Alteration of pulmonary immunity to Listeria monocytogenes by diesel exhaust particles . I. Effects of DEPs on early pulmonary responses

To ascertain whether the change in phagocytic activity was due to a direct interaction of AMs with DEPs, an in vitro assay was also performed. Briefly, 5 x [10.sup.5] AMs isolated from normal male Brown-Norway rats were treated with 25-200 [micro]g/mL DEPs for 2 hr or 100 [micro]g/mL DEPs for 1-24 hr in a rocked culture tube in a humidified incubator (37[degrees]C and 5% C[O.sub.2]). We used dimethylsulfoxide as a solvent control. Viability was assessed by trypan blue exclusion assay, and measurement of phagocytosis was carried out as described above.

Determination of cytokines. BAL cells recovered from rats were suspended in the RPMI-1640 medium (Gibco BRL Life Technologies, Gaithersburg, MD, USA) containing 2 mM glutamine, 100 [micro]g/mL streptomycin, 100 U/mL penicillin, 5 x [10.sup.-5] M 2-[beta]-mercaptoethanol, 5 mM HEPES, and 10% heat-inactivated FBS. Aliquots of 1 mL cell suspensions, adjusted to 4 x [10.sup.6] AMs, were added to each well of 24-well tissue culture plates (Costar, Cambridge, MA, USA) and incubated in a humidified incubator (37[degrees]C and 5% C[O.sub.2]) for 1 hr to allow cell attachment to plastic plate. The nonadherent BAL cells were then removed by rinsing the monolayers three times with RPMI medium. These AM-enriched cells were then treated with or without LPS (1 [micro]g/mL; Sigma Chemical Co.) for 24 hr. The AM-conditioned media were collected and centrifuged (1,200 x g for 4 min) and the supernatants were aliquoted and stored at -70[degrees]C until assayed. To ensure that the number of adherent cells was the same in various culture samples, studies were carried out to determine the cellular protein levels after incubation. The adherent cells were treated with 0.5% Triton X-100 at 37[degrees]C for 30 min, and the media were collected and centrifuged. The protein contents in supernates were determined using Sigma Diagnostic reagents and procedures (Sigma Chemical Co.) on a Cobas Fara II analyzer (Roche Diagnostic Systems). The results did not show a significant difference among the samples from various treatment groups (data not shown).

The concentrations of IL-1[beta], IL-12, and TNF-[alpha] in the culture media were quantified by the enzyme-linked immunosorbent assay (ELISA) using commercial ELISA kits (BioSource International, Inc., Camarillo, CA). The level of detection for each cytokine measured using the ELISA kit was 31.2-2,000 pg/mL for IL-1[beta], 7.8-500 pg/mL for IL-12, and 15.6-1,000 pg/mL for TNF-[alpha]. Absorbance was read at 450 nm with a microplate spectrophotometer reader (SpectraMax 250; Molecular Devices Co., Sunnyvale, CA, USA). The concentrations of these cytokines in the first BAL fluid were also quantified to provide an assessment of in vivo cytokine production by AMs.

Statistical analysis. Results are expressed as mean [+ or -] SE of multiple measurements. Statistical analyses were carried out with the JMP IN statistical program (SAS, Inc., Cary, NC, USA). The significance of the interaction among the different treatment groups for the different parameters at each time point was assessed using an analysis of variance (ANOVA). The significance of difference between individual groups was analyzed using the Tukey-Kramer's honestly significant different (HSD) test. For all analyses, the criterion of significance was set at p < 0.05.