In this study population, ambient concentrations of PAHs and PM2.5 during the last 2 weeks of gestation were associated with decreases in the percentages of T-lymphocytes in cord blood. These associations were stronger for the percentage of CD4+ cells than for the percentage of CD8+ subsets. However, partly because CD4+ are relatively more numerous, the resulting effect of air pollution on the CD4+:CD8+ ratio was essentially null. The association with CD8+ cells was more marked for PAHs, compared with PM2.5. Accompanying these T-cell decreases, the percentage of B-cells (CD19+) increased. Because these outcomes represent distributions of immune phenotype subsets relative to each other, if the percentage of one subset goes down, the percentage of at least one other will increase. It would have been informative to measure absolute lymphocyte counts and functional immune parameters; however, because the quality of these measurements degrades with increasing storage time and transportation, such measurements were deemed not feasible in this project.
The possibility that redistributions of lymphocyte phenotypes may alter susceptibility to infections or inflammatory diseases in otherwise healthy persons, particularly small children, is worthy of investigation in its own right. The longitudinal life-course changes in lymphocyte populations, both absolute and relative, are discussed by Schultz et al. (2000)
, who concluded that the developmental patterns in absolute counts were different from those in relative distributions, and that “discordance between the absolute and relative size of lymphocyte subpopulations emphasizes the consideration of both variables in the assessment of lymphocyte maturation.” Published data suggest that the percentages of many of the immunophenotypic subpopulations differ for cord versus adult blood (Zhao et al. 2002
) but that differences in absolute counts are not always accompanied by differences in percentages (D’Arena et al. 1998
). Percentages of some lymphocyte subtypes change from the fetal to neonatal period (Zhao et al. 2002
). Given that immunologic development is intimately connected to the interactions between the organism and the environment via antigenic challenge, specific chemical exposures could influence these relationships (e.g., exposure to diesel particles can modify the host response to allergen) (Diaz-Sanchez et al. 2003
). Whether alterations in developmental patterns of lymphocytes have clinical implications remains to be established. To shed light on the larger picture, we are addressing, in other work, immunoglobulin production of the neonate and relationships between relative lymphocyte distributions at birth and subsequent early childhood morbidity.
The direction of associations was generally unchanged, whether adjusted for short-term (3 days) or longer-term (45 days) average temperature, season, or both season and short-term temperature, although the precision and magnitude varied. The associations with CD19+ were the most consistent across pollutant metrics ().
Few studies have examined the relationship of air pollution to pediatric immune status. Leonardi et al. (2000)
examined absolute levels of specific lymphocytes in schoolchildren, whereas we assessed percentages of lymphocytes in newborns; hence, our results cannot be compared. Similarly, a cross-sectional study of schoolchildren in two industrial cities suggested a relationship of air pollution with changes in several immune cell fractions, but this report was based on ecologic, not individual-level, data and did not control for any confounders (Skachkova et al. 2001
). Overall, the proportions of T-, B-, and NK lymphocytes in cord blood in our study were not markedly different from those reported in a small Italian study (D’Arena et al. 1998
) and a large Mexican sample (Garcia et al. 1995
). Different laboratory methods and our inclusion of a population-based sample, with no exclusions, can explain any discrepancies.
Consistent with previous findings in this project (Hertz-Picciotto et al. 2002
) and other studies, the length of labor, delivery medication, number of previous pregnancies, maternal education, and time of day of delivery (Levi et al. 1988
) were all related to lymphocyte outcomes at birth. Maternal chronic or severe respiratory diseases during pregnancy did not predict lymphocyte distributions at birth, possibly because these self-reports did not include information on the period in gestation during which they occurred. Although our previous findings using district of residence as a surrogate for chronic exposure might have been confounded by unrecognized interdistrict differences (Hertz-Picciotto et al. 2002
), this problem was eliminated by analysis of short-term temporal variations in PM2.5
and PAHs before birth, in which district and other confounders were controlled. The PM2.5
fraction was selected because it generally displays less spatial heterogeneity than do coarse particles. With only one monitor in each district, fine particles would be subject to less misclassification error. Nevertheless, some error is unavoidable, especially for families living at some distance from the monitors.
Participants in the immune biomarker study were randomly sampled as described above, and the refusal rate was < 5%, supporting the validity of the results based on this sample. Although the distributions of several variables were significantly different in the study sample compared with the full cohort (e.g., the proportion of low-birth-weight infants was 7% in the study sample and 5% in the full cohort), these differences are not likely to have affected either the internal or external validity of the results, because the variables were controlled in the analysis through adjustment for the sampling design or by multivariate adjustment or both. These adjustments combined with the use of sampling weights also provided generalizability to the population that participated in the full cohort study—about 90% of the population in the two districts. Because 14 days before birth could represent different developmental periods for low-birth-weight and preterm newborns, we also conducted a sensitivity analysis in which these infants were excluded and found little change in the results (data not shown).
We furthermore believe our results could be generalized outside the Czech Republic, given that the exposures are similar to those in other countries. Concentrations of PAHs in this investigation were in a range similar to what has been reported from some U.S., European, and Asian cities. For instance, for BaP, our geometric mean was 1.4 ng/m3, which was comparable to levels observed in Pavia (Italy) in 1996 and in Taipei in 1995 and 1996, 1.2 and 1.7 ng/m3, respectively (Naumova et al. 2002
In the Central European Study of Air Quality and Respiratory Health (CESAR, conducted in Bulgaria, Hungary, Czech Republic, Slovak Republic, Poland, and Romania) annual PM2.5
means were between 29 and 67 μg/m3 (Leonardi et al. 2000
). Although the annual average concentration in our study was lower (25 μg/m3), the winter mean was 38 μg/m3, which is higher than the winter means of 18 of the 21 cities analyzed in the European Community Respiratory Health Survey study (Hazenkamp-von Arx et al. 2003
). Most U.S. cities are reported to have lower annual PM2.5
means, although comparable means occurred in Los Angeles (25 μg/m3) and Riverside (29 μg/m3), California, in 2000 (Pinto et al. 2004
The less than daily monitoring during 7 months of the year made it necessary to impute pollution data. Given the schedule of every third day of measurement for 5 months of the year, and every sixth day for 2 months, the probability that missing data are related to levels of PM2.5 or PAH, after conditioning on daily measurements for SO2 and NOx and on the existing data for PM10, PM2.5, and PAH, appears to be extremely small. Thus, the rationale for the assumption of “missing-at-random” is strong.
Confounding due to meteorology and seasonal lymphocyte patterns was accounted for by including 3- or 45-day averages of temperature or season, or both season and temperature in the model. Short-term and longer term time spans of antenatal ambient temperatures were related to a similar extent to T-lymphocyte fractions. Except for CD19+, inclusion of either temperature interval (3- or 45-day average before birth) resulted in less precise estimates of the pollution effects. Season alone does not appear to have this effect; winter by itself does not have any impact once temperature is included. Possibly, our adjustment for temperature could induce bias, because it is highly correlated () with air pollution, particularly within season; however, the direction of such bias is difficult to deduce.
The B-lymphocyte fraction was not related to ambient temperature but was related to both spring and fall seasons. This suggests that the seasonal associations must be due to other variables, perhaps respiratory infections during fall or pollen in spring. Seasonal variations in immunologic parameters occur in healthy children (Afoke et al. 1993
) and are hypothesized to represent adaptive responses to climatic variability and other environmental factors.
We also found that maternal active smoking and/or exposure to second-hand smoke predicted cord blood lymphocyte distributions in preterm low-birth-weight newborns (decreased T-lymphocytes and increased B-lymphocytes). We did not find published results on lymphocyte phenotype fractions in cord blood in relation to cigarette smoking. Studies in adults consistently suggest a rise in either the percentage or absolute count of CD3+ or CD4+ cells in association with cigarette smoke exposure (Santagostino et al. 1999
; Schaberg et al. 1997
; Tollerud et al. 1989
). One of these studies also reports a higher B-cell fraction related to smoking (Santagostino et al. 1999
), which is concordant with our finding.
The pattern in relation to cigarette smoke in our data was similar to what we observed for lymphocyte distributions in relation to ambient air pollution, that is, PAHs. Whether this similarity is due to PAHs or other constituents common to tobacco smoke and ambient air pollution is unclear. Furthermore, lymphocyte changes associated with ambient PAHs in all but the B-cell fractions were greater in births where the mother or others around her smoked (). This association of PAHs beyond the effect of smoke exposure on lymphocyte phenotype fractions is consistent with an impact of these aromatic compounds over a wide range of exposure levels.
The subgroup of children most susceptible to lymphocyte effects from air pollution appears to be those from homes heated by coal. This may be a result of high exposures to PAHs from this source.
Distinct from our earlier work suggesting that chronic, long-term exposure to high ambient air pollution may influence immune development (Hertz-Picciott et al. 2002
), these results demonstrate short-term associations with fine particles and PAHs. Adjustment for temperature does not eliminate these associations. A comparison of the associations with PAHs and with PM2.5
indicates a number of stronger findings for the former. Reductions in CD3+ and CD4+ fractions were larger for PAHs, and no association was observed between PM2.5
and CD8+ fractions. Because the increments used for PAHs and PM2.5
represented approximately a change of two standard deviations for each, these results can be compared.
Our measurements included both the semivolatile and particulate-bound fractions of PAHs. Given this detailed characterization of PAHs and the associations observed here, chemical composition may play a key role in the immune-mediated effects of air pollution. Others have demonstrated that fetal exposures to ambient PAHs occur trans-placentally and can result in mutations in cord blood lymphocytes (Perera et al. 2002
). Moreover, PAHs have been demonstrated to exhibit immunomodulatory properties (Nel et al. 2001
The striking finding of this study is that exposure to ambient PM2.5 and PAHs in late pregnancy is associated with statistically significant changes in the distributions of lymphocyte phenotypes in cord blood. Although the biologic relevance of this finding is not entirely clear, the observation of note is that the fetal immune system may be altered by maternal exposure to these environmental pollutants. Further research on critical windows of vulnerability throughout gestation is warranted, as well as study of whether such changes persist beyond birth and/or are associated with adverse health effects. We have recently completed a follow-up study of these children, which will give the opportunity to relate the changes observed in T-cell and B-cell fractions at birth with subsequent morbidity.