Over the last decade, interest has increased in the neonatal immune system as a model for understanding the role of early immune responses in the development of atopic disease (17
). This study demonstrates that CBMCs of neonates with an increased proliferative response to one allergen also manifest a higher proliferative response to another allergen, as well as to mitogen. Increased proliferation of CBMC in response to stimulation with dust mite Derf1 is associated with increased Derf1-induced secretion of IL-13. In contrast to the paradigm of an increase in Th2 cytokines (IL-13) and a decrease in Th1 (IFN-γ) cytokines shown in some models of allergic diseases, Derf1-induced secretion of IFN-γ is not reduced in this cohorte. Furthermore, an increased proliferative response to the mitogen PHA is associated with increased secretion of IL-13 in response to PHA, but not with reduced secretion of IFN-γ.
This study also investigated whether these immune phenotypes might be differentially expressed at birth. Evidence that some aspects of allergic disposition are present at birth has arisen from several studies (5
). Lymphocytes from cord blood are capable of recognizing and proliferating in response to certain allergens such as milk protein and aeroallergens (17
). Proliferative responses to house dust mite allergen, birch pollen, and rye grass (5
) have been observed in mononuclear cells collected at birth and found already to be present at 22 weeks gestation (25
). The finding in our study of T cell reactivity in response to several indoor allergens and to mitogen evokes several hypotheses. The ability to demonstrate proliferative responses to allergen at birth (22
) suggests the potential occurrence of prior intrauterine exposure to allergen leading to the generation of immunological memory. Whether the responses detected by in vitro
stimulation of CBMC reflect in vivo
primary T cell responses merits further investigation (17
). Besides a genetic atopic predisposition, several allergens may also be transferred through the placenta. IgG-dependent as well as IgG-independent route of transfer may play a role, as well as alternative routes such as via placental fibroids, paracellular pathways or also endocytic mechanism (29
). In addition, the possibility of primary in vitro
T cell responses or cross-reactivity, rather than specific activation of previously sensitized T cells is another potential interpretation (17
). The possibility of endotoxin contamination confounding our results was excluded on the basis of low endotoxin detection and functional assays (see Methods section). A positive proliferative response to a number of stimuli may indicate a heightened immune response in the neonate potentially induced in utero
. Of interest is the report by several studies of an underlying association between CBMC lymphoproliferation and the development of atopic diseases (6
), but also no association between proliferation at birth and atopy up to 24 months of age is reported (41
Increased responses by Th2 lymphocytes to aeroallergens are presumed to be pathogenic in the development of allergic diseases (42
). At birth, possibly because of placenta-derived Th2 trophic factors, allergen-induced mononuclear cell responses are skewed toward a Th2-like phenotype (26
), and IFN-γ responses are particularly low (43
). While IL-13 secretion by CBMCs was correlated with a higher risk of atopic diseases (40
), several studies have associated a reduced IFN-γ response at birth and a potential Th1/Th2 imbalance with the development of atopic manifestation in childhood (7
). The concept of a Th1/Th2 imbalance with the demonstration of decreased Th1 and increased Th2 responses and therefore a pro-allergic phenotype is attractive. However, in one study mitogen-induced cord blood IL-13 responses also appear to be suppressed in children who develop atopic disease (46
). In our study, baseline secretion of IL-13 and IFN-γ by CBMCs was low, but detectable. Further, Derf1-induced cytokine secretion of IL-13 was associated with positive LP to Derf1 stimulation. This Der f1-induced Th2 cytokine pattern is consistent with data showing increased Der f-induced IL-5 secretion in relation to SI positivity in response to Derf stimulation (47
). It is interesting that SI positivity in our study of 135 patients was not correlated with lower IFN-γ production. In addition, Miller found a positive relationship between IFN-γ production and SI positivity in Derf-stimulated CBMCs in 71 patients (47
). Investigations about the role of IFN-γ, a predominant Th1 cytokine, have presented heterogeneous data in studies of atopic disease. Some adult studies report no difference in secretion of IFN-γ in asthmatics compared with controls; others have found lower levels in asthmatics (48
). In contrast, increased levels of IFN-γ have been found in the serum and BAL fluid of asthmatics as compared with controls (49
). Collectively, our data support the concept that immune maturation evokes a complex array of immune responses that are not solely Th1/Th2-mediated.
Another interesting finding in our study results from the data on mitogen stimulation. Increased lymphocyte proliferation induced by mitogen and associated with allergen-induced lymphocyte proliferation or vice versa may account for a generally activated immune system. We have excluded nonspecific activation by blocking MHC II in allergen-stimulated and mitogen-stimulated CBMC and could confirm allergen-specifity (see Methods section). Positivity in response to more than one stimulus may imply that T-cell reactivity reflects the sensitization to indoor antigens in utero but also that infants with a highly positive response to mitogen may be more prone to allergen-induced T-cell reactivity.
A strength of this study is the investigation of unselected samples, with measures of immune responsiveness at birth: both proliferation and supernatant cytokines levels have been assayed in the cord blood of 135 neonates. Further, the lymphoproliferative profile and cytokine responses were evaluated in freshly stimulated, non-cryopreserved samples both with the allergens Derf1, Blag2, and Ova and with the mitogen PHA. The factor that 36.8% mothers of neonates in this study have a history of atopy merits consideration. One limitation of this study may be that information about skin prick test of parents was not available. However, the entity maternal atopy was not a confounding factor for our results and not major focus of the study. Several factors may be associated with lymphocyte proliferation and cytokine secretion, including maternal atopy, smoking, and birth modus (50
). It is interesting that the current literature is diverse indicating either increased proliferative responses in vitro
by CBMCs of neonates with a family history of atopic disease (17
) in comparison to neonates without parental atopy (4
). The logistic multivariate regression revealed that adjusting for potential influencing factors of this study such as gender, race/ethnicity, or smoking further increased the associations between lymphoproliferative responses and cytokine secretion.