Review
Database of bronchoalveolar lavage fluid proteins

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Abstract

Bronchoalveolar lavage during fiberoptic bronchoscopy is extensively used for investigating cellular and biochemical alterations of the epithelial lining fluid in various lung disorders. Two-dimensional electrophoresis (2-DE) offers the possibility to simultaneously display and analyze proteins contained in bronchoalveolar lavage fluid (BALF). We present the current status of 2-DE of BALF samples with an updated listing of the proteins already identified and of their level and/or posttranslational alterations in lung disorders. Alternatives to 2-DE of BALF samples and future prospects of proteomics to unravel lung functions and pathologies are discussed.

Introduction

Bronchoalveolar lavage (BAL) performed during fiberoptic bronchoscopy is a relatively safe technique which has proven useful to collect cells and a wide variety of soluble components from the human lung: phospholipids, nucleic acids and proteins, originating from the thin layer of epithelial lining fluid (ELF) that covers the airways [1]. Centrifugation of BAL allows the separation of cells from the supernatant BAL fluid (BALF) that contains the soluble components of the ELF.

The cellular content of BAL mainly consists of alveolar macrophages (80–95% of the cells), lymphocytes (<10%), neutrophils (<5%), eosinophils (<5%) and sometimes plasma cells. Squamous epithelial cells, bronchial epithelial cells, type II pneumocytes, basophils and mast cells are also found in BAL [2], [3], [4], [5].

Phospholipids, responsible for the decrease of alveolar surface tension, are synthesised by the lung epithelial cells and represent the main components of surfactant (90% [6]). Nucleic acids are found occasionally in BAL during infections by pathogens [7]. Soluble proteins in BALF are very diverse and may originate from a broad range of sources: diffusion from serum across the air–blood barrier, production by pulmonary T cells, synthesis by alveolar macrophages, bronchial epithelial cells, alveolar TI and TII cells, Clara cells, etc. For example, BALF interleukin-10 has four possible origins: production by pulmonary T cells [8], alveolar macrophages [9], bronchial epithelial cells [10], and diffusion from serum across the air–blood barrier.

Careful analysis of each of these BAL components enables accurate diagnosis and follow-up of a number of lung diseases. For example, acute respiratory distress syndrome (ARDS) is characterized by a significant decrease in the percentage of phosphatidylcholine and phosphatidylglycerol and an increase of phosphatidylinositol in total phospholipids (reviewed in Ref. [6]), granulomatous and allergic lung diseases are characterized by an increase in the lymphocyte count [5], Pneumocystis carinii pneumonia can be diagnosed by nested polymerase chain reaction on BALF samples [7].

Due to the variety of origins for proteins present in BAL, differences in the amounts of lung-specific proteins in BAL may result from many different kinds of phenomena. Indeed, reduction of BALF surfactant proteins (SPs) may be caused by reduction in the amount of secreting cells, or by decreased synthesis and/or release by these cells. On the contrary, increase of BALF SPs may result from an increase in the synthesis, from augmented release by secreting cells or from impaired clearance by alveolar macrophages, mucociliary transport, degradation, and absorption into the bloodstream. For instance, increased synthesis and/or release are the most plausible mechanisms explaining BALF surfactant protein A (SP-A, the major protein component of the surfactant) increase in patients with sarcoidosis and hypersensitivity pneumonitis (HP) [11]. BALF SP-A increase in pulmonary alveolar proteinosis (PAP) is also due to impaired removal/degradation of surfactant [12].

Due to the huge diversity of proteins present in BALF as well as the wide variety of origins of each protein considered, analysis of the protein content of BALF is of outstanding interest to diagnose most lung diseases. However, whereas quantification of the expression level of a single protein represents the integration of a multitude of different mechanisms involved in its synthesis, release and/or clearance, measuring changes in the levels of only one particular protein species gives insights only into one particular piece affected in the puzzle of a defined lung disease. Establishing an unambiguous diagnosis of one particular disease, allowing proper treatment and follow-up, requires the combined analysis of a repertoire of protein markers in BALF samples.

Proteomics, a technology-based science which studies levels and post-translational modifications of a large number of proteins simultaneously, their differences between healthy and diseased states and under the influence of environmental factors, fits these requirements [13]. Current interest in the application of proteomics to study human disease is huge and covers a wide variety of biomedical areas including cardiovascular diseases [14], cancer [15] and neurological disorder research [16]. Moreover, proteomics is the only global expression profiling technique applicable to body fluids, which cannot be analyzed via nucleic acid-based approaches [17]. Great developments have thus been encountered in the field of body fluid proteomics, exemplified by the identification of bladder squamous cell carcinoma biomarkers in urine [18] or of the 14-3-3 brain protein in cerebrospinal fluid as a marker for spongiform encephalopathies [19].

In this context, the use of two-dimensional electrophoresis (2-DE) and mapping of BALF as the first step of a proteomic approach is of high interest. The hunt for new lung disease markers in BALF via the 2-DE display and subsequent interpretation of levels and post-translational modifications of the total protein content of BALF, together with the search for differences between healthy versus diseased states is described in this review.

Section snippets

History of proteomics with BALF samples

The origins of differential display proteomics with BALF samples go back to the late 1970s. Searches for disease-associated protein markers were undertaken within lavage effluent proteins displayed using gradient gel electrophoresis or isoelectric focusing in a one-dimensional fashion [20], [21].

The first two-dimensional map displaying the major soluble proteins present in lung lavage was published in 1979 [22]. The identification of 23 serum-derived proteins, accounting for 97% of the total

2-DE of BALF: state of the art

2-DE of BALF samples encountered impressive modernization due to many different technical improvements introduced in the beginning of the 1990s. However, current work still aims at the construction of an exhaustive 2-DE reference database of bronchoalveolar lavage fluid proteins, the search for new disease protein markers, their identification and the understanding of the molecular processes involved in lung diseases.

The most important technical progress brought in in BALF proteomics resides in

Updated database of proteins present in BALF 2-DE maps

Mainly three research groups have published identification of proteins present in the BALF 2-DE map as it stands nowadays (see proteins listed in Table 1 and displayed in Fig. 1).

In 1995, Lindahl et al. published a map comprising about 1000 protein spots with pI values ranging from 4 to 7 [28]. Using a combination of Western blotting, pattern matching with a reference plasma 2-DE map and co-migration with reference standards, they identified 25 different proteins, all originating from plasma.

2-DE mapping of BAL protein alterations in lung disease

The present chapter is dedicated to the description of up- or down-regulated proteins that have been detected on the BALF 2-DE map so far (Table 2).

In 1993, Lenz et al. identified the major surfactant protein, SP-A, as a series of spots located at the acidic side of the map by comparison with the 2-DE pattern of purified SP-A [27]. They showed that BALF SP-A had reduced in expression levels in patient with idiopathic pulmonary fibrosis, which is consistent with previously observed changes in

2-DE mapping of protein alterations in other body fluids in lung disease

Since bronchoalveolar lavage is not the only way of collecting ELF, other techniques have been designed that allow sampling of the distal airways in a less invasive fashion.

Sputum induction using hypertonic saline (HS) has been developed over the last decade, allowing minimally invasive assessment of airway inflammation without subjects having to undergo bronchoscopy [55]. It involves the inhalation of hypertonic saline aerosol, a stimulus known to cause bronchoconstriction in asthmatic

Future directions of BAL fluid proteomics

Efficient proteomics-based diagnosis of lung diseases will focus on different topics, in the near future.

First, completion of the human BALF 2-DE protein map will require a significant increase in the number and intensity of displayed protein spots. Fractionation, specific removal of major serum proteins (with albumin antibodies, for example), solubilization of hydrophobic and/or basic proteins, use of BALF samples from the widest range of pulmonary disorders, narrow-range pH gradients for the

Acknowledgments

Our work was supported by the Commission of the European Communities (QLK4-CT-1999-01308) and the National Funds for Scientific Research. A.B. is a Research Director and R.W. is a Research Associate at the National Funds for Scientific Research.

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