immunization. F'undam. Appl Toxzcol., 1985, 1, 1075-1086. 3. Campos M.G., Segura E, Vargas M.H., Vanda B., Ponce Monter H., Selman M., Montano L.M. - O3-mduced airway hyperresponsiveness to noncholinergic system and other tumuli. J. Appl Physiol., 1992, 73, 354-361. 4. Fujimaki H., Nohara O., Ichmose T., Watanabe N., Salto S. - IL-4 productmn in medmstmal lymph node cells in mice mtratracheally instilled with dmsel exhaust particulates and anUgen. Toxicology, 1994, 92, 261-268. 5. Fujimakl H., Saneyoshi K., Nohara O., Shiraishi E, Imai T. Intranasal instillation of diesel exhaust particulates and antigen in mice modulated cytokine productions in cervical lymph node cells Int. Arch. Allergy Imrnunol., 1995, 108, 268-273. 6. Gavett S.H., Madison S.L., Costa" D.L. - Allergen challenge amplifies pulmonary responses to residual oil fly ash and ambient mr particulate matter samples in BALB/CJ mice. Am.J Resp Cnt. Care Med, 1996, 153, A15. 7. Gershwin LJ., Osebold J.W, Zee xLC. - Immunoglobulin E-containing cells in mouse lung following allergen inhalation and ozone exposure. Int Arch. Allergy AppL Immunol., 1981, 65, 266-277. 8. Hdlam R.R, Blce D.E.. Hahn EE, Schnizlein C.T. - Effects of acute mtrogen dmxide exposure on cellular immunity after lung Immunization. Enwron Res., 1983, 31, 201-211. 9. Matsumura Y - T h e effects of ozone, mtrogen dioxide, and sulfur dioxide on the expernnentally induced allergic respiratory disorder in guinea pNs. 3. The effect on the occurrence of dyspneic attacks. Am. Rev Resp~r. Dzs., 1970. 102, 444-447. 10. Muranaka M., Suzuki S., I(mzmnl K., Takafuji S., Miyamoto T., Ikemon R., Tokiwa H. - Adjuvant activity of diesel-exhaust particulates for the production of IgE antibody in mice. J. Allergy Chn [mmunol., 1986, 77, 616-623. 11. Osebold J.W., Gershwm L.J., Zee Y.C. - Studies on the enhancement of allergic lung sensitization by inhalation of ozone and sulfuric acid aerosol. J Environ Pathol. Tox~col., 1980, 3, 221-234.

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12. OseboldJ W, Owens S.L., Zee Y.C., Dotson W M, LaBarre D D. Immunological alterations in the lungs of mice following ozone exposure' changes m nnmunoglobulin levels and antibodycontaining cells. Arch. Envzron Health, 1979, 34, 258-265. 13. OseboldJ.W., Zee Y.C., Gershwin LJ, - Enhancement of allergic lung senmization m mice by ozone inhalation Proc Soc. Exp Bzol Meal., 1988, 188, 259-264. 14. Rudell B., Sandstr6m T., Stjernberg N, Kolmodm-Hedman B. Controlled diesel exhaust exposure in an exposure chamber. pulmonary effects investigated with bronchoalveolar lavage. J. Aerosol Science, 1990, 21, 411-414. 15. Rudell B., Sandstrom T, Hammarstr6m U., Ledm M.L., Horstedt P, Stjernberg N. - Evaluation of an exposure setup for studying effects of diesel exhaust in human. Int. Arch Occap. Enmron Health, 1994, 66, 77-83. 16. Rudell B., Ledm M.C., Stjernberg N., Lundb/ick B., Sandstrom T Effects on symptoms and lung function in humans experimentally exposed to diesel exhaust. Oceup. Enmron Med., 1996, 53, 658662. 17. Sandstr°m T' - Respirat°ry effects ° f air p°llutants" Experimental studies in humans. Eur. Resp~:J., 1995, 8, 976-995. 18. Sandstrom T., Blomberg A., Helleday R., Rudell B. - A i r pollunon - allergy interacUon' experiences from animal studies. Eur Respir. Rev., 1998, 8, 168-173 19. Suzuki T., Kanoh T., Kanbayashi M., Todome Y., Ohkuni H. The adjuvant activity of pyrene an diesel exhaust on IgE antibody producuon in mice. Arerug~, 1993, 42, 963-968. 20. Takano H., Ichmose T., Mlyabara Y., Yoshlkawa T., Sagai M Diesel exhaust partacles enhance airway responsiveness following allergen exposure m mice. Immunopharm Immunotoxzcol, 1998, 20, 329-336. 21. Takano H., Yoshikawa T, Ichmose T., Mlyabara Y. Imaoka I~, Saga1 M - Diesel exhaust particles enhance antigen-induced airway inflammation and local cytokine expresslon in mice. Am J. Respw. Crit. Care Med., 1997, 156, 36-42. 22. Takafuji S., Suzuki S., Koizumi K., Tadokoro K., Mlyamoto T., Ikemori R., Muranaka M. - Diesel-exhaust particulates inoculated by the intranasal route have an adjuvant activity for IgE production in mice.J. Allergy Chn Immunol., 1987, 79, 639-645 23. Yanai M., Ohrui T, Aikawa T., Okayama H , Sekizawa K., Maeyama K., Sasaki H., Takishlma T. - Ozone increases susceptibility to antigen inhalation in allergic dogs. J Appl Physml., 1990, 68, 2267-2273. -