Morphometric analysis of intralobular, interlobular and pleural lymphatics in normal human lung

Abstract

In spite of their presumed relevance in maintaining interalveolar septal fluid homeostasis, the knowledge of the anatomy of human lung lymphatics is still incomplete. The recent discovery of reliable markers specific for lymphatic endothelium has led to the observation that, contrary to previous assumptions, human lymphatic vessels extend deep inside the pulmonary lobule in association with bronchioles, intralobular arterioles or small pulmonary veins. The aim of this study was to provide a morphometric characterization of lymphatic vessels in the periphery of the human lung. Human lung sections were immunolabelled with the lymphatic marker D2-40, followed by blood vessel staining with von Willebrand Factor. Lymphatic vessels were classified into: intralobular (including those associated with bronchovascular bundles, perivascular, peribronchiolar and interalveolar), pleural (in the connective tissue of the visceral pleura), and interlobular (in interlobular septa). The percentage area occupied by the lymphatic lumen was much greater in the interlobular septa and in the subpleural space than in the lobule. Most of the intralobular lymphatic vessels were in close contact with a blood vessel, either alone or within a bronchovascular bundle, whereas 7% were associated with a bronchiole and < 1% were not connected to blood vessels or bronchioles (interalveolar). Intralobular lymphatic size progressively decreased from bronchovascular through to peribronchiolar, perivascular and interalveolar lymphatics. Lymphatics associated with bronchovascular bundles had similar morphometric characteristics to pleural and interlobular lymphatics. Shape factors were similar across lymphatic populations, except that peribronchiolar lymphatics had a marginally increased roundness and circularity, suggesting a more regular shape due to increased filling, and interlobular lymphatics had greater elongation, due to a greater proportion of conducting lymphatics cut longitudinally. Unsupervised cluster analysis confirmed a marked heterogeneity of lymphatic vessels both within and between groups, with a cluster of smaller vessels specifically represented in perivascular and interalveolar lymphatics within the alveolar interstitium. Our data indicate that intralobular lymphatics are a heterogeneous population, including vessels surrounding the bronchovascular bundle analogous to the conducting vessels present in the pleural and interlobular septa, many small perivascular lymphatics responsible for maintaining fluid balance in the alveolar interstitium, and a minority of intermediate lymphatics draining the peripheral airways. These lymphatic populations could be differentially involved in the pathogenesis of diseases preferentially involving distinct lung compartments.

Morphometry Analysis of Lymphatics in Pulmonary Adenocarcinomas With a Lepidic Growth Pattern

“…These findings are in agreement with other observations showing normal lung lymphatic vessels extending into the interalveolar septa (Kambouchner and Bernaudin 2009;Sozio et al 2012). Although Koukourakis et al (2005) did not observe any intratumoral lymphatic network within a series of lung carcinomas with LYVE-1 antibody, we found lymphatic vessel hot spots within the invasive patterns of lung adenocarcinomas.…”

“…Moreover, it must be underscored that the lymphatic network topography in the lepidic growth fraction is comparable to that in the intralobular distribution in normal lung (Kambouchner and Bernaudin 2009;Sozio et al 2012). In contrast, a higher LVD was observed in the invasive fractions of tumors, suggesting that a different network pattern is associated with invasion.…”

“…However, when considering vascular systems, podoplanin is exclusively expressed by the lymphatic endothelium, thereby explaining why D2-40 is widely considered specific to lymphatic identification. Moreover, using D2-40 as a marker of lymphatic endothelium enabled reappraisal of the organization of the intralobular lymphatic network in normal lung (Kambouchner and Bernaudin, 2009;Sozio et al 2012) and pulmonary interstitial diseases (Kambouchner et al 2011). In this study, only D2-40-immunolabeled lymphatics n, number of analyzed lymphatic vessels; NA, not applicable.…”

“…To identify lymphatic vessels, antivascular endothelial growth factor receptor-3 (VEGFR-3) or antilymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) antibodies were formerly used but we opted for monoclonal antibody D2-40 because of its higher sensitivity and specificity for labeling lymphatic endothelial cells (Evangelou et al 2005;Van der Auwera et al 2006;Kambouchner and Bernaudin 2009;Sozio et al 2012). Anti-VEGFR-3 antibodies were shown to lack specificity and do not clearly discriminate between vascular and lymphatic endothelial cells, due to VEGFR-3 upregulation during tumor angiogenesis (Niki et al 2001).…”

“…In that context, taking advantage of the identification of intralobular lymphatic vessels in normal lung (Kambouchner and Bernaudin 2009;Sozio et al 2012) and using the D2-40 monoclonal antibody as a reliable marker of lymphatic endothelial cells (Evangelou et al 2005; Van der Auwera et al 2006), we evaluated the lymphatic vascular pattern associated with lepidic growth or the invasive component, to obtain insight into lymphangiogenesis in pulmonary adenocarcinomas.…”