Characterization of myeloid and plasmacytoid dendritic cells in human lung

Dendritic cells (DCs) are bone marrow-derived mononuclear cells that play a central role in the initiation of immune responses. Because human lung DCs have been incompletely characterized, we enumerated and phenotyped mononuclear cell populations from excess lung tissue obtained at surgery. Myeloid DCs (MDCs) were identified as CD1c(+)CD11c(+)CD14(-)HLA-DR(+) cells and comprised approximately 2% of low autofluorescent (LAF) mononuclear cells. Plasmacytoid DCs (PDCs) were characterized as CD123(+)CD11c(-)CD14(-)HLA-DR(+) cells and comprised approximately 1.0% of the LAF mononuclear cells. Cells enriched in MDCs expressed CD86, moderate CD80, and little CD40, but cells enriched in PDCs had little to no expression of these three costimulatory molecules. CD11c(+)CD14(-) lineage-negative (MDC-enriched) LAF cells were isolated and shown to be much more potent in stimulating an alloreaction than CD11c(+)CD14(+) lineage-negative (monocyte-enriched) LAF cells. PDC-enriched cells were more capable of responding to a TLR-7 agonist by secreting IFN-alpha than MDC-enriched cells. MDC-enriched cells were either CD123(+) or CD123(-), but both subsets secreted cytokines and chemokines typical of MDC upon stimulation with a TLR-4 agonist and both subsets failed to secrete IFN-alpha upon stimulation with a TLR-7 agonist. By immunohistochemistry, we identified MDCs throughout different anatomical locations of the lung. However, our method did not allow the localization of PDCs with certainty. In conclusion, in the human lung MDCs were twice as numerous and expressed higher levels of costimulatory molecules than PDCs. Our data suggest that both lung DC subsets exert distinct immune modulatory functions.     

Chemokine-like receptor 1 expression and chemerin-directed chemotaxis distinguish plasmacytoid from myeloid dendritic cells in human blood

Plasmacytoid dendritic cells (pDCs) are versatile cells of the immune response, secreting type I IFNs and differentiating into potent immunogenic or tolerogenic APCs. pDCs can express adhesion and chemokine receptors for lymphoid tissues, but are also recruited by unknown mechanisms during tissue inflammation. We use a novel mAb specific for serpentine chemokine-like receptor 1 (CMKLR1) to evaluate its expression by circulating leukocytes in humans. We show that CMKLR1 is expressed by circulating pDCs in human blood, whereas myeloid DCs (mDCs) as well as lymphocytes, monocytes, neutrophils, and eosinophils are negative. We identify a major serum agonist activity for CMKLR1 as chemerin, a proteolytically activated attractant and the sole known ligand for CMKLR1, and we show that chemerin is activated during blood coagulation and attracts pDC but not mDC in ex vivo chemotaxis assays. We conclude that CMKLR1 expression and chemerin-mediated chemotaxis distinguish circulating pDCs from mDCs, providing a potential mechanism for their differential contribution to or regulation of immune responses at sites of bleeding or inflammatory protease activity.     

Differential susceptibility to human immunodeficiency virus type 1 infection of myeloid and plasmacytoid dendritic cells

Human immunodeficiency virus type 1 (HIV-1) infection of dendritic cells (DCs) plays an important role in HIV-1 transmission and pathogenesis. Here, we studied the susceptibility of ex vivo-isolated CD11c+ myeloid DCs (MDCs) and CD123+ plasmacytoid DCs (PDCs) to HIV-1 infection and the function of these cells early after infection. Both DC subsets were susceptible to CCR5- and CXCR4-using HIV-1 isolates (BaL and IIIB, respectively). However, MDCs were more susceptible to HIV-1(BaL) infection than donor-matched PDCs. In addition, HIV-1(BaL) infected MDCs more efficiently than HIV-1(IIIB), whereas PDCs were equally susceptible to both isolates. While exposure to HIV-1 alone resulted in only weak maturation of DCs, Toll-like receptor 7/8 ligation induced full maturation in both infected and uninfected DCs. Maturation did not increase HIV-1 replication in infected DCs, and infected DCs retained their ability to produce tumor necrosis factor alpha after stimulation. Both HIV-1 isolates induced alpha interferon production exclusively in PDCs, irrespective of productive infection. In conclusion, PDCs and MDCs were susceptible to HIV-1 infection, but neither displayed functional defects as a consequence of infection. The difference in susceptibility of PDCs and MDCs to HIV-1 may have implications for HIV-1 transmission and DC-mediated transfer of HIV-1 to T cells.     

Proteomics of bronchoalveolar lavage fluid

Lung diseases are essentially multi-factorial diseases that require a global analysis, and thus, cannot be understood through the sole analysis of individual or small numbers of genes. Proteome analysis has rapidly developed in the post-genome era and is now widely accepted as the obligated complementary technology for genetic profiling. It has been shown to be a powerful tool for the study of human diseases and for identifying novel prognostic, diagnostic and therapeutic markers. During last years, proteomic approaches applied to lung diseases are centred on the analysis of proteins in samples, such as cell cultures, biopsies and physiological fluids like serum and, especially, bronchoalveolar lavage fluid (BALF). BALF is presently the most common way of sampling the components of the epithelial lining fluid (ELF) and the most faithful reflect of the protein composition of the pulmonary airways. This review focuses on the state of the investigations of BALF proteome and its powerful contribution both to a better knowledge of the lung structure at the molecular level and to the study of lung disorders at the clinical level.     

LC-MS analysis of phospholipids and lysophospholipids in human bronchoalveolar lavage fluid

A reversed phase HPLC method was developed for the simultaneous analysis of different phospholipids and lysophospholipids in human bronchoalveolar lavage fluid (BALF). Separation was achieved using a pellicular C8 column at elevated temperatures with an increasing gradient of acetonitrile containing 0.1% formic acid. Detection was carried out by electrospray ionization ion-trap mass spectrometry. Calibration graphs for selected phospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and lysophosphatidylcholine) showed linearity up to 50 ng allowing quantitative determinations. Identification of the individual species within each class was possible with tandem mass spectrometry. Analysis of BALF phospholipids was performed after liquid/liquid extraction with a mixture of chloroform/methanol/acetic acid. Recoveries ranged from 69 to 97% with standard deviations of less than 6%. The limit of detection varied slightly between different classes but was in the range 0.05-0.25 ng total injected amount.     

Cutting edge: differential chemokine production by myeloid and plasmacytoid dendritic cells

To examine the different roles of myeloid dendritic cells (M-DCs) and plasmacytoid dendritic cells (P-DCs) in the induction and regulation of immune response, we have studied chemokine secretion by freshly isolated DC subsets in response to bacterial, viral, and T cell-derived stimuli. M-DCs selectively produced very high levels of the homeostatic chemokines CC chemokine ligand (CCL)17 and CCL22, while P-DCs produced very little if any. In contrast, the proinflammatory chemokine CCL3 was secreted mostly by P-DCs, whereas CCL4 and CXC chemokine ligand 8 were produced by both subsets. The selective production of CCL17 and CCL22 by M-DCs but not P-DCs was confirmed in vivo by immunohistology on human reactive lymph node sections. The high production of CCR4 ligands by M-DCs suggests their capacity to selectively recruit at sites of inflammation T cells with regulatory properties or with a Th2 phenotype, whereas P-DCs, by preferentially secreting CCR1/CCR5 ligands, would mostly recruit effector T cells and, in particular, Th1-type cells.     

Parallel loss of myeloid and plasmacytoid dendritic cells from blood and lymphoid tissue in simian AIDS

The loss of myeloid (mDC) and plasmacytoid dendritic cells (pDC) from the blood of HIV-infected individuals is associated with progressive disease. It has been proposed that DC loss is due to increased recruitment to lymph nodes, although this has not been directly tested. Similarly as in HIV-infected humans, we found that lineage-negative (Lin(-)) HLA-DR(+)CD11c(+)CD123(-) mDC and Lin(-)HLA-DR(+)CD11c(-)CD123(+) pDC were lost from the blood of SIV-infected rhesus macaques with AIDS. In the peripheral lymph nodes of SIV-naive monkeys the majority of mDC were mature cells derived from skin that expressed high levels of HLA-DR, CD83, costimulatory molecules, and the Langerhans cell marker CD1a, whereas pDC expressed low levels of HLA-DR and CD40 and lacked costimulatory molecules, similar to pDC in blood. Surprisingly, both DC subsets were depleted from peripheral and mesenteric lymph nodes and spleens in monkeys with AIDS, although the activation status of the remaining DC subsets was similar to that of DC in health. In peripheral and mesenteric lymph nodes from animals with AIDS there was an accumulation of Lin(-)HLA-DR(moderate)CD11c(-)CD123(-) cells that resembled monocytoid cells but failed to acquire a DC phenotype upon culture, suggesting they were not DC precursors. mDC and pDC from the lymphoid tissues of monkeys with AIDS were prone to spontaneous death in culture, indicating that apoptosis may be a mechanism for their loss in disease. These findings demonstrate that DC are lost from rather than recruited to lymphoid tissue in advanced SIV infection, suggesting that systemic DC depletion plays a direct role in the pathophysiology of AIDS.     

Database of bronchoalveolar lavage fluid proteins

Bronchoalveolar lavage during fiberoptic bronchoscopy is extensively used for investigating cellular and biochemical alterations of the epithelial lining fluid in various lung disorders. Two-dimensional electrophoresis (2-DE) offers the possibility to simultaneously display and analyze proteins contained in bronchoalveolar lavage fluid (BALF). We present the current status of 2-DE of BALF samples with an updated listing of the proteins already identified and of their level and/or posttranslational alterations in lung disorders. Alternatives to 2-DE of BALF samples and future prospects of proteomics to unravel lung functions and pathologies are discussed.     

Galactomannan detection in bronchoalveolar lavage fluid

Prompt diagnosis of invasive pulmonary aspergillosis (IPA) remains a challenge. Galactomannan (GM) assay in serum has been incorporated into diagnostic criteria for IPA, but its performance varies depending on the population in which the test is used. GM assay on bronchoalveolar lavage (BAL) fluid aims to improve upon the test by applying it directly on samples from the target organ. The studies that have examined the utility of BAL-GM are a heterogeneous group, but the results are intriguing, especially in patients who are at risk for IPA from causes other than hematologic malignancy and neutropenia. BAL-GM had sensitivities ranging from 60% to 100% in this group, often far exceeding the performance of serum GM assay. The test shows promise as a useful adjunctive diagnostic modality in the diagnosis of IPA.     

Penicillium marneffei in bronchoalveolar lavage fluid

Penicillium marneffei is a rare human pathogen that may infect either healthy or immunocompromised hosts. In methenamine silver-stained preparations of bronchoalveolar lavage fluid from a patient with dermatomyositis on steroid treatment, round-to-oval intracellular and extracellular microorganisms were found. The finding of occasional septate and elongated forms established the microorganism as probably P marneffei, which was confirmed on culture. Distinguishing this rare fungus from Pneumocystis carinii is important because these two diseases require different forms of treatment.     

Harnessing human plasmacytoid dendritic cells as professional APCs

The plasmacytoid dendritic cell (pDC) constitutes a unique DC subset that links the innate and adaptive arm of the immune system. Whereas the unique capability of pDCs to produce large amounts of type I IFNs in response to pathogen recognition is generally accepted,their antigen-presenting function is often neglected since most studies on antigen presentation are aimed at other DC subsets. Recently, pDCs were demonstrated capable to present antigen leading to protective tumor immunity. In this review, we discuss how pDCs could be exploited in the fight against cancer by analyzing their capacity to capture,process and (cross-) present antigen.     

Impact of neutrophils on antiviral activity of human bronchoalveolar lavage fluid

Surfactant protein D (SP-D) and neutrophils participate in the early innate immune response to influenza A virus (IAV) infection. SP-D increases neutrophil uptake of IAV and modulates neutrophil respiratory burst responses to IAV; however, neutrophil proteases have been shown to degrade SP-D, and human neutrophil peptide defensins bind to SP-D and can cause precipitation of SP-D from bronchoalveolar lavage fluid (BALF). BALF has significant antiviral activity against IAV. We first added neutrophils to BALF during incubation with IAV. Addition of neutrophils to BALF caused significantly greater clearance of IAV from culture supernatants than from BALF alone, and this effect was significantly more pronounced when neutrophils were activated during incubation with the virus. In contrast, if activated neutrophils were incubated with BALF before addition of virus, they reduced antiviral activity of BALF. This effect correlated with depletion of SP-D from BALF. Activation of neutrophils with agonists that induce primary granule release (including release of human neutrophil peptide defensins) caused SP-D depletion, but activation with PMA, which causes only secondary granule release, did not. The ability of activated neutrophils to deplete SP-D from BALF was partially, but not fully, corrected with protease inhibitors but was unaffected by inhibition of neutrophil respiratory burst responses. These results suggest that chronic neutrophilic inflammation (e.g., as in chronic smoking or cystic fibrosis) may reduce SP-D levels and predispose to IAV infection. In contrast, acute inflammation, as occurs in the early phase of IAV infection, may promote neutrophil-mediated viral clearance.     

Expression of Fas antigen in the cells from bronchoalveolar lavage fluid (BALF)

Fas antigen is a cell surface receptor protein that mediates apoptosis expressed in various cells. In this study Fas expression was examined in cells of patients with lung diseases in which changes in the lung immunology were documented. We have performed bronchoalveolar lavage (BAL) in 24 patients with sarcoidosis (8), lung fibrosis (9), primary lung cancer (7), and we compared expression of Fas in BALF cells from all groups and healthy volunteers (6). Fas protein was detected by immunocytochemistry using APAAP technique with an LSAB 2 kit (Dako). Positive reactions for Fas were found in the cytoplasm of epithelial cells, macrophages, neutrophils and lymphocytes (according to the intensity). There were some differences in proportion of positive cells and intensity of reaction between patients with interstitial lung diseases, healthy volunteers as well as patients with lung cancer. Higher expression of Fas in alveolar macrophages was observed in patients with sarcoidosis, lower in patients with lung cancer, lung fibrosis and the lowest in healthy persons. The analysis of Fas antigen expression in the BALF cells may be useful in evaluation of the role of apoptosis in lung homeostasis and pathology.     

Cysteine proteinases in bronchoalveolar epithelial cells and lavage fluid of rat lung

We examined the presence of cathepsins B, H, and L in bronchoalveolar epithelial cells, including alveolar macrophages, and in bronchoalveolar lavage fluid (BALF), using immunocytochemistry and immunoblotting. By light and electron microscopy, immunoreactivity for cathepsins B, H, and L was detected in lysosomes of ciliated and non-ciliated epithelial cells of bronchi and bronchioles, and in macrophages. Immunodeposits for cathepsin H only were demonstrated in lamellar bodies of Type II alveolar epithelial cells, suggesting the cosecretion of surfactants and cathepsin H from the cells into the alveolar space. By immunoblotting, cathepsins B and H were found to be present in BALF. To further investigate the origin of these enzymes in BALF, alveolar macrophages obtained from BALF were cultured for 6 hr in a serum-free medium. Immunoblotting revealed that protein bands corresponding to the pro-form and mature form of cathepsin B and the mature form of cathepsin H were present in the culture medium. From these results, the presence of cathepsins B and H in BALF can be explained by the fact that cathepsin B is secreted from alveolar macrophages and cathepsin H is secreted mainly with surfactants from Type II cells and also from macrophages.     

Metabolomic analysis of bronchoalveolar lavage fluid from cystic fibrosis patients

Metabolite profiles of bronchoalveolar lavage fluid (BALF) from pediatric patients with cystic fibrosis (CF) were correlated to the degree of airway inflammation using nuclear magnetic resonance spectroscopy-based metabolomics. BALF was collected from 11 children with CF during clinically indicated bronchoscopy. The spectra from BALF with high levels of neutrophilic airway inflammation displayed signals from numerous metabolites, whereas the spectra from subjects with low levels of inflammation were very sparse. The metabolites identified in samples taken from subjects with high inflammation include known markers of inflammation such as amino acids and lactate, as well as many novel signals. Statistical analysis highlighted the most important metabolites that distinguished the high- from the low-inflammation groups. This first demonstration of metabolomics of human BALF shows that clear distinctions in the metabolic profiles can be observed between subjects experiencing high versus low inflammation and is a first step toward the goal of discovering novel biomarkers of airway inflammation.     

Human immunodeficiency virus type 1 activates plasmacytoid dendritic cells and concomitantly induces the bystander maturation of myeloid dendritic cells

In this study, we analyzed the phenotypic and physiological consequences of the interaction of plasmacytoid dendritic cells (pDCs) with human immunodeficiency virus type 1 (HIV-1). pDCs are one cellular target of HIV-1 and respond to the virus by producing alpha/beta interferon (IFN-alpha/beta) and chemokines. The outcome of this interaction, notably on the function of bystander myeloid DC (CD11c+ DCs), remains unclear. We therefore evaluated the effects of HIV-1 exposure on these two DC subsets under various conditions. Blood-purified pDCs and CD11c+ DCs were exposed in vitro to HIV-1, after which maturation markers, cytokine production, migratory capacity, and CD4 T-cell stimulatory capacity were analyzed. pDCs exposed to different strains of infectious or even chemically inactivated, nonreplicating HIV-1 strongly upregulated the expression of maturation markers, such as CD83 and functional CCR7, analogous to exposure to R-848, a synthetic agonist of toll-like receptor-7 and -8. In addition, HIV-1-activated pDCs produced cytokines (IFN-alpha and tumor necrosis factor alpha), migrated in response to CCL19 and, in coculture, matured CD11c+ DCs, which are not directly activated by HIV. pDCs also acquired the ability to stimulate na?ve CD4+ T cells, albeit less efficiently than CD11c+ DCs. This HIV-1-induced maturation of both DC subsets may explain their disappearance from the blood of patients with high viral loads and may have important consequences on HIV-1 cellular transmission and HIV-1-specific T-cell responses.     

Activation of plasmacytoid dendritic cells with TLR9 agonists initiates invariant NKT cell-mediated cross-talk with myeloid dendritic cells

CD1d-restricted invariant NK T (iNKT) cells and dendritic cells (DCs) have been shown to play crucial roles in various types of immune responses, including TLR9-dependent antiviral responses initiated by plasmacytoid DCs (pDCs). However, the mechanism by which this occurs is enigmatic because TLRs are absent in iNKT cells and human pDCs do not express CD1d. To explore this process, pDCs were activated with CpG oligodeoxyribonucleotides, which stimulated the secretion of several cytokines such as type I and TNF-alpha. These cytokines and other soluble factors potently induced the expression of activation markers on iNKT cells, selectively enhanced double-negative iNKT cell survival, but did not induce their expansion or production of cytokines. Notably, pDC-derived factors licensed iNKT cells to respond to myeloid DCs: an important downstream cellular target of iNKT cell effector function and a critical contributor to the initiation of adaptive immune responses. This interaction supports the notion that iNKT cells can mediate cross-talk between DC subsets known to express mutually exclusive TLR and cytokine profiles.     

Activation of plasmacytoid dendritic cells

Four years after the discovery of mouse plasmacytoid dendritic cells (pDC), pDC are still very much an 'enigmatic' cell type. It is clear that pDC are potent producers of type I IFN in response to viral, bacterial and even mammalian nucleotides. The role that they play in vivo before and after activation is still under scrutiny. This review concentrates on the pathways to activation of pDC, examining the activating ligands, receptors and signalling molecules that are known to be involved, and the relevance of these activation pathways to human disease.     

Detection of lipid peroxidation in lung and in bronchoalveolar lavage cells and fluid

Inhalation of toxic materials such as asbestos, silica, 100% oxygen, ozone, or nitrogen dioxide may lead to an increased production of reactive oxygen metabolites which may initiate lipid peroxidation. Measurement of lipid peroxidation in cells and fluid obtained by bronchoalveolar lavage (BAL), as well as in lung tissue, may aid in monitoring the development and extent of pulmonary damage after inhalation of a toxic substance. In this study, we employed a sensitive assay for detection of malondialdehyde (MDA), a breakdown product of lipid peroxidation. By separation of the adduct with thiobarbituric acid, using a reverse phase high pressure liquid chromatographic technique, we accurately and sensitively measured the content of MDA in BAL cells, lavage fluid, and lavaged lung tissue homogenates of rats. The amounts of sample required for detection of MDA were small enough possibly to be applied to use with human specimens; in addition, recovery of added MDA was acceptable with all types of samples. Inclusion of a metal chelator in the preparation of samples appeared necessary to prevent metal-catalyzed propagation of lipid peroxidation during the assay. Overall, the method described here using samples from rats may be applicable to detecting lipid peroxidation in BAL samples from humans.     

Hemosiderin-laden macrophages in bronchoalveolar lavage fluid.

Diffuse pulmonary hemorrhage syndromes occasionally present diagnostic problems if the clinical picture is not very typical. The presence of hemosiderin-laden alveolar macrophages in bronchoalveolar lavage (BAL) fluid is a useful diagnostic criterion for this entity. However, in many diffuse interstitial pulmonary diseases (DIPD) there is a lesion at the alveolocapillary barrier, and an exit of red blood cells could occur from the blood vessels, leading to the appearance of siderophages. The aim of this work was dual: to evaluate the presence and number of siderophages in different types of interstitial pulmonary disease and to compare the diagnostic yield of two ways of quantifying hemosiderin-laden macrophages. Three groups of patients--controls (n = 5), DIPD (n = 32) and diffuse pulmonary hemorrhage (n = 3)--were subjected to BAL, and a differential count was made on cytocentrifuged Diff-Quik- and Perl-stained preparations. On the latter, two different measurements were made: the number of macrophages laden with hemosiderin and a quantitative "score." The results of a conventional count (percent of Perl-positive macrophages) showed significant differences between the three groups considered overall. Applying a cutoff value of 20%, the sensitivity of this method was 100% and the specificity, 91.6%. The results of the hemosiderin score showed significant differences between the three groups. Applying a value of 50 as a cutoff, the sensitivity and specificity of the method were 100%. In control patients and carriers of DIPD, the percentage of alveolar macrophages was higher than in healthy subjects. Quantification of the hemosiderin content of alveolar macrophages improved the specificity of the diagnosis of diffuse pulmonary hemorrhage by BAL.