Loss of immunoreactivity for RTI40, a type I cell-specific protein in the alveolar epithelium of rat lungs with bleomycin-induced fibrosis

Abstract

The rat alveolar epithelium comprises three different epithelial cell types: the large squamous type I cells, the cuboidal type II pneumocytes and the bottle-shaped alveolar brush (type III) cells. Various biochemical markers have been used to distinguish type I from type II cells. Most of these markers are not unique for either cell type, but are helpful in defining the different phenotypes of type I and type II cells. Type II pneumocytes may be distinguished from type I cells by their cytokeratin pattern, by the presence of certain enzymes (e.g. alkaline phosphatase, carbonic anhydrase II, carbonyl reductase, protein disulphide isomerase and catalase), by the presence of surfactant apoproteins (SP-A, SP-B, SP-C and SP-D) and by different lectin binding (Maclura pomifera agglutinin (MPA)) (reviewed in [1,2]). Alveolar brush (type III) cells lack surfactant proteins and are selectively decorated with cytokeratin 18-and villin-specific antibodies [3,4]. Type I pneumocytes, in contrast to type II pneumocytes, bind various lectins (Lycopersicon esculentum agglutinin (LEA), Erythrina cristagalli agglutinin (ECA) or Bauhinia purpurea agglutinin (BPA)) [5][6][7], express the leukocytic intercellular adhesion molecule-1 (ICAM-1) [8,9] as well as caveolin, a structural protein of caveolae, and antigens recognized by other type I cell-specific monoclonal antibodies [10][11][12]. One of the type I cell-specific monoclonal antibodies recognizes a 40-42 kDa apical plasma membrane integral protein (RTI40) that was detected in the alveolar fluid of injured lungs [13]. Another type I cellspecific monoclonal antibody is .In the present study, the distribution of RTI40, MEP-1 antigen and the BPA lectin-binding glycoprotein was investigated in bleomycin-injured rat lungs and compared with that in normal adult and foetal lung tissue. The binding to the alveolar surface has been shown to be stable even under conditions of lung injury, compared with the immunoreactions of antibodies against type I cell-specific antigens [7,12]. Materials and methodsMale Wistar rats weighing 350-400 g were given a single dose of 7 units of bleomycin sulphate in phosphatebuffered saline (PBS)·kg body weight -1 by intratracheal instillation. Controls received PBS alone. Different animals were killed at 7, 14, 21, 28, 35 and 42 days after bleomycin application and the lungs were prepared and lavaged via the trachea with PBS [17].Sections of formalin-fixed, paraffin-embedded lung tissue (normal, n=5; injured tissue, 4 weeks after bleomycin administration, n=12) were dewaxed. In addition, sections These results suggest RTI40 as a tool for the evaluation of alveolar epithelial type I cell behaviour during re-epithelialization processes.

In vivo intravital endoscopic confocal fluorescence microscopy of normal and acutely injured rat lungs

“…It also showed cellular hyperplasia, enhanced matrix deposition, and abnormal interstitial cells proliferation. In accordance with the study by Koslowski et al, 29 the in vivo imaging and the ex vivo immunofluorescence showed areas of variable RTI40 protein staining at the alveolar surface (from no to irregulardiscontinuous labeling, with foci of 'normal' or already restored alveolar epithelium) 3 weeks following bleomycin administration. In this regard, taking into consideration that the dose of bleomycin I/T instilled was mild (1.5 vs 7 TU per rat 30 ) and though several brown areas were obviously visible macroscopically, IgGs were topically applied and should not normally have accessed nonatelectatic open airways and Lung in vivo endomicroscopy F Chagnon et al spaces in vivo (ie, less injured areas), whereas they will have contacted these tissues in ex vivo fixated samples.…”

The use of alveolar epithelial type I cell-selective markers to investigate lung injury and repair

“…B. purpurea agglutinin) but demonstrate focal loss of RTI 40 / TIa protein [94]. Other investigations into lung fibrosis also report the loss of ATI-cell-associated proteins, including caveolin-1, RTI 40 /TIa protein, AQP5 and RAGE [30,95,114].…”

“…In contrast, in a model of focal ATI cell necrosis, induced by nitrogen dioxide exposure, BALF concentrations of RTI 40 / TIa protein are elevated only two-fold above control values and alveolar fluid clearance is stimulated [90]. BALF RTI 40 / TIa protein concentrations are also elevated, to varying degrees, in other acute injury models, including hyperoxia [91], Staphylococcus aureus-induced pneumonia [92], acidinstillation followed by ventilator-induced injury [96] and following intratracheal adminstration of bleomycin (table 2) [94]. In the rat model of acid-instillation, RTI 40 /TIa protein concentrations in oedema fluid and plasma were significantly reduced in animals ventilated at low tidal, or protective, volumes in comparison with those ventilated at higher tidal volumes (i.e.…”

Cathepsins in bleomycin-induced lung injury in rat

“…For detection of the type I pneumocyte-specific protein rat type I cell marker (RTI40) in the BALF, proteins were sedimented by centrifugation for 2 h at 100,0006g at 4uC, as described previously [2]. Proteins were redissolved in 360 mM tris-(hydroxymethyl)-aminomethane (Tris)/HCl (pH 6.8) containing 190 mM sodium dodecyl sulphate (SDS), 0.03% bromophenol blue and 55% glycerol (volume/volume) and incubated for 5 min at 95uC.…”

“…Intratracheal administration of bleomycin into the lung of rats causes a sequence of morphological and biochemical changes, such as acute inflammation with oedema and damage to the alveolar epithelium as well as influx of inflammatory cells. Finally, a phase of chronic injury, which is characterised by tissue remodelling with proliferation of fibroblasts and pneumocytes and thickening of the alveolar walls, follows [1][2][3]. This fibrotic process is accompanied by excessive production and accumulation of connective tissue components.…”