Characterization of a population of small macrophages in induced sputum of patients with chronic obstructive pulmonary disease and healthy volunteers

Frankenberger, Marion; Menzel, Manuela; Betz, R.; Kaßner, Gudrun; Weber, Norbert; Kohlhäufl, Martin; Häußinger, K.; Ziegler-Heitbrock, Loems

doi:10.1111/j.1365-2249.2004.02637.x

Cited by 61 publications

(63 citation statements)

References 37 publications

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“…Although not statistically significant, this may be indicative of active inflammation, and could (like eosinophils) be another indicator of inflammatory airway disease. Indeed, one study (28) has shown an increase in a population of small macrophages in another respiratory disorder, chronic obstructive pulmonary disease (COPD).…”

Section: Discussionmentioning

confidence: 99%

Identifying leukocyte populations in fresh and cryopreserved sputum using flow cytometry

Brooks

Dalen

Hermans

et al. 2013

Cytometry Part B Clinical

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Background: Airway inflammation is commonly assessed by sputum induction followed by a differential cell count (DCC) using light microscopy. This method is prone to intercounter variability and poor reproducibility. We aimed to develop a more objective method using flow cytometry (FCM).Methods: Fifty-six sputum inductions were conducted in 41 adults (23 asthmatics). Sputum was processed, a cytospin prepared for DCC, and the remainder immunolabeled for FCM using CD45, CD14, and CD16-specific antibodies to distinguish major leukocyte populations. Aliquots of 15 samples were frozen at 280 C to assess the effects of cryostorage. DCC and FCM were compared, and viability of individual cell populations was determined by FCM.Results: FCM and DCC, and fresh and frozen samples, were significantly correlated, R 5 0.54-0.87; all P < 0.0001, and R 5 0.57 to 1; P < 0.005, respectively. There was a significant neutrophil loss after cryostorage (from median 30.5-17.4% of total leukocytes; P < 0.0001). Cell viability was higher for lymphocytes compared to granulocytes or macrophages (P < 0.001). With the exception of the expected higher levels of eosinophils (P < 0.005), no significant difference in cell differentials or viability was observed between asthmatics and nonasthmatics using either DCC or FCM.Conclusions: FCM is a suitable means of assessing leukocyte populations in induced sputum. Sample storage at 280 C prior to FCM is feasible, but may be detrimental to neutrophils, although good correlations were still observed between fresh and frozen samples. Large differences in viability were found between individual cell populations suggesting that viability dye use may be necessary. V C 2013 International Clinical Cytometry Society

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Section: Discussionmentioning

confidence: 99%

Identifying leukocyte populations in fresh and cryopreserved sputum using flow cytometry

Brooks

Dalen

Hermans

et al. 2013

Cytometry Part B Clinical

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“…Since one of the main functions of macrophages in inflammation is to recruit additional leukocytes, we have studied expression of chemokines, and for this we screened for CCL chemokines that target CCR1-4 in a pilot study on up to five COPD patients (data not shown). Among these CCL 3,4,5,8,11,14,15,17,23,24 and 26 showed no expression, or the difference in expression level between COPD samples with high percentage of small macrophages and control samples with low percentages of small macrophages was less than three-fold (data not shown).…”

Section: Chemokine Gene Expression In Copd Sputum Macrophagesmentioning

confidence: 99%

“…Sputum induction and processing were carried out as described previously (8,9) with some modifications. Briefly, the subjects were asked to inhale sterile saline solutions from 0.9% up to 3% in COPD and to 5% in healthy controls.…”

Section: Sputum Induction and Processingmentioning

confidence: 99%

“…When looking at macrophages in patients with airway disease, a shift from large macrophages to small macrophages has been noted in COPD and in cystic fibrosis (8,9). While these cells express higher levels of CD14 and HLA-DR, they show reduced scavenger receptor expression and reduced phagocytosis (8,9).…”

Section: Introductionmentioning

confidence: 99%

“…While these cells express higher levels of CD14 and HLA-DR, they show reduced scavenger receptor expression and reduced phagocytosis (8,9). The small macrophages may represent cells newly attracted by chemokines from the blood monocyte pool.…”

Section: Introductionmentioning

confidence: 99%

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Chemokine Expression by Small Sputum Macrophages in COPD

Frankenberger

Eder

Hofer

et al. 2011

Mol Med

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Small sputum macrophages represent highly active cells that increase in the airways of patients with inflammatory diseases such as chronic obstructive pulmonary disease (COPD). It has been reported often that levels of cytokines, chemokines and proteases are increased in sputum supernatants of these patients. In COPD, the small sputum macrophages may contribute to these supernatant proteins and recruit additional cells via specific chemokine expression patterns. We therefore investigated the expression profile of chemokines in sputum macrophages obtained from COPD patients in comparison to cells from healthy donors and cells isolated after inhalation of lipopolysaccharide (LPS). We used the minimally invasive procedure of sputum induction and have purified macrophages with the RosetteSep technology. Using macrophage purification and flow cytometry we show that in COPD small sputum macrophages account for 85.9% ± 8.3% compared with 12.9% ± 7.1% of total macrophages in control donors. When looking at chemokine expression we found, for the small macrophages in COPD, increased transcript and protein levels for CCL2, CCL7, CCL13 and CCL22 with a more than 100-fold increase for CCL13 mRNA (P < 0.001). Looking at active smokers without COPD, there is a substantial increase of small macrophages to 60% ± 15% and, here, chemokine expression is increased as well. In a model of airway inflammation healthy volunteers inhaled 20 μg of lipopolysaccharide (LPS), which resulted in an increase of small sputum macrophages from 18% ± 19% to 64% ± 25%. The pattern of chemokine expression was, however, different with an upregulation for CCL2 and CCL7, while CCL13 was downregulated three-fold in the LPS-induced small macrophages. These data demonstrate that sputum macrophages in COPD show induction of a specific set of CCL chemokines, which is distinct from what can be induced by LPS.

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Quantitative analysis of endotoxin‐induced inflammation in human lung cells by Chipcytometry

et al. 2021

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Chipcytometry is a tool that uses iterative staining cycles with multiple antibodies for a detailed characterization of cells. Cell recognition is based on morphological features. Cells fixed on microfluidic chips can be stored and shipped enabling a centralized analysis, which is important for assessments in multi-center clinical trials. The method was initially implemented for the analysis of cells from peripheral blood. We adapted it to more heterogeneous human lung cells from bronchoalveolar lavage (BAL) fluid and induced sputum (IS). We aimed to assess the performance of Chipcytometry to detect and quantify the endotoxin induced inflammatory response in healthy subjects. BAL and IS samples of 10 healthy subjects were collected prior to and following segmental and inhaled endotoxin challenge. Samples were analyzed by Chipcytometry and were compared with flow cytometry, and differential cell count (DCC). Chipcytometry clearly detected the endotoxin induced inflammatory response which was characterized by a massive increase of neutrophils (BAL: 2.5% to 54.7%; IS: 40.5% to 71.1%) and monocytes (BAL: 7.7% to 24.7%; IS: 8.0% to 14.5%). While some differences between detection methods exist, the overall results were comparable. The ability of Chipcytometry to verify fluorescent signals with morphological features improved the precision of rare cell analysis such as of induced sputum lymphocytes. In conclusion, Chipcytometry enables the quantitative analysis of cells from BAL fluid and IS. Advantages over DCC and flow cytometry include the storage of cells on chips, the ability for re-analysis and the mapping of surface marker binding to morphological information. It therefore appears to be a promising method for use in clinical respiratory drug development.

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Characterization of a population of small macrophages in induced sputum of patients with chronic obstructive pulmonary disease and healthy volunteers

Cited by 61 publications

References 37 publications

Identifying leukocyte populations in fresh and cryopreserved sputum using flow cytometry

Identifying leukocyte populations in fresh and cryopreserved sputum using flow cytometry

Chemokine Expression by Small Sputum Macrophages in COPD

Quantitative analysis of endotoxin‐induced inflammation in human lung cells by Chipcytometry

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