Ozone and allergen exposure during postnatal development alters the frequency and airway distribution of CD25+ cells in infant rhesus monkeys

Abstract

The epidemiologic link between air pollutant exposure and asthma has been supported by experimental findings, but the mechanisms are not understood. In this study, we evaluated the impact of combined ozone and house dust mite (HDM) exposure on the immunophenotype of peripheral blood and airway lymphocytes from rhesus macaque monkeys during the postnatal period of development. Starting at 30 days of age, monkeys were exposed to 11 cycles of filtered air, ozone, HDM aerosol, or ozone+HDM aerosol. Each cycle consisted of ozone delivered at 0.5 ppm for 5 days (8 h/day), followed by 9 days of filtered air; animals received HDM aerosol during the last 3 days of each ozone exposure period. Between 2-3 months of age, animals co-exposed to ozone+HDM exhibited a decline in total circulating leukocyte numbers and increased total circulating lymphocyte frequency. At 3 months of age, blood CD4+/CD25+ lymphocytes were increased with ozone+HDM. At 6 months of age, CD4+/CD25+ and CD8+/CD25+ lymphocyte populations increased in both blood and lavage of ozone+HDM animals. Overall volume of CD25+ cells within airway mucosa increased with HDM exposure. Ozone did not have an additive effect on volume of mucosal CD25+ cells in HDM-exposed animals, but did alter the anatomical distribution of this cell type throughout the proximal and distal airways. We conclude that a window of postnatal development is sensitive to air pollutant and allergen exposure, resulting in immunomodulation of peripheral blood and airway lymphocyte frequency and trafficking.

Figure 1. Experimental timeline for ozone and allergen exposure during postnatal development

Infant rhesus monkeys were sensitized to HDM via subcutaneous (SQ) injection with adjuvant at day 14 and day 28. Starting at 30 days of age, monkeys were exposed to 11 cycles of ozone and/or HDM aerosol. Each cycle consisted of ozone exposure for 5 days, followed by 9 days of filtered air (0.5 ppm at 8h/day). HDM aerosol was delivered during the last 3 days of the ozone exposure period. Lavage and tissue specimens were collected at 175 days of age.

Figure 2. Changes in peripheral blood cell counts and lymphocyte frequency as a function of monkey age (months)

Peripheral blood samples were collected from monkeys on a bi-monthly basis, starting at 2 weeks of age. Blood samples for each time point were collected on the fifth day of each cycle, immediately following the last ozone and/or allergen exposure. Each time point represents the mean ± SE values for 5–6 animals. A, Total WBC absolute counts from filtered air control (FA) animals compared with ozone (top graph), HDM (middle graph), and Ozone + HDM (bottom graph). B, Peripheral blood lymphocyte frequency from filtered air control (FA) animals compared with ozone (top graph), HDM (middle graph), and ozone + HDM (bottom graph). Significant effects versus filtered air are indicated by *p<0.05 and **p<0.01, as determined by Bonferonni post tests.

Figure 2. Changes in peripheral blood cell counts and lymphocyte frequency as a function of monkey age (months)

Peripheral blood samples were collected from monkeys on a bi-monthly basis, starting at 2 weeks of age. Blood samples for each time point were collected on the fifth day of each cycle, immediately following the last ozone and/or allergen exposure. Each time point represents the mean ± SE values for 5–6 animals. A, Total WBC absolute counts from filtered air control (FA) animals compared with ozone (top graph), HDM (middle graph), and Ozone + HDM (bottom graph). B, Peripheral blood lymphocyte frequency from filtered air control (FA) animals compared with ozone (top graph), HDM (middle graph), and ozone + HDM (bottom graph). Significant effects versus filtered air are indicated by *p<0.05 and **p<0.01, as determined by Bonferonni post tests.

Figure 3. Time course of CD25 expression on lymphocyte populations in peripheral blood and lavage during postnatal development

Peripheral blood mononuclear cells were evaluated by flow cytometry at approximately 3 months (cycle 5), 4.5 months (cycle 8), and 6 months (necropsy) of age. Lavage cells were evaluated at necropsy only. Each column represents the mean ± SE of values obtained from 3 animals treated with filtered air, ozone, HDM, or ozone + HDM. A, Frequency of CD4+/CD25+ T lymphocytes B, Frequency of CD8+/CD25+ T lymphocytes. Significant effects are indicated by *p<0.05 vs. filtered air; +p<0.05 vs. ozone; #<p<0.05 vs. HDM; **p<0.01 vs. filtered air; ++p<0.01 vs. ozone; ##p<0.001 vs. HDM, as determined by Bonferroni post tests.

Figure 4. Effect of ozone and allergen exposure on distribution of CD4+ cells within airway mucosa of 6 month old monkeys

CD4+ cells were quantitated by immunofluorescence staining of histological sections obtained from trachea and left caudal lobes. Columns represent the average volume ± SE of fluorescence positive staining cells (mm³) from 6 animals treated with filtered air, ozone, HDM or ozone + HDM. Volume of fluorescence positive cells were measured within the epithelial or interstitial compartment with respect to surface area of basal lamina (mm²). Tr, 1, 3, 5, 7 represent cryosections obtained from numbered tissue blocks of trachea and regions from the most proximal (1) to distal (7) intrapulmonary airways of the left caudal lobe. A, Overall volume (sum of five airway generations) of CD4+ cells within the epithelial and interstital compartment of conducting airways. B, Volume of CD4+ cells within the epithelial and interstitial compartment of five airway generations.

Figure 5. Effect of ozone and allergen exposure on distribution of CD25+ cells within airway mucosa of 6 month old monkeys

CD25+ cells were quantitated by immunofluorescence staining of histological sections obtained from trachea and left caudal lobes, as described for Fig 4. Columns represent the average volume ± SE of fluorescence positive staining cells (mm³) from 6 animals treated with filtered air, ozone, HDM or ozone + HDM. A, Overall volume of CD25+ cells (sum of five airway generations) within the epithelial and interstitial compartment of conducting airways. B, Volume of CD25+ cells within the epithelial and interstitial compartment of five airway generations. nd=none detected. Significant effects are indicated by *p<0.05 vs. HDM; +p<0.01 vs. filtered air; #p<0.001 vs. filtered air, as determined by Bonferroni post tests.

Figure 6. Double immunofluorescence staining for CD4+ and CD25+ cells in airway mucosa

Cryosections from a midlevel airway of a representative HDM-exposed infant monkey were double immunostained for CD4 (A, green fluorescence) and CD25 (B, red fluorescence). Overlap of green and red fluorescence shows two CD4+ cells that are not CD25 positive (C, arrows). The dotted line separates the epithelial and interstitial compartments. Scale bar = 20 μm.