Lung dendritic cells at the innate-adaptive immune interface.

This review updates the basic biology of lung DCs and their functions. Lung DCs have taken center stage as cellular therapeutic targets in new vaccine strategies for the treatment of diverse human disorders, including asthma, allergic lung inflammation, lung cancer, and infectious lung disease. The anatomical distribution of lung DCs, as well as the division of labor between their subsets, aids their ability to recognize and endocytose foreign substances and to process antigens. DCs can induce tolerance in or activate naïve T cells, making lung DCs well-suited to their role as lung sentinels. Lung DCs serve as a functional signaling/sensing unit to maintain lung homeostasis and orchestrate host responses to benign and harmful foreign substances.

Innate immunity in allergic disease.

The innate immune system consists of multiple cell types that express germline-encoded pattern recognition receptors that recognize pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs). Allergens are frequently found in forms and mixtures that contain PAMPs and DAMPs. The innate immune system is interposed between the external environment and the internal acquired immune system. It is also an integral part of the airways, gut, and skin. These tissues face continuous exposure to allergens, PAMPs, and DAMPs. Interaction of allergens with the innate immune system normally results in immune tolerance but, in the case of allergic disease, this interaction induces recurring and/or chronic inflammation as well as the loss of immunologic tolerance. Upon activation by allergens, the innate immune response commits the acquired immune response to a variety of outcomes mediated by distinct T-cell subsets, such as T-helper 2, regulatory T, or T-helper 17 cells. New studies highlighted in this review underscore the close relationship between allergens, the innate immune system, and the acquired immune system that promotes homeostasis versus allergic disease.

Chronic beryllium disease: an updated model interaction between innate and acquired immunity.

During the last decade, there have been concerted efforts to reduce beryllium (Be) exposure in the workplace and thereby reduce potential cases of this occupational lung disorder. Despite these efforts, it is estimated that there are at least one million Be-exposed individuals in the U.S. who are potentially at risk for developing chronic beryllium disease (CBD). Previously, we reviewed the current CBD literature and proposed that CBD represents a model interaction between innate and acquired immunity (Sawyer et al., Int Immunopharmacol 2:249-261, 2002). We closed this review with a section on "future directions" that identified key gaps in our understanding of the pathogenesis of CBD. In the intervening period, progress has been made to fill in some of these gaps, and the current review will provide an update on that progress. Based on recent findings, we provide a new hypothesis to explain how Be drives sustained chronic inflammation and granuloma formation in CBD leading to progressive compromised lung function in CBD patients. This paradigm has direct implications for our understanding of the development of an immune response to Be, but is also likely applicable to other immune-mediated lung diseases of known and unknown etiology.

Sulfasalazine and mesalamine modulate beryllium-specific lymphocyte proliferation and inflammatory cytokine production.

Occupational exposure to beryllium (Be) results in Be sensitization (BeS) that can progress to pulmonary granulomatous inflammation associated with chronic Be disease (CBD). Be-specific lymphocytes are present in the blood of patients with BeS and in the blood and lungs of patients with CBD. Sulfasalazine and its active metabolite, mesalamine, are clinically used to ameliorate chronic inflammation associated with inflammatory bowel disease. We tested whether sulfasalazine or mesalamine could decrease Be-stimulated peripheral blood mononuclear cell (PBMC) proliferation in subjects with CBD and BeS and Be-induced cytokine production in CBD bronchoalveolar lavage (BAL) cells. CBD (n = 25), BeS (n = 12) and healthy normal control (n = 6) subjects were enrolled and ex vivo proliferation and cytokine production were assessed in the presence of Be and sulfasalazine or mesalamine. Be-stimulated PBMC proliferation was inhibited by treatment with either sulfasalazine or mesalamine. Be-stimulated CBD BAL cell IFN-γ and TNF-α cytokine production was decreased by treatment with sulfasalazine or mesalamine. Our data suggest that both sulfasalazine and mesalamine interfere with Be-stimulated PBMC proliferation in CBD and BeS and dampens Be-stimulated CBD BAL cell proinflammatory cytokine production. These studies demonstrate that sulfasalazine and mesalamine can disrupt inflammatory pathways critical to the pathogenesis of chronic granulomatous inflammation in CBD, and may serve as novel therapy for human granulomatous lung diseases.

Summary of the 2008 National Institute of Allergy and Infectious Diseases-US Food and Drug Administration Workshop on Food Allergy Clinical Trial Design.

This article summarizes the proceedings of a 2008 Workshop on Food Allergy Clinical Trials Design co-organized by the National Institute of Allergy and Infectious Diseases and the US Food and Drug Administration. The use of food allergens both as therapy and for oral food challenges is associated with a risk of anaphylaxis. Investigators are strongly encouraged to address regulatory considerations by discussing proposed studies with the US Food and Drug Administration. Food allergen administration through the oral or sublingual routes might be less risky than through the subcutaneous route, but this hypothesis has not been proved, and subjects with food allergy might still be at high risk of allergic reactions to such allergen administration. Two distinct mechanisms might lead to beneficial clinical outcomes: desensitization (reversible when food allergen therapy is stopped) and tolerance (persistent benefit even after allergen therapy is stopped). There are important clinical distinctions between desensitization and tolerance. The efficacy of a therapy for food allergy can be evaluated by assessing changes in the dose response to double-blind, placebo-controlled oral food challenges before and after therapy and also by assessing changes in the number of allergic episodes during a longitudinal natural history/exposure study; both approaches have strengths and limitations.

Modulation of lymphocyte proliferation by antioxidants in chronic beryllium disease.

RATIONALE: Occupational exposure to beryllium (Be) can result in chronic granulomatous inflammation characterized by the presence of Be-specific CD4+ T cells. Studies show that oxidative stress plays a role in the pathogenesis of chronic inflammatory disorders. OBJECTIVES: We hypothesized that Be-induced oxidative stress modulates the proliferation of Be-specific CD4+ T cells. METHODS: Thirty-three subjects with chronic beryllium disease (CBD), 15 subjects with beryllium sensitization, and 28 healthy normal control subjects were consecutively enrolled from the Occupational and Environmental Health Clinic of the National Jewish Medical and Research Center. MEASUREMENTS AND MAIN RESULTS: All studies were performed with Ficoll-Hypaque-isolated peripheral blood mononuclear cells from subsets of the study subjects. Decreased intracellular levels of the thiol antioxidants, glutathione and cysteine, were observed in peripheral blood mononuclear cells from subjects with beryllium sensitization and CBD, as compared with healthy control subjects. Beryllium stimulation decreased intracellular thiol antioxidants by more than 40%, accompanied by increased reactive oxygen species levels and the proliferation of Be-specific blood CD4+ T cells from subjects with CBD. Be-induced T-cell proliferation was inhibited by treatment with the thiol antioxidant N-acetylcysteine or the catalytic antioxidant manganese(III) 5,10,15,20-tetrakis(4-benzoic acid)porphyrin (MnTBAP). MnTBAP treatment also inhibited T-cell proliferation in response to the unrelated, MHC class II-restricted antigen tetanus toxoid. Treatment of CBD blood lymphocytes, but not antigen-presenting cells, with MnTBAP decreased Be-induced T-cell proliferation by more than 40%. CONCLUSIONS: Beryllium can mediate a thiol imbalance leading to oxidative stress that may modulate the proliferation and clonal expansion of Be-specific blood CD4+ T cells. These data suggest that Be-induced oxidative stress plays a role in the pathogenesis of granulomatous inflammation in CBD.

TNF polymorphism and bronchoalveolar lavage cell TNF-alpha levels in chronic beryllium disease and beryllium sensitization.

BACKGROUND: Beryllium stimulates TNF-alpha from chronic beryllium disease (CBD) bronchoalveolar lavage (BAL) cells. OBJECTIVE: We sought to relate TNF polymorphisms to beryllium-stimulated TNF-alpha production, to the development of CBD, and to the risk of more severe CBD over time. METHODS: We recruited 147 patients with CBD, 112 beryllium-sensitized subjects, and 323 control subjects; genotyped 5 TNF promoter polymorphisms; and measured beryllium-stimulated and unstimulated BAL cell TNF-alpha production from a subset of subjects. RESULTS: Beryllium-stimulated, but not beryllium-unstimulated, BAL cell TNF-alpha production was significantly increased in patients with CBD compared with that seen in those only sensitized (P = .0002). Those subjects with the TNF -857T allele and the only haplotype (haplotype 4) containing this allele demonstrated significantly lower unstimulated BAL cell TNF-alpha production compared with that seen in noncarriers (P = .009). Patients with CBD alone and combined with sensitized subjects carrying the TNF haplotype 1 compared with those without this haplotype had significantly increased beryllium-stimulated BAL cell TNF-alpha levels (P = .02). We found no significant association between patients with CBD, sensitized subjects, and control subjects with any of the TNF promoter polymorphisms or haplotypes. A greater decrease in Pao(2) at maximum exercise was noted in patients with CBD with the -1031C allele (P = .03) and with haplotypes other than the TNF haplotype 1 (P = .01), 3 (from 5) of which contain the -1031C allele. CONCLUSIONS: The -857T allele and haplotype 1 are associated with BAL cell TNF-alpha production, indicating a potential role of TNF promoter variants in regulation of TNF production in sensitized subjects and patients with CBD. CLINICAL IMPLICATIONS: TNF promoter variants are not risk factors for CBD or sensitization.

Beryllium-induced TNF-alpha production is transcription-dependent in chronic beryllium disease.

Beryllium (Be)-antigen presentation to Be-specific CD4(+) T cells from the lungs of patients with chronic beryllium disease (CBD) results in T cell proliferation and TNF-alpha secretion. We tested the hypothesis that Be-induced, CBD bronchoalveolar lavage (BAL) T cell, transcription-dependent, TNF-alpha secretion was accompanied by specific transcription factor upregulation. After 6 h of Be stimulation, CBD BAL cells produced a median of 883 pg/ml TNF-alpha (range, 608-1,275 pg/ml) versus 198 pg/ml (range, 116-245 pg/ml) by unstimulated cells. After 12 h CBD BAL cells produced a median of 2,963 pg/ml (range, 99-9,424 pg/ml) TNF-alpha versus 55 pg/ml (range, 0-454) by unstimulated cells. Using real-time RT-PCR, Be-stimulated TNF-alpha production at 6 h was preceded by a 5-fold increase in TNF-alpha pre-mRNA copy number:beta-actin copy number (Be median ratio 0.21; unstimulated median ratio 0.04). The median ratio of mature TNF-alpha mRNA:beta-actin mRNA was upregulated 1.4-fold (Be median ratio 0.17; unstimulated median ratio 0.12). Be exposure in the presence of the transcription inhibitor pentoxifylline (PTX) decreased CBD BAL cell TNF-alpha pre-mRNA levels > 60%, whereas treatment with the mRNA splicing inhibitor 2-aminopurine (2AP) decreased levels 40% relative to Be exposure alone. PTX treatment decreased mature TNF-alpha mRNA levels 50% while 2AP decreased levels > 80%, relative to Be exposure alone. Beryllium exposure specifically upregulated transcription factors AP-1 and NF-kappaB. The data suggest that Be exposure induces transcription-dependent TNF-alpha production, potentially due to upregulation of specific transcription factors.

Secondary ion mass spectroscopy demonstrates retention of beryllium in chronic beryllium disease granulomas.

OBJECTIVE: We hypothesized that beryllium (Be) might persist in lung granulomas in patients with chronic beryllium disease (CBD). METHODS: A total of 33 Be-exposed ceramics workers underwent transbronchial biopsy. They were classified based on histopathology and Be-lymphocyte proliferation test as CBD or other categories. Lung tissue sections were analyzed using secondary ion mass spectroscopy. RESULTS: Be was detected in the lungs of all Be-exposed groups. Be levels were increased within the granulomas of patients with CBD compared with the Be levels outside granulomas. Notably, Be was detectable in the lungs of CBD patients who had ceased exposure to Be an average of 9 years previously. CONCLUSIONS: Be was detected in the lungs of all Be-exposed subjects, with the highest levels of persistent Be inside CBD lung granulomas. Be antigen persistence may help explain the chronicity of this granulomatous disorder.

Beryllium-stimulated reactive oxygen species and macrophage apoptosis.

Beryllium (Be), the etiologic agent of chronic beryllium disease, is a toxic metal that induces apoptosis in human alveolar macrophages. We tested the hypothesis that Be stimulates the formation of reactive oxygen species (ROS) which plays a role in Be-induced macrophage apoptosis. Mouse macrophages were exposed to 100 microM BeSO4 in the absence and presence of the catalytic antioxidant MnTBAP (100 microM). Apoptosis was measured as the percentage of TUNEL+ and caspase-8+ cells. ROS production was measured by flow cytometry using the fluorescence probes, dihydroethidine (DHE) and dichlorofluorescein diacetate (DCFH-DA). Be-exposed macrophages had increased TUNEL+ cells (15+/-1% versus controls 1+/-0.2%, P<0.05) and increased caspase-8+ cells (18.7+/-2% versus controls 1.8+/-0.4%, P<0.05). Be-induced caspase-8 activation, and a 4-fold increase in ROS formation, was ameliorated by exposure to MnTBAP. Hydrogen peroxide (30 microM) exposure potentiated Be-induced caspase-8 activation, and was also attenuated by MnTBAP. Our data are the first to demonstrate that Be stimulates macrophage ROS formation which plays an important role in Be-induced macrophage apoptosis.

Beryllium-ferritin: lymphocyte proliferation and macrophage apoptosis in chronic beryllium disease.

A beryllium (Be)-ferritin adduct containing 270 pm of Be stimulated proliferation of bronchoalveolar lavage (BAL) lymphocytes from subjects with chronic beryllium disease (CBD) at concentrations 5-6 logs lower than the amounts of beryllium sulfate (BeSO4) needed to induce proliferation. We observed increased apoptotic CBD BAL macrophages after exposure to both BeSO4 (50 +/- 6%, mean +/- SEM, P <0.05 versus unstimulated controls) and Be-ferritin (40 +/- 2%), whereas only 2.0 +/- 0.2% of BAL lymphocytes underwent activation-induced cell death. Be-ferritin also induced apoptosis in BAL macrophages from subjects with Be sensitization (25 +/- 3%) and in the H36.12j hybrid macrophage cell line (15 +/- 2%). Be-ferritin induced lung macrophage CD95 (Fas) expression and the activation of intracellular caspase-3, -8 and -9. Thus, lung macrophages take up Be-ferritin, delivering physiologically relevant levels of Be that promote Be antigen presentation and macrophage apoptosis. Be-ferritin thereby serves as a "Trojan Horse," triggering proliferation of Be-ferritin-specific CBD BAL T cells. We hypothesize that Be-ferritin exposure may result in persistent antigen exposure inducing Be-specific T cell clonal expansion and T cell helper type 1-type cytokine production and potentially explains the chronicity of CBD and its development years after environmental Be exposure has ceased.

Beryllium-induced tumor necrosis factor-alpha production by CD4+ T cells is mediated by HLA-DP.

Beryllium (Be) presentation to CD4+ T cells from patients with chronic beryllium disease (CBD) results in T cell activation, and these Be-specific CD4+ T cells undergo clonal proliferation and T-helper 1-type cytokine production. In exposed workers, genetic susceptibility to this granulomatous disorder is associated with particular HLA-DPB1 alleles. We hypothesized that these HLA-DP molecules could mediate Be-stimulated tumor necrosis factor-alpha (TNF-alpha) messenger RNA (mRNA) and protein production. Using intracellular cytokine staining, we found that treatment with an anti-HLA-DP, but not anti-HLA-DR, monoclonal antibody inhibited Be-stimulated TNF-alpha expression in lung CD3+ CD4+ T cells. This monoclonal antibody also blocked Be-specific T cell proliferation, increased production of TNF-alpha mature-mRNA transcripts, and increased TNF-alpha protein production by Be-stimulated CBD peripheral blood mononuclear cells and bronchoalveolar lavage (BAL) cells. The Be-stimulated upregulation of TNF-alpha mature-mRNA levels with TNF-alpha protein production was a unique property of CBD BAL cells, and did not occur in BAL cells from Be-sensitized patients without CBD, or sarcoidosis BAL cells. This study identifies HLA-DP as a regulatory component in the activation of T cell receptors on Be-specific CD4+ T cells from CBD patients resulting in proliferation and proinflammatory cytokine production.

Influence of MHC class II in susceptibility to beryllium sensitization and chronic beryllium disease.

A glutamic acid at residue 69(Glu(69)) in the HLA-DPB1 gene (Glu(69)) is associated with chronic beryllium disease (CBD) and possibly beryllium sensitization (BeS). This study tested the hypothesis that MHC class II polymorphisms are important in susceptibility to BeS and CBD and that the Glu(69) variant is related to markers of disease severity. Genomic DNA was obtained from BeS (n = 50), CBD (n = 104), and beryllium-exposed nondiseased (Be-nondiseased) (n = 125) subjects. HLA-DPB1, -DRB1, and -DQB1 genotypes were determined by (sequence-specific primers) PCR. Disease severity was assessed by pulmonary function and exercise testing. A higher frequency of the DPB1 Glu(69) gene was found in CBD and BeS compared with the Be-nondiseased subjects, with odds ratios of 10.1 for CBD vs Be-nondiseased and 9.5 for BeS vs Be-nondiseased. The majority of BeS and CBD subjects displayed non-0201 Glu(69) alleles. Glu(69) homozygosity was higher in the CBD subjects, while BeS subjects were intermediate and Be-nondiseased lowest. DRB1*01 and DQB1*05 phenotypes were reduced in CBD vs Be-nondiseased subjects, while DRB1*13 and DQB1*06 were associated with CBD in the absence of Glu(69). Markers of disease severity, including a lower forced vital capacity, diffusion capacity for carbon monoxide, PaO(2) at rest, maximum workload on exercise testing, and a higher arterial-alveolar gradient at rest, were associated with Glu(69) homozygosity. We conclude that DPB1 Glu69 is a marker of sensitization and not specific for disease. Glu(69) homozygosity acts as a functional marker associated with markers of CBD severity.

Beryllium induces apoptosis in human lung macrophages.

The link between metal-induced apoptosis and granulomatous inflammation in human disease pathogenesis is not established. The presence of TUNEL positive nuclei in chronic beryllium disease (CBD) pulmonary granulomas suggested the possibility that beryllium (Be) could induce apoptosis in adherent macrophages from CBD bronchoalveolar (BAL) cells. Apoptosis was measured in un-stimulated and Be-stimulated BAL adherent macrophages from CBD (n = 21) and Be-sensitized (BeS, n = 16) subjects. Be-stimulated CBD and BeS macrophages underwent caspase-dependent nuclear fragmentation, cytoplasmic membrane blebbing and CD14 loss. Be-stimulated adherent macrophage apoptosis was not due to TNF-alpha production. Apoptosis, CD14 loss and TNF-alpha production were not observed in unstimulated BAL macrophages. Thus, Be-stimulated BAL adherent macrophage apoptosis occurred whether cells were derived from patients with granulomatous inflammation or not, was caspase-mediated and occurred independent of TNF-alpha levels. We conclude that Be-induced human lung adherent macrophage apoptosis could contribute to the host's continued re-exposure to Be resulting in chronic granulomatous inflammation.

Chronic beryllium disease: a model interaction between innate and acquired immunity.

Beryllium (Be) is a lightweight and durable metal useful to a variety of manufacturing processes. With the use of Be in industrial settings, a number of health effects were noted including acute pneumonitis, sensitization to Be, interstitial lung disease and dermatological disease. Interstitial mononuclear cell inflammation and granuloma formation are the primary processes that occur in the lungs of Be-exposed workers, resulting in chronic beryllium disease (CBD). Recent studies have begun to describe the role of Be in the pathogenesis of CBD. These studies reveal that the host's response to Be involves components of the innate immune system or inflammatory responses. Inflammatory responses to Be can establish a state of acquired, Be antigen-specific, cell-mediated immunity. Despite triggering both the innate and acquired immune responses, Be is not eliminated from the host. Rather, it establishes pathways leading to chronic granulomatous inflammation. We will examine recent studies describing the host's cellular and molecular responses to Be, responses that promote granuloma formation.