Bronchoalveolar and serological parameters reflecting the severity of sarcoidosis.

The aim of the present study was to determine which bronchoalveolar lavage fluid (BALF) and serological parameters reflect the severity of newly diagnosed pulmonary sarcoidosis. Seventy-four previously untreated sarcoid patients were categorised into three groups: 10 patients with Löfgren's syndrome, 51 patients with stable disease and 13 patients with progressing disease requiring systemic steroid treatment. Total BALF cell count, percentage of alveolar lymphocytes and lymphocyte CD4/ CD8 ratio were not associated with severity of disease. Interestingly, a significant increase in percentages of BALF neutrophils (5.2 +/- 1.1%) and eosinophils (1.7 +/- 0.6%) was observed in sarcoid patients with progressing disease. Elevated percentages of these two cell types were the only BALF parameters associated with a more frequent necessity for systemic steroid therapy. This association between an elevated percentage of BALF neutrophils and the necessity for steroid treatment was observed in advanced as well as early sarcoidosis (radiological types I and II). Serum levels of soluble interleukin-2 receptor and neopterin were significantly elevated in progressing disease compared to stable disease or Löfgren's syndrome. The present results demonstrate that increased percentages of neutrophils (>3.0%) and eosinophils (>1%) in bronchoalveolar lavage fluid from newly diagnosed pulmonary sarcoidosis is associated with a significantly higher risk of necessity for steroid therapy and may be helpful markers of progressive disease. Furthermore, of the serological parameters investigated, only serum levels of soluble interleukin-2 receptor and neopterin were associated with disease severity.

The cytokine network in sarcoidosis and its clinical relevance.

In recent years, analysis of the cytokine network has substantially improved our knowledge of the immunopathogenesis of sarcoidosis. There is increasing evidence from clinical immunology that analysis of the cytokine network may be helpful for clinicians to assess the extent and activity of sarcoid inflammation. Genetic polymorphisms may contribute to interindividual differences in the regulation of cytokine release. Thus, disease phenotype-associated haplotypes should exist and their analysis might disclose risk profiles of individual patients. Furthermore, serological assessment of cytokines or soluble cytokine receptors may become suitable parameters in clinical practice to detect an ongoing inflammation in chronic sarcoidosis.

[Corrected normal values for serum ACE by genotyping the deletion-/insertion-polymorphism of the ACE gene]. / Korrigierte Normwerte für das Serum-ACE durch Genotypisierung eines Deletions-/Insertions-Polymorphismus des ACE-Gens.

BACKGROUND: In sarcoidosis, serum ACE is widely recognised as a marker of disease activity. Since 1990 a deletion-/insertion polymorphism of the ACE gene is known and a correlation between the genotypes of this polymorphism and serum ACE levels has been observed. Homzygotes for the deletion allele (DD) have the highest levels and homozygotes for the insertion allele (II) the lowest. Heterozygote (DI) persons show intermediate levels. The extent of this influence varies in populations of different ethnic origin. In a large cohort of healthy individuals from North of Germany, genotype-based normal ranges for serum ACE were determined for the population of Germany for the first time. METHODS: In 262 healthy individuals the genotype of the ACE D/I gene polymorphism was determined from genomic DNA by a PCR method. In addition, in serum samples of all these individuals ACE level was measured with a kinetic test. RESULTS: The genotype DD was found in 29.4 % of the individuals examined, the genotype DI in 49.6 % and the genotype II in 21.0 %, respectively. These results are similar to those found in previous investigations in other populations of Central European origin. The mean serum ACE levels (95 % confidence interval) in individuals with the genotypes DD, DI and II are 59.8 U/l (31.8 - 87.8), 47.7 U/l (18.6 - 76.8) and 32.2 U/l (13.7 - 50.7), respectively. Without taking the genotype into account, the average value is 48.0 U/l (15.0 - 80.9). Differences between all genotype groups are highly significant (p < 0.0001). CONCLUSIONS: In sarcoidosis patients, the determination of this ACE gene polymorphism once in the course of the disease allows a better interpretation of the serum ACE levels measured.

Effect of proinflammatory cytokines on interleukin-8 mRNA expression and protein production by isolated human alveolar epithelial cells type II in primary culture.

Alveolar epithelial cells type II (AEC-II) are ideally situated to regulate the recruitment and activation of different types of cells through the production of chemokines in response to inflammatory stimulation from the alveolar space. We hypothesized that these cells are important producers of interleukin-8 (IL-8) in the lung. This lead us to investigate the capacity of isolated human AEC-II cells to release IL-8 and whether this IL-8 release is regulated by proinflammatory cytokines, i.e. IL-1 beta, TNF-alpha and IFN-gamma. We isolated AEC-II from tumor-free sections of human lungs obtained by pneumectomy and purified the cells by magnetic activated cell sorting. For control experiments the AEC-II-like cell line A549 was used. IL-8 concentration was measured by ELISA in supernatants of unstimulated and LPS-, IL-1 beta-, TNF-alpha- and IFN-gamma- stimulated cells. IL-8 mRNA expression was evaluated by RT-PCR. Spontaneous IL-8 mRNA expression and protein secretion by AEC-II were significantly higher in comparison with A549 cells. TNF-alpha increased both IL-8 mRNA expression and protein production, whereas IL-1 beta slightly increased IL-8 release but did not change mRNA expression in AEC-II. LPS and IFN-gamma did not influence IL-8 expression in AEC-II and A549 cells. These results show considerable differences between A549 cell and AEC-II. The latter are capable of producing IL-8 under the control of proinflammatory cytokines. Our findings demonstrate that the modulation of IL-8 release in AEC-II may have an important impact on the immunoreactivity of these cells during pulmonary inflammation in vivo.

Serum level of soluble tumour necrosis factor receptor II (75 kDa) indicates inflammatory activity of sarcoidosis.

OBJECTIVES: Tumour necrosis factor alpha (TNFalpha) is a key cytokine involved in granuloma formation of sarcoidosis. Since soluble TNF receptors (sTNF-R) are known to inhibit TNF effects, we were interested in whether they are elevated in the serum of sarcoidosis patients. METHODS: We determined serum levels of sTNF-R I (55 kDa) and sTNF-R II (75 kDa) in 49 patients with sarcoidosis and 22 controls. The clinical course of the disease was re-evaluated in a follow-up after (mean +/- SE) 6.8 +/- 6.6 months. RESULTS: sTNF-R I (3.1 +/- 1.1 ng mL-1, P < 0.05) and sTNF-R II (5.5 +/- 2.7 ng mL-1, P < 0.0005) were significantly elevated in sarcoidosis compared with controls (2.4 +/- 0.7 and 3.0 +/- 1.3 ng mL-1, respectively). Interestingly, both sTNF receptors were significantly higher in the serum of patients with active compared with inactive sarcoidosis (P < 0.005 and P < 0.0005, respectively). Furthermore, serum sTNF-R II levels were significantly higher in sarcoidosis patients with advanced radiological types II and III. In 10 patients, serum sTNF-R levels were obtained before and after systemic corticosteroid therapy and we observed a significant decrease of sTNF-R II (P < 0.02), whereas sTNF-R I levels were not reduced significantly. CONCLUSIONS: Both types of sTNF receptors are elevated in the serum of sarcoidosis patients with active disease, but only the sTNF-R II seems to be useful for monitoring the inflammatory activity of the disease.

Accessory function and costimulatory molecule expression of alveolar macrophages in patients with pulmonary tuberculosis.

An effective immune response against M. tuberculosis requires a coordinated interaction of alveolar macrophages (AM) and lymphocytes. Secondary signals, such as accessory function (AF) of antigen presenting cells and interaction of costimulatory molecules are also important for T cell activation. In the present study we determined the AF and the expression of CD11a, CD54, CD58, CD80, CD86 and HLA-DR costimulatory molecules by AMs lavaged from patients with pulmonary tuberculosis and controls. We hypothesized that alterations in AF and costimulatory molecule expression may influence the presentation of tuberculosis. Therefore these parameters were also correlated with the radiographic extension of the disease. AMs of patients with tuberculosis exhibited an increased AF and a significantly increased expression of co-stimulatory molecules compared with controls. Furthermore, we observed that the expression of CD54 (ICAM-1) decreased with the course of the disease. We conclude that the infection by M. tuberculosis results in an increased AF of AMs and the activity of AMs remains uninfluenced by the extension of the disease. Clear-cut changes of patterns of costimulatory molecule expression by AMs could not be observed with the progression of tuberculosis.

Different cytokine patterns correlate with the extension of disease in pulmonary tuberculosis.

The relative amounts of different pro- and anti-inflammatory cytokines released at the site of infection by bronchoalveolar lavage (BAL) cells may influence the presentation of tuberculosis. To investigate this hypothesis the in situ release by BAL cells of the following cytokines was measured and correlated with the chest X-ray findings of 43 patients with pulmonary tuberculosis: interleukin (IL)-8, macrophage inflammatory protein-1alpha (MIP-1alpha), IL-6, tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-beta (TGF-beta), interferon-gamma (IFN-gamma), IL-2, IL-4 and IL-5. The release of IL-8 and IL-6 decreased with the progression of the disease, while the release of MIP-1alpha was increased in patients with advanced tuberculosis. The release of TNF-alpha and TGF-beta did not differ between patients with or without cavitary lesions. The Th1 (IFN-gamma and IL-2) and Th2 (IL-4 and IL-5) cytokine release exhibited a gradual increment with the advance of tuberculosis. Thus, our data provide evidence that a Th0 cytokine pattern is predominant at the site of pulmonary tuberculosis. In conclusion, immunoparalysis status could not be observed in our patients with severe tuberculosis.

Time course of the tilorone-induced lysosomal accumulation of sulphated glycosaminoglycans in cultured fibroblasts.

Dicationic amphiphilic drugs such as the immunomodulator tilorone [2,7-bis-[2-(diethylamino)ethoxy]fluoren-9-one] are accumulated in lysosomes and disturb the degradation of sulphated glycosaminoglycans (GAGs) thus leading to generalized lysosomal GAG storage (mainly dermatan sulphate) in vivo and in cultured cells. In the present study, the time course of the tilorone-induced GAG storage was determined in cultured bovine corneal fibroblasts by a radiochemical approach and by morphological examination. In contrast to the rapid lysosomal accumulation of the drug as reported previously, it took approximately 42 h to reach 50% of the GAG storage obtained after 96 h. This is thought to be due to the fact that the temporal development of storage of undigested GAGs depends on the natural delivery of GAGs towards the lysosomal apparatus.

Increased expression of proinflammatory chemokines in bronchoalveolar lavage cells of patients with progressing idiopathic pulmonary fibrosis and sarcoidosis.

BACKGROUND: There is increasing evidence that the proinflammatory chemokines interleukin-8 (IL-8) and macrophage inflammatory protein-1 alpha (MIP-1 alpha) are involved in the pathogenesis of interstitial lung diseases. METHODS: We investigated the release of TNF-alpha, IL-8, MIP-1 alpha by cultured bronchoalveolar lavage (BAL) immune cells of patients with idiopathic pulmonary fibrosis (IPF, n = 24), sarcoidosis (SAR, n = 24), and controls (n = 20) by ELISA. Furthermore, mRNA expression of these cytokines in BAL cells immediately frozen after bronchoscopy was determined. The clinical course of the disease was evaluated and the patients were subdivided into groups with progressing or stable disease. RESULTS: TNF-alpha, IL-8, and MIP-1 alpha were significantly elevated in the supernatants of BAL immune cells of IPF and SAR patients with progressing disease compared to controls (p < 0.005 in both diseases) and also when compared to patients with stable disease (IPF p < 0.005, SAR p < 0.05). Interestingly, the release of TNF-alpha, IL-8, and MIP-1 alpha did not differ significantly between IPF patients with stable disease and controls, whereas in SAR patients with stable disease a difference at a low significance level (p < 0.05) was obtained. In IPF and SAR patients with progressing disease, a clear mRNA signal of TNF-alpha, IL-8, and MIP-1 alpha was detected in BAL immune cells not having been stimulated by adherence to plastic, whereas in patients with stable disease or controls only a weak signal was observed. MIP-1 alpha release correlated positively with percentage of BAL eosinophils in IPF and SAR. Furthermore, the percentage of eosinophils in BAL was significantly elevated in the IPF subgroup with progressing disease. CONCLUSIONS: Our data demonstrate that an exaggerated expression of TNF-alpha, IL-8, and MIP-1 alpha in BAL immune cells is characteristic for IPF and SAR patients who show progressing disease.

Meningeal cells stimulate neuronal migration and the formation of radial glial fascicles from the cerebellar external granular layer.

The cerebellar granule cell layer and partly the Bergmann glial scaffold arise from a secondary subpial proliferative zone, the external granular layer. Their development can be disrupted by selective destruction of meningeal cells. In order to clarify the mechanisms of meningeal control of cortical development, we have investigated the development of early postnatal rat cerebellar slice explants in different coculture set-ups with meningeal cells and other fibroblasts. Fibroblasts of various sources (1) stimulate migration of undifferentiated neurons from the explants by a diffusible factor and (2) trigger the development of a radial phenotype in glial cells by contact-mediated mechanisms involving basal lamina constituents. These data provide further evidence for the involvement of mesenchymal-epithelial interactions in the development of the cerebellar cortex.

Serum level of interleukin 8 is elevated in idiopathic pulmonary fibrosis and indicates disease activity.

It has been shown that interleukin 8 (IL-8) is increased in bronchoalveolar lavage fluid (BALF) of patients with idiopathic pulmonary fibrosis (IPF) and there is increasing evidence that it is involved in the pathogenesis of this disease. To date, no data are available as to whether IL-8 is elevated in sera of IPF patients. We obtained sera from 42 patients with IPF and 20 healthy controls at time of BAL. From 20 of 42 patients with IPF and 12 of 20 controls BALF was available, enabling us to measure IL-8 in serum and BALF of the same time point. IL-8 was significantly elevated in serum (54.7 +/- 7.5 pg/ml, p < 0.0001) and BALF (715.7 +/- 112.4 pg/ml, p < 0.0001) of patients with IPF compared with controls (IL-8 in serum, 5.2 +/- 0.8 pg/ml; IL-8 in BALF, 67.3 +/- 9.7 pg/ml). We observed a significant positive correlation between IL-8 levels in BALF and percentage of BALF neutrophils (p < 0.001) and between serum IL-8 and BALF IL-8 levels (p < 0.005) in patients with IPF. Consequently, the serum IL-8 level correlated positively with the percentage of BAL neutrophils (p < 0.01), indicating that it may reflect the degree of neutrophilic alveolitis in IPF. Furthermore, the serum IL-8 level showed a negative correlation with important indicators of impairment of lung function (DL(CO), TLC, VC) and PaO2. In conclusion, we were able to demonstrate that the degree of neutrophilic alveolitis in IPF is reflected by increased serum levels of IL-8 and we suggest that the serological assessment of IL-8 may provide a useful parameter for clinicians in monitoring patients with IPF.

Sarcoidosis: TNF-alpha release from alveolar macrophages and serum level of sIL-2R are prognostic markers.

At the time of diagnosis, many sarcoidosis patients have no clinical indication for corticosteroid therapy, and prognostic parameters predicting deterioration are missing. In the present study, we investigated parameters derived from bronchoalveolar lavage (BAL) and serum in 77 patients with recently diagnosed sarcoidosis to test their predictive value. Patients were divided into a group with (Group A, n = 37) and a group without (Group B, n = 40) indications for therapy, and the course of the disease was evaluated after 5.7 +/- 0.4 mo. The CD4+/CD8+ lymphocyte ratio and percentage of BAL lymphocytes were of no predictive value. Release of tumor necrosis factor-alpha (TNF-alpha) from cultured alveolar macrophages (AM) was significantly increased in both groups (Group A = 1,872 +/- 428 pg/ml; Group B = 1,561 +/- 449 pg/ml) as compared with controls (220 +/- 37 pg/ml). In Group B, however, patients with a high level of TNF-alpha release had a significantly greater risk of disease progression than did those with normal TNF-alpha release (43.8% versus 8.3%, respectively). From the serologic parameters investigated, consisting of neopterin, angiotensin converting enzyme (ACE), and soluble interleukin-2 receptor (sIL-2R), only the last was of significant predictive value; 42.1% of sarcoidosis patients in Group B with a high level of sIL-2R experienced disease progression, whereas none of those with a normal level did. We conclude that TNF-alpha release and sIL-2R are suitable parameters for predicting disease progression in sarcoid patients who have no indication for therapy at the time of disease diagnosis.