Biomedical subjects

C M Sköld

Publications and source records attributed to C M Sköld.

At least 19 recordsLinked to original sources

PubMed2006

Different inflammatory cell pattern and macrophage phenotype in chronic obstructive pulmonary disease patients, smokers and non-smokers.

Smokers exhibit airway inflammation and increased number of alveolar macrophages (AM), but not all develop chronic obstructive pulmonary disease (COPD). We hypothesized that AMs in COPD patients have an altered functional capacity mirrored in a different phenotype. Sixteen steroid-naive COPD patients [forced expiratory volume in 1 s (FEV(1)) < 70% of predicted] underwent bronchoalveolar lavage (BAL). Age- and smoking-matched non-obstructive smokers (n = 10) and healthy non-smokers (n = 9) served as controls. Nine COPD patients had a BAL cell yield sufficient for flow cytometry analysis, where expression of AM cell surface markers reflecting various functions was determined. AMs from COPD patients showed decreased expression of CD86 (co-stimulation) and CD11a (adhesion) compared to smokers' AMs (P < 0.05). Furthermore, smokers' AMs showed lower (P < 0.05) expression of CD11a compared to non-smokers. AM expression of CD11c was higher in the COPD and smokers groups compared to non-smokers (P < 0.05). The expression of CD54 (adhesion) was lower in smokers' AMs compared to non-smokers (P < 0.05), whereas CD16 was lower (P < 0.05) in COPD patients compared to non-smokers. The AM expression of CD11b, CD14, CD58, CD71, CD80 and human leucocyte antigen (HLA) Class II did not differ between the three groups. The AM phenotype is altered in COPD and further research may develop disease markers. The lower AM expression of CD86 and CD11a in COPD implies a reduced antigen-presenting function. Some alterations were found in smokers compared to non-smokers, thus indicating that changes in AM phenotype may be associated with smoking per se. The functional relevance of our findings remains to be elucidated.

Analysis of Variance↗

PubMed2005

Bronchoalveolar lavage in COPD: fluid recovery correlates with the degree of emphysema.

Bronchoscopy with bronchoalveolar lavage (BAL) is an important research tool for assessing airway inflammation in a variety of inflammatory lung diseases. In chronic obstructive pulmonary disease (COPD), BAL recovery is often low, making analysis of the recovered fluid difficult to interpret. The present authors hypothesised that the degree of emphysema may predict BAL recovery. A total of 20 COPD patients (mean age 57 yrs, range 49-69) with a median (interquartile range) forced expiratory volume in one second (FEV1) of 51 (33-69)% predicted underwent BAL. Matched "healthy" smokers and nonsmokers served as controls. Emphysema index in COPD patients was calculated on computed tomography scan as the percentage of the right lung with pixels <-950 Hounsfield units. The carbon monoxide diffusing capacity of the lung (DL,CO) was determined by the single-breath method. COPD patients had lower BAL recovery than controls. COPD patients with an emphysema index <1 had higher BAL recovery than patients with an emphysema index >1. BAL recovery correlated negatively to emphysema index and positively to DL,CO. However, no correlation was found between recovery and FEV1. In conclusion, the extent of emphysema evaluated by computed tomography-scan index and carbon monoxide diffusing capacity of the lung may predict a low bronchoalveolar lavage recovery in chronic obstructive pulmonary disease patients. These parameters may, therefore, be useful when chronic obstructive pulmonary disease patients are selected for bronchoscopy with bronchoalveloar lavage. The present study underlines the importance of careful phenotyping of chronic obstructive pulmonary disease patients.

Aged↗

PubMed2004

The effect of eosinophils on collagen gel contraction and implications for tissue remodelling.

Asthma is characterized by an eosinophilic inflammation and a subepithelial fibrosis in the airways. Eosinophils contain several cytotoxic substances, such as eosinophil cationic protein (ECP), which can promote inflammation and cause tissue damage. This has generated the hypothesis that eosinophils may drive remodelling of extracellular matrix (ECM). To investigate the role of eosinophils we used an in vitro model for remodelling, the three-dimensional collagen gel contraction assay. Two sources of eosinophils were used in this study, isolated human peripheral eosinophils (purity > 95%) and stimulated [interleukin (IL)-5, IL-3 and granulocyte macrophage-colony stimulating factor (GM-CSF)] HL-60 clone 15 cells. Human eosinophils or HL-60 cells were cast together with human lung fibroblasts (HFL1) in type I collagen gels. Both types of eosinophils augmented fibroblast-mediated collagen gel contraction in a time and concentration-dependent manner. At 48 h, the gel area in HFL1/eosinophil co-culture was 46.5% +/- 0.5 (mean +/- s.e.m.) of initial area and in HFL1 culture 52.3% +/- 0.1 (P < 0.001). Respective figures for HFL1/stimulated HL-60 co-culture and HFL1 culture only were 44.1% +/- 0.5 and 52.4% +/- 0.4 (P < 0.001). The release of ECP was increased when fibroblasts were cultured with eosinophils compared to eosinophils cultured alone. In addition, native ECP added to fibroblast gel cultures also augmented contraction. Our results suggest that eosinophils may interact with mesenchymal cells, promoting remodelling of ECM and that ECP constitutes one potential eosinophil-derived mediator driving this process. We conclude that this may be one important mechanism by which eosinophil-ECM interactions will lead to airway tissue remodelling in asthma.

Asthma↗

PubMed2002

Red blood cells stimulate fibroblast-mediated contraction of three dimensional collagen gels in co-culture.

OBJECTIVE AND DESIGN: Following injury, red blood cells (RBC) may interact with extracellular matrix (ECM). In the present study we hypothesised that RBC, and soluble factors from RBC, might mediate remodelling of ECM by affecting fibroblast-mediated contraction of three dimensional collagen gels. MATERIALS AND METHODS: Human lung fibroblasts (HFL-1), were cultured together with isolated RBC, conditioned medium from RBC (RBC-CM) and hemolysed RBC in type I collagen gels. Gel contraction was determined by an image analyser. RESULTS: Both RBC, RBC-CM and hemolysed RBC stimulated gel contraction by fibroblasts (P < 0.001), compared to fibroblasts alone. The RBC-CM stimulated (P < 0.01) gel contraction in a time and concentration dependent manner. A similar effect was observed when supernatant from hemolysed RBC was tested. The production of fibronectin was increased (P < 0.01) in the co-culture system, compared to fibroblasts cultured alone. CONCLUSIONS: The present study shows that RBC can interact with mesenchymal cells in vitro. The ability of RBC to modulate fibroblast-mediated contraction in vitro, might therefore be an important mechanism regulating repair processes after injury.

Animals↗

PubMed2002

Determination of cardiac involvement in sarcoidosis by magnetic resonance imaging and Doppler echocardiography.

OBJECTIVES: To elucidate whether cardiac magnetic resonance imaging (MRI) could be useful in disclosing structural changes in the myocardium in sarcoidosis patients and to relate echo-Doppler derived indices of left ventricular function to electrocardiogram (ECG) findings. DESIGN: The MRI was performed in 18 consecutive patients with sarcoidosis. Left ventricular ejection fraction (LVEF), i.e. systolic function, was estimated echocardiographically by Simpson's two-dimensional method (n = 16). Diastolic function was estimated by age-corrected Doppler-derived indices: isovolumetric relaxation time (IVRT), deceleration time (DT) and early filling/atrial contraction ratio (E/A ratio). RESULTS: Eleven patients had conduction defects or dysrhythmias (ECG+) whilst seven patients had a normal ECG (ECG-). In two patients, high signalling, contrast-enhanced, isolated regions, suggestive of deposits, were seen in the left ventricular myocardium on MRI. Both these patients had abnormal ECGs and signs of systolic and/or diastolic dysfunction on echocardiography. LVEF was subnormal in seven of 10 of the ECG+ patients and in two of six of the ECG-. Signs of diastolic dysfunction were found in 59% and 56% of the measurements in the ECG+ and ECG- patients, respectively. CONCLUSION: We conclude (i) that myocardial deposits on MRI in sarcoidosis patients have a high specificity for cardiac involvement but a rather low sensitivity; (ii) that a substantial proportion of sarcoidosis patients with abnormal ECGs have echocardiographic signs of systolic and/or diastolic dysfunction.

Adult↗

PubMed2001

Fibroblasts and monocyte macrophages contract and degrade three-dimensional collagen gels in extended co-culture.

BACKGROUND: Inflammatory cells are believed to play a prominent role during tissue repair and remodeling. Since repair processes develop and mature over extended time frames, the present study was designed to evaluate the effect of monocytes and fibroblasts in prolonged culture in three-dimensional collagen gels. METHODS: Blood monocytes from healthy donors and human fetal lung fibroblasts were cast into type I collagen gels and maintained in floating cultures for three weeks. RESULTS: Fibroblast-mediated gel contraction was initially inhibited by the presence of monocytes (P < 0.01). However, with extended co-culture, contraction of the collagen gels was greatly augmented (P < 0.01). In addition, with extended co-culture, degradation of collagen in the gels occurred. The addition of neutrophil elastase to the medium augmented both contraction and degradation (P < 0.01). Prostaglandin E2 production was significantly increased by co-culture and its presence attenuated collagen degradation. CONCLUSION: The current study, therefore, demonstrates that interaction between monocytes and fibroblasts can contract and degrade extracellular matrix in extended culture.

Cell Line↗

PubMed2001

Collaborative interactions between neutrophil elastase and metalloproteinases in extracellular matrix degradation in three-dimensional collagen gels.

BACKGROUND: Extended culture of monocytes and fibroblasts in three-dimensional collagen gels leads to degradation of the gels (see linked study in this issue, "Fibroblasts and monocytes contract and degrade three-dimensional collagen gels in extended co-culture"). The current study, therefore, was designed to evaluate production of matrix-degrading metalloproteinases by these cells in co-culture and to determine if neutrophil elastase could collaborate in the activation of these enzymes. Since co-cultures produce prostaglandin E2 (PGE2), the role of PGE2 in this process was also evaluated. METHODS: Blood monocytes from healthy donors and human fetal lung fibroblasts were cast into type I collagen gels and maintained in floating cultures for three weeks. Matrix metalloproteinases (MMPs) were assessed by gelatin zymography (MMPs 2 and 9) and immunoblotting (MMPs 1 and 3). The role of PGE2 was explored by direct quantification, and by the addition of exogenous indomethacin and/or PGE2. RESULTS: Gelatin zymography and immunoblots revealed that MMPs 1, 2, 3 and 9 were induced by co-cultures of fibroblasts and monocytes. Neutrophil elastase added to the medium resulted in marked conversion of latent MMPs to lower molecular weight forms consistent with active MMPs, and was associated with augmentation of both contraction and degradation (P < 0.01). PGE2 appeared to decrease both MMP production and activation. CONCLUSION: The current study demonstrates that interactions between monocytes and fibroblasts can mediate tissue remodeling.

Blotting, Western↗

PubMed2001

Functional and morphological differences between human alveolar and interstitial macrophages.

Macrophages play an essential role in pulmonary host defense. They are, however, a heterogeneous cell population located in different lung compartments. This study was designed to elucidate differences between two macrophage populations obtained from the human lung, i.e., alveolar macrophages (AM) and interstitial macrophages (IM). Macroscopically tumor-free lung segments from nine patients undergoing lobectomy or pulmectomy were studied. All patients had a diagnosis of primary lung cancer. AM were recovered by bronchoalveolar lavage and IM were isolated by mechanical fragmentation of the lavaged lung segments followed by enzymatic treatment. The cell fractions were analyzed with respect to morphology (transmission electron microscopy) and function (phagocytosis). The cells in the IM fraction were smaller (7.6 +/- 1.8 microm (mean +/- SD) compared with 16.0 +/- 4.1 microm) and morphologically more heterogeneous than those in the AM fraction. Interestingly, a considerable portion of the cells in the IM fraction had a typical AM-like appearance. Despite this, the AM fraction had a higher phagocytic activity compared to IM, with faster attachment and ingestion processes (P <0.001 for both). We conclude that the heterogeneity of human lung macrophages must be taken into consideration when their role in the inflammatory response is studied.

Aged↗

PubMed2001

Co-cultured human mast cells stimulate fibroblast-mediated contraction of collagen gels.

In the current study, we asked whether mast cells might modulate remodeling of extracellular matrix by affecting fibroblast-mediated contraction of three-dimensional collagen gels. Mast cells and human lung fibroblasts were co-cultured in floating type I collagen gels. The area of the gels was measured by an image analyzer. Mast cells in co-culture augmented fibroblast contractility (P < 0.001) in a time- and concentration dependent manner. The tryptase inhibitor bis(5-amidino-2-benzimidazo-lyl)methane (BABIM) were unable to block the augmented fibroblast contractility induced by co-cultured mast cells and tryptase added alone in the culture system had no effect on contractility, suggesting that other mediators besides tryptase might be involved. The amount of collagen in dissolved gels, measured as hydroxyproline, did not change after co-culture indicating that degradation of collagen may not be a major mechanism. Our findings support the hypothesis that the activity of mast cells may drive rearrangement of extracellular matrix and this and could subsequently lead to fibrosis and tissue dysfunction.

Animals↗

PubMed2001

Glucocorticoids and TGF-beta1 synergize in augmenting fibroblast mediated contraction of collagen gels.

TGF-beta plays a central role in the initiation and progression of pulmonary fibrosis. Glucocorticoids are frequently used to treat fibrotic diseases, but beneficial effects are often modest. Both TGF-beta and glucocorticoids have been reported to increase fibroblast contraction of native collagen gels, a model of fibrotic tissue remodeling. Therefore, we sought to determine how glucocorticoids interact with TGF-beta in this system. In this study, human fetal lung fibroblasts (HFL-1) were pretreated with or without TGF-beta for 72 h before they were cast into type I collagen gels. Various concentrations of glucocorticoids (budesonide or hydrocortisone) were added at the time of casting. Gel size was then monitored at different times after gel release. The surrounding media were collected for the assay of prostaglandin E2 (PGE2) and the cell lysates were analyzed for cyclooxygenase (COX) expression by immunoblot. Glucocorticoids alone significantly enhanced fibroblast-mediated contraction of collagen gels (P < 0.01) and dose-dependently inhibited PGE2 release by HFL-1 fibroblasts. TGF-beta significantly augmented gel contraction but also induced a 30% increase in PGE2 release and increased the expression of COX-1. Glucocorticoids inhibited TGF-beta1 induced-PGE2 release, and enhanced TGF-beta augmented gel contraction without significantly affecting TGF-beta augmented COX-1 expression. Indomethacin, a COX inhibitor, increased TGF-beta augmented gel contraction but had no further effect when added together with glucocorticoids. Thus, glucocorticoids can synergize with TGF-beta in augmenting fibroblast mediated collagen gel contraction through the inhibition of PGE2 production. Such interactions between glucocorticoids and TGF-beta may account, in part, for the lack of response of fibrotic diseases to glucocorticoids.

Budesonide↗

PubMed2001

Interactions between monocytes and smooth-muscle cells can lead to extracellular matrix degradation.

BACKGROUND: Chronic infiltration of the airway wall with inflammatory cells characterizes both asthma and chronic bronchitis. Remodeling of the airway wall is also a feature of both diseases. OBJECTIVE: We hypothesized that collagen degradation may take place during coculture of monocytes with smooth-muscle cells (SMCs) and that this degradation might be altered by agents that modify the inflammatory regimen. METHODS: Monocytes (4.5 x 10(5)/mL) were cast into collagen gels containing human airway SMCs (4.5 x 10(5)/mL) and released into serum-free Dulbecco's modified Eagle's medium containing neutrophil elastase. Collagen content was quantified as total insoluble hydroxyproline on day 5. Zymography and immunoblotting were used to detect matrix metalloproteinases. RESULTS: Monocytes cocultured with SMCs in 3-dimensional native type I collagen gels produced TNF-alpha and IL-1beta and resulted in collagen degradation (30.5 vs 17.9 mg per gel) through inducing matrix metalloproteinase 1, 2, and 9 by means of SMCs. PGE(2) was significantly increased in coculture (0.9 vs 10.5 ng/mL). Indomethacin (1 micromol/L) completely inhibited PGE(2) production but augmented collagen degradation (17.9 vs 2.3 microg per gel), and this was blocked by the addition of exogenous PGE(2). Dexamethasone also inhibited collagen degradation in coculture. CONCLUSION: The current study supports the concept that interactions among cells present in the airway inflammatory milieu that characterize airway disease can lead to alterations in tissue structure and suggests mechanisms by which therapeutic strategies can be designed to modify tissue remodeling.

Cell Communication↗

PubMed2001

Synergistic neutrophil elastase-cytokine interaction degrades collagen in three-dimensional culture.

Proteolytic degradation of extracellular matrix is thought to play an important role in many lung disorders. In the current study, human lung fibroblasts were cast into type I collagen gels and floated in medium containing elastase, cytomix (combination of tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma), or both. After 5 days, gel collagen content was determined by measuring hydroxyproline. Elastase alone did not result in collagen degradation, but in the presence of fibroblasts, elastase reduced hydroxyproline content to 75.2% (P < 0.01), whereas cytomix alone resulted in reduction of hydroxyproline content to 93% (P < 0.05). The combination of elastase and cytomix reduced hydroxyproline content to 5.2% (P < 0.01). alpha(1)-Proteinase inhibitor blocked this synergy. Gelatin zymography and Western blot revealed that matrix metalloproteinase (MMP)-1, -3, and -9 were induced by cytomix and activated in the presence of elastase. Tissue inhibitor of metalloproteinase (TIMP)-1 and -2 were also induced by cytomix but were cleaved by elastase. We conclude that a synergistic interaction between cytomix and elastase, mediated through cytokine induction of MMP production and elastase-induced activation of latent MMPs and degradation of TIMPs, can result in a dramatic augmentation of collagen degradation. These findings support the notion that interaction among inflammatory mediators secreted by mononuclear cells and neutrophils can induce tissue cells to degrade extracellular matrix. Such a mechanism may contribute to the protease-anti-protease imbalance in emphysema.

Animals↗

PubMed2001

Cigarette smoke inhibits human bronchial epithelial cell repair processes.

By interfering with the ability of airway epithelial cells to support repair processes, cigarette smoke could contribute to alterations of airway structures and functions that characterize chronic obstructive pulmonary disease (COPD). The current study assessed the ability of cigarette smoke extract (CSE) to alter human airway epithelial cell chemotaxis, proliferation, and contraction of three-dimensional collagen gels, a model of extracellular matrix remodeling. The volatile components contained in cigarette smoke, acetaldehyde and acrolein, were able to inhibit all three processes. Nonvolatile components contained within lyophilized CSE also inhibited chemotaxis but displayed no activity in the other two bioassays. CSE also inhibited the ability of airway epithelial cells to release transforming growth factor (TGF)-beta and fibronectin. Exogenous fibronectin was unable to restore epithelial cell contraction of collagen gels. Exogenous TGF-beta partially restored the ability of airway epithelial cells to contract collagen gels and to produce fibronectin. This supports a role for inhibition of TGF-beta release in mediating the inhibitory effects of cigarette smoke. Taken together, the results of the current study suggest that epithelial cells present in the airways of smokers may be altered in their ability to support repair responses, which may contribute to architectural disruptions present in the airways in COPD associated with cigarette smoking.

Acetaldehyde↗

PubMed2001

ECG-abnormalities in Japanese and Swedish patients with sarcoidosis. A comparison.

BACKGROUND AND AIM OF THE WORK: ECG abnormalities are more common in patients with sarcoidosis than in controls. The incidence of cardiac sarcoid granulomas in the Japanese population has been found to be higher than that seen in Caucasians. We compared the prevalence of ECG abnormalities in Japanese and Swedish patients with sarcoidosis. METHODS: Twelve-lead routine ECG's were compared between consecutive patients (134 Japanese and 149 Swedish) of similar age with histologically verified sarcoidosis or a high clinical probability of the disease and a history of no more than 12 months before the ECG. RESULTS: There was no statistically significant difference in the prevalence of first degree AV block, ST-T-segment abnormalities, right bundle branch block (RBBB) and left anterior block (LAH) in Japanese and Swedish patients. Among the Swedes, the results were compared with those of a smaller group (n = 29) of older patients with a longer disease course. In these few patients LAH and RBBB occurred more frequently than in patients with a recent diagnosis. CONCLUSIONS: ECG abnormalities in patients with sarcoidosis seem to be of similar frequency in Japan and Sweden. ST-T changes and first degree AV block would appear early in the course of the disease, whereas more pronounced conduction defects may appear later.

Cardiomyopathies↗

PubMed2000

Blood monocytes attenuate lung fibroblast contraction of three-dimensional collagen gels in coculture.

Mononuclear phagocytes can interact with mesenchymal cells and extracellular matrix components that are crucial for connective tissue rearrangement. We asked whether blood monocytes can alter matrix remodeling mediated by human lung fibroblasts cultured in a three-dimensional collagen gel. Blood monocytes from healthy donors (>95% pure) were cast into type I collagen gels that contained lung fibroblasts. Monocytes in coculture inhibited the fibroblast-mediated gel contractility in a time- and concentration-dependent manner. The concentration of PGE(2), a well-known inhibitor of gel contraction, was higher (P < 0.01) in media from coculture; this media attenuated fibroblast gel contraction, whereas conditioned media from either cell type cultured alone did not. Three-dimensional cultured monocytes responded to conditioned media from cocultures by producing interleukin-1beta and tumor necrosis factor-alpha, whereas fibroblasts increased synthesis of PGE(2). Antibodies to interleukin-1beta and tumor necrosis factor-alpha blocked the monocyte inhibitory effect and reduced the amount of PGE(2) produced. The ability of monocytes to block the fibroblast contraction of matrix may be an important mechanism in regulating tissue remodeling.

Cell Culture Techniques↗

PubMed2000

Rat alveolar and interstitial macrophages in the fibrosing stage following quartz exposure.

Exposure to quartz induces pulmonary inflammation and development of fibrosis. In order to study the fibrosing process, we investigated morphology, function and phenotype of alveolar (AMs) and interstitial (IMs) macrophages at an early stage of fibrosis in rats. Rats were exposed by intratracheal instillations of 10 mg quartz (n=8) or saline (n=8) and studied 3 months later. AMs were obtained by bronchoalveolar lavage and IMs by mechanical fragmentation, followed by enzymatic digestion of lung tissue. Histology revealed subacute silicosis, with early focal fibrosis and alveolar lipoproteinosis. AM quartz exposure increased phagocytic activity and expression of major histocompatibility complex (MHC) Ia antigens, the latter being associated with cellular antigen presenting capacity. IM had an even more pronounced expression of MHC than AM after quartz exposure. Both macrophage fractions had a higher expression of OX-42 (complement receptor 3, CR3) than controls, but the increase in the IM fraction might be explained by the remaining AM in the IM fraction. Exposed AM adhered less to extracellular matrix components (vitronectin and fibronectin) than controls. In contrast, the adhesion of IM to vitronectin increased after exposure. Besides increased adhesion, the effects on IM were scarce. Our results therefore do not support the hypothesis that IM has a key role in the process of inflammation, including fibrosis.

Animals↗

PubMed2000

Clara cell secretory protein. Levels in BAL fluid after smoking cessation.

STUDY OBJECTIVES: The bronchiolar Clara cell is a major target for tobacco smoke exposure. To improve our understanding of the putative regenerative/repair mechanism(s) in the bronchiolar epithelium, we measured the levels of the Clara cell secretory protein (CCSP) in BAL fluid in healthy volunteers following smoking cessation. DESIGN: BAL was performed before smoking cessation, and at 1, 3, 6, 9, and 15 months following smoking cessation, in eight healthy volunteers with a previous mean cigarette consumption of 19 pack-years. The levels of CCSP in BAL fluid were assessed in immunoblotting experiments using an antibody against human CCSP. RESULTS: Significantly (p < 0.05) higher levels of CCSP in BAL fluid were observed at 3, 6, and 9 months after smoking cessation, while the levels of CCSP in BAL fluid at 15 months after smoking cessation were the same as those before smoking cessation. CONCLUSIONS: Despite the long history of smoking among patients in the present study group, signs of early regeneration in the bronchiolar epithelium were noted, in that the levels of CCSP in BAL fluid were elevated at the indicated time points following smoking cessation. Furthermore, we propose that the insult to the bronchiolar epithelium made by cigarette smoking caused the levels of CCSP in the BAL fluid at 15 months after smoking cessation to return to the levels noted before smoking cessation. The present study suggests a role for CCSP as a marker for nonciliated bronchiolar cell function.

Adult↗

PubMed1999

Two-colour flow-cytometric analysis of pulmonary alveolar macrophages from smokers.

The study of alveolar macrophages (AM) from smokers by flow cytometry (FCM) has been limited by strong autofluorescence and the lack of reliable markers to identify macrophages. Crystal violet quenching was reported to be effective in reducing autofluorescence of AM. CD68 is a marker for macrophages in immunohistochemistry, but has been less useful in FCM because of poor surface expression. This study evaluated the effectiveness of a method for two-colour FCM analysis of AM combined with membrane permeabilization and crystal violet quenching. Bronchoalveolar lavage cells, fixed in 4% paraformaldehyde and permeabilized using 0.5% Triton X100, were incubated with fluorescent-labelled antibodies for 30 min and quenched with a saturated crystal violet solution. Two-colour FCM was then performed using forward/side scatter gating to select AM. Autofluorescence at 525 nm (fluorescein isothiocyanate) and 575 nm (phycoerythrin) markedly decreased after quenching. After permeabilization, 97.1+/-2.8% of the gated cells were CD68+, while 53.9+/-18.6% of the AM were positive without permeabilization. CD68+ cells were sorted and proved to be AM morphologically. Analysis of CD71 (transferrin receptor) expression by FCM correlated with immunocytochemistry (r=0.77, p<0.05). The permeabilization/quenching technique, therefore, represents a satisfactory means to evaluate alveolar macrophages by flow cytometry.

Adult↗