Diesel exhaust particles stimulate human airway epithelial cells to produce cytokines relevant to airway inflammation in vitro Takayuki Ohtoshi, MDa,

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Diesel exhaust particles stimulate human airway epithelial cells to produce cytokines relevant to airway inflammation in vitro Takayuki Ohtoshi, MDa, Hajime Takizawa, MD, PhDa, Hitoshi Okazaki, MDa, Shin Kawasaki, MDa, Naonobu Takeuchi, MDb, Ken Ohta, MD, PhDc, Koji Ito, MD, PhDa Journal of Allergy and Clinical Immunology Volume 101, Issue 6, Pages 778-785 (June 1998) DOI: 10.1016/S0091-6749(98)70307-0 Copyright © 1998 Mosby, Inc. Terms and Conditions

Fig. 1 Effect of SPM on release of GM-CSF by human primary nasal epithelial cells in vitro. Human primary nasal epithelial cells were isolated from resected nasal polyps and cultured in hormonally defined Ham' F12 medium until confluence. A, Cells were treated with and without 250 μg/ml SPM. Supernatants were collected after different time periods, and concentration of immunoreactive GM-CSF was evaluated. There was a time-dependent increase in GM-CSF levels with SPM (250 μg/ml) compared with that of untreated cells. Results were representative among three different experiments. B, Different doses of SPM were added to cells, and concentration of GM-CSF was measured after 24 hours. SPM at doses of 250 and 2500 μg/ml showed a significant stimulatory effect on GM-CSF production. *p < 0.01, n = 3 (ANOVA). Journal of Allergy and Clinical Immunology 1998 101, 778-785DOI: (10.1016/S0091-6749(98)70307-0) Copyright © 1998 Mosby, Inc. Terms and Conditions

Fig. 2 Effect of SPM on IL-8 by human primary nasal epithelial cells in vitro. Cells were treated with different doses of SPM, and release of IL-8 into media was evaluated after 24 hours. SPM did not show statistically significant effect on IL-8 release. Journal of Allergy and Clinical Immunology 1998 101, 778-785DOI: (10.1016/S0091-6749(98)70307-0) Copyright © 1998 Mosby, Inc. Terms and Conditions

Fig. 3 Effect of DEPs on production of GM-CSF by human airway epithelial cells. A, On confluence, the primary nasal epithelial cells were treated with DEPs (10 and 50 μg/ml), and supernatants were harvested after different time periods. DEPs showed a time-dependent stimulatory effect of GM-CSF production. Results were representative among three different experiments. B, Dose-dependency of DEPs on GM-CSF release from nasal epithelial cells. DEPs (10 to 50 μg/ml) showed a significant stimulatory effect on GM-CSF release after 24 and 48 hours. *p < 0.01, n = 3 (ANOVA). C, DEPs showed a stimulatory effect on GM-CSF release from primary bronchial epithelial cells after 24 hours. *p < 0.01, n = 3 (ANOVA). Addition of protein synthesis inhibitor cycloheximide (CHX) (10 μg/ml) blocked increase in GM-CSF levels stimulated by DEPs, showing a de novo protein synthesis process as in B and C. Journal of Allergy and Clinical Immunology 1998 101, 778-785DOI: (10.1016/S0091-6749(98)70307-0) Copyright © 1998 Mosby, Inc. Terms and Conditions

Fig. 4 Effect of DEPs on IL-8 release by human airway epithelial cells. A, DEPs showed stimulatory effect on IL-8 release by primary nasal epithelial cells in dose-dependent fashion after 24 and 48 hours. *p < 0.01, n = 3 (ANOVA). B, DEPs also showed stimulation on BEAS-2B cells after 24 hours. Addition of protein synthesis inhibitor cycloheximide (CHX) (10 μg/ml) blocked the increase in IL-8 levels stimulated by DEPs, showing de novo protein synthesis process. C, Effect of charcoal and graphite on the IL-8 release from BEAS-2B cells. Neither charcoal nor graphite showed significant effect on IL-8 release (24 hours) at noncytotoxic concentrations (10 and 50 μg/ml). Journal of Allergy and Clinical Immunology 1998 101, 778-785DOI: (10.1016/S0091-6749(98)70307-0) Copyright © 1998 Mosby, Inc. Terms and Conditions

Fig. 5 Effect of benzo(a)pyrene on GM-CSF release from BEAS-2B cells. Prototype aromatic hydrocarbon benzo(a)pyrene had significant stimulatory effect on GM-CSF release from bronchial epithelial cells in dose-dependent fashion after 24 hours in vitro. *p < 0.01 compared with controls, n = 3 (ANOVA). Journal of Allergy and Clinical Immunology 1998 101, 778-785DOI: (10.1016/S0091-6749(98)70307-0) Copyright © 1998 Mosby, Inc. Terms and Conditions

Fig. 6 Directional characteristics of IL-8 release by human bronchial epithelial cell line BEAS-2B on stimulus of IL-1β and DEPs. Transformed human bronchial epithelial BEAS-2B cells were plated on upper side of double-chamber plate and cultured until confluence. A, Human recombinant IL-1β (5 ng/ml), a potent stimulatory agent on cytokine production by epithelial cells, showed an upregulation of IL-8 production to both sides of chambers when it was added to upper and lower chambers. *p < 0.01 to control of upper-side release, **p < 0.01 to control of lower-side release. B, BEAS-2B cells were cultured, and DEPs (10 μg/ml) were added to either side of plate. DEPs showed significant stimulatory effect only when added to upper side of plate. *p < 0.01 to control of upper-side release, **p < 0.01 to control of lower-side release. Journal of Allergy and Clinical Immunology 1998 101, 778-785DOI: (10.1016/S0091-6749(98)70307-0) Copyright © 1998 Mosby, Inc. Terms and Conditions