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DOI: 10.1369/jhc.4A6438.2004
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Journal of Histochemistry and Cytochemistry
Volume 52 (12): 1537-1542, 2004
Copyright ©The Histochemical Society, Inc.

Combined Smooth Muscle and Melanocytic Differentiation in Lymphangioleiomyomatosis

Xiaoning Zhe and Lucia Schuger

Department of Pathology, Wayne State University, School of Medicine, Detroit, Michigan

Correspondence to: Lucia Schuger, MD, Dept. of Pathology, Wayne State University, 540 E. Canfield St., Rm. 9248, Detroit, MI 48201. E-mail: lschuger{at}med.wayne.edu


   Summary
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 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Pulmonary lymphangioleiomyomatosis (LAM) is characterized by abnormal proliferation of immature-looking smooth muscle (SM)-like cells (LAM cells), leading to lung destruction and cyst formation. In addition to expressing some SM markers, scattered LAM cells express the melanocytic maker gp100, which is recognized by antibody HMB45, suggesting that at least a few LAM cells may have melanocytic differentiation. Here we immunostained 26 LAM samples for several melanocyte-related proteins. These studies showed that all LAM cells express tetraspanin CD63, a melanoma-associated protein that belongs to the transmembrane 4 superfamily. The majority of LAM cells also immunoreacted with PNL2, an antibody against a yet uncharacterized melanocytic antigen. Furthermore, we examined the co-expression of PNL2 and Ki-67, an indicator of cell proliferation, and found that PNL2-positive LAM cells showed a significantly lower proliferation rate compared with their negative counterparts. Our findings shed new light on the nature of the LAM cells by demonstrating their combined SM and melanocytic differentiation and the existence of subpopulations with different proliferative potential. Furthermore, these studies provided two new antibodies useful in the diagnosis of LAM.

(J Histochem Cytochem 52:1537–1542, 2004)

Key Words: lymphangioleiomyomatosis • CD63 • PNL2 • lung


   Introduction
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 Summary
 Introduction
Materials and Methods
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 Discussion
 Literature Cited
 
LYMPHANGIOLEIOMYOMATOSIS (LAM) is a rare pulmonary disorder that affects primarily women, with a mean age of onset in the thirties (Corrin et al. 1975Go; Kalassian et al. 1997Go). The disease is characterized by an abnormal proliferation of elongated or oval smooth muscle (SM)-like cells (LAM cells) in the pulmonary interstitium (Bernstein et al. 1995Go; Maziak et al. 1996Go). Although LAM cells lack significant cellular atypia, mitotic activity, or metastatic potential, over time these cells proliferate to obstruct and destroy the lung parenchyma, leading to progressive loss of pulmonary function and eventually to death (Sullivan 1998Go). LAM occurs either as an isolated disorder or in association with tuberous sclerosis complex (TSC). TSC is an autosomal dominant inherited disease caused by a mutation in either the TSC1 or the TSC2 tumor suppressor gene, and both have been associated with TSC-LAM. The critical role of TSC2 is strongly supported by the findings of Carsillo et al. (2000)Go, who demonstrated mutations in the TSC2 gene in LAM cells from sporadic LAM cases.

LAM cells have a phenotype consistent with that of immature SM cells. Accordingly, they express SM {alpha}-actin as well as some other markers of SM differentiation (Matsui et al. 2000Go,2001Go). On this basis, LAM cells have been generally considered a variant of SM cells. However, unlike muscle, scattered LAM cells express gp100, a melanocyte-related protein immunorecognized by antibody HMB45 (Adema et al. 1994Go). gp100 expression suggests that a least some LAM cells feature partial melanocytic differentiation. We decided to test this hypothesis further by using a panel of antibodies against melanocyte-related proteins to immunostain 26 LAM samples. The melanocyte-related proteins and antibodies were Melan A (Kawakami et al. 1997Go), melanoma-associated antigen-1 (MAGE-1) (Carrel et al. 1996Go), PNL2 (Rochaix et al. 2003Go), HMB50 (Esclamado et al. 1986Go), and CD63 (Vennegoor and Rumke 1986Go). Here we show that LAM cells expressed some of these melanocyte markers. More specifically, our studies demonstrated that essentially all LAM cells immunoreacted with antibody against CD63, a tetraspanin protein found primarily in melanoma cells (Atkinson et al. 1984Go,1985Go). In addition, the majority of the cells immunoreacted with antibody PNL2, a newly described antibody against a yet unknown melanocyte-specific protein (Rochaix et al. 2003Go). Furthermore, we found that the mitotic activity was concentrated in PNL2-negative cells, thereby indicating the existence of LAM cell subpopulations with different proliferation capabilities.

Our study contributes to the demonstration that LAM lesions have a dual smooth muscle/melanocytic differentiation.


   Materials and Methods
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 Materials and Methods
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Clinical Information
In compliance with the required regulations and consents, 14 formalin-fixed, paraffin-embedded LAM specimens (12 from open lung biopsy samples and two from lung transplant samples) were obtained from Pathology Departments nationwide coordinated by the LAM Foundation. Twelve additional LAM samples were obtained from the NHLBI LAM Registry (all were lung transplant samples). Of the total 26 cases, the age range was between 25 and 79 years. The initial complaint included pneumothorax (six cases), hemoptysis (two cases), pleural effusion (two cases), and diverse pulmonary symptoms (14 cases). Two cases also presented with TSC. Three cases of idiopathic pulmonary fibrosis and three cases of organizing pneumonia were selected from our pathology archival material.

Immunohistochemistry
Serial 5-µm-thick sections from each case were immunostained with the following antibodies: PNL2, HMB45, SM {alpha}-actin, Melan A (all from Dako Cytomation California; Carpinteria, CA), CD63, Ki-67 (both from Chemicon International; Temecula, CA), MAGE-1, and HMB50 (both from NeoMarkers; Fremont, CA). Except for SM {alpha}-actin and CD63, antigen retrieval (0.01 M citrate buffer, pH 6.0) was performed before primary antibody incubation. Antibodies were all used at a concentration of 4 µg/ml. Staining was completed using a commercial peroxidase–anti-peroxidase kit following the manufacturer's instructions (ABC kit from Vector Laboratories; Burlington, CA) as previously described (Yang et al. 1998Go; Zhang et al. 1999Go). Controls for these experiments included omission of the first antibody and substitution with preimmune mouse or goat IgG (Sigma; St Louis, MO).

Double Immunostaining and Determination of Cell Proliferation
Double immunostaining for PNL2 and Ki-67 was carried out by using a substrate combination of Vector Blue and AEC (Vector Laboratories) with the same ABC system used for single staining. A brief counterstaining with 0.1% Mayer's hematoxylin solution (Sigma) generated a light-blue nuclear background different from the intensive Vector Blue immunosignals. Ki-67-positive cells in LAM lesions were counted respectively from both PNL2-positive and -negative subgroups in all the 26 cases. Statistical analysis was performed by Student's t-test.


   Results
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 Materials and Methods
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Discussion
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Essentially all LAM cells in 26 of 26 cases immunoreacted with antibody CD63, whereas ~80% of LAM cells immunoreacted with antibody PNL2 (Figure 1). This generalized immunoreactivity contrasted with the scattered positivity observed when antibody HMB45 was used. As expected, the alveolar structures, airways, blood vessels, and their surrounding fibroconnective tissue did not immunoreact with these two antibodies (Figures 1 and 2), nor did other two diffuse pulmonary conditions tested here, i.e., idiopathic pulmonary fibrosis and organizing pneumonia (not shown).



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  		Figure 1

Serial sections of an LAM lesion immunostained for SM {alpha}-actin, HMB45, CD63, and PNL2 (all reddish-brown). Alveolar walls surrounding the lesion are negative. Bar = 100 µm.

 


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  		Figure 2

Higher magnification of an LAM lesion immunostained for SM {alpha}-actin, HMB45, and CD63. Arrows point to vascular SM and adventitia. Inset within the CD63 picture shows two LAM cells with their nuclei surrounded by the granular positive CD63 immunostaining. Bar = 50 µm.

 
A higher magnification of an LAM lesion is shown in Figure 2. The lesion is immunostained with antibodies against SM {alpha}-actin (upper panel), HMB45 (middle panel), and CD63 (lower panel). CD63 is clearly present in all LAM cells. The pattern of immunostaining is granular and is distributed in the cytoplasm (Figure 2 inset). The blood vessel wall included in the figure is negative for CD63. As expected, its SM wall is positive for SM {alpha}-actin and its fibroconnective adventitial layer is negative. CD63-positive cells within the blood vessel represent granulocytes. Some subtypes of granulocytes, particularly eosinophils, have been found to immunoreact with CD63 (Calafat et al. 1997Go; Matsumoto et al. 1999aGo).

Figure 3 represents a higher magnification of an LAM lesion immunostained with antibodies against SM {alpha}-actin (upper panel), HMB45 (middle panel), and PNL2 (lower panel). Note that most but not all LAM cells immunoreact with PNL2. In addition, the immunoreactivity is cytoplasmic and granular (Figure 3 inset). Immunostainings with antibodies against Melan-A, HMB50, and MAGE-1 were noncontributory because they stained most of the lung tissue nonspecifically (not shown).



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  		Figure 3

Higher magnification of an LAM lesion immunostained for SM {alpha}-actin, HMB45, and PNL2. Inset within the PNL2 picture shows two LAM cells with their nuclei surrounded by the coarsely granular positive PNL2 immunostaining. Bar = 50 µm.

 
Because not all LAM cells immunoreacted with PNL2, we sought to determine the proliferation level of PNL2-positive cells compared with PNL2-negative cells. For these studies we used the antibody Ki-67, a marker of cell proliferation (Schluter et al. 1993Go) and counted up to 200 PNL2-positive cells and 50 PNL2-negative LAM cells per case. The average percentage of Ki-67 positivity in PNL2-positive cells was 9.6 ± 2.9%, while the Ki-67 positivity reached 76.8 ± 15.2% in the PNL2-negative subgroup, which comprised less than 20% of the whole LAM cell population (Figure 4). Therefore, we confirmed that LAM cells have low mitotic activity, as previously shown (Matsumoto et al. 1999bGo), and demonstrated that PNL2- positive cells have the lowest mitotic rate.



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  		Figure 4

Histogram representing the percentage of Ki-67-positive nuclei in PNL2-positive and -negative cells. The right panel is a picture of a LAM lesion immunostained for PNL2 (reddish cytoplasm) and Ki-67 (bright blue, arrow). Note that the cell positive for Ki-67 does not immunoreact with PNL2. The nuclei are counterstained with hematoxylin (pale blue, arrowheads). Bar = 30 µm.

 

   Discussion
Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
Literature Cited
 
LAM is characterized by the abnormal proliferation of elongated cells (LAM cells) in the pulmonary interstitium, which eventually obstruct and destroy the lung parenchyma. Although LAM cells are considered an immature form of SM cells, here we showed that all LAM cells in 26 cases immunoreacted with CD63 and most of them immunoreacted with PNL2, both markers for melanocyte differentiation (Vennegoor and Rumke 1986Go; Rochaix et al. 2003Go). Moreover, these two markers were not present in some other diffuse lung diseases, such as pulmonary fibrosis or organizing pneumonia. It should be stressed that CD63 is also present in platelets, macrophages/histiocytes, and some subtypes of white blood cells (Calafat et al. 1997Go; Cieutat et al. 1998Go; Heijnen et al. 1998Go; Matsumoto et al. 1999aGo). Although LAM cells do not have a phenotype consistent with that of platelets or white blood cells, the possibility that LAM are a type of histiocyte cannot be completely excluded on the basis of morphology alone. Such a possibility, however, was ruled out by their lack of immunoreactivity with CD68, a histiocyte-specific antibody (not shown) and also by the fact that PNL2 immunoreacts exclusively with melanocytes and melanoma cells (Rochaix et al. 2003Go).

Our studies indicated that LAM cells do not constitute a homogeneous cell population but that they can be divided at least into three subtypes, those that immunoreact with CD63 alone, those that also immunoreact with PNL2, and those few that express gp100. LAM cell subpopulations differed in their ability to proliferate and therefore in their potential to advance the course of the disease. The cells with the lowest mitotic activity were those positive for PNL2, suggesting that this is a relatively more stable or more differentiated cell subpopulation. We did not determine whether or not HMB45-positive cells immunoreact with PNL2, but Matsumoto et al. (1999b)Go demonstrated that HMB45-positive cells have a 6% mitotic index, which is similar to what we found in PNL-2-positive cells.

This study therefore identified two reliable antibodies to diagnose LAM and, more importantly, it shed new light on the LAM cell and how we view it. Because there are no normal cells with this specific immunophenotype, LAM cells should no longer be considered a variant of SM cells. On the contrary, LAM cells must be regarded as a fully abnormal type with the unique characteristic of having dual SM/melanocyte differentiation. Interestingly, this type of differentiation suggests a neural crest cell origin. Neural crest cells originate at the dorsalmost region of the neural tube and migrate far from their source of origin to specific places in the embryo where they give rise to a variety of tissues, including all melanocytes and certain visceral and vascular SM (Etchevers et al. 2001Go,2002Go). Furthermore, it has been shown that neural crest cells can differentiate into SM cells in vitro in the presence of transforming growth factor (TGF)-ß1 (Shah et al. 1996Go), folic acid (Boot et al. 2003Go), or when a specific type of culture medium is used (Jain et al. 1998Go). Because neural crest cells can also differentiate into non-proliferative cell types, such as neurons and adrenal medullary cells, redirection of LAM cells into a mature non-proliferative type may represent a potential approach to treatment of LAM.


   Acknowledgments
 
Supported by NHLBI grants HL-48730 and HL-67100 (to LS) and by a fellowship from the LAM Foundation.

We thank Dr Gerald Beck (Department of Biostatistics and Epidemiology, Cleveland Clinic Foundation) and Ms Sue Byrnes (Director of The LAM Foundation) for facilitating the obtainment of LAM tissue and for providing the pertaining clinical data.


   Footnotes
 
Received for publication June 10, 2004; accepted August 3, 2004


   Literature Cited
Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 

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