Interindividual variability in the expression of surfactant protein A and B in the human lung during development

Abstract

The surfactant complex, thanks to its multiple actions including decrease of surface- tension and antimicrobial activity, plays a fundamental role in newborn survival, lowering the risk of respiratory distress syndrome. The aim of this work was to determine if the synthesis of two surfactant proteins (SP), SPA and pro-SPB, shows some inter-individual variability during lung development in the intrauterine life. Immunoreactivity for SPA and pro-SPB was investigated in the lungs of 40 subjects, including 15 fetuses, ranging from 14 to 22 weeks of gestation, and 25 neonates, from 24 to 41 weeks. Lung samples were formalin fixed, paraffin-embedded and routinely processed. SPA and pro-SPB were detected utilizing commercial antibodies. A semi-quantitative grading system (1 to 4) was applied, based on the number of reactive cells and the intensity of immunostaining. Surfactant protein immunostaining was found in three compartments: bronchi, bronchioles and alveoli, starting from 14 weeks of gestation in the bronchial epithelium and from the 21st week in the alveolar spaces. Differences were found regarding SPA and pro-SPB expression in the vast majority of subjects: in some lungs, SPA was more expressed whereas in others pro-SPB showed an higher degree of immunoreactivity. The expression of both surfactant proteins was not strictly correlated with gestational age. Whereas the highest levels of reactivity were detected in at term neonates, on the other hand one case with grade 3 was detected at 22 weeks and one negative case for both proteins was observed at 31 weeks. Our data clearly show a marked inter-individual variability regarding the production of SPA and pro-SPB and suggest the existence of other epigenetic factors, acting during gestation, that might influence surfactant production and, consequently, the survival potential of neonates at birth.

Figure 1.

Examples of different degrees of reactivity for surfactant proteins (SPA and pro-SPB). A) Grade 1; scale bar: 50 µm. B) Grade 2; scale bar: 50 µm. C) Grade 3; scale bar: 100 µm. D) Grade 4; scale bar: 25 µm. Arrows pointing to the most representative positive staining.

Figure 2.

Immunohistochemical expression of SPA in bronchial epithelial cells, in bronchiolar epithelial cells and in alveolar epithelium at different magnification. A) Bronchial epithelial cells; scale bar: 50 µm. B) Bronchial epithelial cells; scale bar: 25 µm. C) Bronchial epithelial cells; scale bar: 200 µm. D) Bronchial epithelial cells; scale bar: 25 µm. E) Bronchiolar epithelial cells; scale bar: 100 µm. F) Bronchiolar epithelial cells; scale bar: 50 µm. G) Bronchiolar epithelial cells scale; bar: 100 µm. H) Bronchiolar epithelial cells; scale bar: 50 µm); I,J) Alveolar epithelium at 25 weeks of gestational age with different alveolar dimension; scale bar: 50 µm. K,L) Alveolar epithelium at 22 weeks of gestational age with different alveolar shape and branching; scale bar: 50 µm. Arrows pointing to the most representative positive staining.

Figure 3.

Pro-SPB immunoreactivity in bronchial epithelial cells, in bronchiolar epithelial cells and in alveolar epithelium at different magnification. A) Bronchial epithelial cells; scale bar: 50 µm. B) Bronchial epithelial cells; scale bar: 25 µm. C) Bronchial epithelial cells; scale bar: 100 µm. D) Bronchial epithelial cells; scale bar: 50 µm. E) Bronchial epithelial cells; scale bar: 100 µm. F) Bronchiolar epithelial cells; scale bar: 50 µm. G) Bronchiolar epithelial cells; scale bar: 50 µm. H) Bronchiolar epithelial cells; scale bar: 100 µm. I) Alveolar epithelium; scale bar: 100 µm. J) Alveolar epithelium at 25 weeks of gestational age with different alveolar dimension; scale bar: 50 µm. K, L) Alveolar epithelium with different alveolar shape; scale bar: 50 µm. Arrows pointing to the most representative positive staining.

Figure 4.

Histogram graphical representation of SPA and pro-SPB expression in bronchi at different gestational age showind the higher degree of reactivity of pro-SPB in 11 cases, SPA in 6 cases and the differences between the two surfactant proteins.

Figure 5.

Histogram graphical representation of SPA and pro-SPB expression in bronchioles at different gestational age showing the marked differences between SPA and pro-SPB in the same lung were also observed in the small airways and the inter-individual variability.

Figure 6.

Histogram graphical representation of SPA and pro-SPB expression in alveolar epithelium at different gestational age with the highest values of SPA expression in the last weeks of gestation and showing the irregular pro-SPB expression with the highest values from 25 to 39 weeks of gestation.

Figure 7.

Bar graphical representation showing the percentage of SPA immunohistochemical expression into three groups of gestational age: 14-21 weeks, 22-30 weeks and 31-41 weeks. The highest reactivity were observed from the 31^st week.