Lymphangioleiomyomatosis

Study objectives: Lymphangioleiomyomatosis (LAM) is a rare disorder of unknown etiology, affecting almost exclusively women of childbearing age, that is associated with the proliferation of spindle cells and cystic changes in the affected lung. The underlying processes that contribute to this disease are poorly understood. Transforming growth factor (TGF)-[[beta].sub.1] is a potent cytokine that promotes mesenchymal cell proliferation and regulates the synthesis of extracellular matrix (ECM) components, particularly fibronectins. Herein, we evaluate the expression of TGF[[beta].sub.1] and matrix-associated fibronectin in lung specimens demonstrating LAM.

Design: Lung biopsy specimens that were confirmed to contain pathologic LAM cells were obtained from 13 patients. The specimens were submitted to immunohistochemical evaluation for TGF[[beta].sub.1] and fibronectin, as well as the typical markers of LAM cells. Healthy lung parenchyma surrounding resected neoplasms was studied in a parallel fashion as control tissues.

Measurements and results: In an 13 LAM cases and in healthy lung parenchyma, we demonstrated that TGF[[beta].sub.1] localized consistently to airway epithelial cells. However, in LAM tissues, matrix-associated TGF[[beta].sub.1] was also consistently found ill regions containing pathologic LAM cells. Notably, more abundant TGF[[beta].sub.1] was observed in highly cellular areas compared to the walls of chronic cystic regions in LAM tissues. Fibronectin, a matrix component that is strongly expressed in response to active TGF[[beta].sub.1] was found to consistently colocalize with this protein in these highly cellular regions, supporting TGF[[beta].sub.1] activity in these regions. The markers of proliferating LAM cells, including proliferating cell nuclear antigen, were also markedly present in these highly cellular LAM regions.

Conclusion: These studies suggest that the proliferation of aberrant LAM cells may be associated with altered regional expression of TGF[[beta].sub.1] and related ECM proteins.

Key words: fibronectins; lymphangiolciomyomatosis; transforming growth factor-[beta]

Abbreviations: ECM = extracellular matrix; LAM = lymphangioleiomyomatosis; PCNA = proliferating cell nuclear antigen; TGF = transforming growth factor

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Lymphangiolciomyomatosis (LAM) is a rare, progressive lung disease that is characterized by the proliferation of abnormal smooth muscle cells (LAM cells) in the lungs and along the axial lymphatics of the chest and abdomen. (1-5) The pulmonary parenchymal changes associated with this peribronchial, perivascular, and perilymphatic LAM cell proliferation result in the formation of thin-walled cysts, and the obstruction of lymphatic channels and conducting airways with progressive respiratory failure. (6,7) Despite recognition that this disorder occurs primarily ill women of childbearing age and is often linked to genetic mutations responsible for tuberous sclerosis, both the etiology and effective management of LAM remain elusive. (3,8-12)

In LAM, the lung parenchyma exhibits 0.5 to 2.0 cm cystic structures lined with hyperplastic type II pneumocytes, intermixed with variable sized fascicles of LAM cells comprising a portion of the cyst wall. LAM cells are also found in the adjacent affected lung tissue. (3,13,14) The following two types of LAM cells are demonstrable in these fascicles: small, spindle-shaped cells, which predominate hE the central areas; and larger, epithelioid cells located chiefly in the periphery. (15-17) While the LAM cells tend to associate with pulmonary lymphatics, the cellular arrangement within loci is typically haphazard, and there is marked heterogeneity within and between LAM foci. (10,14,18,19) It is noteworthy that, despite clinical findings of significant airway obstruction and diminished gas exchange capacity, pathologic review does not exhibit extensive airway involvement with LAM cells or inflammatory cell infiltration. (1,2,5-7,11,18,20,21)

The potential contribution of the extracellular matrix (ECM) to LAM cell proliferation and disease pathogenesis has not been clearly elucidated. Transforming growth factor (TGF)-[[beta].sub.1] is an important cytokine that is known to strongly influence cell cycle progression and ECM expression. TGF[[beta].sub.1] contributes to the progression of many interstitial lung diseases as diverse as usual interstitial pneumonia and pulmonary Langerhans cell histiocytosis. (22,23) This potent agent promotes the proliferation of mesenchymal cells, with associated induction of ECM components, particularly fibronectins and collagens. (23,24) In disease states, the enhanced expression of TGF[[beta].sub.1] contributes to dysregulated cellular proliferation and excessive matrix deposition. (23,25,26)

In this study, we assessed the potential contribution of TGF[[beta].sub.1] to the pathogenesis of LAM by immunohistochemically defining the regional expression of TGF[[beta].sub.1] and fibronectin, a TGF[[beta].sub.1]-induced protein. Given the proliferation-inducing qualities of TGF[[beta].sub.1] and fibronectin, the overexpression of these ECM components may contribute to the dysregulated proliferation of LAM cells. For comparison, we delineated cellular regions of interest by immunohistochemical staining for typical markers of LAM cells, including HMB-45, proliferating cell nuclear antigen (PCNA), and [alpha]-smooth muscle actin. It has been shown previously that the morphologically different LAM cell types also display distinct staining patterns, with stronger HMB-45 staining in epithelioid LAM cells and more PCNA identified in spindle cells. (16,27) Our overall objective was to detect regional differences in TGF[[beta].sub.1] and fibronectin expression, comparing areas of abundant proliferating LAM cells to more chronic cystic regions in available LAM tissues.

MATERIALS AND METHODS

Patient Selection and Lung Sections

After obtaining appropriate study approval from the institutional review board, 13 patients with histologic evidence of LAM from the case flies of Mayo Clinic Rochester were investigated (Table 1). The clinical records mad radiographs of these women were reviewed to confirm that their presentations mad clinical courses were typical of LAM, prior to the review of die tissue obtained by clinically indicated biopsies. Eleven lung specimens were obtained by open lung biopsy, one by video assisted thoracoscopic biopsy, and one by transbronchial biopsy. All specimens were immediately fixed in 10% phosphate buffered formalin and were embedded in paraffin. The specimens were all histologically re-reviewed by an experienced pulmonary pathologist (TVC) to confirm the presence of pathologic LAM cells in each case. Serial 5-[micro]m sections were obtained from the paraffin blocks, mounted on glass slides, deparaffinized with xylene, rehydrated with graded alcohols, and submitted to immunohistochemistry. (25) Tissue also was obtained from four patients undergoing lung resections for non-small cell carcinoma. These specimens were identically handled, mad tumor-free parenchymal regions were used for normal control lung tissue.

Immunohistochemistry

All materials were of reagent grade (unless otherwise specified, all from Sigma Chemical Co; St. Louis, MO). All antibodies used for immunohistochemistry were commercially available. To confirm firm the specificity of tissue staining, all specimens were simultaneously stained in an identical fashion, substituting nonimmune Igs for the primary antibody.

The tissue localization of TGF[[beta].sub.1] was evaluated using a monoclonal mouse anybody generated against human TGF[[beta].sub.1] derived from platelets (Chemicon, Inc; Temecula, CA). Immunohistochemistry for TGF[[beta].sub.1] was performed as previously described. (25) Briefly, the deparaffinized tissue sections were incubated in methanol, containing 0.5% hydrogen peroxide to quench endogenous peroxidase activity, and in 2% normal horse serum to reduce nonspecific binding of antibodies. The sections were next incubated with primary anti-TGF[[beta].sub.1] antibody (10 [micro]g/mL) for 1 h, washed, and subsequently reacted with biotinylated rabbit antimouse antibody (2 [micro]g/mL) for 30 min (Vector Laboratories; Burlingame, CA). The sections were washed again and were incubated with peroxidase-conjugated streptavidin (2 [micro]g/mL) for 30 min (Vector Laboratories). After rinsing, bound antibodies were demonstrated using 3-amino-9-ethylcarbazole substrate in the presence of N,N-dimethylformamide, sodium acetate, and 0.03% hydrogen peroxide for 15 min. After substrate development, sections were counterstained in Mayer hematoxylin for 6 min.

Aside from the modifications noted below, immunohistochemistry tests for fibronectin, [alpha]-smooth muscle actin, and PCNA were performed similarly to that for TGF[[beta].sub.1]. Following deparaffinization, the specimens used in fibronectin detection were boiled for 20 min at 100[degrees]C in 10 mmol/L citric acid at pH 6.0. After quenching with methanol-hydrogen peroxide, and incubation with normal blocking serum, the sections were incubated with mouse monoclonal antibodies recognizing human matrix-associated fibronectin (MAB88904; Chemicon) at a concentration 4 [micro]g/mL for 2 h. The detection of [alpha]-smooth muscle actin was performed utilizing a mouse monoclonal antibody against a synthetic N[H.sub.2] terminus decapeptide of [alpha]-smooth muscle actin (Cymbus Biotechnology Ltd; Chandlers Ford, Hants, UK) for 1 h at a concentration of 5 [micro]g/mL. The PCNA identification was executed with a mouse monoclonal antibody generated against rat PCNA (Chemicon), incubating at 5 [micro]g/mL for 2 h. This antibody has previously been demonstrated to have significant affinity for human PCNA. (28) No counterstain was applied to the PCNA-stained specimens.

HMB-45 immunohistochemistry, testing was performed utilizing a mouse monoclonal antibody against metastatic, human, malignant melanoma (1 [micro]g/mL; Dako Corp; Carpinteria, CA) and a commercially available streptavidin-biotin detection system (Dako). Sections of malignant melanoma were simultaneously stained as positive controls.

The degrees of reactivity to each immunostain were assessed semiquantitatively using a visual analog scale, as previously described. (29) Staining was graded as follows: 0, no staining; 1, staining in 1 to 2 quadrants of each x 400 microscopic field; 2, staining noted diffusely in most fields; and 3, intense staining noted diffusely. Intermediate scores were allowed for samples with variable staining or intensity, which approximates more than one designation (eg, 0.5 for staining in rare fields or 2.5 for variably intense staining). Areas of LAM-involved tissue were reviewed and scored independently by two graders (S.E.E. and A.H.L.), with distinct scores given for predominantly hypercellular and for predominantly cystic regions. Interrater disagreement was uncommon but was handled by conjoint review of the slides in question. Consensus scores are reported. For TGF[[beta].sub.1] and fibronectin samples, data analysis was performed using a statistical software package (JMP; SAS Institute; Cary, NC), comparing cell-dense areas to cystic areas with the Wilcoxon rank sum test.

RESULTS

Identification of LAM Cells With HMB-45, [alpha]-Smooth Muscle Actin, and PCNA

All studied lung specimens from affected patients revealed LAM cells by routine histology. Immunohistochemical evaluation of these specimens also demonstrated reactivity to both HMB-45, a marker of LAM cells, and PCNA, an antigen associated with cellular proliferation, throughout regions of abnormal tissue (Fig 1). Furthermore, in all LAM cases, the LAM cells also expressed a-smooth muscle actin (Fig 1).

[FIGURE 1 OMITTED]

The degree of immunoreactivity to the various epitopes studied was assessed semiquantitatively in various regions of abnormal tissue. Evaluation included both highly cellular regions of lung tissue with numerous LAM cells and less cellular regions, with the latter tending to show fewer LAM cells and marked thin-walled chronic cystic changes. As demonstrated in Table 2, highly cellular LAM cell loci were found to have the greatest intensity of HMB-45 and the cell proliferative marker PCNA. Nonetheless, cells expressing both of these epitopes were identified even in the most cystic regions of abnormal tissue where a paucity of LAM cells was present.

Localization of TGF[[beta].sub.1] and Matrix-Associated Fibronectin

As anticipated, TGF[[beta].sub.1] was consistently localized to the bronchial epithelium and to the vascular endothelium in all the specimens reviewed. However, in LAM tissues we also identified extensive ECM-associated TGF[[beta].sub.1] staining, most markedly throughout the highly cellular LAM cell foci (Fig 2). Of the 13 LAM cases studied, the intensity of staining for TGF[[beta].sub.1] within these cell-dense LAM fascicles was scored as [greater than or equal to] 2 in 10 cases and was scored at least 1 in 3 cases (Table 2). HMB-45--positive and PCNA-positive regions with intermediate LAM cell density tended to demonstrate a proportionally intermediate intensity of staining for TGF[[beta].sub.1] (Table 2).

[FIGURE 2 OMITTED]

In contrast, the intensity of TGF[[beta].sub.1] staining in the LAM cell-poor cystic regions of tissue was markedly less than in the more cellular regions (Fig 2). No cystic region examined received a score of > 1 for TGF[[beta].sub.1] expression, with seven specimens being essentially devoid of TGF[[beta].sub.1] immunoreactivity in these chronic cystic regions (Table 2). One LAM specimen had insufficient cystic tissue for adequate characterization.

Immunohistochemical investigation for fibronectin, a TGF[[beta].sub.1]-induced protein, demonstrated abundant expression, which faithfully colocalized with TGF[[beta].sub.1] within these LAM cell foci. As with TGF[[beta].sub.1], the intensity of fibronectin expression was proportionally related to tissue LAM cell density. Cellular areas rich in LAM cells demonstrated robust fibronectin deposition. In contrast, the more cystic LAM cell-poor regions exhibited relatively little matrix-associated fibronectin compared to the more cellular regions, with most samples exhibiting limited fibronectin expression in these cystic regions (Table 2). Consistently, the differential staining between cellular regions (ie, fibronectin-rich) and cystic regions (ie, fibronectin-poor) of LAM tissues was very readily distinguishable in all of the LAM cases evaluated. In addition, statistical analysis of the TGF[[beta].sub.1] and fibronectin stain scoring also was performed. Wilcoxon rank sum testing revealed significantly different TGF[[beta].sub.1] and fibronectin deposition in cystic vs cellular LAM tissue regions for both of these immunostains (p < 0.001 for each stain comparing cystic to cellular regions). As with TGF[[beta].sub.1], regions of intermediate LAM cell density revealed transitional fibronectin expression.

As previously reported with the LC and CC TGF[[beta].sub.1] antibodies, normal lung parenchyma displayed TGF[[beta].sub.1] and fibronectin staining in the airspace epithelium and vascular endothelium (30-32) (Fig 3). However, in contradistinction to the LAM-involved tissue, there was scant (if any) interstitial matrix-associated deposition of TGF[[beta].sub.1] or fibronectin observed in the normal parenchyma from the control cases.

[FIGURE 3 OMITTED]

Taken together, these findings indicate that LAM cells are associated with abundant TGF[[beta].sub.1] and fibronectin deposition in the ECM surrounding fascicles of LAM cells. Furthermore, the number of these mediators varies with the numbers of LAM cells present, being greatest in cellular fascicles and markedly less in the walls of cysts that contain few LAM cells. As has been reported elsewhere, (25,31) the morphologically normal lung tissue did not demonstrate substantial ECM-associated TGF[[beta].sub.1], only TGF[[beta].sub.1] associated with epithelial and vascular structures.

DISCUSSION

In this immunohistochemical investigation of LAM-involved lung tissue, we observed extensive ECM-associated TGF[[beta].sub.1] expression in regions of proliferative LAM cell foci. In addition to displaying the characteristic LAM cell markers HMB-45 and [alpha]-smooth muscle actin, these cells also exhibit increased PCNA expression, suggesting a correlation between TGF[[beta].sub.1] deposition and pathologic LAM cell proliferation. Conversely, markedly cystic regions of LAM tissue, containing fewer LAM cells, revealed substantially reduced TGF[[beta].sub.1] expression. Utilizing ECM fibronectin deposition as a biologically relevant marker for local TGF[[beta].sub.1] activity, a similar fibronectin expression pattern was observed.

The contribution of the ECM to the pathogenesis of LAM is poorly understood at the present time. A central process in LAM pathogenesis seems to be the dysregulated proliferation of cells of an apparent mesenchymal smooth muscle lineage. Given the known stimulatory effect of TGF[[beta].sub.1] and fibronectin on mesenchymal cell proliferation, we postulate that TGF[[beta].sub.1] may promote disease progression. We further propose that the TGF[[beta].sub.1]-induced deposition of such ECM components as fibronectin may result in tissue remodeling and perhaps further contribute to disease pathogenesis. An earlier survey (33) of TGF[[beta].sub.1] deposition in interstitial lung disease supports our observations of the association of TGF[[beta].sub.1] with pathologic LAM cells in a single case of LAM. This earlier study, however, did not comment on the regional expression of TGF[[beta].sub.1] in cystic and cellular regions, and did not contain information on fibronectin matrix deposition or proliferative markers such as PCNA.

LAM cells are immunohistochemically reactive to such smooth muscle-specific antigens as desmin, vimentin, [alpha]-smooth muscle actin, and smooth muscle myosin heavy chains I and II. (13) Curiously, LAM cells also characteristically react with HMB-45, a monoclonal antibody directed against gp100, a melanocyte antigen found in premelanosomes. (12,14,34,35) The epitope against which HMB-45 reacts in LAM cells has not been identified, but the ability of HMB-45 to discriminate between LAM and other entities with smooth muscle proliferation such as benign metastasizing leiomyomatosis has rendered HMB-45 a useful diagnostic adjunct for LAM. (13,15,35) While most LAM cell foci contain HMB-45-positive cells, it has been suggested that only 17 to 67% of individual LAM cells are HMB-45-positive, with peripheral, epithelioid cells possibly more likely to react positively to this marker than central, spindle cells. (1,16)

Consistent with the dysregulated cellular proliferation that is characteristic of this disorder, prior investigators (16,27) also have demonstrated that LAM cells express proteins involved in cellular propagation at higher levels than surrounding normal lung tissue. Specifically, PCNA is extensively expressed, particularly in spindle cells. Interestingly, an inverse relationship has been suggested between the proliferative state (demonstrated by PCNA in spindle cells) and HMB-45 positivity (found mainly in epithelioid cells). (16,27) Moreover, PCNA expression may well be associated with the active expansion of LAM cell populations within diseased tissue. While the present study was not designed to investigate a relationship between PCNA and TGF[[beta].sub.1], we observed substantially greater TGF[[beta].sub.1] and fibronectin expression in regions with strong PCNA reactivity. Given the proliferative implications of both findings, this seems to be a logical association. No direct conclusions can be drawn from our study regarding the comparative staining of HMB-45 vs PCNA, since our approach focused on overall LAM cell density rather than on specific cell morphology.

The basis of pathologic and clinical progression in LAM remains an enigma. Later clinical progression has been proposed as probably being more related to the development of cystic changes and airflow obstruction. However, Matsui et al (36) have shown that disease prognosis correlates both with the extent of cystic changes as well as with the extent of lung involvement by LAM cells. Thus, progression in this disease appears to be related to the proliferation of LAM cells and the eventual formation of cysts.

The lack of inflammatory, cell infiltration and the robust local expression of matrix metalloproteinases suggest that the LAM cells themselves contribute to their extracellular environment. (20,36) Matrix metalloproteinases break down elastin and may promote the eventual formation of cystic spaces. (36) Such tissue destruction may be modulated by local cellular proliferation and the concurrent formation of new ECM. Preliminary studies (37-39) also have proposed that increased expression of basic fibroblast growth factor and platelet-derived growth factor are associated with LAM cell foci. More recent investigations (40) have noted alterations in insulin-like growth factors and nitric oxide production. However, the role of ECM in promoting LAM cell proliferation and disease progression requires additional investigations.

ECM-associated fibronectin exhibits diverse actions, ranging from roles in cellular adhesion and migration, to the regulation of cellular proliferation, differentiation, and communication. (41) Concordant with these actions, altered fibronectin levels have been demonstrated in various disease states, including interstitial lung diseases other than LAM. (30,42) In addition, ECM-associated fibronectin is strongly expressed in response to biologically active TGF[[beta].sub.1], making it a useful in situ marker of local TGF[[beta].sub.1] activity. (25) The assembly of fibronectin matrices in abnormal tissues further enhances the local proliferation of mesenchymal cells during disease progression.

Although disease progression in LAM is not generally accompanied by the deposition of dense fibrotic materials that are rich in collagens, our observations that robust TGF[[beta].sub.1]-associated and ECM-associated fibronectin expression are present in cellular, proliferative LAM-affected lung tissue and the coexpression of the cell proliferative marker PCNA lend support to their roles as contributing to the pathogenesis of LAM, through modulating LAM cell proliferation.

TGF[[beta].sub.1] contributes to the development of other diverse, proliferative lung diseases, including usual interstitial pneumonia, sarcoidosis, and pulmonary Langerhans cell histiocytosis. (22-25) While enhanced TGF[[beta].sub.1] expression is not by itself "specific" for any of these disorders, what is striking, and is of potential importance to the pathogenesis of LAM, is our observation that LAM tissues exhibit dramatic regional differences in TGF[[beta].sub.1] expression. Significantly, the marked expression of TGF[[beta].sub.1] was observed in proliferative LAM lesions characterized by numerous LAM cells, and a paucity of this growth factor was present in more chronic cystic changes. These findings may suggest a novel therapeutic approach with which to pharmacologically treat this disease, perhaps at an earlier, more amenable stage.

A precise mechanism for TGF[[beta].sub.1] effects in LAM is not postulated herein, owing to its pluripotent actions. TGF[[beta].sub.1] induces proliferation among many mesenchymal cell types, which the authors postulate also may occur in LAM cells, given their apparent mesenchymal origin. However, in the absence of acceptable cell line or animal models, we cannot fully exclude that TGF[[beta].sub.1] allows permissive LAM cell hyperplasia by inhibiting the growth of other cell types. Similarly, though suggestive, the striking expression of cellular LAM-associated fibronectin cannot be conclusively determined to be due only to the effects of TGF[[beta].sub.1] alone using such an immunohistochemistry approach, as other factors also may contribute to ECM deposition.

Currently, the treatment options for LAM are disappointingly few and of limited efficacy. Hormonal manipulations have not consistently resulted in clinical improvement, and the options for those with advanced progressive disease may be limited to lung transplantation. New therapeutic approaches are clearly needed. The development of new treatment approaches has been hampered by the limited quantities of tissues, few cellular systems, and the absence of appropriate animal models that are typical of LAM. Under these constraints, descriptive analyses of human tissue, such as this, may provide important new hypotheses for investigation. Therapeutic agents with activity that is antagonistic to TGF[[beta].sub.1] currently are being developed. These agents include decorin, losartan, antibody-based strategies, cytokine-signaling antagonists, and, potentially, gene therapeutic approaches. (43,51) Further study of the role of TGF[[beta].sub.1] and ECM in LAM offers the potential for greater understanding and new management options for this devastating disease.

ACKNOWLEDGMENT: The authors thank the members of the Mayo Interstitial Lung Disease focus group for the identification and clinical management of these patients with LAM. The authors further appreciate the technical advice of Drs. Zvezdana Vuk-Pavlovic and Arthur Andrews in initiating the immunohistochemical assays.

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* From the Thoracic Diseases Research Unit (Drs. Evans, Ryu, and Limper), Division of Pulmonary & Critical Care. Department of Interned Medicine Mayo Clinic and Foundation. Rochester, MN; and the Department of Laboratory Medicine and Pathology (Dr. Colby), Mayo Clinic Scottsdale AZ.

This work was supported by funds from the Robert N. Brewer Family Foundation and by funds from the Mayo Foundation to Dr. Limper.

Manuscript received September 12, 2002; revision accepted July 18, 2003.

Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (e-mail: permissions@chestnet.org).

Correspondence to: Andrew H. Limper, MD, FCCP, 8-24 Stabile Building, Mayo Clinic, Rochester, MN, 55905; e-mail limper. andrew@mayo.edu

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