Lipofibroblast Phenotype in Pulmonary Interstitial Glycogenosis

To the Editor:

In a 2002 publication in the Journal, Canakis and colleagues defined a novel form of childhood interstitial lung disease in neonates who presented with respiratory distress in the first days to weeks of life. They termed the disorder “pulmonary interstitial glycogenosis” on the basis of the finding of increased glycogen-laden mesenchymal cells in the interstitium (1).

Subsequently, pulmonary interstitial glycogenosis has been observed in various conditions associated with neonatal hypoxemia and injury, most frequently deficient lung growth, but also meconium aspiration, pulmonary hypertension, and congenital lung lesions (2). The clinical presentation ranges from indolent tachypnea and hypoxemia to refractory pulmonary hypertension and respiratory failure. Some infants improve with corticosteroids without apparent long-term morbidity, whereas mortality occurs in association with comorbid conditions (1, 3).

Despite growing experience, pulmonary interstitial glycogenosis remains a histologic descriptor of unknown etiology. Lung biopsy reveals focal or diffuse expansion of alveolar septa by spindle-shaped cells containing periodic acid–Schiff–positive, diastase-labile material consistent with glycogen. Diffuse expression of vimentin and focal smooth muscle actin positivity suggest these cells have a fibroblast phenotype. Electron microscopy shows these mesenchymal cells are primitive, with few cytoplasmic organelles and abundant monoparticulate glycogen; some cells have prominent anastomosing cisternae of endoplasmic reticulum consistent with fibroblast differentiation. Canakis and colleagues acknowledged uncertainty about the nature of these cells while noting some interstitial cells contained droplets of neutral lipid, in addition to glycogen (1).

Similar to the original description of pulmonary interstitial glycogenosis, we have also observed lipid droplets in some of these glycogenated cells, reminiscent of lipid-containing fibroblasts/lipofibroblasts, as described in the rodent lung. Lipofibroblasts play a critical role in rodent lung maturation by stimulating alveolarization and lipid homeostasis and can be detected by adipocyte differentiation-related protein (ADRP), a protein necessary for lipid uptake that localizes to intracellular neutral lipid droplets (4). Although the presence of lipofibroblasts in normal human lung has been debated (1, 5, 6), we sought to assess the presence of lipofibroblasts in pulmonary interstitial glycogenosis cases.

Methods

Patients with biopsy-proven pulmonary interstitial glycogenosis were identified from a pathology database under a protocol approved by Seattle Children’s Hospital Institutional Review Board. Five cases were chosen for study based on the requirement of having both frozen lung tissue and material available for electron microscopy. Paraffin sections were stained with hematoxylin and eosin, periodic acid–Schiff with and without diastase treatment, and Movat stain. Lipid content on frozen sections was assessed by oil-red-O staining followed by cytokeratin immunostaining by indirect immunoperoxidase. Pulmonary lipofibroblasts were detected by double immunofluorescence staining for ADRP and vimentin.

Results

The clinical characteristics of cases are summarized in Table 1. Lung biopsies showed expansion of the alveolar walls by spindled cells with vacuolated cytoplasm and indistinct cell membranes (Figure 1A). Four cases also had deficient alveolarization, reflective of poor lung growth. In all cases, electron microscopy (Figures 1B and 1C) and oil-red-O staining (Figure 1D) showed rare or small foci of lipid-containing mesenchymal cells within the alveolar wall, as well as colocalization for ADRP and vimentin (Figures 1E and 1F), consistent with lipofibroblasts.

Table 1.

Characteristics of Patients with Pulmonary Interstitial Glycogenosis

	Case 1	Case 2	Case 3	Case 4	Case 5
Sex	Male	Male	Female	Female	Male
Gestational age, wk	38	37	41	35	36
Pre-/neonatal complications	Pneumothorax	Severe chylothorax	Total anomalous pulmonary venous return/vein stenosis	VACTERL/double outlet right ventricle	Preeclampsia, pulmonary hypertension
O2 or mechanical ventilation at birth	Yes	Yes	Yes	Yes	Yes
Age at lung biopsy	5 d	4 wk	7 wk	4 wk	3 d
Status at lung biopsy	Mechanical ventilation	Mechanical ventilation	Mechanical ventilation (ECMO postoperative)	Mechanical ventilation	Mechanical ventilation (ECMO postoperative)
Additional indication for surgery	n/a; only unknown lung disease	Thoracic duct ligation	Cardiac surgery	Cardiac surgery	n/a; only unknown lung disease
Other histologic findings	Deficient growth	Deficient growth	Pulmonary hypertension,* lymphangiectasia	Deficient growth, pulmonary hypertension	Deficient growth, pulmonary hypertension
Outcome	9 mo; tachypnea, cough	5.5 yr; asymptomatic	Died 9 wk; pulmonary hypertension	4 yr; intermittent asthma	4 mo; asymptomatic

Definition of abbreviations: ECMO = extracorporeal membrane oxygenation; n/a = not applicable; VACTERL = vetebral defects, anal atresia, cardiac malformations, tracheoesophageal fistula and/or esophageal atresia, renal dysplasia, limb anomalies.

^*Pulmonary hypertensive histologic findings were defined by presence of medial hypertrophy of pulmonary arteries ± intimal hyperplasia.

Figure 1.

Lipofibroblast phenotype in pulmonary interstitial glycogenosis. Characteristic spindle cells expand the alveolar interstitium in the biopsy from case 4, which also showed poor lung growth and hypertensive change of the pulmonary arteries (A). Transmission electron microscopy of interstitial cells in the alveolar wall from case 3 (B, original magnification, ×8,500) and case 2 (C, original magnification ×18,200), showing some of these cells contain lipid bodies (arrows), as well as dispersed cytoplasmic glycogen. Oil-red-O staining (D) demonstrates lipid droplets of variable size in scattered alveolar interstitial cells; cytokeratin (Dako M3515) immunostaining denotes epithelial cells. (E and F) Some interstitial cells in pulmonary interstitial glycogenosis express adipocyte differentiation–related protein (Fitzgerald 10R-A117ax; E developed with 3,3′-diaminobenzidine; F developed with fluorescein isothiocyanate-green), which colocalizes with vimentin (red; Dako M7020; nuclei in blue, 4′,6-diamidino-2-phenylindole counterstain). No significant oil-red-O positive or adipocyte differentiation-related protein immunopositive interstitial cells were seen within lung biopsies from age-matched patients without histologic findings of pulmonary interstitial glycogenosis (data not shown).

Discussion

Interstitial lipofibroblasts in these pulmonary interstitial glycogenosis cases were confirmed by electron microscopy, oil-red-O staining, and coexpression of ADRP and vimentin. Pulmonary lipofibroblasts have been well studied in the rodent lung, where they are present in the alveolar wall during the period of septation and diminish by apoptosis after alveolarization is complete (7, 8). Lipofibroblasts mediate lipid transfer to type 2 alveolar epithelial cells for surfactant phospholipid synthesis and protect against oxidant injury (9). Disruption of lipofibroblast differentiation pathways, including parathyroid hormone–related peptide and peroxisome proliferator–activated receptor-γ, results in differentiation of the lipofibroblast to a myofibroblast phenotype, as seen in models of neonatal hyperoxic lung injury (9).

There is clinical and pathologic evidence to support that pulmonary interstitial glycogenosis is a self-limited entity, including that the mesenchymal cells have transient proliferative capacity (10). Although the impetus for the mesenchymal cell proliferation in pulmonary interstitial glycogenosis is unclear, we and others have suggested that accumulation of these cells is a nonspecific feature of the neonatal lung, reacting to injurious conditions or reflective of delay in neonatal transition. Canakis and colleagues speculated that pulmonary interstitial glycogenosis reflects a delay or aberration in the maturation of pulmonary mesenchymal cells that do not normally contain abundant glycogen (1). Glucocorticoids increase lipid trafficking from the lipofibroblast to type II alveolar epithelial cells in culture, increasing surfactant synthesis. We speculate that the anecdotal rapid clinical response to pulse steroids in infants with pulmonary interstitial glycogenosis may be attributable to this mechanism (1).

Determining the significance of the scattered lipofibroblasts in pulmonary interstitial glycogenosis is critical as the disorder predominates in neonates who often have conditions affecting lung development. Of interest, pulmonary interstitial glycogenosis has not been recognized in children older than 1 year, implying a developmental process or limited window of time when these cells may play a role. Further studies analyzing human lung will be necessary to determine the fate of the lipofibroblasts in pulmonary interstitial glycogenosis and whether they serve a similar function in lung development and repair, as they do in rodent lung. Recognition of lipofibroblasts in pulmonary interstitial glycogenosis confirms that this idiopathic, rare disorder is related to lung development and may inform broader understanding of fundamental developmental processes.