Ambient ultrafine particles provide a strong adjuvant effect in the secondary immune response: implication for traffic-related asthma flares

Abstract

We have previously demonstrated that intranasal administration of ambient ultrafine particles (UFP) acts as an adjuvant for primary allergic sensitization to ovalbumin (OVA) in Balb/c mice. It is important to find out whether inhaled UFP exert the same effect on the secondary immune response as a way of explaining asthma flares in already-sensitized individuals due to traffic exposure near a freeway. The objective of this study is to determine whether inhalation exposure to ambient UFP near an urban freeway could enhance the secondary immune response to OVA in already-sensitized mice. Prior OVA-sensitized animals were exposed to concentrated ambient UFP at the time of secondary OVA challenge in our mobile animal laboratory in Los Angeles. OVA-specific antibody production, airway morphometry, allergic airway inflammation, cytokine gene expression, and oxidative stress marker were assessed. As few as five ambient UFP exposures were sufficient to promote the OVA recall immune response, including generating allergic airway inflammation in smaller and more distal airways compared with the adjuvant effect of intranasally instilled UFP on the primary immune response. The secondary immune response was characterized by the T helper 2 and IL-17 cytokine gene expression in the lung. In summary, our results demonstrated that inhalation of prooxidative ambient UFP could effectively boost the secondary immune response to an experimental allergen, indicating that vehicular traffic exposure could exacerbate allergic inflammation in already-sensitized subjects.

Fig. 1.

Allergic sensitization, ovalbumin (OVA) challenge, and ultrafine particles (UFP) inhalation protocol. Animals were intranasally sensitized with saline, OVA, or OVA + UFP followed by inhalation exposure to filtered air (FA) or concentrated ambient UFP in the presence of 1% aerosolized OVA. Necropsy (Nec) was conducted on day 26.

Fig. 2.

Inhalation of UFP during OVA challenge enhanced eosinophilic airway inflammation and OVA-specific antibody production in OVA/UFP-sensitized mice compared with those in the same sensitization group receiving FA instead of UFP. A: bronchoalveolar lavage (BAL) eosinophil count; *P < 0.05 compared with saline and OVA sensitization groups; **P < 0.05 compared with OVA/FA challenge from the corresponding sensitization group. B: OVA-IgE; *P < 0.05 compared with saline and OVA sensitization groups; **P < 0.05 compared with OVA/FA challenge from the corresponding sensitization group. C: OVA-IgG1; *P < 0.05 compared with saline and OVA sensitization groups. Challenge with OVA/FA or OVA/UFP led to similar level of increase in BAL lymphocyte count in OVA/UFP-sensitized animals, but had no effect on monocyte count (data not shown).

Fig. 3.

RT-PCR showing that ambient UFP exposure during OVA challenge enhanced Th2 and Muc5ac gene expression. A: eotaxin; B: IL-5; C: IL-13; D: Muc5ac. *P < 0.05 compared with respective saline sensitization; **P < 0.05 compared with respective OVA sensitization; #P < 0.05 compared with respective OVA/FA.

Fig. 4.

Inhalation of ambient UFP during OVA challenge enhanced IL-17a gene expression and neutrophil influx in the lungs of OVA/UFP-sensitized mice. A: RT-PCR analysis of IL-17a expression in the lung. B: BAL neutrophil count. *P < 0.05 compared with respective saline sensitization; **P < 0.05 compared with respective OVA sensitization; #P < 0.05 compared with respective OVA/FA.

Fig. 5.

Schematic highlighting of the intrapulmonary impact sites during UFP inhalation. Arrows indicate the potential target site of UFP during the secondary immune response with alveolar duct and alveolus being the major targets. OVA/UFP/CAP only: OVA/UFP sensitization followed by challenge with OVA/ultrafine concentrated ambient particle inhalation.

Fig. 6.

Immunohistochemical staining of eosinophilic major basic protein (MBP) demonstrating that OVA/UFP challenge induced more severe allergic inflammation in the alveolar ducts (ad) and alveoli (a) than in the proximal axial airway (aa; generation 5) in OVA/UFP-sensitized mice. A and B: OVA sensitization followed by OVA/FA challenge. C and D: OVA sensitization followed by OVA/UFP challenge. E and F: OVA/UFP sensitization followed by OVA/FA challenge. G and H: OVA/UFP sensitization followed by OVA/UFP challenge. Respiratory epithelium lining axial airway, ep; pulmonary artery, pa; blood vessel, bv. Arrows indicate positive staining of MBP in eosinophils.

Fig. 7.

OVA/UFP-enhanced Ym1 expression in the lung. A: immunohistochemistry showing Ym1 expression in alveolar macrophages/giant cells in centriacini, along with inflammation in preterminal (ptb), terminal bronchioles (tb), and alveolar ducts (ad). Arrows indicate positive staining of Ym1 protein. 1: OVA sensitization followed by OVA/FA challenge. 2: OVA/UFP sensitization followed by OVA/FA challenge. 3: OVA sensitization followed by OVA/UFP challenge. 4: OVA/UFP sensitization followed by OVA/UFP challenge. Pulmonary arteries, pa; asterisk, peribronchiolar inflammation. B: Ym1 gene expression in the lung. *P < 0.05 compared with respective saline sensitization; **P < 0.05 compared with respective OVA sensitization; #P < 0.05 compared with respective OVA/FA.