Combined Smooth Muscle and Melanocytic Differentiation in Lymphangioleiomyomatosis
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
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Summary |
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(J Histochem Cytochem 52:15371542, 2004)
Key Words: lymphangioleiomyomatosis CD63 PNL2 lung
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Introduction |
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LAM cells have a phenotype consistent with that of immature SM cells. Accordingly, they express SM -actin as well as some other markers of SM differentiation (Matsui et al. 2000
,2001
). 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. 1994
). 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. 1997
), melanoma-associated antigen-1 (MAGE-1) (Carrel et al. 1996
), PNL2 (Rochaix et al. 2003
), HMB50 (Esclamado et al. 1986
), and CD63 (Vennegoor and Rumke 1986
). 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. 1984
,1985
). 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. 2003
). 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.
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Materials and Methods |
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Immunohistochemistry
Serial 5-µm-thick sections from each case were immunostained with the following antibodies: PNL2, HMB45, SM -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
-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 peroxidaseanti-peroxidase kit following the manufacturer's instructions (ABC kit from Vector Laboratories; Burlington, CA) as previously described (Yang et al. 1998
; Zhang et al. 1999
). 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.
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Results |
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Figure 3 represents a higher magnification of an LAM lesion immunostained with antibodies against SM -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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Discussion |
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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) 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. 2001,2002
). 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. 1996
), folic acid (Boot et al. 2003
), or when a specific type of culture medium is used (Jain et al. 1998
). 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.
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Acknowledgments |
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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.
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Footnotes |
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Literature Cited |
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