Ultrastructure of bronchial biopsies from patients with allergic and non-allergic asthma.

Epithelial damage is commonly found in airways of asthma patients. The aim of this study was to investigate epithelial damage in allergic and non-allergic asthma at the ultrastructural level. Bronchial biopsies obtained from patients with allergic asthma (n=11), non-allergic asthma (n=7), and healthy controls (n=5) were studied by transmission electron microscopy. Epithelial damage was found to be extensive in both asthma groups. Both in basal and in columnar cells, relative desmosome length was reduced by 30-40%. In columnar cells, half-desmosomes (i.e., desmosomes of which only one side was present) were frequently noticed. Eosinophils showing piece-meal degranulation were commonly observed in allergic asthma. Degranulating mast cells were more often observed in allergic asthma. Goblet cell hyperplasia was only found in allergic asthma. Lymphocytes were increased in both groups. In both groups, the lamina densa of the basal lamina was thicker than the control by about 40-50%. In allergic asthma the lamina densa was irregular with focal thickening. While there was always a tendency for changes (epithelial damage, desmosomes, degranulating mast cells, basal lamina) to be more extensive in allergic asthma compared to non-allergic asthma, there was no significant difference between the two groups in this respect. Reduced desmosomal contact may be an important factor in the epithelial shedding observed in patients with asthma.

Inflammation and structural changes in the airways of patients with primary Sjögren's syndrome.

The present study aimed to compare the cellular pattern and structural changes in the airways of patients with primary Sjögren's syndrome (pSS) with healthy controls. Bronchial biopsy specimens were obtained from seven subjects with pSS and seven healthy controls. All the patients with pSS had increased bronchial responsiveness to methacholine. In the biopsies inflammatory cells, cytokine-producing cells, tenascin and laminin were visual zed by immunostaining. Patients with pSS had a higher number of neutrophils and mast cells than healthy controls, while the number of eosinophils was similar in the two groups. The number of IL-8-positive cells was higher in pSS butthe numbers of IL-4-and IL-5-positive cells were not significantly different between pSS and healthy controls. The numbers of T cells in patients with pSS were higher than in healthy controls, while the numbers of CD25-positive cells were similar to the healthy controls. The degree of epithelial integrity in patients with pSS was significantly lower than in the control group and the tenascin and laminin layers were significantly thicker in the pSS group. There was a correlation between the number of mast cells and the thickness of the tenascin and laminin layers in pSS. In conclusion, we found that the cellular pattern in the bronchial mucosa of patients with pSS displayed large numbers of neutrophils, mast cells and T-lymphocytes. These changes in inflammatory cell numbers seemed to relate to the observed increased epithelial damage and structural changes of the subepithelium. The structural findings, but not the pattern of inflammatory cells, are shared with atopic asthma and may relate to the increased bronchial hyper-responsiveness seen in both diseases.

Inflammation and structural changes in the airways of patients with atopic and nonatopic asthma. BHR Group.

The aim of the present study was to compare the cellular pattern and structural changes in the airway walls of atopic and nonatopic patients with asthma. Bronchial biopsy specimens were obtained from 13 atopic subjects with asthma, nine nonatopic patients with asthma, and seven healthy control subjects and investigated using immunohistochemical methods. The number of eosinophils increased in both asthma groups, but significantly more in the atopic group. The number of mast cells increased similarly in the two asthma groups, whereas the number of neutrophils increased only in the nonatopic asthma group. The number of T-lymphocytes (CD3-, CD4-, CD8-, CD-25-positive cells) was higher in patients with atopic asthma compared with nonatopic asthma. Interleukin-4 (IL-4) and IL-5-positive cells were more frequently found in the atopic asthma group, whereas cells staining for IL-8 were more frequent in the nonatopic group. The degree of epithelial damage was significantly higher in the atopic asthma group compared with the control subjects and the nonatopic asthmatics. The tenascin and laminin layer was significantly thicker in the atopic group compared with the group of nonatopic asthmatics. In the atopic group, there was a significant negative correlation between epithelial integrity (defined as the relative length of intact epithelium) and the eosinophil count and also between the number of CD25-positive cells and epithelial integrity. The number of mast cells correlated positively with the thickness of tenascin- and laminin-positive layers. In conclusion, we provide evidence of different patterns of involvement of inflammatory cells in atopic and nonatopic patients with asthma. There were also structural differences in the bronchial mucous membrane between atopic asthma and nonatopic asthma. This suggests that there are differences in the extent of the immunopathologic response of these clinically distinct forms of asthma.

Combined fluorine-18-FDG and carbon-11-methionine PET for diagnosis of tumors in lung and mediastinum.

UNLABELLED: We evaluated the value of PET using 18F-fluorodeoxyglucose (FDG) and 11C-methionine, individually or in combination, to distinguish malignant from benign tumors and to identify or exclude mediastinal metastases. METHODS: Seventeen patients with a tumor in the lung or mediastinum were evaluated with 18F-FDG and 11C-methionine PET. For morphological comparison, we used CT, and all findings were confirmed by histology of surgical resection specimens (n = 16) or by cytology (n = 1). RESULTS: All tumors were visualized equally well with both tracers, and there were no false-positive results. In 2 patients with a malignant tumor, coexisting pneumonia was correctly diagnosed as an inflammatory lesion because of its wedge-like shape. PET correctly excluded hilar invasion and mediastinal lymph node metastases in 10 of 14 patients with primary lung tumor. PET identified mediastinal metastases in 4 of 4 patients. CT failed to detect mediastinal tumor spread in 2 patients and gave a false-positive reading in 2 others. Significantly higher uptake (SUV) and transport rate (slope) values were obtained from malignant than benign lesions with both tracers. No major differences were seen in either the levels of significance or accuracy when the two tracers were compared. Slope values did not add further information to what was obtained with SUV. Density correction of SUV and slope values, to avoid the influence of surrounding air as well as tumor heterogeneity, increased these differences somewhat. Both tracers distinguished malignant from benign lesions with a 93% sensitivity and an accuracy of 89%-95%, but sensitivity improved to 100% when values from both tracers were combined. CONCLUSION: Fluorine-18-FDG and 11C-methionine PET visualized all tumors equally well and detected mediastinal spread better than CT. For differentiation purposes, the problems of false-positive and false-negative PET findings could not be safely overcome in a limited number of cases either by the use of both tracers, by the additional use of slope values or by lesion density correction.

Effects of IL-2 treatment on different compartments of the irradiated rat lung analysed by bronchoalveolar lavage and lung tissue morphology.

In recent years, interleukin-2 (IL-2) has been used as an immunomodulatory agent in the treatment of various malignant tumours. However, this treatment has been limited by serious side-effects, including toxic reactions in the lung. The effects of IL-2 treatment on inflammatory cell populations in the normal and irradiated rat lung were investigated in this study. IL-2 was continuously administered as a subcutaneous infusion over a 6 week study period. Irradiation was given in a single dose (25 Gy) the day after starting IL-2 treatment. Evaluation with bronchoalveolar lavage fluid (BALF) analysis and lung tissue morphology was made 6 weeks after irradiation. In nonirradiated rats, IL-2 treatment induced significant increases in the total number of inflammatory cells in the perivascular, interstitial and peribronchial tissues as well as in the alveolar space. These increases were not reflected in BALF; on the contrary, a significant decrease of the total numbers of inflammatory cells was found in BALF. Irradiation alone caused a more pronounced inflammatory response was significant increases of all inflammatory cells in all lung compartments, which was also reflected in BALF. Concomitant treatment with IL-2 and irradiation induced an enhanced accumulation of inflammatory cells in the perivascular and peribronchial tissues compared with irradiation alone. Thus, both irradiation and IL-2 treatment induce inflammatory reactions in the lung, but there were signs of synergistic effects seen in this study. Furthermore, the results also emphasize the difficulties in making sophisticated conclusions from BALF analyses alone.

Characterization of the mechanism involved in bleomycin-induced increased hyaluronan production in rat lung.

The molecular mechanisms behind the accumulation of hyaluronan during bleomycin-induced lung injury in rats were investigated. The stimulatory effects of bronchoalveolar lavage fluid (BALF) and alveolar macrophage (AM)-conditioned media on hyaluronan synthesis in normal rat lung fibroblast cultures were studied as well as the hyaluronan binding activity on AM. BALF obtained on days 1 and 5 after bleomycin instillation exhibited hyaluronan stimulatory activity similar to that of 10% fetal serum; the activity returned to control values on day 14 after bleomycin treatment. Conditioned media from cultures of AM obtained from bleomycin-treated rats exhibited stimulatory effects higher than that of media from AM of control rats and equal to or higher than that of 10% fetal calf serum. The stimulatory activity in BALF was significantly inhibited by neutralizing antibodies against transforming growth factor-beta; the activity in AM-conditioned media was only partially affected. Neutralizing antibodies against platelet-derived growth factor-BB or -AA had no such inhibiting effect. Interestingly, AM from bleomycin-treated rats exhibited low hyaluronan binding activity. [3H]Hyaluronan binding by AM on days 1 and 5 after bleomycin administration was about 2-fold and 4-fold lower, respectively, compared with that by AM derived from saline-treated rats. This decrease was normalized 14 days after bleomycin treatment. In conclusion, our results indicate that factors with high potential to stimulate hyaluronan synthesis in rat lung fibroblasts are accumulated in BALF from bleomycin-treated rats and that AM are likely to be one source of such stimulatory factors.(ABSTRACT TRUNCATED AT 250 WORDS)

Phenotypic and functional activation of alveolar macrophages, T lymphocytes and NK cells in patients with systemic sclerosis and primary Sjögren's syndrome.

OBJECTIVES: Attempts to differentiate between the pathogenesis of the severe pulmonary manifestations observed in systemic sclerosis (SSc) and the mild form in primary Sjögren's syndrome (pSS) were performed by studying cell populations recovered during bronchoalveolar lavage (BAL). METHODS AND RESULTS: Two-colour flow cytometric analysis of BAL fluid lymphocytes showed a similar degree of phenotypic activation (DR+) of CD4+ and CD8+ T lymphocyte subsets and CD16+ NK cells in patients with SSc (n = 13) and pSS (n = 11) groups and healthy controls (n = 11). Alveolar macrophages expressed the CD14 antigen at significantly increased densities in patients with SSc. Alveolar macrophage activation in SSc was also suggested by increased IL-6 concentrations in neat BAL fluid and increases in macrophage production of TNF alpha and EGF in vitro. SSc patients also had increased proportions of neutrophils and eosinophils in BAL fluid. No correlations were found between any cellular subsets or cytokine levels in BAL fluid and lung status at the time of lavage in SSc or pSS patients or the subsequent course of the pulmonary function in SSc patients. CONCLUSION: It is concluded that the phenotypical activation of alveolar helper/inducer (DR+CD4+) and suppressor/cytotoxic (DR+CD8+) T lymphocytes and NK (DR+CD16+) cells is not a prerequisite for the development of lung fibrosis in SSc or bronchial hyper-responsiveness in pSS. Alveolar macrophage activation may contribute to the development of lung fibrosis in SSc.

Alveolar accumulation of fibronectin and hyaluronan precedes bleomycin-induced pulmonary fibrosis in the rat.

The development of bleomycin-induced pulmonary fibrosis in rats was studied over a period of 30 days after an intratracheal instillation of bleomycin. Fibronectin was visualized in histological sections and quantified in bronchoalveolar lavage fluid (BALF) and related to simultaneous measurements of hyaluronan, collagen and albumin in BALF and/or lung tissue extracts. An increase in BALF fibronectin levels was noted after 3 days and the peak value a sixty fold increase was noted at day 7. Thereafter, the fibronectin levels declined and reached control values on day 21. A pronounced, patchily distributed staining for fibronectin appeared in the injured alveolar tissue parallel to the increased lavage fluid fibronectin levels on days 3-7. A fainter, streakily distributed fibronectin staining remained within the alveolar walls in areas with proliferating fibroblasts on days 14-30. Albumin in BALF increased to a peak level, 20 times control values, after 3 days and then rapidly declined. Thus, the ratio of fibronectin to albumin increased to a peak value of 43 times control values on day 7, indicating that plasma leakage cannot be the only source of the observed increase in lavage fibronectin. Lung tissue hydroxyproline increased between days 7 and 30, whereas extractable hyaluronan in lung tissue and bronchoalveolar lavage fluid peaked on days 3-7 and then gradually declined towards normal values on days 21-30. These data demonstrate that fibronectin accumulates in the alveolar tissue during the early inflammatory phase of the bleomycin-induced lung injury, parallelling hyaluronan accumulation and preceding the development of pulmonary fibrosis.

High frequency of IgM antibodies to coxsackie B virus in sarcoidosis patients and patients with asbestos-related lesions.

By using radioimmunoassay (RIA) for detection of IgM antibodies to Coxsackie B viruses (CBV), the occurrence of these antibodies was investigated in patients with sarcoidosis and asbestos-related lesions. Sixty-one per cent of the patients with sarcoidosis, all patients with benign asbestos pleural effusion, and 67% of those with diffuse asbestos-related pleural thickening showed CBV-IgM. Patients with healed sarcoidosis or pleural plaques were all negative, and among the "healthy" controls seven per cent had CBV-IgM. Thus, there was a high frequency of CBV-IgM in patients with sarcoidosis and in those with asbestos-related diseases. Since the titres could be the effect of an unspecific polyclonal stimulation of the B cells, sera were tested for antibodies to rubella and cytomegalovirus, but without any remarkable results.

Eosinophil activation in systemic sclerosis.

Circulating levels of eosinophil cationic protein (ECP) were increased 4-fold in patients with systemic sclerosis (SSc) compared with those in healthy control subjects. There was no correlation between the ECP concentrations and laboratory indices of inflammatory activity or visceral involvement. Mean ECP levels were higher in patients with a history of occupational exposure to silica, even though patients who had no such history also had ECP levels higher than normal. The patients had increased bronchoalveolar levels of ECP, which correlated with impaired lung functioning. Skin infiltration with activated eosinophils and extracellular deposits of ECP were present in skin biopsy samples from the SSc patients. We conclude that eosinophil activation is part of the inflammatory process in SSc.

Alveolar accumulation of hyaluronan and alveolar cellular response in bleomycin-induced alveolitis.

Hyaluronan (HA) accumulating in the alveolar interstitial tissue of rats injured by a single intratracheal instillation of bleomycin has been visualized histologically and assayed. HA was present already by Day 1 after bleomycin treatment, increased to a maximum value on Days 3 and 7 and then declined. A time-dependent relationship between this early connective tissue response and the invasion of inflammatory cells in the alveolar tissue was apparent. The dominating invading cells by Day 1 were granulocytes showing positive staining for the monoclonal antibody OX-42 reflecting the C3b receptor. The numbers of macrophages expressing class II antigens started to increase on Day 1, reaching a maximum on Days 3-7 and then declined. Macrophages were the dominating OX-42+ cells by Day 7. The appearance of W3/13+ cells ("pan-T-lymphocytes") showed a similar pattern to that for the class II expressing macrophages. The number of cells expressing CD4 antigen increased until Day 3 and levelled off on Day 30 whilst the largest number of cells expressing CD8 antigen was seen on Day 30. Few cells expressing B-cell phenotype outside lymph nodules were identified. Alveolar lining epithelial cells, probably epithelial type II cells, expressed class II antigens by Days 3-14. The time-related accumulation of HA and the appearance of T-cells, macrophages and granulocytes expressing signs of activation suggests that these cells may be involved in the early connective tissue response of the lung injured by bleomycin.

Accumulation of hyaluronan in bronchoalveolar lavage fluid is independent of iron-, complement- and granulocyte-depletion in bleomycin-induced alveolitis in the rat.

Previous studies on bleomycin-induced alveolitis in rats have demonstrated a transient histological accumulation of hyaluronan (hyaluronate or hyaluronic acid) in the alveolar interstitium, corresponding to increases in hyaluronan (HA) levels in bronchoalveolar lavage (BAL) fluid and lung tissue extracts. The accumulation of HA was related to the influx of inflammatory cells, especially polymorphonucleated cells (PMNs) in BAL fluid and the increase in lung water. In this study we have investigated the influence of iron, complement and PMN dependent mechanisms on the early connective response of the lung in the bleomycin rat model. Iron depletion had no effect on HA or the cellular composition of lavage fluid recovered on day 4 post bleomycin. Treatment of bleomycin-injured rats with cobra venom factor (CVF), totally neutralized complement haemolytic activity but had no effect on lavage HA or the cell invasion in BAL. Treatment with anti-neutrophil serum (ANS), reduced blood and lavage PMN by 70-80%, but had no influence on HA levels in BAL. These results suggest that regulatory mechanisms other than those dependent on iron, complement activation or PMN recruitment are responsible for HA accumulation in this fibrosing alveolitis animal model.

High-dose corticosteroids during bleomycin-induced alveolitis in the rat do not suppress the accumulation of hyaluronan (hyaluronic acid) in lung tissue.

A single intratracheal injection of bleomycin in rats induced, 4 days later, a considerable accumulation of hyaluronan (hyaluronate, hyaluronic acid) in the lung tissue. This connective tissue reaction was quantified biochemically by analysing hyaluronan (HA) in bronchoalveolar lavage fluid (BAL) and lung tissue extracts. The molecular weight of the HA recovered during lavage was 0.2-0.3 X 10(6) daltons. The HA accumulation was related to an increase in lung water content and associated with an increased influx of eosinophils, neutrophils and lymphocytes into BAL fluid. High-dose corticosteroid treatment (prednisolone 15 mg.kg-1 bw per day) to bleomycin injured rats had no effect on the lung tissue content of HA, the recovery of HA during BAL or the molecular weight of HA accumulated in the alveolar space. Furthermore, steroids did not influence the increased lung water content, or the appearance of inflammatory cells in lavage fluid. These findings indicate that the early connective tissue response to the bleomycin lung injury is mediated by a mechanism which is unaffected by systemic high-dose corticosteroids.

Lung accumulation of hyaluronan parallels pulmonary edema in experimental alveolitis.

The glycosaminoglycan hyaluronan (HA, hyaluronate or hyaluronic acid) was quantified in rat lung during the development of bleomycin-induced lung injury. Extracted lung HA was measured by a radiometric assay. In control rats, the HA lung content was 95 +/- 5 (SE) micrograms/g of freeze-dried and homogenized lung tissue. After a single intratracheal instillation of bleomycin, the HA content of the lung increased significantly on day 1. Peak HA concentrations on days 3-7 averaged 70% higher than normal lung HA concentrations. From day 7 the HA concentrations progressively declined and had returned to normal by day 30. Qualitative assessment of lung HA has previously demonstrated that HA is accumulated in the edematous interstitial alveolar space during the alveolitis phase of bleomycin injury. Since high-molecular-weight HA has unique hydrophilic properties, another aim of the study was to elucidate the possible link between the increase in lung HA and the development of interstitial-alveolar edema postbleomycin. HA recovered by bronchoalveolar lavage from bleomycin-injured lungs increased with increasing total lung HA and had a mean molecular size of 220,000 +/- 50,000 (SE), indicating the minimum size of HA accumulated in the alveolar space. The relative lung water increased significantly on days 3-7 after bleomycin administration. A close relationship (P less than 0.001) and time dependence between the increase in relative lung water and the increase in lung HA were found.

Hyaluronan (hyaluronic acid) in bronchoalveolar lavage fluid during the development of bleomycin-induced alveolitis in the rat.

Hyaluronan (hyaluronate or hyaluronic acid) was measured in bronchoalveolar lavage (BAL) fluid from rats during a 30-day period after a single intratracheal dose of bleomycin. An increase of hyaluronan (HA) in BAL fluid was apparent by 3 days after bleomycin administration. Peak HA values were reached on Day 5 and were about 75 times higher than in control animals. Analyses of urea in BAL and HA in serum indicated that the increased levels of HA in BAL, postbleomycin, could not be explained by plasma leakage. The HA recovered by lavage decreased progressively after Day 5 and returned to normal 3 wk after bleomycin treatment. The HA content and an increased influx of PMN in BAL fluid were correlated and showed a similar time dependence, suggesting that enhanced synthesis of HA in the smaller airways is linked to the early inflammatory phase of the bleomycin injury.

Accumulation of hyaluronic acid in the alveolar interstitial tissue in bleomycin-induced alveolitis.

By using biotin-labeled proteoglycan core protein and an avidin-enzyme system, hyaluronic acid (HA) was visualized in the lungs of rats at different times (4, 10, and 20 days) after bleomycin injury. Four days after an intratracheal injection of bleomycin, HA was accumulated in the edematous alveolar septa of the focal areas with lung tissue injury. An interstitial cellular infiltrate of mainly lymphocytes was present. In normal rat lung, HA was not seen in the alveolar tissue but confined to peribronchial and perivascular spaces. Ten and twenty days after bleomycin administration, increasing numbers of macrophages were apparent in the alveolar space. Proliferating fibroblasts and deposition of collagen in the alveolar tissue were observed while the diffuse HA accumulation was becoming less prominent in the alveolar interstitial tissue. HA was more distinctly located in the surroundings of proliferating fibroblasts. A few scattered alveolar macrophages showed a positive staining for HA. An increased water content of the lung was most apparent 4 days after bleomycin administration. The accumulation of HA, a glycosaminoglycan with unique qualities to immobilize water, in the alveolar interstitium suggests a role for HA in the alveolar interstitial edema. The appearance of HA in alveolar macrophages might indicate that macrophage phagocytosis contributes to the elimination of HA from inflamed lung tissue.