Working while unwell: Workplace impairment in people with severe asthma.

BACKGROUND: Severe asthma affects quality of life; however, its impact on workplace productivity is poorly understood. OBJECTIVE: To compare workplace productivity-absenteeism and presenteeism-and impairment in daily activities in severe and non-severe asthma over time and identify characteristics associated with presenteeism in severe asthma. METHODS: The Severe Asthma Web-based Database is an ongoing observational registry from Australia, New Zealand and Singapore. At April 2017, 434 patients with severe asthma and 102 with non-severe asthma were enrolled (18-88 years; 59% female). Participants provided comprehensive clinical and questionnaire data at baseline and were followed-up every 6 months for 24 months. Absenteeism (percentage of time not at work), presenteeism (self-reported impairment at work) and impairment in daily activities outside work due to health problems in the last week were calculated. RESULTS: At baseline, 61.4% of participants with severe asthma and 66.2% with non-severe asthma under 65 years were employed. At younger ages (30-50 years), fewer severe asthma participants were employed (69% vs 100%). Presenteeism and impairment in daily activity were more frequently reported in severe asthma and in participants with poorer asthma control, poorer lung function and more past-year exacerbations (P < .01). Over time, deteriorating asthma control was associated with increasing presenteeism. Although absenteeism was not different between severe and non-severe asthma, worse asthma control was associated with absenteeism (P < .001). In participants with severe asthma, presenteeism was reported more frequently in those with poorer asthma control, poorer asthma-related quality of life and symptoms of depression or anxiety (P < .01). CONCLUSION AND CLINICAL RELEVANCE: Severe asthma was associated with impairment at work and outside the workplace. Improving asthma control and mental health may be important targets for optimizing workplace productivity in severe asthma. Presenteeism and absenteeism may represent key metrics for assessing intervention efficacy in people with severe asthma of working age.

Sputum cytology during late-phase responses to inhalation challenge with different allergens.

BACKGROUND: In mouse models of allergic asthma, exposure to different allergens can trigger distinct inflammatory subtypes in the airways. We investigated whether this observation extends to humans. METHODS: We compared the frequency of sputum inflammatory subtypes between mild allergic asthma subjects (n = 129) exposed to different allergens in inhalation challenge tests. These tests were performed using a standardized protocol as part of clinical trials of experimental treatments for asthma, prior to drug randomization. Five allergen types were represented: the house dust mites Dermatophagoides pteronyssinus and Dermatophagoides farinae, ragweed, grass, and cat. RESULTS: Of 118 individuals with a sputum sample collected before allergen challenge (baseline), 45 (38%) had paucigranulocytic, 51 (43%) eosinophilic, 11 (9%) neutrophilic, and 11 (9%) mixed granulocytic sputum. Of note, most individuals with baseline paucigranulocytic sputum developed eosinophilic (48%) or mixed granulocytic (43%) sputum 7 hours after allergen challenge, highlighting the dynamic nature of sputum inflammatory subtype in asthma. Overall, there was no difference in the frequency of sputum inflammatory subtypes following challenge with different allergen types. Similar results were observed at 24 hours after allergen challenge. CONCLUSIONS: Unlike reported in mice, in humans the sputum inflammatory subtype observed after an allergen-induced asthma exacerbation is unlikely to be influenced by the type of allergen used.

Real-life effectiveness of omalizumab in severe allergic asthma above the recommended dosing range criteria.

BACKGROUND: Omalizumab (Xolair) dosing in severe allergic asthma is based on serum IgE and bodyweight. In Australia, patients eligible for omalizumab but exceeding recommended ranges for IgE (30-1500 IU/mL) and bodyweight (30-150 kg) may still receive a ceiling dose of 750 mg/4 weeks. About 62% of patients receiving government-subsidized omalizumab are enrolled in the Australian Xolair Registry (AXR). OBJECTIVES: To determine whether AXR participants above the recommended dosing ranges benefit from omalizumab and to compare their response to within-range participants. METHODS: Data were stratified according to dose range status (above-range or within-range). Further sub-analyses were conducted according to the reason for being above the dosing range (IgE only vs. IgE and weight). RESULTS: Data for 179 participants were analysed. About 55 (31%) were above recommended dosing criteria; other characteristics were similar to within-range participants. Above-range participants had higher baseline IgE [812 (IQR 632, 1747) IU/mL vs. 209 (IQR 134, 306) IU/mL] and received higher doses of omalizumab [750 (IQR 650, 750) mg] compared to within-range participants [450 (IQR, 300, 600) mg]. At 6 months, improvements in Juniper 5-item Asthma Control Questionnaire (ACQ-5, 3.61 down to 2.01 for above-range, 3.47 down to 1.93 for within-range, P < 0.0001 for both) and Asthma Quality of Life Questionnaire (AQLQ mean score (3.22 up to 4.41 for above-range, 3.71 up to 4.88 for within-range, P < 0.0001) were observed in both groups. Forced expiratory volume in one second (FEV1 ) improved among above-range participants. There was no difference in response between above-range and within-range participants. Above-range participants due to either IgE alone or IgE and weight had similar improvements in ACQ-5, AQLQ and FEV1 . CONCLUSIONS AND CLINICAL RELEVANCE: Patients with severe allergic asthma above recommended dosing criteria for omalizumab have significantly improved symptom control, quality of life and lung function to a similar degree to within-range participants, achieved without dose escalation above 750 mg.

Sex hormones and systemic inflammation are modulators of the obese-asthma phenotype.

BACKGROUND: Both systemic inflammation and sex hormones have been proposed as potential mediators of the obese-asthma phenotype. The aim of this study was to examine the associations between sex hormones, oral contraceptive pill (OCP) use, systemic inflammation and airway inflammation in adults with asthma. METHODS: Obese (n = 39) and nonobese (n = 42) females and obese (n = 24) and nonobese (n = 25) males with asthma were recruited. Females were further categorized as reproductive-aged (<50 years old; n = 36) or older (>50 years old; n = 45). Thirteen (36.1%) reproductive-aged females were using the OCP. Participants had induced sputum cell counts measured and blood analysed for sex hormones and inflammatory markers. RESULTS: Obese reproductive-aged females had higher sputum %neutrophils than nonobese reproductive-aged females (45.4 ± 24.3% vs 27.5 ± 17.5%, P = 0.016); however, there was no difference in sputum neutrophils in obese compared with nonobese males (P = 0.620) or older females (P = 0.087). Multiple linear regression analysis found testosterone and OCP use to be negative predictors of sputum %neutrophils, while C-reactive protein and IL-6 were positive predictors of sputum %neutrophils. BMI and age were not significant predictors in the multivariate model. Reproductive-aged females using the OCP had significantly lower sputum %neutrophils than those not using the OCP (23.2 ± 12.6% vs 42.1 ± 23.8%, P = 0.015). CONCLUSIONS: This study suggests that sex hormones and systemic inflammation may be mediating the obese-asthma phenotype. The observation that OCP use was associated with lower sputum %neutrophils in reproductive-aged females warrants further investigation.

Identification of STOML2 as a putative novel asthma risk gene associated with IL6R.

BACKGROUND: Functional variants in the interleukin-6 receptor gene (IL6R) are associated with asthma risk. We hypothesized that genes co-expressed with IL6R might also be regulated by genetic polymorphisms that are associated with asthma risk. The aim of this study was to identify such genes. METHODS: To identify genes whose expression was correlated with that of IL6R, we analyzed gene expression levels generated for 373 human lymphoblastoid cell lines by the Geuvadis consortium and for 38 hematopoietic cell types by the Differentiation Map Portal (DMAP) project. Genes correlated with IL6R were then screened for nearby single nucleotide polymorphisms (SNPs) that were significantly associated with both variation in gene expression levels (eSNPs) and asthma risk. RESULTS: We identified 90 genes with expression levels correlated with those of IL6R and that also had a nearby eSNP associated with disease risk in a published asthma GWAS (N = 20 776). For 16 (18%) genes, the association between the eSNP and asthma risk replicated with the same direction of effect in a further independent published asthma GWAS (N = 27 378). Among the top replicated associations (FDR < 0.05) were eSNPs for four known (IL18R1, IL18RAP, BCL6, and STAT6) and one putative novel asthma risk gene, stomatin-like protein 2 (STOML2). The expression of STOML2 was negatively correlated with IL6R, while eSNPs that increased the expression of STOML2 were associated with an increased asthma risk. CONCLUSION: The expression of STOML2, a gene that plays a key role in mitochondrial function and T-cell activation, is associated with both IL-6 signaling and asthma risk.

Increased Peripheral Blood Pro-Inflammatory/Cytotoxic Lymphocytes in Children with Bronchiectasis.

OBJECTIVE: Bronchiectasis (BE) in children is common in some communities including Indigenous children in Australia. Relatively little is known about the nature of systemic inflammation in these children, especially the contribution of specific pro-inflammatory and cytotoxic lymphocyte subsets: T-cells, natural killer (NK) cells and NKT-like cells. We have shown that these cells produce increased cytotoxic (granzyme b and perforin) and inflammatory (IFNγ and TNFα) mediators in several adult chronic lung diseases and hypothesised that similar changes would be evident in children with BE. METHODS: Intracellular cytotoxic mediators perforin and granzyme b and pro-inflammatory cytokines were measured in T cell subsets, NKT-like and NK cells from blood and bronchoalveolar samples from 12 children with BE and 10 aged-matched control children using flow cytometry. RESULTS: There was a significant increase in the percentage of CD8+ T cells and T and NKT-like subsets expressing perforin/granzyme and IFNγ and TNFα in blood in BE compared with controls. There was a further increase in the percentage of pro-inflammatory cytotoxic T cells in Indigenous compared with non-Indigenous children. There was no change in any of these mediators in BAL. CONCLUSIONS: Childhood bronchiectasis is associated with increased systemic pro-inflammatory/cytotoxic lymphocytes in the peripheral blood. Future studies need to examine the extent to which elevated levels of pro-inflammatory cytotoxic cells predict future co-morbidities.

IgE+ B cells are scarce, but allergen-specific B cells with a memory phenotype circulate in patients with allergic rhinitis.

BACKGROUND: Despite the critical role of immunoglobulin E (IgE) in allergy, circulating IgE+ B cells are scarce. Here, we describe in patients with allergic rhinitis B cells with a memory phenotype responding to a prototypic aeroallergen. METHODS: Fifteen allergic rhinitis patients with grass pollen allergy and 13 control subjects were examined. Blood mononuclear cells stained with carboxyfluorescein diacetate succinimidyl ester (CFSE) were cultured with Bahia grass pollen. Proliferation and phenotype were assessed by multicolour flow cytometry. RESULTS: In blood of allergic rhinitis patients with high serum IgE to grass pollen, most IgE(hi) cells were CD123+ HLA-DR(-) basophils, with IgE for the major pollen allergen (Pas n 1). Both B and T cells from pollen-allergic donors showed higher proliferation to grass pollen than nonallergic donors (P = 0.002, and 0.010, respectively), whereas responses to vaccine antigens and mitogen did not differ between groups. Allergen-driven B cells that divided rapidly (CD19(mid) CD3(-) CFSE(lo) ) showed higher CD27 (P = 0.008) and lower CD19 (P = 0.004) and CD20 (P = 0.004) expression than B cells that were slow to respond to allergen (CD19(hi) CD3(-) CFSE(mid) ). Moreover, rapidly dividing allergen-driven B cells (CD19(mid) CFSE(lo) CD27(hi) ) showed higher expression of the plasmablast marker CD38 compared with B cells (CD19(hi) CFSE(mid) CD27(lo) ) that were slow to divide. CONCLUSION: Patients with pollen allergy but not control donors have a population of circulating allergen-specific B cells with the phenotype and functional properties of adaptive memory B-cell responses. These cells could provide precursors for allergen-specific IgE production upon allergen re-exposure.

Full blood count parameters for the detection of asthma inflammatory phenotypes.

BACKGROUND: In asthma, the airway inflammatory phenotype influences clinical characteristics and treatment response. Although induced sputum is the gold standard test for phenotyping asthma, a more accessible method is needed for clinical practice. OBJECTIVE: To investigate whether white blood cell counts and/or their derived ratios can predict sputum eosinophils or neutrophils in uncontrolled asthma. METHODS: This cross-sectional study evaluated 164 treated but uncontrolled asthmatic patients with sputum induction and blood collection. Receiver-operating characteristic (ROC) curves were used to assess the relationship between blood and sputum parameters. RESULTS: There was a significant positive relationship between blood eosinophil parameters and the percentage of sputum eosinophil count. A weak but significant correlation was found between sputum neutrophil percentage and blood neutrophil percentage (r = 0.219, P = 0.005). ROC curve analysis identified that blood eosinophil percentage count was the best predictor for eosinophilic asthma, with an area under the curve (AUC) of 0.907 (P < 0.001). The optimum cut-point for blood eosinophil percentage was 2.7%, and this yielded a sensitivity of 92.2% and a specificity of 75.8%. The absolute blood eosinophil count was also highly predictive with an AUC of 0.898 (P < 0.0001) at a blood eosinophil cut-off of 0.26 × 10(9) /L. The blood eosinophil/lymphocyte ratio (ELR) and eosinophil/neutrophil ratio (ENR) were increased in eosinophilic asthma, and the neutrophil/lymphocyte ratio (NLR) was increased in neutrophilic asthma. Neutrophilic asthma could also be detected by blood neutrophil percentages and NLR, but with less accuracy. CONCLUSIONS AND CLINICAL RELEVANCE: Blood eosinophil counts and derived ratios (ELR and ENR) can accurately predict eosinophilic asthma in patients with persistent uncontrolled asthma despite treatment. Blood neutrophil parameters are poor surrogates for the proportion of sputum neutrophils. Blood counts may be a useful aid in the monitoring of uncontrolled asthma.

Functional immunoglobulin E cross-reactivity between Pas n 1 of Bahia grass pollen and other group 1 grass pollen allergens.

BACKGROUND: Grass pollens are major triggers of allergic rhinitis and asthma, but the immunological relationships between pollen allergens of the subtropical Bahia grass, Paspalum notatum, and temperate grasses are unresolved. OBJECTIVE: To assess serum IgE cross-reactivity between subtropical P. notatum and temperate Lolium perenne (Ryegrass) pollen allergens. METHODS: Serum IgE reactivities of grass pollen-allergic patients with P. notatum, L. perenne and Cynodon dactylon (Bermuda grass) pollen extracts and their respective purified group 1 allergens, Pas n 1, Lol p 1 and Cyn d 1, were compared by immunoblotting, ELISA and basophil activation. RESULTS: In a cohort of 51 patients from a temperate region, a high frequency of IgE reactivity with each grass pollen was detected, but reactivity with L. perenne pollen was substantially greater than with P. notatum and C. dactylon pollen. Similarly, serum IgE reactivity with Lol p 1 was greater than with Pas n 1 or Cyn d 1. For seven of eight sera studied in detail, asymmetric serum IgE cross-reactivity was observed; L. perenne pollen inhibited IgE reactivity with P. notatum pollen but not the converse, and IgE reactivity with Pas n 1 was inhibited by Lol p 1 but IgE reactivity with Lol p 1 was not inhibited by Pas n 1 or Cyn d 1. Importantly, P. notatum pollen and Pas n 1 activated basophils in grass pollen-allergic patients from a temperate region, although stimulation was greater by pollen of L. perenne than P. notatum or C. dactylon, and by Lol p 1 than Pas n 1 or Cyn d 1. In contrast, a cohort of 47 patients from a subtropical region showed similar IgE reactivity with P. notatum and L. perenne pollen, and reciprocal cross-inhibition of IgE reactivity between L. perenne and P. notatum. CONCLUSIONS: Pollen allergens of the subtropical P. notatum, including Pas n 1, show clinically relevant IgE cross-reactivity with pollen allergens of L. perenne but also species-specific IgE reactivity.

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 (FEV(1)) 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 FEV(1) (r(2) = 0.530, p<0.001), while CRP and SAA were inversely proportional to FEV(1). Multiple linear regression analysis showed that only sRAGE was a strong predictor of FEV(1). 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.

Toll-like receptor 7 function is reduced in adolescents with asthma.

Anti-viral innate immune responses may be impaired in asthma, although the mechanisms are not well understood. Toll-like receptors (TLRs) 7 and 3 are particularly relevant for initiating responses to common respiratory viruses, as they recognise single-stranded viral RNA and double-stranded viral RNA, respectively. The aim of the present study was to investigate TLR7 and TLR3 function in 14-yr-old adolescents with asthma. Blood mononuclear cells obtained from 17 atopic asthmatics, 29 atopic, non-asthmatics and 21 healthy, non-atopic individuals, were stimulated with the TLR7 agonist imiquimod and the TLR3 agonist poly I:C. Expression of anti-viral molecules was measured by real-time PCR. Concentrations of interferon-gamma-inducible cytokine protein (IP)-10 and interleukin (IL)-6 were measured by ELISA. TLR7-induced myxovirus resistance protein A and 2'5' oligoadenylate synthetase mRNA expression and protein levels of IP-10 were significantly lower in asthma subjects compared with healthy subjects (p = 0.041, p = 0.003 and p = 0.001 respectively). There was a significant negative correlation between total serum immunoglobulin E and IP-10 following TLR7 stimulation. However, TLR3-induced responses did not vary with asthma or atopy. IL-10 mRNA and IL-6 protein synthesis were similar in asthmatic and control subjects. In conclusion, TLR7 function is reduced in adolescents with asthma and this may contribute to susceptibility to respiratory viral infections.

Alveolar macrophages and CC chemokines are increased in children with cystic fibrosis.

Airway inflammation is an important component of cystic fibrosis (CF) lung disease. We sought to determine whether alveolar macrophages were involved in early CF lung disease. Children with CF (median age 3.1 yrs) participated in a surveillance programme that included annual bronchoalveolar lavage (BAL). Control samples were obtained from non-CF children (median age 3.1 yrs; n = 24) investigated for persistent respiratory symptoms. Pulmonary infection was detected in 31% (16 out of 51) and 38% (nine out of 24) of children from the CF and non-CF groups, respectively. Alveolar macrophages in BAL were increased in CF compared with non-CF in the absence of infection (223x10(3) versus 85x10(3) cells.mL(-1); p = 0.001) and were associated with elevations in the CC chemokines (macrophage inflammatory protein (MIP)-3alpha (chemokine (C-C motif) ligand (CCL)20; 355.8 versus 46.0 pg.mL(-1); p<0.001), monocyte chemotactic protein-1 (CCL2; 263.5 versus 25.3 pg.mL(-1); p<0.001), MIP-1alpha (CCL3; 38.2 versus 4.9 pg.mL(-1); p<0.001) and MIP-1beta (CCL4; 326.6 versus 27.5 pg.mL(-1); p<0.001)). Total cell counts and neutrophil numbers increased in the presence of infection; however, there was no additional effect of CF. Alveolar macrophages and CC chemokines are elevated in the lungs in young children with CF even in the absence of pulmonary infection. Longitudinal studies are required to determine the clinical relevance of these findings.

Maternal reactivity to fetal alloantigens is related to newborn immune responses and subsequent allergic disease.

BACKGROUND: Maternal allergy confers stronger allergy risk (than paternal allergy) suggesting that maternal patterns of immune response can directly influence immune development in offspring. Women prone to allergic immune responses to allergens may also have altered immune responses to other antigens including fetal antigens. OBJECTIVE: This exploratory study examines relationships between maternal immune responses to fetal antigens and the subsequent risk of allergy. METHODS: Mononuclear cells (MNC) were collected from 36 mother-infant pairs to compare maternal (and fetal) cellular immune responses to alloantigens (fetal, maternal or unrelated donor [URD]), and allergens in allergic (18 pairs) and non-allergic (18 pairs) mothers. Thirty children had documented allergic outcomes at 6 years of age. RESULTS: In this population, allergic outcomes in the offspring were associated more strongly with materno-fetal immune interactions than with a maternal family history of allergy. Specifically, allergic disease at 6 years of age was associated with significantly higher maternal responses to fetal alloantigens (lymphoproliferation, P=0.008; IL-13, P=0.02 and IFN-gamma, P=0.015), whereas associations with maternal allergy did not reach significance (P=0.07). Fetal IFN-gamma alloantigen responses were significantly correlated with the degree of human lymphocyte antigen (HLA) mismatch (maternal HLA class II antibodies) (tau=0.3, P=0.03). The capacity of the fetus to produce IL-13 (tau=0.4, P=0.001) and IL-10 (tau=0.3, P=0.029) was directly related to the level of these cytokines produced by the mother in response to fetal antigens. Allergic mothers showed a non-significant trend for stronger lymphoproliferation to fetal alloantigens. The number of previous pregnancies (gravidity) was associated with stronger maternal responses to fetal alloantigens, as shown by lymphoproliferation (Kendall tau=0.3, P=0.04) and IFN-gamma (tau=0.3, P=0.04) synthesis, but did not affect fetal responses to the various stimuli. CONCLUSIONS: Maternal responses to fetal antigens were related to fetal immune responses and subsequent allergy. This novel observation suggests that events at the materno-fetal interface have an important influence on early immune development and should be investigated further.

Cytosine-phosphate-guanine motifs fail to promote T-helper type 1-polarized responses in human neonatal mononuclear cells.

BACKGROUND: The T-helper type 1 (Th1) trophic properties of bacterial cytosine-phosphate-guanine (CpG) motifs have made them logical adjuvants both for the suppression of Th2-mediated allergic disease in early life and for promoting vaccine responses in neonates who have relatively immature Th1 function. However, little is known about their effects on immature immune responses in this period. OBJECTIVES: To compare the effects of CpG on adult and neonatal cellular immune responses to various stimuli. METHODS: The immune responses of mononuclear cells (MC) derived from neonates (n=25) and their mothers (n=25) were compared in vitro. These were stimulated with house dust mite (HDM), CpG B, CpG C, non-CpG oligodeoxynucleotides (ODN) or diphtheria toxoid (DT) in optimized conditions. In parallel cultures, CpGs were combined with HDM or DT antigens to assess the effect of the various ODN on these antigen-specific responses. Lymphoproliferation and cytokine responses IL-13, IFN-gamma, IL-6, IL-10, TNF-alpha) were measured for all of the cultures described above. RESULTS: Although neonates showed attenuated lymphoproliferation to CpG, the production of antigen-presenting cell-derived cytokines such as IL-6 and IL-10 and the up-regulation of major histocompatibility complex class II (HLA-DR) were detected at adult levels. T cell-derived cytokines (IL-13 and IFN-gamma) were not detectable in response to CpG alone. Most neonates also failed to produce detectable IFN-gamma to HDM or DT (unlike adults), but readily produced IL-13 to these stimuli. The addition of CpG resulted in an increase in neonatal IFN-gamma production in response to HDM (P=0.011) and a similar but non-significant trend with DT. However, rather than inhibiting Th2 IL-13 responses, the addition of CpGs was associated with a significant increase in the IL-13 responses to HDM (P=0.016) and DT (P=0.03), effects not seen in adults. CONCLUSIONS: This study provides further evidence that neonatal MC responses to CpG are functionally different from adults, and do not show clear Th1 polarization. The CpG associated increase in Th2 responses may reflect a potentiation of the normal neonatal Th2 propensity, or non-specific activation of neonatal MC.

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. METHODS: The capacity of circulating neonatal (and maternal) APC to produce IL-12 p70 in response to LPS (and IFN-gamma) stimulation was assessed in a group of 60 children with previously well-characterized immune responses to allergens and atopic outcomes. The IL-12 responses were compared with allergen-induced lymphoproliferation (to house dust mite (HDM) ovalbumin (OVA), cat and beta-lactoglobulin (BLG)) and IL-13 and IFN-gamma cytokine responses (to OVA, HDM and phytohaemaglutinin (PHA)) in the neonatal and postnatal periods. IL-12 responses were also compared according to atopic risk and atopic outcomes (doctor-diagnosed asthma, eczema, food allergies and sensitization as evidenced by skin prick testing) at 6 years clinical follow-up. RESULTS: Maternal peripheral blood mononuclear cells (PBMC) synthesized significantly greater amounts of IL-12 than neonatal PBMC, though within maternal-infant pairs IL-12 responses were significantly correlated (r = 0.4, P = 0.019). Moreover, neonatal IL-12 responses were positively correlated with neonatal allergen proliferation for HDM (r = 0.6, P < 0.0001), OVA (r = 0.55, P < 0.0001), cat (r = 0.5, P = 0.003) and BLG (r = 0.55, P = 0.001), but negatively correlated with neonatal IL-13 responses to both allergens tested (HDM: r = - 0.4, P = 0.03 and OVA: r = - 0.5, P = 0.001). Both neonatal and maternal IL-12 responses were positively correlated with postnatal IFN-gamma responses to HDM at 12, 18 and 24 months of age (responses after age of 2 years were not assessed). There was no relationship between atopic risk and IL-12 capacity in the neonatal period, but there was a (non-significant) trend for neonatal IL-12 responses to be lower in the high-risk children who developed clinical allergy at 6 years (compared with the low risk group) although the number in this analysis was small. CONCLUSIONS: Reduced APC IL-12 production in the perinatal period was associated with reduced T cell activation (lymphoproliferation), stronger neonatal Th2 responses, and weaker Th1 responses to allergen in the postnatal period. This supports the notion that variations in APC function in early life may contribute to altered allergen-specific cytokine responses associated with later allergy.

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. OBJECTIVES: To enumerate changes in the numbers of circulating CD33(+) myeloid DC in asthmatics, before and after allergen challenge. METHODS: Blood DC numbers were enumerated by flow cytometry before and at 3, 6 and 24 h after inhaled allergen and diluent in 10 mild, allergic asthmatic subjects. RESULTS: Blood DC numbers rapidly fell from 3.42 +/- 0.30 x 10(7)/L at baseline, to 2.10 +/- 0.17 x 10(7)/L by 3 h post-challenge (P < 0.01), and remained significantly below baseline values at both 6 and 24 h following allergen challenge. No such changes in DC numbers were noted after diluent challenge. A similar, early fall in circulating lymphocytes was also noted post-allergen challenge, whereas changes in circulating eosinophil and neutrophil numbers occurred more slowly. CONCLUSIONS: A significant proportion of myeloid DC rapidly 'disappear' from the circulation following allergen inhalation, suggesting that margination of circulating myeloid DC, and their recruitment into the airway mucosa, is an important feature of the immune response to inhaled allergen.

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 (22.4 v 7.8 cells/mm(2)). 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.

Simplified quantitation of myeloid dendritic cells in peripheral blood using flow cytometry.

BACKGROUND: Recognition of the importance of dendritic cells (DC) in the initiation of T-cell-dependent immune responses has led to increasing interest in methods for the identification of DC within the circulation. We sought to develop a flow cytometric method that would allow the reliable enumeration of absolute myeloid DC counts in minimally manipulated blood samples. METHODS: Myeloid DC were identified by three-color staining of whole blood leukocytes as a discrete population of mononuclear cells expressing high levels of HLA-DR and CD33, yet having little or no expression of CD14 and CD16. This method was analyzed for reproducibility and variation in blood DC number during typical clinical day hours and after exercise. The new method was compared to an established commercial kit method. RESULTS: FACS sorting of the CD33(+) DC showed that they morphologically resembled immature DC, and developed cytoplasmic projections typical of mature DC following overnight culture in granulocyte macrophage-colony stimulating factor (GM-CSF). Within peripheral blood, these DC were found at a mean concentration of 17. 4 +/- 5.4 x 10(6) per liter, corresponding to 0.93 +/- 0.27% of mononuclear cells. Comparison of duplicate samples stained and analyzed in parallel showed that the intrasample variability was very low, with an intraclass correlation coefficient of 0.95. The frequency of CD33(+) myeloid DC and their light scatter characteristics were similar to that of CD11c(+) myeloid cells. Four-color FACS analysis revealed complete identity of CD11c(hi), HLA-DR(+) DC with CD33(+), HLA-DR(+) DC. Only rare CD33(+) DC coexpressed CD123 and HLA-DR. Numbers of blood myeloid DC, identified by CD33 staining, showed no significant variation during standard laboratory hours. However, their numbers rose significantly during vigorous exercise, in parallel to other blood cells. CONCLUSIONS: The method described herein is rapid, reproducible, requires only small volumes of blood, can be readily used by a clinical immunology laboratory, and requires fewer antibodies than a currently available commercial method.

Reduced expression of hemopoietic cytokine receptors on cord blood progenitor cells in neonates at risk for atopy.

BACKGROUND: Recent findings point to an association between allergic asthma in adults and increased responsiveness of myeloid progenitor cells to certain hemopoietic growth factors. However, it is not clear at what age these changes in progenitor cells first become manifest, although increasing evidence suggests that the allergic phenotype may begin to emerge in very early life. OBJECTIVE: We sought to compare expression of hemopoietic cytokine receptors on CD34(+) progenitor cells in cord blood from normal infants ("low risk" for subsequent atopy) and infants with at least one atopic first degree relative ("at risk" for subsequent atopy). METHODS: Cord blood was obtained from 21 neonates. Nonadherent mononuclear cells were stained with mAbs directed against CD45, CD34, and the alpha-chains of the GM-CSF, IL-3, and IL-5 receptors and analyzed by flow cytometry. RESULTS: No differences in absolute CD34(+) numbers were observed between the 2 groups. However, expression of GM-CSF receptor on CD34(+) cells was reduced in the "at-risk" compared with the "low- risk" group (P =.021), although no significant differences were noted between the 2 groups with respect to IL-3 and IL-5 receptor expression. CONCLUSION: The functional sequelae of reduced GM-CSF receptor expression on CD34(+) cells remain to be determined. Nonetheless, these findings show an association between genetic risk for atopy and changes in the expression of hemopoietic cytokine receptors on cord blood progenitor cells and support the notion that the allergic phenotype may begin to evolve in the perinatal period.

Selective inhibition of T cell proliferation but not expression of effector function by human alveolar macrophages.

BACKGROUND: Alveolar macrophages are thought to play an important part in regulating lung immune responses. While it is clear that human alveolar macrophages suppress T cell proliferation in vitro, the mechanisms by which this is achieved are not clear, nor is it known whether alveolar macrophages also inhibit other aspects of T cell function. METHODS: Peripheral blood mononuclear cells were stimulated with phytohaemagglutinin or house dust mite allergen, and cultured with variable numbers of autologous alveolar macrophages obtained by bronchoalveolar lavage from 20 normal subjects. RESULTS: Alveolar macrophages induced a reversible inhibition of T cell proliferation in response to both mitogen and allergen stimulation, with the latter being considerably more susceptible to inhibition. This was achieved via heterogenous mechanisms, involving both soluble factors derived from alveolar macrophages and cell-cell contact. Despite inhibiting proliferation, alveolar macrophages had little or no effect on T cell calcium flux, the characteristic changes in CD3, CD2, CD28 and interleukin-2 (IL-2) receptor expression which accompany normal T cell activation, and IL-2 and interferon gamma secretion. In contrast, alveolar macrophages inhibited the tyrosine phosphorylation of proteins which may be involved in IL-2 receptor-associated signal transduction. CONCLUSIONS: The immunoregulatory properties of alveolar macrophages are relatively selective, allowing T cell activation and cytokine secretion while inhibiting T cell proliferation within the lung.