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Rationale: Endothelial colony forming cells (ECFCs) or late blood outgrowth endothelial cells can be isolated from human cord or peripheral blood, display properties of endothelial progenitors, home into ischemic tissues and support neovascularization in ischemic disease models. Objective: To assess the functions of CYTL1 (cytokine-like 1), a factor we found preferentially produced by ECFCs, in regard of vessel formation. Methods and Results: We show by transcriptomic analysis that ECFCs are distinguished from endothelial cells of the vessel wall by production of high amounts of CYTL1. Modulation of expression demonstrates that the factor confers increased angiogenic sprouting capabilities to ECFCs and can also trigger sprouting of mature endothelial cells. The data further display that CYTL1 can be induced by hypoxia and that it functions largely independent of VEGF-A (vascular endothelial growth factor-A). By recombinant production of CYTL1 we confirm that the peptide is indeed a strong proangiogenic factor and induces sprouting in cellular assays and functional vessel formation in animal models comparable to VEGF-A. Mass spectroscopy corroborates that CYTL1 is specifically O-glycosylated on 2 neighboring threonines in the C-terminal part and this modification is important for its proangiogenic bioactivity. Further analyses show that the factor does not upregulate proinflammatory genes and strongly induces several metallothionein genes encoding anti-inflammatory and antiapoptotic proteins. Conclusions: We conclude that CYTL1 can mediate proangiogenic functions ascribed to endothelial progenitors such as ECFCs in vivo and may be a candidate to support vessel formation and tissue regeneration in ischemic pathologies.

Interleukin-3 is a growth and differentiation factor for various hematopoietic cells. IL-3 also enhances stimulus-dependent release of mediators and cytokine production by mature basophils. Function of IL-3 has not been studied in horses because of lack of horse-specific reagents. Our aim was to produce recombinant equine IL-3 and test its effect on sulfidoleukotriene and cytokine production by equine peripheral blood leukocytes (PBL). Equine IL-3 was cloned, expressed in E. coli and purified. PBL of 19 healthy and 20 insect bite hypersensitivity (IBH)-affected horses were stimulated with Culicoides nubeculosus extract with or without IL-3. Sulfidoleukotriene (sLT) production was measured in supernatants by ELISA and mRNA expression of IL-4, IL-13 and thymic stromal lymphopoietin (TSLP) assessed in cell lysate by quantitative real-time PCR. Recombinant equine IL-3 (req-IL-3) had a dose dependent effect on sLT production by stimulated equine PBL and significantly increased IL-4, IL-13 and TSLP expression compared to non-primed cells. IL-3 priming significantly increased Culicoides-induced sLT production in IBH-affected but not in non-affected horses and was particularly effective in young IBH-affected horses (≤ 3 years). A functionally active recombinant equine IL-3 has been produced which will be useful for future immunological studies in horses. It will also allow improving the sensitivity of cellular in vitro tests for allergy diagnosis in horses.

Background: T helper (Th)17/22 cells may play a role in allergic asthma. Recent findings showed increased mRNA expression for IL-22 in peripheral blood mononuclear cells (PBMC) from patients with asthma. Thereby, the role of the natural antagonist of IL-22, IL-22 binding protein (IL-22 BP), a soluble receptor for IL-22, remains to be elucidated. In this study, we investigate the expression of mRNA for IL-22 BP in individuals with asthma. Methods: We assessed the effect of allergen inhalation challenge on mRNA-expression for IL-22 and IL-22 BP, and IL-22 receptor in mononuclear cells of the broncheoalveolar fluid (BALF) from individuals with allergic asthmatic. Furthermore, in vitro we investigated mRNA expression for IL-22 and IL-22 BP, and IL 22 receptor after costimulation of peripheral blood mononuclear cells (PBMC) from individuals with asthma and normal controls with Th2 cytokines IL-4, IL-9, and GM-CSF in a time-dependent manner. Results: The expression of mRNA for IL-22-BP in PBMCs from healthy controls and individuals with asthma could be found after co-stimulation with IL-4 after a minimum of 12 hours, which hold on for 96 hours. The expression of mRNA for IL-22 BP in subjects with atopic asthma was discovered in mononuclear cells of the BALF after segmental allergen inhalation challenge. Our findings suggest a possible role for IL-22 BP in allergen-induced airway responses.

Background aims The potential of cultured adipose-derived stem cells (ASC) in regenerative medicine and new cell therapeutic concepts has been shown recently by many investigations. However, while the method of isolation of ASC from liposuction aspirates depending on plastic adhesion is well established, a standard expansion medium optimally maintaining the undifferentiated state has not been described. Methods We cultured ASC in five commonly used culture media (two laboratory-made media and three commercially available media) and compared them with a standard medium. We analyzed the effects on cell morphology, proliferation, hepatocyte growth factor (HGF) expression, stem cell marker profile and differentiation potential. Proliferation was measured with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and a fluorescent assay. Release of HGF was assessed by an immunoassay. Expression of characteristic stem cell-related transcription factors and markers was evaluated by quantitative polymerase chain reaction (qPCR) (Nanog, Sox-2, Rex-1, nestin and Oct-4) and flow cytometry (CD44, CD73, CD90, CD105 and CD166), and differentiation was shown by adipogenic medium. Results The morphology and expansion of ASC were significantly affected by the media used, whereas none of the media influenced the ASC potential to differentiate into adipocytes. Furthermore, two of the media induced an increase in expression of transcription factors, an increased secretion of HGF and a decrease in CD105 expression. Conclusions Culture of ASC in one of these two media before using the cells in cell therapeutic approaches may have a benefit on their regenerative potential.

High hopes are put into the use of mesenchymal stem cells (MSCs) in various approaches for tissue engineering and regenerative medicine. MSCs are derived from different tissues with only small differences in their phenotype or their differentiation potential, but higher differences in the cell yield. Since fat is easily accessible and contains a high amount of MSCs to be isolated, adipose-derived stem cells (ASCs) are very promising for clinical approaches. ASCs are not a completely homogeneous cell population. Our study was initiated to explore an easy and convenient method to reduce heterogeneity. We tested different isolation methods: (1) the standard isolation method for ASCs based on plastic attachment, (2) the standard method with an initial washing step after 60 min of adherence and (3) immunomagnetic isolation by 4 typical markers (CD49a, CD90, CD105 and CD271). Cells isolated by these methods were evaluated using quantitative PCR and flow cytometry as well as by their differentiation potential. Washing led to a significantly lower expression of desmin, smA and six2, and a higher expression of the stem cell markers nestin, oct-4 and sall-1, compared to standard isolated cells, while the immunomagnetically isolated cells showed no significant changes. All cells independent of the isolation method could be induced to differentiate into adipocytes and osteoblasts. Our study demonstrates that a simple washing step reduces heterogeneity of cultured ASCs according to PCR analysis, whereas the immunomagnetic isolation only showed minor advantages compared to the standard method, but the disadvantage of significantly lower cell yields in the primary isolates.

Extracellular ATP serves as a danger signal to alert the immune system of tissue damage by acting on P2X or P2Y receptors. Here we show that allergen challenge causes acute accumulation of ATP in the airways of asthmatic subjects and mice with experimentally induced asthma. All the cardinal features of asthma, including eosinophilic airway inflammation, Th2 cytokine production and bronchial hyper-reactivity, were abrogated when lung ATP levels were locally neutralized using apyrase or when mice were treated with broad-spectrum P2-receptor antagonists. In addition to these effects of ATP in established inflammation, Th2 sensitization to inhaled antigen was enhanced by endogenous or exogenous ATP. The adjuvant effects of ATP were due to the recruitment and activation of lung myeloid dendritic cells that induced Th2 responses in the mediastinal nodes. Together these data show that purinergic signaling has a key role in allergen-driven lung inflammation that is likely to be amenable to therapeutic intervention.

Allergic asthma is a chronic inflammatory T helper 2 (Th2)-associated disease. There is evidence that the atopic milieu affects the development of CD8(+) T cells in patients. We therefore analysed activation and differentiation states of CD8(+) T cells in asymptomatic patients regarding the cytomegalovirus serological status. Memory CD8(+) T cells (CCR5(high)CD3(+)CD8(+)), memory/effector cells (CD27(+)CD28(-)CD3(+)CD8(+)), effector cells (CD27(-)CD28(-)CD3(+)CD8(+)) and activated CD8(+) T cells (CD11b(+)CD3(+)CD8(+)) were identified by flow cytometry in peripheral blood of 19 (seven cytomegalovirus (CMV)(+)/12 CMV(-)) patients with allergic asthma (AA) and 21 (seven CMV(+)/14 CMV(-)) healthy controls (HC). Effector and activated CD8(+) T cells were significantly elevated in CMV(+) HC compared to CMV(-) HC. There was a non-significant trend for reduced percentages of effector CD8(+) T cells in CMV(+) AA (median: 10.4%, range: 4.4-33.8%) compared to CMV(+) HC (median: 23.1%, range: 10.7-54.1%; P = 0.128) and in CMV(-) AA (median: 4.1%, range: 0.6-13.4%) compared to CMV(-) HC (median: 5.7%, range: 0.2-17.0%; P = 0.085). Activated CD8(+) T cells were reduced significantly in CMV(+) AA (median: 17.0%, range: 6.0-29.4%) compared to CMV(+) HC (median: 40.4%, range: 18.9-67.0%; P = 0.004) and showed a non-significant trend in CMV(-) AA (median: 15.0%, range: 2.9-24.0%) compared to CMV(-) HC (median: 20.2%, range: 5.8-71.0%; P = 0.060). Activated CD8(+) T cells are significantly reduced in CMV(+) patients with allergic asthma. Furthermore, a trend for an impaired terminal CD8(+) T cell differentiation is observed in CMV(+) and CMV(-) patients with asthma.

Background: Dendritic cells control pulmonary immune reactions. Characteristics of dendritic cells in human bronchoalveolar lavage fluid (BALF) after allergen challenge are unknown. Methods: 7 patients with allergic asthma (median 23 years, range 19–25 years) underwent segmental challenge and were lavaged 10 min and 24 h after challenge. Dendritic cell subsets and surface markers in BALF and in peripheral blood were analysed using four-colour flow cytometry. Results: Plasmacytoid dendritic cells (pDCs, median 0.06%, range 0.01–0.08%) and myeloid dendritic cells (mDCs, median 0.47%, range 0.27–0.87%) were detectable in BALF from control segments. CD1a-positive dendritic cells in BALF were identified as a subpopulation of mDCs. Both pDCs (median 0.56%, range 0.09–1.83%) and mDCs (median 1.82%, range 0.95–2.29%) increased significantly in BALF 24 h (p = 0.018 compared with the control segments for pDCs and mDCs), but not 10 min, after allergen challenge. The percentage increase in pDCs was higher than that of mDCs after allergen challenge, as reflected by an enhanced pDC:mDC ratio after allergen challenge. In peripheral blood, there was a significant decrease in mDCs (p = 0.038) and a trend to a decrease in pDCs (p = 0.068) 24 h after allergen challenge. Analysis of dendritic cell surface molecules showed that after allergen challenge, BALF dendritic cells have a less mature phenotype compared with BALF dendritic cells from control segments. Conclusion: Using a comprehensive strategy to analyse dendritic cell subsets in human BALF, we have shown for the first time that both myeloid and plasmacytoid dendritic cells accumulate in the airway lumen after allergen challenge in patients with asthma.

Background: Allergic asthma has been linked to an increase in T-helper type 2-like cytokines and T cells, but there is growing evidence for a role of lymphocyte-mediated cytotoxic mechanisms in the pathogenesis of asthma. Therefore, we investigated the cytotoxic potential of different lymphocyte subpopulations in patients with allergic asthma. Methods: Granzyme A, B, K, and perforin expression in peripheral blood lymphocytes was analyzed using flow cytometry. Soluble granzymes were measured in serum using specific enzyme-linked immunosorbent assays. Results: Asthmatics had significantly decreased percentages of granzyme and perforin-positive CD4 T cells compared with non-atopic controls. In patients with asthma, the granzyme B and perforin-positive subset of CD8+ T cells and natural killer T cells, which represent more differentiated cell populations, were significantly reduced, while this was not observed in the less differentiated granzyme K+ subsets. In addition, the serum concentrations of granzyme B were significantly reduced in patients with asthma, while granzyme K concentrations were not different. Interestingly, there was a negative correlation between granzyme A, B and perforin expression in T cell subsets as well as serum granzyme B concentrations and total serum immunglobulin E. In CD3-negative natural killer cells, no differences in granzyme or perforin expression between patients with asthma and controls were detected. Conclusion: In allergic asthma, cytotoxic T lymphocyte subsets of a more differentiated phenotype are significantly decreased and this is correlated to serum immunglobulin E levels.

Uridine nucleotides and UDP-glucose are endogenous molecules, which are released into the extracellular environment in a lytic manner after cell damage, as well as by regulated nonlytic mechanisms. Recently, a UDP-glucose-specific G(i) protein-coupled P2Y receptor, namely P2Y(14), has been cloned. In this study, we demonstrated expression of the P2Y(14) mRNA in human lung epithelial cells and in the epithelial cell lines A549 and BEAS-2B. Evidence of functional expression of the P2Y(14) receptor in these cell lines was provided by calcium measurements after stimulation with uridine 5'-diphosphoglucose (UDP-glc). Experiments with pertussis toxin and the Ca(2+)-chelator EGTA revealed participation of pertussis toxin-sensitive G(i/o)-proteins in the mobilization of Ca(2+)-ions from intracellular stores by UDP-glc. Moreover, UDP-glc increased secretion of the potent neutrophil chemoattractant CXCL8/IL-8 in A549 and BEAS-2B cells in a pertussis toxin-sensitive manner. Moreover, reverse transcription and quantitative polymerase chain reaction revealed that UDP-glc modulated mRNA levels of IL-8/CXCL8. However, stimulation of A549 and BEAS-2B cells with UDP-glc neither modified basal nor cytokine-induced secretion of the CXC-chemokines CXCL9/MIG, CXCL10/IP-10, and CXCL11/I-TAC. In addition, UDP-glc did not affect proliferation of the two cell lines. In summary, our data provide evidence for a distinct physiologic role of P2Y(14) in the selective release of specific chemokines from human airway epithelial cells.

Granzymes (Gzm) are a group of serine proteases which are stored in the granules of cytotoxic lymphocytes. In humans, five granzymes have been characterized to date at the molecular level. While GzmA and GzmB have been extensively studied, little is known about GzmH, GzmK and GzmM. In this study, we describe the generation of mAbs against human GzmK and GzmM by genetic immunization. The obtained anti-GzmK and anti-GzmM mAbs are not cross-reactive with GzmA, GzmB, GzmM and GzmA, GzmB, GzmK, respectively, and show a granular staining pattern in human lymphocytes. Flow cytometric analysis of peripheral blood lymphocytes revealed that GzmA, GzmM and perforin show a similar distribution. They are expressed in almost all CD16+CD56+ NK cells, CD3+CD56+ NKT cells and gammadelta T cells as well as in 20-30% of all CD3+CD8+ TC cells. Surprisingly, GzmK was not detected in the highly cytotoxic CD16+CD56+ NK cells but was preferentially expressed in lymphocytes of the T cell lineage, staining 20% of CD3+CD8+ TC cells, 50% of CD3+CD56+ NKT cells and 40% of gammadelta T cells, as well as 60% of the small sub-population of CD56bright+ NK cells. Our data suggest that human granzymes are differentially expressed in distinct sub-populations of peripheral blood lymphocytes.

NK cells and cytotoxic T lymphocytes can induce apoptosis in virus-infected and transformed target cells via the granule exocytosis pathway. The key components of the cytolytic granules are perforin and several serine esterases, termed granzymes. While the cellular distribution of human granzymes A (GrA) and B (GrB) has been well characterized much less is known about the expression pattern of human granzyme K (GrK). In this study GrA, GrB, and GrK expression was analyzed in human peripheral blood lymphocytes using flow cytometry. There was a distinct population of GrK expressing CD8+ T cells with a CD27+/CD28+/CCR5high/CCR7-/perforin-/low/IFN-gamma+ memory-like phenotype, while all CD56bright NK cells were also positive for GrK. In addition, GrK was also expressed in subpopulations of CD56+ T cells, CD4+ T cells, and TCRgammadelta+ T cells. In contrast, GrB was primarily expressed in CD56dim NK cells and differentiated memory CD8+ T cells with the CD27-/low/CD28-/low/CCR5-/low/CCR7-/CD11b+/perforinhigh phenotype. Only few CD8+ T cells expressed both GrB and GrK. GrA was found to be co-expressed in all GrB- and GrK-expressing T cells. Our findings suggest that granzyme expression during the differentiation process of memory CD8+ T cells might be as follows: GrA+/GrB-/GrK+ --> GrA+/GrB+/GrK+ --> GrA+/GrB+/GrK-.

Summary Background Atopic asthma is linked to a T-helper type 2 dominated pathogenesis, but there is increasing evidence of Th1/Tc1-mediated processes in the aetiopathology of asthma. Killer-specific secretory protein of 37 kDa (Ksp37) is expressed in cytotoxic lymphocytes, selectively in the effector subsets of CD8+- and CD4+ T lymphocytes and in CD16+/CD56dim natural killer cells and γ/δ T cells. This effector cell-specific expression of Ksp37 and its coexpression with perforin suggest that Ksp37 might be involved in processes mediated by cytotoxic cells. Objective We hypothesize that Ksp37 could indicate the involvement of cytotoxic lymphocytes in the pathogenesis of atopic asthma, and investigated Ksp37 concentration in bronchoalveolar lavage fluid (BALF) collected 10 min, 18, 42 or 162 h after segmental allergen provocation and in serum of patients with atopic asthma (n=25). Methods Ksp37 concentrations in BALF and serum were detected by ELISA. Flow cytometric analysis was used to assess numbers and cell subsets in BALF. Results Ksp37 increased significantly in BALF 10 min, 18 and 42 h, but not 162 h after allergen challenge compared with saline-challenged controls, while Ksp37 serum levels did not change significantly at all time-points. In addition, the increase in Ksp37 concentrations in BALF correlated with the corresponding numbers of lymphocytes. Conclusions We conclude that Ksp37 level increased in BALF 10 min, 18 and 42 h after allergen challenge but not in peripheral blood. Our findings suggest that segmental allergen challenge in asthma is associated with an increase in Ksp37 concentrations in BALF and an influx of potentially cytotoxic T lymphocytes into the lungs.

Summary Background The interaction of chemokines with their receptors strongly influences the migration of leucocytes. Objective In order to assess the contribution of these molecules to the local recruitment of T cells in bronchial asthma, we analysed the expression of 14 chemokine receptors on lung-derived T cells. Methods Chemokine-receptor expression by T cells derived from the peripheral blood, the bronchoalveolar lavage fluid and the bronchial mucosa was analysed by flow cytometry and immunohistochemistry. Expression profiles in healthy and mildly asthmatic individuals were compared, the latter prior and after segmental allergen provocation. Results Compared with peripheral blood, alveolar T cells expressed significantly more CCR2, CCR5, CCR6, CXCR3 and CCR4. However, no differences were observed between healthy controls and unchallenged asthmatics. In patients developing significant inflammatory responses following specific allergen challenge, a marked increase in the percentage of CCR4+ and CCR7+, and reduced numbers of CXCR3-bearing alveolar T cells were detected. Following specific allergen challenge, chemokine-receptor expression profiles of T cells from the alveolar space and the mucosa or the submucosa were similar, excluding a particular subcompartmentalization of the chemokine/chemokine-receptor system. Conclusion The expression of certain chemokine receptors by lung T cells suggests a contribution to the physiological recruitment of T cells to the lungs, both in healthy controls and unchallenged mild asthmatics. However, strong allergen-induced airway responses were associated with a specific chemokine-receptor profile, suggesting the involvement of certain chemokine receptors in the pathogenesis of allergic bronchial inflammation.

Interleukin-13 (IL-13) is critical for the development of allergic asthma and is involved in the activation of eosinophils within the airways. IL-13 exerts its activity on target cells via the dimeric IL-13 receptor (IL-13R), which comprises the IL-13 receptor alpha1-chain (IL-13Ralpha1) as a specific component. The aim of this study was to investigate the expression of the IL-13Ralpha1-chain on primary human eosinophilic granulocytes. Furthermore, it addresses the regulatory influence of cytokines on the level of surface abundance of this receptor subunit. Expression of IL-13- and IL-4-receptor subunits in purified primary human eosinophils was monitored at the messenger RNA level by reverse transcription polymerase chain reaction and at the protein level by flow cytometry. For the analysis of IL-13Ralpha1 surface expression, a new monoclonal antibody, which was generated using genetic immunization, was employed. Different cytokines with established activity on eosinophils were studied with regard to their influence on IL-13Ralpha1 in vitro by flow cytometry. Whereas IL-13 and IL-4 had inhibitory effects on IL-13Ralpha1 expression on eosinophils, interferon-gamma, tumour necrosis factor-alpha, and, to the largest extent, transforming growth factor-beta, enhanced the expression of this receptor subunit. A positive regulatory response evoked by transforming growth factor-beta and interferon-gamma does not prevent inhibitory effects caused by IL-13. These findings suggest a regulatory cytokine network influencing the reactivity of eosinophils to IL-13.

Summary Background IL-5 is a specific cytokine for eosinophil accumulation, activation and prolongation of survival and can be recovered in elevated concentrations from the bronchoalveolar compartment in atopic asthma following allergen challenge. Objective The action of IL-5 is mediated via the specific IL-5 receptor-α (IL-5Rα). Although in vitro data suggest that IL-5R expression is regulated by cytokines such as IL-3, IL-5 and GM-CSF, IL-5R regulation in vivo and its kinetics following allergen provocation are incompletely understood. Methods We investigated IL-5R regulation in vivo following segmental allergen provocation (SAP) with an individually standardized dose of allergen in 12 patients with atopic asthma. Lavage was performed 10 min and 18 h (eight patients) and 10 min and 42 h (eight patients) after allergen challenge. In addition to differential cell counts, IL-5Rα was measured by flow cytometry and IL-5 concentrations in bronchoalveolar lavage (BAL) fluid were determined by ELISA. Results IL-5Rα expression decreased significantly on peripheral blood and on BAL eosinophils 18 and 42 h after SAP. In contrast, IL-5 concentrations increased significantly in BAL fluid 18 and 42 h after SAP. In four and two patients, respectively, there were detectable IL-5 concentrations in serum 18 or 42 h after allergen exposure. Conclusions Although there was no correlation between IL-5 concentrations and IL-5Rα expression on eosinophils in BAL, our data support previous in vitro and in vivo findings of a negative feedback mechanism between IL-5 concentrations and IL-5Rα expression on eosinophils.

SUMMARYKiller-specific secretory protein of 37 kDa (Ksp37), identified as a Th1/Tc1 specific secretory protein is expressed preferentially in cytotoxic T lymphocytes (CTL) and natural killer (NK) cells and might be involved in essential processes of CTL-mediated immunity. Although extrinsic asthma is linked currently to a Th2-dominated pathogenesis, there is increasing evidence for Th1/Tc1-mediated processes in the aetiopathology of asthma. CTL from patients with asthma have been shown to express cytokines and effector molecules which were different from healthy controls. We hypothesized that Ksp37 could indicate the involvement of CTL in the pathogenesis of extrinsic asthma. We therefore investigated Ksp37 expression in PBMC from patients with mild extrinsic asthma (n = 7) and healthy controls (n = 7). Flow cytometric analysis was used to quantify Ksp37+ cells and to investigate cellular Ksp37 expression as relative mean fluorescence intensities (MFI). We found a significantly (P = 0·016) higher percentage of Ksp37+ cells within the total lymphocyte population obtained from patients with mild extrinsic asthma compared with healthy controls. Subdifferentiation revealed a significant difference limited exclusively to the CD8+ subset (P = 0·010). In addition, Ksp37 secretion from cultured peripheral blood mononuclear cells (PBMC) and MFI of Ksp37+ lymphocytes were increased in patients with asthma compared with healthy controls. We conclude that mild extrinsic asthma appears to be associated with an increased expression of the Tc1 related protein Ksp37. The functional role of Ksp37 in the pathogenesis of asthma remains to be elucidated.

SUMMARYAsthma has been linked to a chronic, T-cell-mediated bronchial inflammation. Because other T-lymphocyte-mediated, chronic inflammatory disorders have been associated with elevated granzyme B (grB) expression we tested the hypothesis that atopic asthma might be associated with elevated grB levels in the bronchoalveolar compartment. Therefore we performed intracellular grB staining in lymphocytes from bronchoalveolar lavage (BAL) collected 42 h after segmental allergen provocation (SAP) in allergic patients with bronchial asthma. There was a significant increase in CD3+, CD8+, and CD16/56+ lymphocytes expressing grB in BAL 42 h after SAP as compared to saline challenged controls. However, compared to peripheral blood the percentages of these lymphocyte subsets detected as grB+ in BAL remained significantly lower. Measurement of extracellular grB in BAL fluids by a particle immunoassay revealed significantly elevated grB levels in the allergen challenged bronchoalveolar compartment 42 h following SAP in six of the eight patients (range

Extracellular nucleotides are the focus of increasing attention for their role as extracellular mediators since they are released into the extracellular environment in a regulated manner and/or as a consequence of cell damage. Here, we show that human eosinophils stimulated with different nucleotides release eosinophil cationic protein (ECP) and the chemokine interleukin 8 (IL-8), and that release of these two proteins has a different nucleotide requirement. Release of ECP was triggered in a dose-dependent manner by ATP, UTP and UDP, but not by 2′-&3′-o-(4-benzoyl-benzoyl)adenosine 5′-triphosphate (BzATP), ADP and α,β-methylene adenosine 5′ triphosphate (α,β-meATP). Release of IL-8 was triggered by UDP, ATP, α,β-meATP and BzATP, but not by UTP or ADP. Pretreatment with pertussis toxin abrogated nucleotide-stimulated ECP but not IL-8 release. Release of IL-8 stimulated by BzATP was fully blocked by the P2X7 blocker KN-62, while release triggered by ATP was only partially inhibited. IL-8 secretion due to UDP was fully insensitive to KN-62 inhibition. Priming of eosinophils with GM-CSF increased IL-8 secretion irrespectively of the nucleotide used as a stimulant. It is concluded that extracellular nucleotides trigger secretion of ECP by stimulating a receptor of the P2Y subfamily (possibly P2Y2), while, on the contrary, nucleotide-stimulated secretion of IL-8 can be due to activation of both P2Y (P2Y6) and P2X (P2X1 and P2X7) receptors. British Journal of Pharmacology (2003) 138, 1244–1250. doi:10.1038/sj.bjp.0705145

Extracellular nucleotides elicit multiple responses in eosinophils but no information on expression of purinergic receptors in these cells is available so far. In the present study we show that human eosinophils express the following P2Y and P2X subtypes: P2Y(1), P2Y(2), P2Y(4), P2Y(6), P2Y(11), and P2X(1), P2X(4), P2X(7), whose stimulation results in intracellular Ca(2+) increase and production of large amounts of reactive oxygen intermediates. These events are stimulated or inhibited, respectively, by P2 receptor agonists or antagonists.

The cause of asthma which has been linked to a chronic, T-cell-mediated bronchial inflammation, remains unclear. A number of other T-lymphocyte-mediated, chronic inflammatory disorders have been associated with autoimmunity and there are data indicating that autoimmune phenomena might also be present in asthma. Expression of perforin, a cytotoxic molecule produced by lymphocytes, has been implicated in the pathogenesis of autoimmune disease. We therefore tested the hypothesis that allergic and intrinsic asthma might be associated with an increase in lymphocytes producing perforin by comparing the expression of intracellular perforin in peripheral blood lymphocytes of patients with extrinsic asthma (n = 13), intrinsic asthma (n = 7), and healthy (control subjects (n = 18). Lymphocytes were identified using flow cytometry and subdivided into CD3(+), CD4(+), CD8(+), CD16(+), and CD56(+) subpopulations after staining with appropriate monoclonal antibodies. The percentage of perforin-positive total lymphocytes as significantly elevated in patients with allergic as well as intrinsic asthma when compared with normal control subjects. Analysis of lymphocyte subpopulations also revealed a significant increase in the percentage of CD3(+), CD4(+), CD8(+), and CD56(+) cells expressing perforin in allergic asthma and a significant increase in the percentage of CD4(+), and CD56(+) cells in intrinsic asthma when compare with healthy control subjects. Perforin expression in CD4(+) cells in intrinsic asthma was also significantly elevated compared with allergic asthma. We conclude that allergic and intrinsic asthma is associated with increased expression of perforin in T-lymphocyte subsets.

Study objective: To assess the postoperative course of pleural leukocyte counts and cytokine concentrations in patients with malignant and nonmalignant lung disease who underwent thoracic surgery. Patients and interventions: A total of 21 patients undergoing thoracic surgery were included in the study. Twelve patients had a malignant disease, and 9 had a nonmalignant disease. Six patients underwent video-assisted thoracoscopy and 15 underwent thoracotomy. Pleural drainage fluid from the chest tubes was collected postoperatively at 0h, 3h, 6h, 12h, 24h, 48h, 72h, and 96 h. The same schedule, as well as one additional preoperative sample, was applied for blood collections. Results: A trend toward lower concentrations of tumor necrosis factor-a (TNF-a), granulocytemacrophage colony-stimulating factor, and interleukin-10 was observed in patients with malignant disease compared to those without malignancy. These differences achieved significance for TNF-a in the drainage fluid of those patients with nonmalignant disease who had undergone formal thoracotomy. Patients with malignant disease showed significantly lower macrophage fractions in drainage fluid and lymphocyte fractions in serum. All patients with complications had malignant disease and showed the lowest cytokine concentrations, as well as the lowest fractions of both macrophages in drainage fluid and lymphocytes in serum. Conclusion: The data suggest that malignancy may lead to impairment of the wound-healing process via modification of the inflammatory cell infiltrate and locally released cytokines. (CHEST 1999; 115:1604 ‐1610)

Increased concentrations of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-4, and IL-13 have been measured in bronchoalveolar lavage fluid (BALF) of patients with asthma following allergen provocation. In addition, these cytokines have also been reported to activate eosinophils in vitro. Although cytokine interactions have been postulated in the activation of eosinophils, the combined effects of cytokines on eosinophil activation remain poorly understood. Because activation of eosinophils has been regarded as a crucial event in the pathogenesis of asthmatic inflammation, we tested the hypothesis that IL-4 and IL-13 could enhance the effects of TNF-alpha on eosinophil activation. For this purpose, eosinophils from normal donors were purified and cultured in the presence of IL-4 or IL-13 and TNF-alpha. Eosinophil survival and surface expression of CD69 were assessed by flow cytometry. There was a concentration- and time-dependent upregulation in CD69 expression as well as eosinophil survival when eosinophils were incubated with IL-13, IL-4, or TNF-alpha. However, eosinophil viability and CD69 expression increased synergistically when eosinophils were incubated with IL-13 or IL-4 in the presence of TNF-alpha. This synergistic effect of IL-4 and IL-13 on CD69 expression was not limited to TNF-alpha but was also observed with IL-5. Our study provides evidence that IL-4 can activate eosinophils in a similar fashion as does IL-13. Furthermore, this study shows that the addition of IL-4 or IL-13 to TNF-alpha or IL-5 has synergistic effects on eosinophil activation, suggesting that the combined effects of different cytokines present in BALF following allergen provocation can enhance eosinophil activation in vitro.

CD95 (Fas, APO-1) is a cell surface receptor expressed on many cells including eosinophils which mediates apoptosis when ligated by agonistic antibodies or its natural ligand FasL. Since inhibition of apoptosis may play an important role in controlling tissue eosinophilia, we investigated the expression of CD95 on purified peripheral blood eosinophils from normal donors. Freshly isolated eosinophils expressed CD95 on the cell surface as well as CD95-specific mRNA at low levels which did not change during 24-h culture. Incubation of eosinophils with IL-3, IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF) did not modulate the basal expression of CD95. IFN-gamma as well as TNF-alpha, however, induced a significant, dose- and time-dependent increase in CD95 mRNA and cell surface expression as measured by reverse transcription-PCR and flow cytometry. Co-stimulation with IFN-gamma and TNF-alpha had synergistic effects on the CD95 surface expression on eosinophils. Addition of IL-3, IL-5 or GM-CSF to IFN-gamma- and TNF-alpha-stimulated eosinophils caused in a reduction of CD95 expression. Functional activity for CD95 following incubation with IFN-gamma and TNF-alpha was demonstrated by increased apoptosis in response to cross-linking with FasL. From these data, we conclude that IFN-gamma and TNF-alpha can up-regulate cell surface expression of CD95 on eosinophils, which leads to an increased susceptibility of eosinophils to Fas-mediated apoptosis. Thus, our results suggest that receptors involved in eosinophil apoptosis can be regulated by antagonistic cytokines.

Allergic asthma is associated with the recruitment of activated inflammatory cells after allergen challenge. Surface expression of CD14 has been proposed as a marker of cell activation and differentiation. We therefore measured CD14 expression on activated macrophages and granulocytes as well as soluble CD14 (sCD14) concentrations in peripheral blood and bronchoalveolar lavage (BAL) following segmental allergen provocation (SAP) with individually standardized doses of allergen in eight patients with allergic asthma. Two segments of the right lung were challenged with allergen. Two segments of the left lung, into which saline was instilled, served as controls. CD14 expression on macrophages and granulocytes was determined by flow-cytometry and concentrations of interleukins and sCD14 were analysed by enzyme-linked immunosorbent assay (ELISA) 10 min and 18 h after challenge. Soluble CD14 concentrations remained unchanged in BAL fluid after saline challenge and 10 min after SAP, but increased significantly 18 h after SAP. Although macrophage numbers increased 18h after SAP, CD14 expression on these cells did not change. Unlike macrophages, granulocyte numbers correlated with sCD14 levels 18 h after SAP while their CD14 expression decreased significantly. Furthermore, sCD14 correlated with interleukin (IL)-13 concentrations 18 h after SAP. An increase in soluble CD14 can be observed 18 h but not 10 min after segmental allergen provocation suggesting local release of this surface antigen. Our findings imply that CD14-mediated cell activation following segmental allergen provocation could play a role in asthmatic inflammation.

Mit dem Aufkommen spezifischer Antikorper wurde der klassischen Histologie/Cytologie ein neues Werkzeug zur Identifikation verschiedenster Zell- und Gewebebausteine, insbesondere deren Proteinkomponenten, zur Verfugung gestellt. Daraufhin entwickelte sich ein Arbeitsgebiet, das je nach Autor unterschiedlich betitelt wird – meist mit Immunhistologie, Immunhistochemie oder Immuncytochemie. Obwohl die Begriffe implizieren, dass auf histologischer Ebene bzw. auf zellularer Ebene gearbeitet wird, werden die Begriffe in der Literatur zumeist nicht streng voneinander getrennt verwendet. Samtlichen Methoden dieser Branche ist die Art der Detektion relevanter Proteine gemein. Sie erfolgt unter Ausnutzung spezifischer Antigen-Antikorper-Bindungen. Daher wird im Folgenden zusammenfassend von in situ-Immunlokalisation die Rede sein. Die Auswertung erfolgt im Allgemeinen lichtmikroskopisch und/oder elektronenmikroskopisch, wobei sogar eine „gewisse“ Quantifizierung der relevanten Proteine moglich ist – sofern die Praparation es hergibt und die erforderliche mikroskopische Ausstattung inkl. adaquater Imagingsysteme vorhanden ist. Die Aufgabe des Antikorpers ist es – wie immer – sein spezifisches Antigen im Gewebe bzw. in/an der Zelle zu finden und zu binden. Damit allein ist es noch nicht getan, denn nackte Antikorper sieht man ja bekanntermasen irrsinnig schlecht. Daher markiert man den Antikorper mit irgendetwas Wahrnehmbarem, woruber dann der Antigen-Nachweis erfolgt. Als Markierung kommen Fluorochrome und Enzyme sowie korpuskulare Anhangsel zum Einsatz. Deren Visualisierung erfordert unterschiedliche Detektionssysteme. Fluorochrome absorbieren und emittieren Licht bestimmter Wellenlange – folglich benotigt man ein Fluoreszenzmikroskop. Enzyme benotigen ein spezifisches Substrat, das nach seiner Umsetzung die relevanten Strukturen farblich markiert – hier ist lediglich ein Lichtmikroskop vonnoten, ebenso bei korpuskularen Markierungen, im Wesentlichen kolloidales Gold. Letzteres wird auch gern genutzt, um elektronenoptisch noch tiefere, subzellulare Einblicke zu erhaschen.

Bestimmt kann jeder von uns die Frage nach seiner Korpergrose und nach seinem Gewicht – auch wenn so mancher nicht gerne daruber spricht – genau beantworten. Wird man aber nach einer anderen, und dazu sogar noch praktisch unveranderlichen, individuellen Eigenschaft, namlich der der Blutgruppe gefragt, mussen sich mit Sicherheit viele unter uns eingestehen, dass sie dies nicht mit Gewissheit beantworten konnen.

„Statistik… nein danke!“ Man lehnt sich wohl nicht gerad’ weit aus dem Fenster bei der Behauptung, dass derartige Gedanken uns allen schon einmal durch den Kopf gingen. Andererseits steht es auser Frage, dass man im Laufe eines Laborlebens nicht an der Statistik vorbeikommt. An diese Tatsache wird man immer dann erinnert, wenn Fragen durch das Labor schallen wie z. B.: „Welchen Signifikanztest muss ich hier eigentlich anwenden?“ oder „Muss ich hier das arithmetische Mittel oder den Median berechnen?“ Die Reaktionen der Kollegen reichen von verschamtem Abwenden bis hin zum genervten Augenrollen – glucklich darf sich wahnen, wer auf statistische Kompetenz trifft. Im folgenden Kapitel soll der Versuch unternommen werden, einen fur das alltagliche Laborleben relevanten Ausschnitt aus dem weiten Gebiet der Statistik komprimiert, verstandlich und dabei auch noch interessant aufzubereiten.

Subcutaneous adipose tissue is ubiquitous and easily accessible in large quantities with a minimal invasive procedure by liposuction aspiration, qualifying it as a good source for stem cell isolation. Adipose-derived mesenchymal stromal/stem cells (ASCs) can easily be isolated by a relatively simple method, yielding a high amount of stem cells, which is essential for stem cell-based therapies and tissue engineering. Several studies have provided evidence that ASCs in situ reside in the wall surrounding the vasculature, and that blood vessels in virtually all organs and tissues harbour ubiquitous (mesenchymal) stem cells in their perivascular niche. Nevertheless, the exact localization of ASCs in native adipose tissue is still under debate. ASCs are isolated by their capacity to adhere to cell culture plastic and are maintained in culture for up to 10–15 passages. Cultured cells are characterized by their expression of a panel of characteristic markers and their capacity to differentiate into cells from meso-, ecto- and endodermal lineages. ASCs possess a high plasticity and differentiate into various cell types, including endothelial cells in vitro and in vivo. This review summarizes and discusses the current knowledge about ASCs and their differentiation potential into endothelial cells in vitro and in vivo.

„Tot oder lebendig“ – dieser Ausdruck zierte fruher die Steckbriefe vieler Gesetzloser und ist auch heutzutage fur jeden Experimentator, der sich mit Zellen beschaftigt, noch aktuell. Die unterschiedlichsten Fragestellungen erfordern die Bestimmung der Viabilitat von Zellen. Dazu gehoren beispielsweise Cytotoxizitatsstudien. Interessiert man sich fur die Aktivitat bestimmter Enzyme, so ist es fur eine Standardisierung der Enzymaktivitat unerlasslich, die Anzahl lebender Zellen zu bestimmen. Auch die Messung sezernierter Zellmetabolite in Kulturuberstanden erfordert eine Viabilitatsbestimmung der Zellen, da diese im Todesfall ihr Innerstes in das Medium entlassen. Diese „Innereien“ konnen cytotoxisch wirken und sehr stabile Enzyme wie zum Beispiel Proteasen und Nucleasen enthalten, die bei ihrer Freisetzung die Messergebnisse gewaltig durcheinander bringen und dem Experimentator schlaflose Nachte und Albtraume bescheren konnen.

Sie sind uberrascht, den Begriff „ubernaturlich“ in einem naturwissenschaftlichen Methodenbuch zu lesen? Glauben Sie uns, das ist gar nicht so weit hergeholt. Aber lassen Sie uns am Anfang beginnen.

Histamine H1 receptor antagonists (antihistamines) are widely used for treatment of allergic conditions such as rhinitis, urticaria and atopic dermatitis. A number of first- and second-generation compounds are available; all prevent the pro-inflammatory pharmacological actions of histamine related to allergy and inflammation through competitive antagonism at H1 receptors. In addition, both in vitro and animal studies suggest that second-generation antihistamines may also show anti-inflammatory effects unrelated to H1 receptor antagonism. These effects include inhibition of the functions of several effector and regulator cells of inflammation, such as degranulation of mast cells, oxygen radical release from eosinophils and neutrophils, lipid mediator generation from eosinophils, and eosinophil cell migration. Although their precise mode of action is incompletely understood, H1 antagonists may interfere with cellular transmembrane signalling processes, including Ca++ transmembrane flux and intracellular mobilisation, accumulation of intracellular adenosine 3′,5′-monophosphate (cyclic AMP), and the activities of both protein kinase C and NADPH oxidase. Whether or not H1 antagonists mediate these effects via binding to histamine receptor subtypes other than H1 remains, as yet, unclear. This review discusses the clinical effects and the putative mode of anti-inflammatory action unrelated to H1 receptor blockade of this class of drugs.

Thymic stromal lymphopoietin (TSLP) is an interleukin-7 (IL-7)-like cytokine with a pivotal role in development and maintenance of atopic diseases such as allergic asthma and atopic dermatitis. Moreover, recent studies show an involvement of TSLP in the progression of various cancers. TSLP signaling is mediated by the TSLP receptor (TSLPR), a heterodimeric type I cytokine receptor. It consists of the IL-7 receptor alpha chain (IL-7Rα), which is shared with the IL-7 receptor, and the TSLPRα chain as a specific subunit. Blocking signal release by TSLP without affecting IL-7 function is a potentially interesting option for the treatment of atopic diseases or certain tumors. By employing the extracellular domain of human TSLPRα chain (hTSLPRα(ex)) as an antigen, we generated a set of monoclonal antibodies. Several binders to native and/or denatured receptor protein were identified and characterized by cytometry and Western blot analysis. A screen based on a STAT3-driven reporter gene assay in murine pro-B cells expressing a functional hTSLPR yielded two hybridoma clones with specific antagonistic properties towards hTSLP, but not IL-7. Kinetic studies measuring blockade of hTSLP-dependent STAT phosphorylation in a TSLP-responsive cell line revealed an inhibitory constant in the nanomolar range.

Regulatory CD4+ T cells (Tregs) control immune responses using secretion of anti-inflammatory cytokines and/or cytotoxic mechanisms and play a central role in the outcomes of several immune pathologies. Previous studies suggest an impaired function of Tregs in allergy, especially during allergen seasons, but the underlying mechanism is not known. Therefore, we analysed the impact of the T helper type 2 cytokine interleukin (IL)-4 on in vitro generated adaptive Tregs (aTregs), which have been reported to use the granzyme B (GrB)/perforin pathway to kill autologous immune cells. aTregs were generated by co-ligation of CD3 and CD46 on CD4+ T lymphocytes and granzyme expression was analysed using flow cytometry. To quantify GrB and perforin expression as well as IL-10 secretion in response to IL-4, specific enzyme-linked immunosorbent assays were performed in cell lysates and/or culture supernatants. Using a flow cytometry-based cytotoxicity assay the impact of IL-4 on the cytotoxic potential of aTregs was investigated. While IL-4 did not affect IL-10 secretion and perforin expression in aTregs, a significant suppression of GrB synthesis was detected in the presence of IL-4. In addition, IL-4-mediated suppression of GrB led to impaired cytotoxicity of aTregs against K562 target cells. In conclusion, our data suggest that IL-4 might play a role in impaired aTreg function in allergy.

Activated macrophages have been characterized as M1 and M2 according to their inflammatory response pattern. Here we analyzed the M2 marker expression and intracellular signal transduction in the course of cytokine-driven differentiation. We found elevated spontaneous production of the chemokines CCL17, CCL18 and CCL22 and increased expression of CD206 by alveolar macrophages from patients with lung fibrosis. Stimulation of normal human AM with Th2 cytokines IL-4 and/or IL-10 in vitro revealed IL-4 as the most powerful inducer of M2-phenotype in AM and monocytes. Importantly, IL-10 enhanced IL-4-induced expression of CCL18 and IL-1RA in a synergistic fashion. IL-4/IL-10 stimulation induces a strong activation of STAT3 in AM from fibrosis patients. These results suggest an important role for M2 polarized AM in the pathogenesis of pulmonary fibrosis and indicate that both IL-4 and IL-10 account for human AM phenotype shift to M2, as seen in patients with fibrotic interstitial lung diseases.

Background: Granzymes are a sub-family of serine proteases involved in the pathogenesis of many inflammatory disorders. In contrast to granzyme A and B, the role of granzyme K (GrK) in human lung diseases is unknown. Therefore, we investigated the release and expression of GrK in allergic asthma, chronic obstructive pulmonary disease (COPD), and bronchopneumonia. Methods: Soluble GrK was quantified using an enzyme-linked immunosorbent assay in bronchoalveolar lavage fluid of patients with allergic asthma (before and after segmental allergen challenge), and patients with mild COPD, pneumonia, and healthy controls. The molecular form of GrK was analyzed by Western blot. Flow-cytometry was performed to determine the cellular expression of GrK. Results: Compared with healthy controls, there were normal levels of soluble GrK in bronchoalveolar lavage fluid of patients with COPD, and patients with allergic asthma before allergen challenge. In contrast, soluble GrK was strongly increased in bronchoalveolar lavage fluid of patients with acute bronchopneumonia. In patients with allergic asthma, there was a significant increase in soluble GrK as well as in GrK expressing CD8+ T cells in bronchoalveolar lavage fluid 24 hours and 72 hours after allergen challenge. After allergen challenge, soluble GrK correlated with the percentage of GrK expressing CD8+ T cells. Finally, we show that the endobronchial release of the CCR5 ligand CCL3 might be a mechanism for the recruitment of GrK+CD8+ T cells after allergen challenge. Conclusion: These data provide first evidence that the expression of GrK is upregulated in acute airway inflammation, both in infectious and non-infectious diseases.

BACKGROUND IL-13 promotes acute allergic asthma and is discussed to play a role in late asthmatic features such as fibrotic processes and airway remodelling. The contributions of IL-13-mediated mechanisms to subepithelial events related to fibrosis are not yet settled. OBJECTIVE We investigated the impact of IL-13 on lung epithelial cells as apoptotic effector and on lung fibroblasts as inducer of pro-fibrotic gene expression. METHODS Using the two lung epithelial cell lines A549 and BEAS-2B as well as primary lung epithelial cells, we investigated the capability of IL-13 to induce apoptosis by both flow-cytometry and ELISA. The ability of IL-13 to increase the expression of pro-fibrotic genes and to exert influence on the expression of its own receptor was investigated by real-time quantitative PCR measurement of mRNAs encoding collagen I, collagen III, basic fibroblast growth factor (bFGF), alpha-smooth muscle actin (alpha-SMA) and the IL-13 receptor alpha1 (IL-13Ralpha1) chain in human primary lung fibroblasts. The specificity of IL-13-mediated cellular responses was confirmed by means of an inhibitory monoclonal antibody directed to the IL-13 receptor. RESULTS IL-13 induces apoptosis in lung epithelial cell lines as well as in primary lung epithelial cells. Furthermore, IL-13 increases the expression of mRNA for alpha-SMA and collagen III, but not for bFGF in human primary lung fibroblasts. The susceptibility of lung fibroblasts to IL-13-induced up-regulation of pro-fibrotic genes is associated with the regulation of IL-13 receptor expression. IL-13-dependent fibrosis-associated effects could be inhibited by antibody-mediated blockade of the IL-13Ralpha1 subunit. CONCLUSION Our findings indicate a function of IL-13 as a mediator in fibrotic processes leading to loss of functional airway tissue in asthma. They also highlight the therapeutic potential of specifically targeting the interaction between IL-13 and its receptor.

Airway dendritic cells (DCs) are key regulators of pulmonary immune responses. However, information is limited regarding the characteristics of airway DCs in human lung diseases. Plasmacytoid DCs (pDCs) and myeloid DCs (mDCs) were analysed using four-colour flow cytometry in bronchoalveolar lavage fluid (BALF) from nonsmoking controls and patients with sarcoidosis, idiopathic pulmonary fibrosis (IPF) and pneumonia (in the presence or absence of immunosuppression). Compared with controls, immunocompetent patients with pneumonia displayed strongly enhanced pDC counts in BALF. In contrast, pDC counts in BALF from immunocompromised patients with pneumonia were even lower than in controls. This discrepancy was not explained by a different chemotactic milieu in the airways; all patients with pneumonia were characterised by strongly increased concentrations of the pDC-attracting chemokine, CXC chemokine ligand 10, in BALF. Patients with IPF were characterised by normal percentages of DC subtypes. However, the mDCs of patients with IPF were not as mature (CD83-positive) as those of controls. Patients with sarcoidosis displayed a unique increase in CD1a-negative mDCs in the airways. In addition, there was altered expression of costimulatory molecules (increased CD80 and decreased CD86 expression) on mDCs in patients with sarcoidosis. These data suggest that inflammatory diseases of the human lung are associated with a differential phenotype and recruitment of airway dendritic cells.