Discrimination of resident and infiltrated alveolar macrophages by flow cytometry in influenza A virus-infected mice

Laser flow cytometric analysis was used in conjunction with in vivo labeling with the lipophilic fluorescent dye DiIC18(5)-DS to discriminate resident alveolar macrophages from newly infiltrating monocytes/macrophages in mice with and without pulmonary influenza A virus infection. Leukocytes in bronchoalveolar lavage (BAL) and peripheral blood were analyzed by 2-color flow cytometry as a function of time following intravenous injection of DiIC18(5)-DS. At 4 hours, dye-positive leukocytes were present in both BAL and blood of normal mice, indicating that DiIC18(5)-DS rapidly crossed the pulmonary endothelial-epithelial barrier. At 4 days after dye injection, 98% of BAL cells were DiIC18(5)-DS positive, and almost all of these were monocytes/macrophages based on labeling with fluorescein isothiocyanate (FITC)-conjugated antibody to the Mac-3 marker. Only 3.2% +/- 0.3% of peripheral blood monocytes (approximately 0.16% of total peripheral blood leukocytes) were DiIC18(5)-DS positive at 6 days after injection, whereas > 95% of BAL leukocytes were strongly dye-positive on days 6 to 28. When DiIC18(5)-DS was injected in mice 6 days prior to intranasal challenge with influenza A, flow cytometry indicated that 57.8% 5.6% and 60.7% +/- 8.5% of macrophages/monocytes in BAL were newly infiltrated (i.e., DiIC18(5)-DS negative, Mac-3 positive) at 4 and 7 days, respectively, post viral infection. The discrimination of subpopulations of resident and newly recruited macrophages in BAL should facilitate future mechanistic studies on pulmonary infection and inflammatory lung injury.     

Differential cell analysis in bronchoalveolar lavage fluid from pulmonary lesions of patients with tuberculosis.

To obtain information on the cellular reactions to Mycobacterium (M) tuberculosis in the lung, we analyzed the cells in bronchoalveolar lavage (BAL) fluid from pulmonary lesions in comparison with those in BAL fluid from nonaffected regions of the lungs, and control lungs, and in peripheral blood of patients with tuberculosis. Neutrophils and lymphocytes were increased in number in BAL fluid from affected lesions of the lungs of patients with miliary tuberculosis and patients with active pulmonary tuberculosis compared with those in BAL fluid from control patients, but the number of alveolar macrophages was decreased in BAL fluid from tuberculous lesions. However, the numbers of these cells were not changed in the BAL fluid from nonaffected regions of the lungs of patients with active or inactive pulmonary tuberculosis. The numbers of lymphocytes were decreased and the numbers of monocytes were increased in peripheral blood from patients with miliary tuberculosis and with active tuberculosis, indicating inverse changes in the numbers of lymphocytes and monocytes in the peripheral blood to those in the BAL fluid of patients with tuberculosis. These results indicate characteristic redistributions of immune or inflammatory cells in response to infection with M tuberculosis and suggest that these changes are important for understanding the pathophysiology of pulmonary tuberculosis.     

Altered immunological reactivity in alveolar macrophages from patients with sarcoidosis

Lung macrophages may play an important role in the pathogenesis of pulmonary sarcoidosis. In this study, the ability of pulmonary macrophages and blood monocytes from sarcoidosis patients, normal controls and disease controls to provide the accessory signal necessary for the concanavalin A-induced activation of normal blood T cells was examined. Blood monocytes from all groups supplied a significantly greater accessory signal than lung macrophages. The accessory capacity of lavage macrophages from sarcoidosis patients varied over a wide range and correlations were sought between these values and other parameters of disease activity. Whilst there was no correlation with clinical parameters, accessory function of alveolar macrophages correlated significantly with the percentage of T helper cells in bronchoalveolar lavage (BAL) fluid (p less than 0.05) and, more closely, with the T helper:T suppressor ratio in BAL fluid (p less than 0.01). This interrelationship between macrophage activity and the T cell infiltrate favours the probability that both cell types participate in the sarcoid disease process and raises the possibility that T cells of both helper and suppressor phenotypes contribute to the pathogenesis.     

Evaluation of bronchoalveolar cells in pulmonary paracoccidioidomycosis.

To investigate the local immune response, the cellular infiltrate and cytokine levels were analysed in bronchoalveolar lavage (BAL) from patients with pulmonary paracoccidioidomycosis. The group consisted of 19 patients aged 34-65 yrs. The diagnosis was confirmed by demonstration of the fungus in the sputum or BAL fluid and by serological tests. Cytospin preparations showed an increased number of lymphocytes and neutrophils in BAL. A higher number of CD8+ lymphocytes were observed in BAL compared with peripheral blood. Alveolar macrophages (AM) expressed approximately three-fold more major histocompatibility class II, intercellular adhesion molecule-1 and B7-2 molecules on their surfaces than their circulating counterparts, indicating that they had differentiated into activated macrophages inside the lungs. Cultured AM produced higher levels of interleukin (IL)-6, tumour necrosis factor (TNF)-alpha and macrophage inflammatory protein (MIP)-1alpha than peripheral blood monocytes. BAL fluid contained low but detectable amounts of IL-6, TNF-alpha and MIP-1alpha, and specific antibodies to Paracoccidioides brasiliensis, mainly of the immunoglobulin G2 isotype. As macrophage inflammatory protein-1alpha was shown to selectively attract CD8+ T-cells and this population was elevated in bronchoalveolar lavage, the data suggest that, besides macrophages, CD8+ T-cells may have an important role in the pathogenesis of pulmonary paracoccidioidomycosis.     

Lung and blood mononuclear cell responses of tuberculosis patients to mycobacterial proteins.

The differences in specificity of human lung and peripheral lymphocytes for mycobacterial antigens (Ag) need to be evaluated in order to identify vaccine candidates against pulmonary tuberculosis (TB). Therefore, the present study examined the response to low molecular weight secretory proteins of Mycobacterium tuberculosis in bronchoalveolar lavage (BAL) and peripheral blood mononuclear cells (PBMCs) from minimal pulmonary TB and non-TB patients. Ag85A, Ag85B, culture filtrate protein (CFP)-31, CFP-22.5, CFP-21, M. tuberculosis protein-64 and an as yet uncharacterised 19 kDa protein were found to be predominantly recognised by BAL cells of TB patients on the basis of lymphocyte proliferation and significant interferon-gamma release. However, recognition of CFP-8, 6-kDa early secreted antigenic target, CFP-10, CFP-14.5, M. tuberculosis secretory protein-17 and five other as yet uncharacterised low molecular weight polypeptides was found to be high on the basis of lymphocyte proliferation at the level of PBMCs. Furthermore, BAL macrophages, and not blood monocytes, were found to produce nitric oxide (NO) in response to mycobacterial Ags. Among polypeptides predominantly recognised by BAL lymphocytes, only Ag85A and Ag85B were found to induce both NO and interleukin-12 (p40) by alveolar macrophages. In conclusion, the present results indicate heterogeneity in antigen recognition by bronchoalveolar lavage cells and peripheral mononuclear blood cells of minimal tuberculosis patients, and also suggest the utility of antigen 85 complex polypeptides for the development of a future mucosal antituberculous vaccine.     

A serial immunologic and histopathologic study of lung injury induced by trimellitic anhydride

Trimellitic anhydride (TMA) can induce immunologic lung disease in exposed workers. We have developed a rat model of TMA lung injury characterized by lung hemorrhage and an immune response to trimellityl (TM) haptenized lung proteins. The model is similar to the pulmonary disease-anemia syndrome (PDA) seen in workers exposed to TMA fumes. Sprague-Dawley rats, 15 per exposure period, inhaled micronized TMA powder, 100 micrograms/m3, 6 h/day, for 2,6, or 10 days and were sacrificed. At each time period, total, IgG, IgA, and IgM antibody to TM-rat serum albumin (TM-RSA) were measured by radiolabeled antigen binding and enzyme-linked immunosorbent assay (ELISA) in serum and bronchoalveolar lavage fluid (BAL). Hemorrhagic lung foci, weight, and displacement volume were determined, and lungs were examined by light and electron microscopy. There was no lung injury or antibody response at 2 days. There was minimal lung injury at 6 days with low levels of antibody in BAL and serum. At 10 days, there was a marked increase in hemorrhagic foci and in BAL and serum antibody levels. BAL antibody levels at 6 and 10 days had higher correlations with measures of lung injury than corresponding serum levels. There was minimal ultrastructural change at 6 days. By Day 10, there was marked intraalveolar hemorrhage, alveolar septal inflammatory nodules, abundant alveolar macrophages, and evidence of endothelial and epithelial cell injury. These results indicate that the immune response to inhaled TMA occurs parallel with the development of lung lesions, and antibody levels in BAL and serum are highly correlated with lung injury.     

Time course of cigarette smoke-induced pulmonary inflammation in mice.

Inflammation of the airways and lung parenchyma plays a major role in the pathogenesis of chronic obstructive pulmonary disease. In the present study a murine model of tobacco smoke-induced emphysema was used to investigate the time course of airway and pulmonary inflammatory response, with a special emphasis on pulmonary dendritic cell (DC) populations. Groups of mice were exposed to either cigarette smoke or to control air for up to 24 weeks. In response to cigarette smoke, inflammatory cells (i.e. neutrophils, macrophages and lymphocytes) progressively accumulated both in the airways and lung parenchyma of mice. Furthermore, a clear infiltration of DCs was observed in airways (10-fold increase) and lung parenchyma (1.5-fold increase) of cigarette-exposed mice at 24 weeks. Flow cytometric analysis of bronchoalveolar lavage (BAL) DCs of smoke-exposed mice showed upregulation of major histocompatability complex II molecules and costimulatory molecules CD40 and CD86, compared with BAL DCs of air-exposed mice. Morphometric analysis of lung histology demonstrated a significant increase in mean linear intercept and alveolar wall destruction after 24 weeks of smoke exposure. In conclusion, the time course of the changes in inflammatory and dendritic cells in both bronchoalveolar lavage and the pulmonary compartment of cigarette smoke-exposed mice was carefully characterised.     

Alveolar macrophages from bronchoalveolar lavage of patients with pulmonary histiocytosis X: Determination of phenotypic and functional changes

In recent years the alveolar macrophage has been found to play a central role in interstitial lung disease. Pulmonary histiocytosis X is characterized by infiltrating fibroblasts, mononuclear cells, and CD-1-positive Langerhans cells. Bronchoalveolar lavage (BAL) fluid displays an increase of CD-1-positive cells and a remarkable exaggeration of the total cell count with only slight changes in the differential cell count. Changes of alveolar macrophage phenotype and functional activity occurring in pulmonary histiocytosis X have not yet been characterized. The BAL fluid of nine patients with histologically proven isolated pulmonary histiocytosis X was compared with that of 16 control patients. Immunophenotyping of alveolar macrophages by monoclonal maturation and differentiation markers of monocyte/ macrophage lineage cells [Ki-M2, Ki-M6 (CD-68), Ki-M8, Ki-M1 (CD- 11c)] revealed a significant increase of immature macrophages with a more monocyte-like phenotype. The proliferation marker Ki-67 revealed an increased proportion of proliferating macrophages. Functional analysis by measuring oxygen radical release revealed an increase both in baseline and stimulated luminol-enhanced chemiluminescence. Fibronectin production was elevated in alveolar macrophage supernatants from pulmonary histiocytosis X patients. These findings are consistent with phenotypic changes of alveolar macrophages in other interstitial lung diseases such as sarcoidosis and idiopathic pulmonary fibrosis. Local proliferation and the fresh influx of blood monocytes seem to be responsible for the increase in immature and functionally activated alveolar macrophages. The increase in oxygen radical release and fibronectin production suggests an augmented tissue injuring and fibrosing capacity of alveolar macrophages in pulmonary histiocytosis X.     

Studies of bronchoalveolar lavage cells and fluids in pulmonary sarcoidosis. I. Enhanced capacity of bronchoalveolar lavage cells from patients with pulmonary sarcoidosis to induce angiogenesis in vivo

Both allogeneic immunocompetent CD4+ lymphocytes and activated macrophages of mice can induce neovascularization when inoculated intradermally into host animals. Because sarcoidosis is associated with an increase in both activated macrophages and CD4+ effector lymphocytes in the lung, we carried out experiments in which cells obtained by bronchoalveolar lavage (BAL) of patients with pulmonary sarcoidosis were tested in a murine intradermal angiogenesis assay. BAL cells from patients with pulmonary sarcoidosis induced a significantly greater degree of angiogenesis than those from normal volunteers or from patients with other lung diseases. Moreover, the degree of angiogenesis induced by BAL cells from patients with sarcoidosis correlated positively with the severity of the disease. When BAL cells were separated into macrophage and lymphocyte subpopulations by flow cytometric techniques, the observed angiogenic activity was restricted primarily or exclusively to macrophages; lymphocytes were unable to induce angiogenesis in this xenogeneic assay system. These experiments suggest that pulmonary macrophages may play a role in the pathogenesis of sarcoidosis by inducing changes in the pulmonary microvasculature. Moreover, we hypothesize that these vascular changes may be induced not only in the lung but also in other organ systems such as skin, muscle, and eye in which microangiopathies are associated with sarcoid disease.     

Phenotype of blood monocytes and alveolar macrophages in interstitial lung disease

The immunologic phenotype of the monocyte-macrophage cell populations in bronchoalveolar lavage (BAL) fluid and monocytes in peripheral blood (PB) were studied in 20 patients with sarcoidosis, 18 with idiopathic pulmonary fibrosis (IPF), seven with extrinsic allergic alveolitis (EAA), and 12 healthy volunteers. There were no significant differences in expression of the immunologic markers CD13(My7), CD14(My4), and Monocyte-2 on blood monocytes between the patient groups and healthy volunteers, but there were marked differences between groups in the expression of the three markers on BAL macrophages. The percentage of Monocyte-2+ macrophages was increased in BAL in subjects with sarcoidosis, EAA, and IPF compared with healthy volunteers, greatest in EAA. This increase is probably due to increased recruitment of blood monocytes into alveoli, since the cells had a monocytic morphology on phase contrast microscopy (in normal subjects the majority of blood monocytes, but few alveolar macrophages, express the Monocyte-2 antigen). Patients with IPF had a significantly lower percentage of CD13(My7)+ macrophages in BAL than the other three groups. Compared with IPF patients and healthy volunteers, patients with EAA had a significantly higher percentage of CD14(My4)+ macrophages, whereas in sarcoidosis patients the numbers were reduced. These observations suggest an increased influx of blood monocytes into the alveoli in interstitial lung disorders. Phenotypic differences were found between the BAL macrophage populations of the various interstitial diseases. These differences in alveolar macrophage phenotype may be due to local factors, depending on the type of inflammation.     

There is no activation of O2- production by alveolar macrophages and neutrophil polymorphonuclear leukocytes in rat lung transplants during the reimplantation response and acute rejection.

Activation of phagocytes (alveolar macrophages [AM] and neutrophil polymorphonuclear leukocytes [PMN]) can cause tissue damage in inflammatory lung diseases. In this study we investigated whether phagocytes contribute to the development of tissue damage in lung grafts histologically observed during two different processes: the reimplantation response and the acute rejection. Therefore, the number and profile of bronchoalveolar lavage (BAL) and blood phagocytes and their in vitro spontaneous and serum-treated-zymosan (STZ)-stimulated O2- production were assessed after allogeneic (BN to LEW) and syngeneic (LEW to LEW) transplantation of the left lung in rats. BAL PMN numbers increased during the reimplantation response, whereas during the late phase of the rejection process BAL AM and PMN numbers were increased. The O2- production by the BAL phagocytes and blood PMN were not increased at any stage. Strikingly, the STZ-stimulated O2- production by the BAL phagocytes was significantly impaired during acute rejection. Our data suggest that activation of the O2- production by bronchoalveolar phagocytes does not play an important role in the development of tissue damage in lung transplants during the reimplantation response and acute rejection. The impaired O2- production by alveolar phagocytes during acute lung rejection may contribute to the increased susceptibility for pulmonary infections after lung transplantation.     

Interferon-gamma enhances the pulmonary CXC chemokine response to intratracheal lipopolysaccharide challenge

CXC chemokines are major chemoattractants for pulmonary polymorphonuclear leukocyte (PMNL) recruitment. To study the effects of interferon (IFN)-gamma on the pulmonary chemokine response to lipopolysaccharide (LPS) challenge, rats were treated with intratracheal IFN-gamma (1x10(5) U/rat) 24 h before an intratracheal LPS (100 microg/rat) challenge. Intratracheal LPS caused significant increases in both cytokine-induced neutrophil chemoattractant (CINC) and macrophage inflammatory protein-2 in bronchoalveolar lavage (BAL) fluid and pulmonary PMNL recruitment. IFN-gamma enhanced these responses. IFN-gamma also increased LPS-induced tumor necrosis factor (TNF)-alpha in BAL fluid. LPS-induced TNF-alpha and CINC mRNA expression in alveolar macrophages was increased by IFN-gamma. CD11b/c and CD18 expression on circulating PMNLs was not affected by IFN-gamma, nor was the chemotaxis of these cells. IFN-gamma increases the pulmonary CXC chemokine response, which may serve as one mechanism underlying enhanced PMNL delivery into the lung.     

Levels of elastase activity in bronchoalveolar lavage fluids of healthy smokers and nonsmokers

Elastase activity was measured in concentrated, cell-free bronchoalveolar lavage (BAL), using the synthetic substrate butyloxycarbonyl-L-alanyl-L-alanyl-L-prolyl-L-valyl-amino-methylcoumarin. The BAL fluids obtained from young, asymptomatic smokers with normal urine desmosine concentrations 1 h after they had smoked 2 cigarettes showed significant increases in elastase levels compared with those in nonsmoking control subjects [nanomoles substrate hydrolyzed (3 h) per milligram lavage albumin = mean 2.7 +/- 1.9 SD (11 smokers) versus 0.5 +/- 0.4 (11 nonsmokers), p less than 0.01]. Repeated BAL samples were obtained at later times from one smoker with a high initial enzyme value and from one nonsmoking control subject. Elastase activity varied over time, but both subjects consistently remained within their respective group ranges. Inhibition studies on pooled BAL from smokers showed that the elastase activity present had properties of both serine and metalloenzymes, suggesting that neutrophils and/or monocytes (serine enzyme) as well as macrophages (metalloenzyme) contributed to the observed activity. Lung lavage cells obtained from 2 of the smokers and 2 of the nonsmokers were stained with both a chromogenic substrate and by indirect immunofluorescence for the serine enzyme. Positively stained neutrophils were readily found in smokers' lavages, but no, or only rare, positive mononuclear cells could be identified. By contrast, peripheral blood mononuclear cells from all 4 subjects stained positively with either method. These results show that some asymptomatic smokers have significantly more elastase activity in their bronchopulmonary secretions than do nonsmokers (as measured with a low molecular weight synthetic substrate). Furthermore, the enzyme activity recovered in smokers' BAL appears to be derived mainly from neutrophils (serine enzyme) and macrophages (metalloenzyme), rather than from monocytes.     

Inflammatory events after fibrin microembolization. Alterations in alveolar macrophage and neutrophil function

We performed bronchoalveolar lavage (BAL) 0.5 to 24 h after thrombin-induced pulmonary microembolization in spontaneously breathing sheep to examine the inflammatory events that occur after pulmonary intravascular coagulation. Neutrophil alveolitis was evident as early as 0.5 h after microembolization and was maximal at 4 h (4.9 +/- 1.5% neutrophils of total BAL cells at baseline versus 26.2 +/- 2.8% at 4 h post-thrombin). Neutrophils obtained both at baseline (isolated from peripheral blood) and at 0.5 to 24 h after thrombin (isolated from BAL) did not demonstrate significant basal production of superoxide anion (O2-) and produced similar amounts of O2- upon challenge with phorbol myristate acetate (PMA) 200 micrograms/ml. The basal O2- production by alveolar macrophages was also not increased. However, alveolar macrophages recovered after fibrin microembolization produced greater amounts of O2- (29.1 +/- 6.3 nm O2-/10(6) cells at 0.5 h) after challenge with PMA compared with alveolar macrophages recovered prior to embolization (10.6 +/- 1.6 nm O2-/10(6) cells baseline), suggesting that thrombin-induced microembolization primes alveolar macrophages and enhances their O2- generation. Neutrophil chemotactic activity was detected in BAL fluid at 0.5 h post-microembolization and reached a peak level at 2 h. Alveolar macrophages were a source of the chemotactic activity since conditioned medium obtained from 2-h post-thrombin macrophages induced neutrophil chemotaxis, whereas baseline cells did not. The addition of the thrombin to macrophages did not result in the generation of chemotactic activity from baseline macrophages, indicating that macrophages were activated during the process of intravascular coagulation rather than by thrombin per se. Post-thrombin BAL fluid also stimulated O2- generation from sheep neutrophils.(ABSTRACT TRUNCATED AT 250 WORDS)     

Local inflammatory responses following bronchial endotoxin instillation in humans

To study local lung inflammation, 34 subjects had endotoxin (1-4 ng/kg) instilled into a lung segment and saline instilled into a contralateral segment followed by bronchoalveolar lavage (BAL) at 2 h, 6 h, 24 h, or 48 h. Endotoxin instillation resulted in a focal inflammatory response with a distinct time course. An early phase (2 h to 6 h) revealed an increase in neutrophils (p = 0.0001) with elevated cytokines (tumor necrosis factor [TNF]-alpha, TNF receptors [TNFR], interleukin [IL]-1beta, IL-1 receptor antagonist, IL-6, granulocyte-colony-stimulating factor [G-CSF], all p < or = 0.002, but no change in IL-10) and chemokines (IL-8, epithelial neutrophil activating protein-78, monocyte chemotactic protein-1, macrophage inflammatory protein [MIP]-1alpha, MIP-1beta, all p < or = 0.001, but no change in growth-regulated peptide-alpha). A later phase (24 h to 48 h) showed increased neutrophils, macrophages, monocytes, and lymphocytes (all p < or = 0.02), and a return to basal levels of most mediators. Elevated levels of inflammatory markers (TNFR(1), TNFR(2), L-selectin, lactoferrin, and myeloperoxidase) persisted in the BAL at 48 h (p < or = 0.001). Increased permeability to albumin occurred throughout both phases (p = 0.001). Blood C-reactive protein, serum amyloid A, IL-6, IL-1ra, G-CSF, but not TNF-alpha increased by 8 h (all p < or = 0.008). The local pulmonary inflammatory response to endotoxin has a unique qualitative and temporal profile of inflammation compared with previous reports of intravenous endotoxin challenges. This model provides a means to investigate factors that initiate, amplify, and resolve local lung inflammation.     

Phenotypic and functional activation of alveolar macrophages,T lymphocytes and NK cells in patients with systemic sclerosis and primary Sj?gren'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.     

Bronchoalveolar mastocytosis and lymphocytosis after nitrogen dioxide exposure in man: a time-kinetic study

The combination of environmental chamber exposure and bronchoalveolar lavage (BAL) was used to study the time-course of the cell response in the human lung to nitrogen dioxide (NO2). Healthy subjects were exposed for 20 min to 7 mg NO2.m-3 (4 ppm), a concentration which occurs indoors in industries and is below the peak exposure limit for work places in most countries, 10 mg.m-3 (5.5 ppm). BAL was performed in all subjects several weeks before exposure and 4, 8, 24 and 72 h after exposure, in eight subjects at each time. Mastocytosis and lymphocytosis were found in BAL fluid 4-24 h after exposure, with normalization after 72 h. A mild increase in lysozyme positive macrophages was found 24-72 h after exposure. The time-course of the human pulmonary cell response to NO2, demonstrated in BAL fluid, represents a new and previously not reported finding after exposure to this common air pollutant. Our findings are diverging from results obtained in animal studies, using approximately the same NO2 concentrations, indicating that the results from the animal studies may not be transferable to man.     

Prognostic role of eosinophils in pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) and pulmonary fibrosis associated with a collagen vascular disorder (PF-CVD) are chronic inflammatory lung disorders which may be characterized in various subgroups of patients by increased numbers of macrophages, neutrophils, lymphocytes, and/or eosinophils. Previous studies have suggested that the cell populations recovered with bronchoalveolar lavage (BAL) may be important in predicting disease progression and response to therapy. We evaluated this hypothesis in 27 patients by determining if the cell populations recovered with BAL differed between patients who improved, remained stable, or worsened in their pulmonary functions (as defined by at least a 15 percent change in forced vital capacity) over a six-month observation period. The findings suggested that BAL eosinophilia may be a marker of progressive lung disease in patients with IPF and PF-CVD.     

Pretreatment with olprinone hydrochloride, a phosphodiesterase III inhibitor, attenuates lipopolysaccharide-induced lung injury via an anti-inflammatory effect

PURPOSE: Acute respiratory distress syndrome is characterized by neutrophil accumulation in the lungs and the activation of several cytokines produced by macrophages. Olprinone hydrochloride, a specific phosphodiesterase III inhibitor, has anti-inflammatory effects and inhibits the activation of macrophages, in addition to its inotropic and vasodilatory effects. The purpose of this study was to examine the beneficial effects of olprinone on lipopolysaccharide (LPS)-induced pulmonary inflammation. MATERIALS AND METHODS: Lung inflammation was produced by intravenous LPS injection into rats. The rats were divided into four groups: a vehicle group in which normal saline was injected, an olprinone group in which olprinone was injected at a dose of 0.2mg/kg, a dexamethasone group in which dexamethasone was injected at a dose of 5mg/kg, and a control group. In each group, drug was injected intraperitoneally 30 min before the intravenous administration of LPS. The blood was obtained at 1h and then animals were sacrificed at 6h and blood and lung specimen were obtained for cytokine analysis and pathological examination. On another set of experiment, bronchioloalveolar lavage (BAL) was performed for cytokine analysis of BAL fluid. The macrophages isolated from normal rat by BAL were cultured in vitro with the presence of LPS and olprinone or dexamethasone, and supernatant was collected. The levels of several cytokines in the serum, in the BAL fluid, and in the culture supernatant were determined. RESULTS: The animals injected with LPS were found to have an influx of neutrophils in the lungs, and inflammatory cytokines, such as TNF-alpha and IL-6, and anti-inflammatory cytokine IL-10 were produced. Pretreatment with olprinone or dexamethasone significantly inhibited the LPS-induced neutrophil influx into the lungs, suppressed inflammatory cytokines TNF-alpha and IL-6. The level of anti-inflammatory cytokine IL-10 increased in an olprinone group. The inhibition of TNF-alpha and IL-6, and the augmentation of IL-10 release were also observed in in vitro culture of isolated rat alveolar macrophages when olprinone (10(-5)mol/ml) and LPS (10 microg/ml) were cultured together. However, the level of IL-10 in serum and culture supernatant was suppressed in a dexamesathone group. CONCLUSION: LPS-induced lung inflammation is strongly inhibited by olprinone accompanying the enhancement of IL-10 and the inhibition of inflammatory cytokines. Results of the in vitro experiment suggest that alveolar macrophages may play an important role in ameliorating LPS-induced lung inflammation and the mechanism of its effect is different from that of steroid.     

Bronchoalveolar lavage (BAL) in newly diagnosed patients with collagen vascular diseases

Collagen vascular diseases (CVD) are commonly associated with interstitial lung diseases. Bronchoalveolar lavage (BAL) fluid analysis has important diagnostic value when considered in conjunction with other information. The present study was undertaken in newly diagnosed patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) at presentation to characterise BAL cellular constituents and elucidate the cellular picture in patients with and without pulmonary symptoms and in those with and without radiological (high resolution computed tomography) features of interstitial lung disease. All the patients were non-smokers and had not received any form of treatment for their diseases. The means of percentages of lymphocytes, neutrophils, and macrophages were 23.3%, 6.2%, 70.5% respectively. There was a significant BAL lymphocyte predominance in patients with pulmonary symptoms, and a lymphocyte and neutrophil predominance in those having radiological evidence of interstitial lung disease.