John W. Upham

Development of Interleukin-12-Producing Capacity throughout Childhood

ABSTRACT Increasing evidence indicates that the capacity to induce protective Th1 immune responses is impaired in early childhood, an observation that can be partially attributed to deficiencies in antigen-presenting-cell function. Synthesis of interleukin 12 (IL-12), a key Th1-trophic cytokine, is markedly reduced in the neonatal period, though there is a paucity of knowledge concerning the ontogeny of IL-12-synthetic capacity throughout the childhood years. Hence, we examined the production of bioactive IL-12 p70 by circulating mononuclear cells in a population of healthy individuals. As expected, the capacity to synthesize IL-12 p70 in response to either lipopolysaccharide or heat-killed Staphylococcus aureus was markedly impaired at birth, even after priming of cells with gamma interferon. Surprisingly however, IL-12 p70 synthesis by peripheral blood mononuclear cells from both 5- and 12-year-old children was still substantially below that seen in adults, and this did not appear to be related to excessive production of IL-10. In contrast, dendritic cells from adults and neonates, derived from monocytes with granulocyte-macrophage colony-stimulating factor and IL-4, synthesized equivalent amounts of IL-12 p70 in response to microbial stimulation. This indicates that the impaired capacity for IL-12 synthesis in childhood is not an intrinsic property of circulating mononuclear cells but rather can be readily overcome in response to appropriate maturational stimuli. Because IL-12 arose predominantly from circulating HLA-DR+ cells that lacked B-cell- and monocyte-specific markers, we propose that the slow maturation of IL-12-synthetic capacity in the childhood years can be attributed to deficiencies in the number and/or function of dendritic cells.

Rapid dendritic cell recruitment to the bronchial mucosa of patients with atopic asthma in response to local allergen challenge

BACKGROUND Airway dendritic cells (DC) play an important role in chronic allergic airway inflammation in experimental animals, but a similar role for DC in human allergic asthma has been difficult to define. This pilot study was undertaken to elucidate the role of DC in allergic asthma by examining their potential to migrate to the lower airways in response to bronchial challenge with specific allergen. METHODS Bronchial biopsy specimens were obtained from seven patients with allergic asthma before and 4–5 hours after allergen challenge. Multicolour immunofluorescence staining was performed on mucosal cryosections to identify changes in the number and phenotypes of DC. RESULTS A dramatic increase in the number of CD1c+HLA-DR+ DC were observed in the lamina propria after challenge compared with baseline (22.4v 7.8 cells/mm2). The rapid accumulation (within 4–5 hours) of these cells strongly suggests that they were directly recruited from peripheral blood. CONCLUSION We have shown for the first time that a specific DC subset rapidly emigrates into the human bronchial mucosa during allergic inflammation. While this study is based on relatively few patients, the consistency of the overall results strongly suggests that the rapid population dynamics of human airway DC closely parallel those in animal models of acute inflammation. These findings support suggestions that DC have an important role in human airway allergy.

Functional maturation of CD4+CD25+CTLA4+CD45RA+ T regulatory cells in human neonatal T cell responses to environmental antigens/allergens.

A number of laboratories have reported cord blood T cell responses to ubiquitous environmental Ags, including allergens, by proliferation and cytokine secretion. Moreover, the magnitude of these responses has been linked with risk for subsequent expression of allergy. These findings have been widely interpreted as evidence for transplacental priming and the development of fetal T memory cells against Ags present in the maternal environment. However, we present findings below that suggest that neonatal T cell responses to allergens (and other Ags) differ markedly from those occurring in later life. Notably, in contrast to allergen-responsive adult CD4+ T cell cultures, responding neonatal T cell cultures display high levels of apoptosis. Comparable responses were observed against a range of microbial Ags and against a parasite Ag absent from the local environment, but not against autoantigen. A notable finding was the appearance in these cultures of CD4+CD25+CTLA4+ T cells that de novo develop MLR-suppressive activity. These cells moreover expressed CD45RA and CD38, hallmarks of recent thymic emigrants. CFSE-labeling studies indicate that the CD4+CD25+ cells observed at the end of the culture period were present in the day 0 starting populations, but they were not suppressive in MLR responses. Collectively, these findings suggest that a significant component of the reactivity of human neonatal CD4+ T cells toward nominal Ag (allergen) represents a default response by recent thymic emigrants, providing an initial burst of short-lived cellular immunity in the absence of conventional T cell memory, which is limited in intensity and duration via the parallel activation of regulatory T cells.

Inhalant allergen‐specific T‐cell reactivity is detectable in close to 100% of atopic and normal individuals: covert responses are unmasked by serum‐free medium

Background It is widely held that in vitro T cell responses to allergens are more prominent in atopic than in normal individuals, though this conclusion is based upon culture techniques which fail to detect proliferative responses in a significant minority of atopies and many normals.

TLR4 Polymorphisms Mediate Impaired Responses to Respiratory Syncytial Virus and Lipopolysaccharide

Severe bronchiolitis following respiratory syncytial virus (RSV) infection occurs in only a small subset of infected infants and the basis for variations in disease severity is not understood. Innate immune responses to RSV are mediated by TLR-4, and the 299Gly and 399Ile alleles of the TLR4 gene have been linked epidemiologically with increased severity of RSV disease in children. We hypothesized that cellular immune responses to RSV mediated by these variant forms of the receptor are defective relative to responses mediated via the common form of the receptor. Human bronchial epithelial cells were transfected with TLR4 constructs encoding the common TLR4 gene sequence (299Asp/399Thr), or the 299Gly or 399Ile alleles, and cytokine responses to in vitro RSV challenge were analyzed in the different transfected cells. Follow-up studies compared RSV-induced responses in PBMC from children expressing these same TLR4 genotypes. Human bronchial epithelial expressing 299Gly or 399Ile displayed normal levels of intracellular TLR4 but failed to efficiently translocate the receptor to the cell surface. This was associated with reduced NF-κB signaling post-TLR4 engagement, reduced production of IFNs, IL-8, IL-10, IL-12p35, IL-18, and CCL8, and the absence of acute-phase TNF-α. These findings were mirrored by blunted PBMC responses to RSV in children expressing the same TLR4 variants. Compromised first-line defense against RSV at the airway-epithelial surface of children expressing these TLR4 variants may thus confer increased susceptibility to severe infections with this virus.

Postnatal Development of Monocyte Cytokine Responses to Bacterial Lipopolysaccharide

Early childhood is a period of heightened susceptibility to infection due to immaturity of the immune system, and the nature of these developmental deficiencies is only partially understood. In this study, we focused on the ontogeny of the innate immune system by investigating the capacity of mononuclear cells to secrete a wide spectrum of inflammatory cytokines in response to interferon (IFN)-γ priming and lipopolysaccharide (LPS) stimulation, namely IL-6, IL-10, IL-12, IL-18, IL-23, tumor necrosis factor (TNF)-α, and myxovirus resistance protein A, induced by type-I IFN, at several time points between birth (cord blood) and adulthood. Competence to produce all these cytokines followed a similar developmental pattern, with slow postnatal up-regulation from the response observed in cord blood. Unexpectedly, IL-6, IL-10, TNF-α, and IFN-γ showed slow postnatal up-regulation but also elevated cord blood responses equal to or greater than the adult level. This was transient and not observed at 2 mo of age, and was not related to predelivery stress of the newborns. Variations in Toll-like receptor (TLR)4 function may account for these age related differences in cytokine responses, as TLR4 expression on neonatal monocytes post LPS stimulation was elevated and sustained relative to infants and adults.

Rapid response of circulating myeloid dendritic cells to inhaled allergen in asthmatic subjects

Background Dendritic cells (DC) are thought to play a key role in the initiation and maintenance of T cell immunity to inhaled antigens. While the density of DC within the bronchial mucosa is increased in stable asthma, there is little information currently available concerning the effects of allergen inhalation on DC in subjects with asthma.

Reduced soluble receptor for advanced glycation end-products in COPD

The soluble receptor for advanced glycation end-products (sRAGE) has anti-inflammatory properties, and deficiency of circulating sRAGE is associated with various human diseases. Whether sRAGE concentrations are reduced in chronic obstructive pulmonary disease (COPD) has not been determined. The aim of this study was to determine plasma levels of sRAGE in COPD patients and establish whether sRAGE varies in relation to forced expiratory volume in 1 s (FEV1) and other inflammatory markers. 61 COPD patients and 42 healthy controls were recruited. Plasma sRAGE, C-reactive protein (CRP) and serum amyloid A (SAA) were measured in patients with stable COPD. A subgroup had measurements during acute exacerbations of COPD (AECOPD). sRAGE was significantly lower in stable COPD than in healthy controls (p<0.001), while CRP (p<0.001) and SAA (p = 0.015) were higher in stable COPD than in healthy controls. Multiple linear regression confirmed that COPD was negatively associated with sRAGE (p<0.001). Plasma sRAGE was positively correlated with FEV1 (r2 = 0.530, p<0.001), while CRP and SAA were inversely proportional to FEV1. Multiple linear regression analysis showed that only sRAGE was a strong predictor of FEV1. AECOPD were associated with even lower sRAGE levels that increased with convalescence. Circulating sRAGE is lower in COPD and shows a strong correlation to the degree of airflow limitation.

Neonatal interleukin-12 capacity is associated with variations in allergen-specific immune responses in the neonatal and postnatal periods

Background and objectives A reduced capacity of antigen presenting cells (APC) to provide pro‐T helper 1 (Th1) signals, such as IL‐12, to T cells during early life may be implicated in the development of T helper 2 (Th2)‐mediated allergic disease. In this study we examined the relationships between the capacity for IL‐12 responses in the neonatal period and atopic risk (family allergy), in vitro T cell responses to allergens, and the subsequent development of allergic disease at 6 years

Airway epithelial cells regulate the functional phenotype of locally differentiating dendritic cells: Implications for the pathogenesis of infectious and allergic airway disease

Atopic asthma pathogenesis is driven by the combined effects of airway inflammation generated during responses to viral infections and aeroallergens, and both these pathways are regulated by dendritic cells (DC) that differentiate locally from monocytic precursors. These DCs normally exhibit a sentinel phenotype characterized by active Ag sampling but attenuated presentation capability, which limits the intensity of local expression of adaptive immunity. How this tight control of airway DC functions is normally maintained, and why it breaks down in some atopics leading to immunopathological changes in airway tissues, is unknown. We postulated that signals from adjacent airway epithelial cells (AEC) contribute to regulation of local differentiation of DC. We tested this in a coculture model containing both cell types in a GM-CSF-IL-4-enriched cytokine milieu characteristic of the atopic asthmatic airway mucosa. We demonstrate that contact with AEC during DC differentiation up-regulates expression of the function-associated markers MHC class II, CD40, CD80, TLR3, and TLR4 on DCs with concomitant up-regulation of Ag uptake/processing. Moreover, the AEC-conditioned DCs displayed increased LPS responsiveness evidenced by higher production of IL-12, IL-6, IL-10, and TNF-α. The Th2 memory-activating properties of AEC-conditioned DCs were also selectively attenuated. Data from microarray and blocking experiments implicate AEC-derived type 1 IFNs and IL-6 in modulation of DC differentiation. Collectively, these findings suggest that resting AECs modulate local DC differentiation to optimize antimicrobial defenses in the airways and in the process down-modulate capacity for expression of potentially damaging Th2 immunity.