Functional characterization of small and large alveolar macrophages in sarcoidosis and idiopathic pulmonary fibrosis compared with non-fibrosis interstitial lung diseases

Abstract

BACKGROUND:

Sarcoidosis is a granulomatous disease that mostly affects the lungs. Advanced tissue injury caused by this disease can progress to pulmonary fibrosis with similar characteristics shared with idiopathic pulmonary fibrosis (IPF). The initial presentations of both sarcoidosis and IPF may be shared with other interstitial lung diseases (ILDs). Two populations of macrophages have been described in the alveolar space: small alveolar macrophages (AMs) and large alveolar macrophages. Despite their protective function, these cells may also play a role in the initiation and maintenance of inflammation leading to fibrosis.

OBJECTIVE:

The aim of this study was the functional characterization of small and large AM subpopulations in sarcoidosis and IPF as a pathology with respectively mild and advanced tissue injury causing fibrosis, in comparison with non-fibrosis ILDs.

METHODS:

Activation and adhesion surface markers as well as functions of small and large AMs isolated from bronchoalveolar lavage (BAL) were assessed by Flow Cytometry within patients with confirmed sarcoidosis ( $n=$ 14), IPF ( $n=$ 6), and non-fibrosis ILDs ( $n=$ 9).

RESULTS:

Our results showed that small AMs are immunologically more active, which may be important for airway inflammation. They are also proportionally more abundant in IPF, and therefore they may be more involved in a fibrosis process associated with the down-regulation of HLA-DR, LeuCAM, and CD62L expression. In Sarcoidosis, the inflammatory process appears to be associated with up-regulation of CD38 expression and oxidative burst activity.

CONCLUSION:

A relevant potential of the activation and adhesion markers as well as oxidative burst activity expressed on small and large AMs, in the perspective of differential diagnosis of sarcoidosis and IPF.

Keywords

Sarcoidosis idiopathic pulmonary fibrosis non-fibrosis ILDs small alveolar macrophages large alveolar macrophages

1. Introduction

Sarcoidosis is a multiorgan granulomatous disease of unknown cause and poorly understood pathogenesis that affects the pulmonary sites in more than 90% of cases. The clinical course varies generally. Spontaneous resolution is observed in most occurrences of the disease, but 20–25% of cases evolve into pulmonary fibrosis [1]. The evolvement of sarcoidosis has been generally studied using clinical, chest radiographic, and pulmonary function tests [2]. According to the chest radiographic classification of sarcoidosis, the presence of stage IV designates overt pulmonary fibrosis that is generally associated with reduced pulmonary function and a poorer prognosis accompanied by elevated morbidity and mortality [3]. A subset of cases with sarcoidosis progressing to pulmonary fibrosis represents a potential shared disease mechanism and morphological aberrations with idiopathic pulmonary fibrosis (IPF). Although the radiologic manifestations of these two disorders are different in most cases, the approach to the diagnosis of these diseases is similar, thus becoming difficult to differentiate sarcoidosis from IPF.

In IPF and sarcoidosis, the alveolar macrophages (AMs), with all their protective functions, can also play an important role in the initiation and maintenance of inflammation leading to the development of fibrosis. The total amount of these cells is increased, likely caused by an increased influx and recruitment of blood monocytes to the alveoli. Furthermore, AMs seem to play a key role in the pathogenesis of both types of diseases by secreting a variety of pro-inflammatory cytokines, growth factors, and oxygen radicals.

Macrophages exhibit a range of phenotypes. At one extreme, there is the M1 polarization, which is defined by pro-inflammatory and cytotoxic properties, while at the other extreme; there are M2 macrophages, which have anti-inflammatory functions. This model is simplistic. Nevertheless, it is clear that macrophages display remarkable plasticity and may form heterogeneous subpopulations with diverse physiological functions.

Two macrophage subpopulations, small and large, described in the alveolar space have been suggested. In chronic lung diseases, the normal distribution of AMs appears to be changed. The number of small cells is significantly increased in the alveolar space during chronic inflammatory lung diseases such as interstitial pulmonary fibrosis and collagen vascular diseases [4]. There have been reports of a population of macrophages that were smaller and less granulated in the sputum and alveolar space of adults with chronic obstructive pulmonary disease (COPD) and had high expression of HLA-DR and CD14, as well as increased expression of tumor necrosis factor $\alpha$ (TNF $\alpha)$ . These small macrophages accounted for 45% of all macrophages in sputum from adults with COPD compared to 7% from control subjects [5, 6]. Small macrophages were also described in the sputum of adults with cystic fibrosis (CF), representing 73% of all macrophages compared to 16% in healthy individuals [7]. It is reasonable to postulate that these two types of cells participate differently in lung diseases. In the present study, we address this question in sarcoidosis and IPF, two of the most commonly known interstitial lung diseases (ILDs). The aim of this study was to functional characterization of small and large AM subpopulations in sarcoidosis and IPF as a pathology with respectively mild and advanced tissue injury causing fibrosis, in comparison with non-fibrosis ILDs.

2. Materials and methods

2.1 Study subjects

Table 1
Demographic and clinical characteristics of patients

	Sarcoidosis	IPF	Non-fibrosis ILDs
Basic characteristics
No of individuals ( $n$ )	14	6	9
Mean age, yr $\pm$ SD	51.6 $\pm$ 2.8	62.3 $\pm$ 3.4	64.2 $\pm$ 2.5
Sex, F/M	10/4	0/6	3/6
Smoking (smokers/never/ex-smokers)	2/12/0	6/0/0	4/3/2
Chest X-ray stage (I/II/III/IV/n.d)	1/7/5/1	n.d	n.d
Clinical symptoms
Caught	6	1	4
Sputum	1	0	1
Dyspnea	6	5	4
Fever	1	0	1

n.d: not done F: Female M: Male SD: Standard deviation.

Twenty-nine patients with a suspected ILD diagnosis (mean age $\pm$ standard deviation, 57.7 $\pm$ 2.0 years; 44.8% female) who underwent bronchoalveolar lavage (BAL) as part of their initial diagnostic workup were enrolled in this study. Patients were divided based on the diagnosis reached by the attending clinician into three groups for comparison, namely sarcoidosis ( $n=$ 14), IPF ( $n=$ 6), and non-fibrosis ILDs ( $n=$ 9). All patients are from the Unit of Respiratory Disease, Pneumo-Phtisiology Department, Bronchoscopy Section, Moulay Youssef Hospital of Rabat, Morocco. Informed consent was obtained from patients according to the National Ethics Committee. Demographic and clinical data of the entire population were extracted from clinical files and are shown in Table 1.

The diagnosis of sarcoidosis was considered highly specific and certain if clinical presentations and thoracic imaging were consistent and compatible with pulmonary sarcoidosis and there were non-caseating granulomas in endobronchial or transbronchial biopsy specimens or from endobronchial ultrasound-guided transbronchial needle aspirations of enlarged hilar or mediastinal lymph nodes [8]. Patients were stratified by findings on chest radiograph at presentation into stages 0–IV, according to Scadding: stage 0 – normal, stage I – mediastinal and bilateral hilar lymphadenopathy without lung involvement, stage II – lymphadenopathy and lung involvement, stage III – only lung involvement, stage IV – lung fibrosis [9]. The diagnosis of IPF was based on surgical lung biopsy features or in accordance with the ATS/ERS International Multidisciplinary Consensus Classification of Idiopathic Interstitial Pneumonia [10].

A group of nine patients had a diagnosis of tuberculosis ( $n=$ 4), hypersensitivity pneumonitis (HP) ( $n=$ 1), Connective tissue disease ( $n=$ 3), and scleroderma ( $n=$ 1).

Data on smoking status were available for all patients. There were 14 ever-smokers (12 current smokers and 2 ex-smokers) and 15 never-smokers. Exclusion based on missing data.

2.2 Bronchoalveolar lavage and preparation of cells

Patients underwent bronchoscopy with BAL in the morning. Under local anesthesia, BAL was performed by the instillation of sterile physiological saline (NaCl) solution at 37 ${}^{\circ}$ C in aliquots of generally 10–20 ml, and then the solution was gently aspirated using negative pressure. The overall fluid recovery is immediately placed on ice until use. The first fraction was used for microbiological analyses when appropriate, while the second and third fractions were pooled and filtered in the lab through a cell strainer with 100- $\mu$ m to remove debris and mucus. Cells were centrifuged at 400x g for 10 min, washed twice, and re-suspended in phosphate-buffered saline (PBS). The recovered volume was determined and stored at 4 ${}^{\circ}$ C until processing.

Figure 1.

Measurement of oxidative burst in small and large AMs by flow cytometer. (a) Gating on singlet cells. (b) Representative dot plot showing the difference between small and large AMs. (c) A representative histogram of functional AM activity in different conditions of stimulation. AMs are producing different amounts of reactive oxidants. Red – unstimulated AMs; blue – AMs stimulated with PMA; orange – AMs stimulated with E.coli.

2.3 Measurement of cell-surface markers

All BAL samples were tested on the day of BAL within 2 hours of collection. For phenotypic analysis, cell suspensions were adjusted to $10^{6}$ cells/ml and labeled with the following combination of monoclonal antibodies: CD38-PE, HLA-DR-PerCP, CD25-FITC, CD11c-PE, CD18-FITC or with respective corresponding isotype antibodies (BD Bioscience). After labeling, the samples were lysed and fixed. Cells were acquired on a FACSCanto II Flow Cytometer using BD FACSDiva software. Following a previously established nomenclature describing lung macrophages, two distinct small and large AM subpopulations were identified based on their size and granularity [5, 6] (Fig. 1a,b). Data were analyzed using FlowJo software version 10 and were expressed as the percentage of positive cells or median fluorescence intensity (MFI) rate corresponding to the MFI of cells labeled with monoclonal antibodies divided by the MFI of respective cells labeled with corresponding isotype antibodies.

2.4 Oxidative burst measurement

The quantitative determination of AM oxidative burst was performed. The respiratory burst was stimulated by the addition of mitogen phorbol 12-myristate 13-acetate (PMA) as a strong stimulus and opsonized Escherichia coli (E. coli) as a particular stimulator (BD Biosciences). This stimulation induces granulocytes to produce reactive oxygen metabolites. The radical formation was measured at 37 ${}^{\circ}$ C by conversion of dihydrorhodamine 123 fluorogenic substrate (e.g., dihydrorhodamine [DHR] – 123 or 6-carboxy-2, 7-dichlorohydrofluorescien diacetate) to the fluorescent rhodamine 123. A sample without stimulus served as negative background control. The addition of the lysing solution stopped the reaction. Following further washing, DNA in all remaining cells is stained with propidium iodide (PI) to permit leukocyte identification and could discriminate viable cells from dust particles and damaged cells. The samples were then analyzed via the FACSCanto II Flow Cytometer using BD FACSDiva software Data were analyzed using FlowJo software version 10 and were expressed as MFI rate corresponding to the MFI of the stimulated cells divided by the MFI of the respective cells in rest status (Fig. 1c).

2.5 Statistical analysis

All statistical analysis was performed using IBM SPSS Statistics 21. Data are represented as percentages or MFI rates. The results are reported as the mean $\pm$ Standard Error of the Mean (SEM). The Paired t-test was used to compare the results of small and large AMs gated separately for each group. The non-parametric Mann-Whitney U-test was used to compare cell surface marker expression and function data between patient groups. $P<$ 0.05 was considered significant. Graphs were performed using GraphPad Prism 8.

Figure 2.

Differential percentage of small and large AMs in the BAL within patients with IPF, sarcoidosis, and non-fibrosis ILDs. ${}^{*}p<$ 0.05.

3. Results

3.1 Relative representation of small and large macrophages

We investigated the proportion of small and large AMs in the BAL of patients diagnosed with sarcoidosis ( $n=$ 14), IPF ( $n=$ 6), and non-fibrosis ILDs ( $n=$ 9). For further analysis of these subsets of cells, a gate that includes the entire AM population (“large” and “small” AMs) was first applied. The proportion of small and large AMs in BAL of each group is shown in Fig. 2 ( ${}^{*}p<$ 0.05).

The proportion of large AMs was significantly higher than that of small AMs in patients with sarcoidosis and non-fibrotic ILD (76.8% vs 19.0% and 83.8% vs 14.1%, respectively), while no significant difference was shown in large and small AMs within IPF patients (65.6% vs 31.5%, respectively) (Fig. 2).

3.2 Cell surface functional markers expression on large and small AMs

The expression density of activation and adhesion markers on the surface of AM subpopulations was measured. Large and small AMs were first compared for each group individually. HLA-DR was significantly higher in small cells for all patient groups (sarcoidosis, IPF, and non-fibrotic ILDs) (100.2 $\pm$ 13.5, 17.4 $\pm$ 2.9, and 108.1 $\pm$ 19.8, respectively) compared to large AMs (6.4 $\pm$ 2.06, 1.1 $\pm$ 0.1 and 4.2 $\pm$ 1.1, respectively). Furthermore, CD38 showed a significantly increased density on small AMs (15.2 $\pm$ 3.4) for sarcoidosis, (4.5 $\pm$ 1.09) for IPF, and (9.7 $\pm$ 1.9) for non-fibrosis ILDs compared to the same cells (3.2 $\pm$ 0.3) for sarcoidosis, (1.4 $\pm$ 0.2) for IPF and (2.4 $\pm$ 0.2) for non-fibrosis ILDs. Without ignoring CD11c which represented significantly higher proportions on small AMs (101.4 $\pm$ 15.4) for sarcoidosis, (19.6 $\pm$ 5.0) for IPF, and (94.3 $\pm$ 12.5) for non-fibrosis ILDs compared to large AMs (46.6 $\pm$ 15.9) for sarcoidosis, (13.2 $\pm$ 3.3) for IPF and (21.2 $\pm$ 3.4) for non-fibrosis ILDs. In addition, the CD62L marker showed similarly higher expression on small AMs than on large ones, but only within patients with non-fibrotic ILDs (29.3 $\pm$ 5.6 and 7.1 $\pm$ 1.4, respectively) (data not shown).

The expression density of each measured marker on the surface of small and large AMs was also compared between patient groups. HLA-DR expression was significantly higher in small and large AMs within patients with sarcoidosis (100.3 $\pm$ 27.8 and 6.5 $\pm$ 1.8, respectively) as well as non-fibrotic ILD (108.1 $\pm$ 36.0 and 4.2 $\pm$ 1.4, respectively) compared with IPF patients (17.5 $\pm$ 7.1 and 1.2 $\pm$ 0.5, respectively).

To evaluate whether the differences in the expression of other activation markers, the CD38 molecule was tested. This marker revealed a significantly higher density on small and large sarcoid AMs (15.3 $\pm$ 4.2 and 3.2 $\pm$ 0.9, respectively) compared to IPF (4.5 $\pm$ 1.8 and 1.5 $\pm$ 0.6, respectively) and non-fibrosis ILDs (6.2 $\pm$ 1.0 and 2.1 $\pm$ 0.6, respectively). On the other hand, CD25 expression on small and large AMs showed no significant difference between the three patient groups.

The expression of particular beta2-integrins CD11c and CD18 that appear to be involved in cell-cell interactions was investigated. We observed a higher intensity of these markers in small AMs expressed within both patients with sarcoidosis and non-fibrosis ILDs (60.6 $\pm$ 16.8 and 86.6 $\pm$ 28.9 for CD18, 101.5 $\pm$ 28.1 and 94.4 $\pm$ 31.5 for CD11c, respectively) compared to those with IPF (32.5 $\pm$ 13.3 for CD18 and 19.6 $\pm$ 8.0 for CD11c). Large AMs showed higher CD11c expression in sarcoidosis patients (46.6 $\pm$ 12.9) compared with IPF (13.3 $\pm$ 5.4).

Figure 3.

Expression of activation and adhesion markers on large and small AM subpopulations in the BAL within patients with IPF, sarcoidosis and non-fibrosis ILDs. (a)(b): Small Macrophages, (c)(d): Large Macrophages. Data are expressed as the MFIrate of each marker and represent as mean (Min-Max).

The expression of selectin CD62L on small AMs showed significant differences between the three groups of patients. The lower level was expressed within IPF (4.4 $\pm$ 1.9), followed by sarcoidosis (14.5 $\pm$ 3.8) and then non-fibrotic ILDs (29.3 $\pm$ 5.6), $p<$ 0.05 in all cases). The expression of this marker on the surface of large AM showed significantly higher levels within sarcoidosis patients (10.6 $\pm$ 1.5) and non-fibrosis ILDs (7.1 $\pm$ 1.4) compared to IPF patients (1.9 $\pm$ 0.5) (Fig. 3).

3.3 Oxidative burst function

Figure 4.

Assessment of AM subpopulations oxidative burst in the BAL within patients with IPF, sarcoidosis and non-fibrosis ILDs. ${}^{*}p<$ 0.05.

We also assessed the functional aspect by measuring the oxidative burst activity of small and large AMs in twelve individuals from three patients groups.

Following stimulation by PMA, this activity was significantly higher within sarcoidosis patients in both small and large AMs (27.79 $\pm$ 12.43 and 30.62 $\pm$ 13.69, respectively) compared with IPF patients (2.44 $\pm$ 1.21 and 5.06 $\pm$ 2.53, respectively) and non-fibrosis ILDs (2.67 $\pm$ 1.54 and 2.83 $\pm$ 1.63, respectively). E.coli Expression was significantly higher on small AMs comparing sarcoidosis (4.09 $\pm$ 1.83) with IPF (2.05 $\pm$ 1.03) and non-fibrosis ILDs patients (2.02 $\pm$ 1.17) (Fig. 4).

4. Discussion

During tissue damage following mechanical or toxic injury, an inflammatory response is induced in response to pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) released by invading organisms and dead and dying cells, respectively [11]. When the wound healing process is highly organized and controlled, the inflammatory response resolves over time and restores normal tissue architecture. However, if the wound healing response is either dysregulated or chronic, it can develop fibrosis or scarring, ultimately leading to organ failure, cell damage, and death [12].

Several cell types involved in tissue repair tightly regulate the wound-healing responses. This complex inflammatory response is characterized by the recruitment, proliferation, and activation of a collection of cells, including neutrophils, macrophages, natural killer (NK) cells, T cells, B cells, fibroblasts, epithelial and endothelial cells, that together make up the cellular response that orchestrates tissue repair [13]. Because of their highly flexible programming [14], macrophages have been shown to exhibit critical regulatory activity at all stages of repair and fibrosis [15]. Consequently, because they represent the potentially important key to the inflammatory and fibrosis process, there has been a great deal of interest over the past few years in deciphering the contributions of the different macrophage subpopulations and phenotypes that control the initiation, maintenance, and resolution of wound healing responses and fibrosis development in across different organs.

Macrophage polarization plays a crucial role in chronic inflammatory diseases. Overall, the persistence, resolution, or progression of granulomas and the conversion to fibrosis is a balance between M1/M2 polarization [16]. M1 macrophages are associated with pro-inflammatory functions, and an exacerbation of tissue inflammation initiates the pro-fibrotic process [12]. On the other hand, M2 macrophages have anti-inflammatory properties due to their ability to secrete interleukin (IL)-10, arginase, TGF $\beta$ , and HO-1 [17].

Nevertheless, the division into two populations (M1 and M2) is simplistic. Macrophages display remarkable phenotypic heterogeneity in different tissue environments.

Several reports have suggested that two macrophage subpopulations, small and large, are present in the alveolar space [4]. They have been described in settings of cystic fibrosis [7], acute respiratory distress syndrome [18], sputum samples of COPD patients [5, 6], as well as in the lower airways of patients with ILDs [19]. The presence of small and large AMs means that the cells do not belong strictly to two subpopulations but rather are at some intermediate stage of polarization between pro- and anti-inflammatory populations [20].

As far as we know, few studies have investigated these two AM subpopulations in sarcoidosis and IPF. However, according to these reports, their involvement in inflammatory tissue damage is still unclear.

The aim of this study was the functional characterization of these lung macrophage subpopulations in sarcoidosis and IPF as a pathology with respectively mild and advanced tissue injury causing fibrosis, in comparison with non-fibrosis ILDs.

In our study, two distinct populations were identified as small and large AMs based on size and granularity in line with the nomenclature previously established to describe lung macrophages [5, 6, 21].

Our results show that small macrophages are more represented in IPF patients than in sarcoidosis and non-fibrosis ILDs; This indicates that the increased degree of inflammation and tissue damage could be related to the local predominance of these small AMs [18]. It also suggests that these cells may be crucial towards inflammation in the lung [7]. As for the origin of these small AMs, the most likely explanation is that these cells are monocytes, newly migrated from the blood into the lung in a chemokine-dependent manner. CCL2 is a chemokine that can drive the recruitment of monocytes into the lung [22]. Elevated levels of CCL2 were found in IPF [23]. Hence, CCL2 may be responsible for the increase of small AMs in the disease. However, in a published study, the authors postulated that the greater part of AMs recovered by BAL in sarcoidosis are mature and activated cells [24].

To characterize functionally these AM subpopulations in this study, cells were first studied through the expression of various activation and adhesion surface markers. The comparison of small and large AMs within each group individually showed that small cells expressed significantly more HLA-DR, CD38, and CD11c than large AMs. Furthermore, CD62L expression was higher in small AMs than in large AMs in non-fibrosis ILDs (data not shown). These data suggest that small AMs may be the immunologically active cells, and thus may be more involved in the airway inflammation process toward fibrosis.

Subsequently, the two AM subpopulations were compared separately between the study patient groups. Activation profiling results showed that individuals with IPF had a lower intensity of HLA-DR expression on small and large AMs, whereas patients with sarcoidosis did not differ significantly from those with non-fibrosis ILDs. HLA-DR is involved in antigen presentation to CD4+ T-cells, which means that HLA-DR intensity expression may vary in AM due to the presence of T-activated lymphocytes. The uncontrolled activation and accumulation of pulmonary T lymphocytes is considered a major factor in the pathogenesis of sarcoidosis [25].

On the other hand, our results suggest that the fibrotic process leads to a decrease in the antigen-presentation capacity of small and large AMs. This is in agreement with Komatsu et al. who revealed reduced expression of HLA-DR in pulmonary fibrosis [26]. In IPF, alveolar epithelial micro-injury leads to an adverse wound-healing response characterized by the progressive accumulation of scar tissue. A dysregulated alveolar repair response is associated with the secretion of inflammatory mediators, including cytokines and chemokines, leading to platelet activation and mediating the recruitment of inflammatory cells such as macrophages, lymphocytes, and neutrophils. These inflammatory cells release pro-fibrotic cytokines, recruit and activate fibroblasts, potentiate collagen synthesis and accumulation of extracellular matrix (ECM) components, as well as promote fibroblast to myofibroblast differentiation [27]. The overall decrease in immune cell number and function in IPF may be attributed to the replacement of parenchymal tissue with fibrotic scar tissue [28]. Nevertheless, HLA-DR antigen expression on AM subsets in IPF has also been reported by Kiemle-Kallee et al. to be within the normal range [29]. The difference between this and our observation is probably due to the measurement of global AMs with no discrimination between small and large cells.

CD38 is broadly expressed in activated T cells and was recently found to be expressed in other immune cell types, including macrophages [30]. More recently, CD38 was considered an inflammatory marker for macrophages in human inflammatory processes [30]. In our study, a significantly higher expression of CD38 was observed within sarcoidosis patients while no significant difference was found for this marker within IPF compared to non-fibrotic ILDs. We also show that this marker differs significantly regarding small and large AMs. This finding is concordant with the recent use of CD38 as a marker to determine sarcoidosis activity and predict the disease course [31].

The surface receptor for IL-2 (IL-2R) or CD25 is another activation marker found in a variety of cell types, including T and B cells. However, it was recently found that IL-2R is also expressed in activated macrophages [32]. There was no significant difference in CD25 expression by large and small AMs between the three disease groups. This result is consistent with the suppressive activity of AM communally shown by Fireman et al. within patients with both sarcoidosis and IPF mainly by cell-to-cell contact [32].

Activation of inflammatory functions can also be expressed through adhesion molecules. Expression of L-selectin (CD62L) and leukocyte cell adhesion molecule (LeuCAM) p150,95 (CD11c/CD18) integrins are temporally coordinated to ensure that the processes of leukocyte adhesion, rolling, and diapedesis can occur during emigration upon the initiation of an inflammatory response. Adhesion molecules play an important role in pulmonary inflammation, several studies have described their modulation of expression on human AMs [33, 34, 35].

Our results showed lower expression of CD11c/CD18 and CD62L molecules on small AMs within patients with IPF. This seems to have no impact on the AM migration to the alveoli during this disease. Indeed, sarcoidosis and non-fibrotic ILD patients had a lower representation of small AMs compared with IPF, despite higher expression of CD11c/CD18 and CD62L molecules. Even integrin and selectin molecules are involved in the trans-endothelial migration of macrophages; we can hypothesize that the expression of these adhesion molecules is downregulated when the cells reach the target tissue. Hoogsteden et al. [36] found that in sarcoidosis and IPF, the percentage of AM-positive for CD11c was lower than that of peripheral blood monocytes (PBM) cells. This indicates that the expression of these cell surface adhesion molecules is downregulated during PBM maturation and migration into the alveoli.

Furthermore, LeuCAM is known to be involved in cell activation in addition to its role in cell recruitment [37]. Increased expression of LeuCAM on AM in sarcoidosis may be involved in AM activation. In macrophages, secretion of superoxide anion can be interpreted as a sign of cellular activation.

Also known as the respiratory burst, superoxide anions involve NADPH oxidase to generate the main reactive oxygen species (ROS), superoxide radical (O ${}_{2}^{-})$ , and hydrogen peroxide (H ${}_{2}$ O ${}_{2})$ , promoting, directly or indirectly, the death of the microbe [38]. However, when inflammation becomes chronic, it induces persistent activation of macrophages and neutrophils, which become a persistent source of oxidative damage to DNA and cell components and induce fibrosis.

Conflicting data exist concerning the oxidative burst potential of sarcoid AM. Increased oxidative metabolism after in vitro stimulation has been reported by Aerts et al. [39]. However, Nielsen et al. [40] reported a pronounced decrease observed in sarcoid patients with active alveolitis, while Greening and Lowrie found a normal function [41]. Our results showed a significantly increased secretion of oxidants in small AMs after PMA and E.coli stimulation for sarcoid patients compared with the other two groups, indicating activated small AMs. However, previous studies also demonstrated an interaction between LeuCAM and O2-secretion, suggesting that intact CD11/CD18 molecules are necessary for the production of oxygen metabolites [42]. In addition, it has been found that in the genetic deficiency of the CD11/CD18 epitope (leukocyte adhesion deficiency), phagocytic cells are not able to perform a regular respiratory burst, resulting in an increased frequency of bacterial infections [42]. Large AMs also exhibit high oxidative potential even at moderate levels in sarcoidosis patients as they were detected only in PMA-induced activity. However, some results do not necessarily negate the idea of macrophage activation in pulmonary sarcoidosis. Differences in the methods used to estimate oxidant production could explain the divergent results [43], and the macrophage activation level seems to vary also depending on the used test [44].

Nonetheless, some authors have reported PMA-induced oxidative burst release of AM in vitro. Fels et al. [45] used low doses of PMA to observe increased sensitivity of sarcoid AMs for PMA stimulation. Aerts et al. [39] related this increase in oxidative activity to the activated state of AM in sarcoidosis; they reported that the release of superoxide anion and related radicals may be important in the pathogenesis of pulmonary sarcoidosis.

In chronic lung diseases, this killing activity appears to be dysfunctional, causing specific bacterial populations to induce the underlying physiopathology of the disease [46]; this results in increased numbers of apoptotic cells. If macrophages fail to clear these cells, secondary necrosis occurs, promoting a pro-inflammatory phenotype [47]. Failure to resolve inflammation may be a key to the chronic inflammation that occurs in many lung diseases, including sarcoidosis and IPF.

5. Conclusion

Taken together, our results underline the role of the immunologically active cells of small AMs in airway inflammation. However, we emphasized the involvement of small AMs in the fibrosis process. Our study also suggests that the use of CD38 maker and oxidative burst activity can give a specific characterization to sarcoidosis. This finding revels new aspects of differential diagnosis. Nevertheless, all these interesting findings need to be confirmed in large-scale studies establishing as well as the relationship between M1/M2 and Small/Large repartition of AMs.

Footnotes

Acknowledgments

The authors thank all study participants and the health professional for facilitating this work’s realization.

Conflict of interest

The authors declare that there is no conflict of interest regarding the publication of this paper.

Authors contributions

F.S. and S.E. conceived of the presented idea. A.E, H.T, N.N, and C.K verified the analytical methods. J.B and A.I supervised the findings of this work. All authors discussed the results and contributed to the final manuscript.

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Functional characterization of small and large alveolar macrophages in sarcoidosis and idiopathic pulmonary fibrosis compared with non-fibrosis interstitial lung diseases

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSION:

Keywords

1. Introduction

2. Materials and methods

2.1 Study subjects

Table 1 Demographic and clinical characteristics of patients

2.4 Oxidative burst measurement

2.5 Statistical analysis

3.1 Relative representation of small and large macrophages

3.2 Cell surface functional markers expression on large and small AMs

5. Conclusion

Footnotes

Acknowledgments

Conflict of interest

Authors contributions

References

Table 1
Demographic and clinical characteristics of patients