Lung fibroblasts from patients with emphysema show markers of senescence in vitro.

BACKGROUND: The loss of alveolar walls is a hallmark of emphysema. As fibroblasts play an important role in the maintenance of alveolar structure, a change in fibroblast phenotype could be involved in the pathogenesis of this disease. In a previous study we found a reduced in vitro proliferation rate and number of population doublings of parenchymal lung fibroblasts from patients with emphysema and we hypothesized that these findings could be related to a premature cellular aging of these cells. In this study, we therefore compared cellular senescence markers and expression of respective genes between lung fibroblasts from patients with emphysema and control patients without COPD. METHODS: Primary lung fibroblasts were obtained from 13 patients with moderate to severe lung emphysema (E) and 15 controls (C) undergoing surgery for lung tumor resection or volume reduction (n = 2). Fibroblasts (8E/9C) were stained for senescence-associated beta-galactosidase (SA-beta-Gal). In independent cultures, DNA from lung fibroblasts (7E/8C) was assessed for mean telomere length. Two exploratory 12 k cDNA microarrays were used to assess gene expression in pooled fibroblasts (3E/3C). Subsequently, expression of selected genes was evaluated by quantitative PCR (qPCR) in fibroblasts of individual patients (10E/9C) and protein concentration was analyzed in the cell culture supernatant. RESULTS: The median (quartiles) percentage of fibroblasts positive for SA-beta-Gal was 4.4 (3.2;4.7) % in controls and 16.0 (10.0;24.8) % in emphysema (p = 0.001), while telomere length was not different. Among the candidates for differentially expressed genes in the array (factor > or = 3), 15 were upregulated and 121 downregulated in emphysema. qPCR confirmed the upregulation of insulin-like growth factor-binding protein (IGFBP)-3 and IGFBP-rP1 (p = 0.029, p = 0.0002), while expression of IGFBP-5, -rP2 (CTGF), -rP4 (Cyr61), FOSL1, LOXL2, OAZ1 and CDK4 was not different between groups. In line with the gene expression we found increased cell culture supernatant concentrations of IGFBP-3 (p = 0.006) in emphysema. CONCLUSION: These data support the hypothesis that premature aging of lung fibroblasts occurs in emphysema, via a telomere-independent mechanism. The upregulation of the senescence-associated IGFBP-3 and -rP1 in emphysema suggests that inhibition of the action of insulin and insulin-like growth factors could be involved in the reduced in vitro-proliferation rate.

Solitary follicular thyroid carcinoma of the skull base and its differentiation from ectopic adenoma--review, use of galectin-3 and report of a new case.

With only four histologically proven cases in the literature, solitary skull base metastasis of thyroid carcinoma is extremely rare. Having treated another patient harboring a lesion with osseous destruction in the petroclival region and downward soft tissue extension we analyzed this case in conjunction with previous reports. In contrast to parenchymal brain metastasis that usually consists of the papillary type, histological examination revealed differentiated follicular tumors in all cases. All were located around the clivus. The radiographic picture resembled that of chordomas or chondrosarcomas. In the tissue obtained during thyroidectomy no evidence of primary malignancy was found in any of the cases according to standard histological criteria. In our case, a recently developed immunocytological marker - galectin-3 - was applied to differentiate between ectopic thyroid adenoma and carcinoma. The results were indicative of anaplastic growth. Tumor remnants responded well to postoperative 131I internal radiation and TSH suppression therapy. Distant metastasis of follicular thyroid carcinoma has to be considered in the differential diagnosis of destructive skull base lesions. Histological evaluation should include immunohistochemistry or clonal analysis to differentiate between adenomatous and carcinomatous growth and initiate effective radiotherapy early. Prognosis is by far not as poor as in brain metastases and appears to depend largely on location, size and histological appearance.

Surfactant protein levels in bronchoalveolar lavage after segmental allergen challenge in patients with asthma.

BACKGROUND: Allergic asthma is associated with airway inflammation and dysfunction of pulmonary surfactant. Because surfactant proteins (SP) account for immunomodulatory functions as well as biophysical functions, we hypothesized that the allergic response in asthma might be accompanied by a dysregulation of SPs. METHODS: We measured levels of SP-A, SP-B, SP-C and SP-D by enzyme-linked immunosorbent assay in bronchoalveolar lavage (BAL) fluid of 23 asthma patients and 10 healthy control subjects under well-controlled conditions before and 24 h after segmental allergen provocation. These data were related to surfactant function, Th(2) cytokine levels in BAL fluid and to the degree of eosinophilic inflammation. RESULTS: In patients with asthma, allergen challenge increased BAL levels of SP-B, SP-C and SP-D while SP-A was decreased. For SP-B and SP-D, a moderate increase was also observed after saline challenge. In contrast, no alterations were observed in healthy control subjects. Levels of SP-B and SP-C in asthmatics correlated with the ratio of small to large surfactant aggregates (SA/LA ratio) and correlated negatively with BAL surface activity. Furthermore, increased SP-C but not SP-B levels after allergen challenge correlated with eosinophil numbers, interleukin (IL)-5, and IL-13 in BAL while increased SP-D levels only correlated with eosinophil numbers. CONCLUSIONS: This study demonstrates significant alterations of all SPs in BAL fluid after allergen challenge of which SP-C was most closely related to surfactant dysfunction and the degree of the allergic inflammation.

Surfactant protein D inhibits early airway response in Aspergillus fumigatus-sensitized mice.

BACKGROUND: The surfactant protein SP-D has been reported to reduce bronchial hyper-responsiveness, blood eosinophilia, and T-helper type 2 cytokines in models of allergic asthma. However, little is known about the functional effect of SP-D on the early airway response upon allergen inhalation, which is an important feature of this disease. OBJECTIVE: We investigated whether SP-D is able to reduce the immediate allergen-induced mediator release and the early bronchial obstruction in addition to its effects on airway inflammation and bronchial hyperresponsiveness in an Aspergillus fumigatus mouse asthma model. METHODS: A. fumigatus-sensitized mice were treated with a recombinant fragment of human SP-D or placebo. Lung functions were measured in orotracheally intubated, spontaneously breathing animals using body plethysmography. In addition, passively sensitized precision-cut lung slices (PCLS) were used to determine the effect of SP-D on allergen-induced histamine release. RESULTS: SP-D inhibited the allergen-induced early airway response and reduced airway hyperresponsiveness compared with placebo. Eosinophilia in bronchoalveolar lavage and lung tissue was reduced after SP-D treatment, possibly by reducing eotaxin levels in the lung. Furthermore, SP-D treatment reduced the allergen-induced histamine release from PCLS. CONCLUSION: These data suggest that SP-D not only reduces allergen-induced eosinophilic inflammation and airway hyperresponsiveness but also provides protection against early airway obstruction by inhibition of early mediator release.

Surfactant proteins SP-A and SP-D as modulators of the allergic inflammation in asthma.

Surfactant proteins A and D (SP-A and SP-D) are members of the collectin family and bind to various motifs of microorganisms, particles and allergens. They play an important role in the first-line defense within the lung. Recent research has highlighted that these proteins not only augment innate immune responses to invading microorganisms but also act on adaptive immune functions like dendritic cell maturation and T cell proliferation. Both SP-A and SP-D downregulate the eosinophilic inflammation in murine asthma models and shift the cytokine profile towards a T helper cell type 1 response. In addition, they are effective at alleviating bronchial hyperresponsiveness. Although our knowledge about surfactant proteins as modulators of the allergic inflammatory reaction in asthma is still limited, the idea that surfactant proteins play a role in asthma has attracted increasing attention. In this review, the impact of the lung collectins SP-A and SP-D on asthmatic allergic inflammation and vice versa will be discussed.

Complement factors C3a and C5a are increased in bronchoalveolar lavage fluid after segmental allergen provocation in subjects with asthma.

Allergic asthma is thought to be the result of an inappropriate specific immune response against common environmental antigens. However, studies of animal asthma models have also linked the innate immune system, in particular complement factors C3a and C5, to murine airway hyperresponsiveness. Because the possible role of these anaphylatoxins in patients with asthma is not understood, we tested the hypothesis that C3a and C5a will increase in the bronchoalveolar lavage (BAL) fluid of patients with asthma after segmental allergen provocation. In a group of 15 subjects with mild asthma we found a significant upregulation of C3a and C5a 24 h after allergen challenge compared with baseline values (p < 0.01). In a control group of healthy volunteers the concentrations remained basically unchanged. Furthermore, we found a strong correlation between both anaphylatoxins and the number of eosinophils (p < 0.01) and, to a lesser degree, with the number of neutrophils (p < 0.05) in BAL fluid. These data suggest a contribution of anaphylatoxins C3a and C5a to the pathogenesis in asthma. However, the pathogenic role of these substances in relation to asthma remains to be elucidated, for example, by using anaphylatoxin receptor blockers as a possible new therapeutic principle.

Local release of eosinophil peroxidase following segmental allergen provocation in asthma.

BACKGROUND: Eosinophil peroxidase (EPO) is an eosinophilic basic protein, which leads to increased permeability and damage of bronchial epithelial cells in asthma. OBJECTIVE: As little is known about its local expression and release in humans the intracellular expression in lung and peripheral eosinophils and the concentrations of EPO in bronchoalveolar lavage (BAL) fluid and serum was investigated in patients with asthma. METHODS: Twelve mild atopic asthmatic and nine control subjects underwent segmental sham and allergen challenge. EPO concentrations in BAL fluid and serum were determined by immunoassay and flow cytometry was used to determine the intracellular expression of EPO in BAL-derived and peripheral eosinophils. RESULTS: In asthmatic patients a large increase in BAL eosinophils--total cells: median 9.5 x 10(6) (range: 0.5 to 455.0 x 10(6)); relative: 38% (1 to 91%)--was detectable 24 h following allergen challenge, but peripheral blood eosinophil counts did not change. Concentrations of EPO in BAL fluid increased from 1 microg/L (1.0 to 6.8 microg/L) to 42 microg/L (5.6 to 379.6 microg/L; P < 0.01) after allergen but not after saline challenge (1.5 microg/L; 1.0 to 21.9 microg/L), whereas in control subjects all measurements were below the detection limit. Serum concentrations of EPO increased slightly from 18.3 microg/L (3.0 to 56.8 microg/L) to 27 microg/L (3.8 to 133.9 microg/L; P < 0.05) 24 h after allergen challenge in asthmatic patients. Furthermore, the intracellular expression of EPO (measured as mean fluorescence intensity) was decreased in BAL eosinophils compared with blood eosinophils (mean fluorescence intensity 29 (7 to 71) vs. 48 (20 to 85); P < 0.01) after allergen challenge. CONCLUSION: The finding of increased EPO concentrations in the BAL fluid and decreased intracellular EPO expression in pulmonary eosinophils of asthmatic patients reflects the allergen-triggered release of EPO into the bronchial space.

Cytokine profile of bronchoalveolar lavage-derived CD4(+), CD8(+), and gammadelta T cells in people with asthma after segmental allergen challenge.

T cell-derived cytokines play an important role in the pathogenesis of allergic asthma, but little is known about the cytokine profile of their different subsets. The aim of the present study was to investigate the cytokine production potential of CD4(+), CD8(+), or gammadelta(+) T cells derived from the bronchoalveolar space of mild atopic asthmatic subjects (n = 11) and nonatopic control subjects (n = 9) before and 24 h after segmental allergen challenge. The cytokine production was determined using the technique of intracellular cytokine detection by flow cytometry. Comparing asthmatic with control subjects we found no difference in the percentage of CD4(+), CD8(+), or gammadelta T cells in the bronchoalveolar lavage fluid before and after allergen challenge. Before allergen challenge the proportion of cells producing the cytokines interferon (IFN)-gamma, interleukin (IL)-2, IL-4, IL-5, and IL-13 was not different in CD4(+) and CD8(+) cells. The major difference between the groups was an increased percentage of positive-staining cells for the T helper-(Th)2-cytokines IL-5 and IL-13 in the gammadelta T-cell subset. After allergen challenge, all T-cell subsets revealed a decreased proportion of cells producing the Th1-type cytokines IFN-gamma and IL-2. The percentage of IL-4- and IL-5-positive cells did not change in all subsets, and there was a decreased proportion of IL-13- positive cells in the CD4(+) subset. These findings indicate an increased Th2-cytokine profile in gammadelta T cells. After allergen challenge, the dysbalance between Th1 and Th2 cytokines was further accentuated by a reduction in Th1 cytokine-producing T cells.

Dysfunction of pulmonary surfactant in asthmatics after segmental allergen challenge.

Increased airway resistance in asthma may be partly due to poor function of pulmonary surfactant. This study investigated the inflammatory changes of bronchoalveolar lavage fluid (BALF) and the performance of BALF surfactant in healthy control subjects (n = 9) and patients with mild allergic asthma (n = 15) before and after segmental challenge. BALF was obtained for baseline values, and 24 h after challenge with saline solution in one lung segment and with allergen in another. Cell counts, phospholipid and protein concentrations, and ratios of small to large surfactant aggregates (SA/LA) were analyzed. Surface tension was determined with a pulsating bubble surfactometer, and the ability of the BALF surfactant to maintain airway patency was assessed with a capillary surfactometer. Baseline values of control subjects and asthmatics were not different. Challenge with saline and antigen raised total inflammatory cells in both control subjects and asthmatics. Allergen challenge of asthmatics, but not of healthy volunteers, significantly increased eosinophils, proteins, SA/ LA, and surface tension at minimum bubble size, and diminished the time the capillary tube is open. In conclusion, allergen challenge in asthmatics induced surfactant dysfunction, probably mainly because of inhibiting proteins. During an asthma attack, narrow conducting airways may become blocked, which might contribute to an increased airway resistance.

Enhanced expression of fas ligand (CD95L) on T cells after segmental allergen provocation in asthma.

BACKGROUND: Little is known about the termination of the T-cell driven inflammation found in patients with allergic asthma. OBJECTIVE: Because signals delivered through Fas/Fas ligand can lead to T-cell apoptosis, we investigated the expression of Fas and Fas ligand on peripheral blood- and bronchoalveolar lavage fluid (BALF)-derived T cells and the percentage of apoptotic BALF cells in asthma. METHODS: Nine atopic subjects with mild asthma and 9 control subjects underwent segmental sham and allergen challenge. Flow cytometry was used to determine the T-cell expression of Fas and Fas ligand, and the terminal dUTP nick end labeled technique was applied to detect apoptotic BALF cells. RESULTS: In asthmatic and control subjects almost all T cells in the BALF expressed Fas antigen without changes after saline or allergen challenge. A small percentage of T cells in BALF expressed the Fas ligand. In asthmatic subjects, but not in control subjects, there was a significant increase in Fas ligand after allergen challenge (CD3: 0.8% +/- 0.6% [baseline] vs 3.2% +/- 1.2% [allergen challenge]; CD4: 1.8% +/- 0.0% vs 4.3% +/- 1.8%; CD8: 2.8% +/- 2.4% vs 9.1% +/- 4.8%) but not after saline challenge, with a significant correlation to the percentage of BALF eosinophils. Apoptotic BALF cells were localized exclusively in macrophages at a very low frequency (0.03% to 0.15%) and without changes after saline or allergen challenge in both groups. CONCLUSION: In asthma there is an upregulation of Fas ligand on T cells in BALF after allergen challenge. Because there is no evidence for increased apoptosis, this phenomenon may reflect antigen-induced T-cell activation rather than apoptosis.

Interleukin 16 and T-cell chemoattractant activity in bronchoalveolar lavage 24 hours after allergen challenge in asthma.

IL-16 has been shown to be one of the earliest CD4(+) cell chemoattractants present in BAL 4-6 h after antigen challenge but little is known about its persistence and biological activity after 6 h. We determined the concentration of IL-16 using ELISA and the T-cell chemoattractant activity using a modified Boyden chamber assay in unconcentrated BAL fluid from 13 patients with mild asthma and 9 nonatopic control subjects at baseline and 24 h after segmental allergen or saline challenge. Furthermore, the percentage of IL-16-producing T cells was determined in the different samples of BAL fluid using a flow cytometric intracellular cytokine assay. Although no substantial levels of IL-16 protein were detectable in BAL fluid from control subjects and patients with asthma at baseline and after saline challenge, IL-16 concentrations were significantly elevated in patients with asthma after allergen challenge (median, 97 pg/ml; range, 38-362 pg/ml; p < 0.01). Furthermore, there was an increased T-cell chemoattractant activity after allergen challenge in patients with asthma (p < 0.01), which could be blocked by preincubation with anti-IL-16 antibodies and which correlated significantly with the IL-16 protein levels (R = 0.90, p < 0.01) and with the level of Fas ligand expression on BAL CD4(+) cells (R = 0. 80, p < 0.05). A high percentage (mean 70-90%) of CD4(+) and CD8(+) cells stained positively for IL-16 in both patients with asthma and control subjects without differences after allergen or saline challenge. These data demonstrate that the increased chemotactic activity for T cells in patients with asthma is mainly attributable to IL-16. Although T cells by themselves are able to produce IL-16, other cells, such as epithelial cells, have to be considered as further sources for this cytokine in patients with asthma.