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Angiotensin-Converting Enzyme, Transforming Growth Factor [[Beta].sub.1], and Interleukin 11 in the Osteolytic Lesions of Langerhans Cell Histiocytosis.

Osteolytic lesions are common in patients with either localized or multisystem involvement in Langerhans cell histiocytosis (LCH).[1] Although the skull is the more frequently affected site, virtually any bone in the body can be involved.[2] Relatively recent studies have shown that human melanoma and breast cancer cells can induce bone resorption in a murine calvaria culture system through both the direct elaboration of interleukin 11 (IL-11) and by enhancing its production via the activation of the latent transforming growth factor [Beta] (TGF-[Beta])[3,4]; therefore, it was hypothesized that these 2 cytokines may be involved in the pathogenesis of the osteolytic lesions in LCH. Moreover, in light of the evidence that angiotensin II directly stimulates the transcription and bioactivation of TGF-[Beta],[5] it was also considered that the angiotensin system may be part of this pathogenetic sequence. The purpose of this report is threefold: first, to document the expression of IL-11 and the latency-associated peptide (LAP) of TGF-[[Beta].sub.1] in osteolytic lesions from patients with LCH; second, to provide immunohistochemical evidence for the presence of the angiotensin system in such lesions; and third, to develop a possible pathogenetic sequence that incorporates these factors in osteoclastogenesis and osteolysis in LCH.


Study Population

Five patients with histologically confirmed LCH comprise the study population. These patients consist of 1 male and 4 females, ranging in age from 7 to 31 years. Radiographically, 3 had calvarial and 1 each had osteolytic lesions of the ulna and radius, respectively. All 5 patients presented with symptoms of localized bone disease. In 3 patients, the apparent monostotic nature of the process was established by either total body bone scan using technetium Tc 99m or a skeletal survey using conventional radiographs. Brightfield microscopy on representative hematoxylin-eosin-stained sections from the surgically obtained lesional tissue in all 5 cases coincided with the clinical presentation and radiographic findings in supporting a diagnosis of LCH. Additionally, transmission electron microscopy was performed in 2 cases and revealed the presence of Birbeck granules in the LCH histiocytes.


Representative sections of archival, paraffin-embedded material from each of the 5 patients were reacted in immunohistochemical procedures for the detection of the following antigens: [CD.sub.1a], S100 protein, IL-11, LAP of TGF-[[Beta].sub.1], and angiotensin-converting enzyme (ACE), respectively.

Mouse monoclonal antihuman [CD.sub.1a] (Immunotech, Westbrook, Me), antihuman S100 protein (BioGenex, San Ramon, Calif), and antihuman ACE (Accurate Chemical & Scientific Corp, Westbury, NY) antibodies are all commercially available for use in immunohistochemistry.

Mouse monoclonal antihuman IL-11 antibody (clone 22315.1, IgG2a,k, R&D Systems, Inc, Minneapolis, Minn) is reactive in direct enzyme-linked immunosorbent assay with recombinant (baculovirus/Sf1) human sequence IL-11. A neutralization (block-off) experiment was performed using a known immunoreactive tissue substrate from 1 of the test cases and this anti-IL-11 antibody, following incubation in a solution containing recombinant human IL-11 in excess. The recombinant human IL-11 effectively neutralized the anti-IL-11 antibody, thereby affirming its specificity in immunohistochemistry.

Goat polyclonal antibody reactive with LAP of human TGF- [[Beta].sub.1] (catalog No. AB-246-NA, R&D Systems) was used in this study. This antibody against LAP previously has been shown to react with the latent TGF-[[Beta].sub.1] in immunohistochemical applications.[6]

Briefly, the general immunohistochemical procedure involved antigen enhancement methods and detection systems as previously described.[7] All primary antibodies were used at a concentration of 20 [micro]g/mL or as per the manufacturer's recommendation. Sections were processed using the procedure described herein or in reaction minus the primary antibody and with nonimmune serum for the negative control. Positive controls were run for each of the aforementioned antibodies using tissues previously established to be immunoreactive for the antigen in question. Specifically, these include a skin biopsy specimen with a [CD.sub.1a] antigen-expressing LCH; a "sausage" control containing an S100 protein-expressing melanoma; placenta and decidua basalis with IL-11 antigen-positive intermediate trophoblasts and decidual cells; tonsillar tissue containing LAP antigen-expressing squamous epithelium and endothelial cells; and a "sausage" control with endothelial cells immunoreactive for ACE antigen. Double immunostaining for [CD.sub.1a] and IL-11 antigens was performed on a representative case, also as previously described.[7]


A reading of representative hematoxylin-eosin-stained sections revealed the following features common to all cases: (1) the presence of variable numbers of multinucleated osteoclast-like giant cells within the lesion; (2) a companion population of mononuclear histiocytes, some with deep nuclear clefts or grooves, the karyomorphism of Langerhans cells; and (3) a component of neutrophilic and eosinophilic granulocytes in varying proportions.

Immunoreactivity for S100 protein or [CD.sub.1a] clearly identified LCH histiocytes in proximity to the osteoclast-like giant cells. In addition, IL-11 antigen was detected in the cytoplasm of LCH-type histiocytes in all cases, and double immunostaining revealed IL-11 and [CD.sub.1a] coexpression in such cells. Occasional endothelial cells were also positive for IL-11 antigen. A similar pattern of immunoreactivity was noted for LAP of TGF-[[Beta].sub.1], namely cytoplasmic positivity in Langerhans-type histiocytes and occasional endothelial cells. In contrast, ACE antigen was detected in a plasmalemmal distribution in all cases, was most prominent in histiocytes that lacked the karyomorphism of Langerhans cells, and was occasionally noted on osteoclast-like giant cells and endothelial cells.

All these observations and immunoreactivities, except for the double immunostaining, are illustrated in Figures 1 and 2.



The detection in this study of IL-11 antigen in the bony lesions of LCH is consistent with the growing body of evidence implicating this cytokine in osteolysis.[3,4,8] Both endothelial cells and LCH histiocytes showed immunoreactivity. The association of IL-11 production in bone-derived endothelial cells and osteolytic lesions has been reported[9]; the demonstration of IL-11 antigen in LCH histiocytes has not been previously established, at least to my knowledge, from a review of the literature. Mechanistically, IL-11 is thought to effect bone resorption by promoting osteoclastogenesis through enhanced osteoclast differentiation.[4,8] The latter may be mediated by a direct stimulatory effect on progenitors or indirectly by increasing prostaglandin [E.sub.2] production.[4,10,11] The presence of variable numbers of multinucleated osteoclast-like giant cells, as a morphologic concomitant in these LCH lesions, supports such a pathogenic sequence. Additionally, IL-11 could be contributing to the bony lesions in LCH by stimulating osteoblast-mediated degradation of type I collagen in osteoid, thereby interfering in remodeling and repair.[8]

Similarly, the demonstration of LAP of TGF-[[Beta].sub.1] in lesional histiocytes and occasional endothelial cells in this series of cases is also consistent with osteolysis, given the role of TGF-[[Beta].sub.1] in the regulation of IL-11 synthesis. That is to say, activation of latent TGF-[[Beta].sub.1] could enhance IL-11 gene transcription and production of this protein, leading to bone resorption.[3,12,13] Parenthetically, TGF-[[Beta].sub.1] appears to be an essential factor for development of Langerhans cells[14] and, therefore, may contribute to the expanded population of LCH histiocytes in these osteolytic lesions.

One of the known bioactivators of TGF-[[Beta].sub.1] is angiotensin II.[5] Because of this established fact and in light of the relatively recent observation of Rosenstein and coworkers that documented an elevation of serum ACE in a patient with LCH and osteolytic mandibular and maxillary lesions,[15] the detection of ACE in this study was not surprising. Furthermore, the immunoreactivity for ACE antigen on non-Langerhans cell histiocytes, occasional osteoclast-like giant cells, and endothelial cells in this series of cases accords with earlier reports that collectively document its presence on monocytes, macrophages, and endothelial cells.[16,17]

Finally, other putative osteoclastogenic factors, such as IL-6[18] and parathyroid hormone,[19] could play a role in the osteolysis of LCH. However, because IL-6 does not appear to be present in LCH histiocytes, such a contribution would have to come from the accompanying reactive cells within the lesions.[20] Similarly, because parathyroid hormone levels have been shown to rise during clodronate-associated resolution of multifocal eosinophilic granuloma of bone,[21] it is unlikely that parathyroid hormone per se is responsible for the osteolytic lesions in LCH.

In summary, the findings in this study suggest a pathogenetic sequence for the osteolytic lesions in LCH that involves ACE-catalyzed angiotensin II formation, leading to the activation of latent TGF-[[Beta].sub.1] and, in turn, to the enhanced production of IL-11, resulting in osteoclastogenesis. Moreover, such a possible sequence offers novel and relatively safe therapeutic options for the treatment of the osteolytic lesions in LCH. These options include possible use of 1 or more of the following: (1) bisphosphonates, known antagonists of osteoclastic function[22]; (2) nonsteroidal anti-inflammatory agents (eg, indomethacin) to limit the level of IL-Il-induced prostaglandin [E.sub.2] production[4]; and (3) ACE inhibitors.[5]

I thank Glen Kauwell for his help with the immunohistochemical studies and Dr Harvey Gaylord for assisting in the neutralization study involving IL-11.


[1.] Dehner LP. Pediatric Surgical Pathology. Baltimore, Md: Williams & Wilkins; 1987:970.

[2.] Slater JM, Swarm OJ. Eosinophilic granuloma of bone. Med Pediatr Oncol. 1980;8:151-164.

[3.] Morinaga Y, Fujita N, Ohishi K, Tsuruo T. Stimulation of interleukin-11 production from osteoblast-like cells by transforming growth factor-beta and tumor cell factors. Int J Cancer. 1997;71:422-428.

[4.] Morinaga Y, Fujita N, Ohishi K, Zhang Y, Tsuruo T. Suppression of interleukin-11-mediated bone resorption by cyclooxygenase inhibitors. J Cell Physiol. 1998;175:247-254.

[5.] Wolf G. Link between angiotensin II and TGF-beta in the kidney. Miner Electrolyte Metab. 1998;24:174-180.

[6.] Ehrhart EJ, Segarini P, Tsang ML-S, Carroll AG, Barcellos-Hoff MH. Latent transforming growth factor beta 1 activation in situ: quantitative and functional evidence after low-dose gamma-irradiation. FASEB J. 1997;11:991-1002.

[7.] Brown RE. Histogenesis of Reed-Sternberg and dendritic interdigitating cells in nodular sclerosing Hodgkin's disease: immunohistochemical evidence for monocytoid precursors. Ann Clin Lab Sci. 1997;27:329-337.

[8.] Hill PA, Tumber A, Papaioannov S, Meikle MC. The cellular actions of interleukin-11 on bone resorption in vitro. Endocrinology. 1998;139:1564-1572.

[9.] Zhang Y, Fugita N, Oh-hara T, et al. Production of interleukin-11 in bone-derived endothelial cells and its role in the formation of osteolytic bone metastasis. Oncogene. 1998;16:693-703.

[10.] Galvin RJ, Bryan P, Horn JW, Rippy MK, Thomas JE. Development and characterization of a porcine model to study osteoclast differentiation and activity. Bone. 1996;19:271-279.

[11.] Arenzana-Seisdedos F, Barbey S, Virelizier IL, Kornprobst M, Nezelof C. Histiocytosis X purified ([T6.sup.+]) cells from bone granuloma produce interleukin 1 and prostaglandin [E.sub.2] in culture. J Clin Invest. 1986;77:326-329.

[12.] Elias JA, Tang W, Horowitz MC. Cytokine and hormonal stimulation of human osteosarcoma interleukin-11 production. Endocrinology. 1995;136:489-498.

[13.] Tang W, Yang L, Yang YC, Leng SX, Elias JA. Transforming growth factor-beta stimulates interleukin-11 transcription via complex activating protein-1-dependent pathways. J Biol Chem. 1998;273:5506-5513.

[14.] Borkowski TA, Letterio JJ, Farr AG, Udey MC. A role for endogenous transforming growth factor [beta.sub.1] in Langerhans cell biology: the skin of transforming growth factor beta 1 null mice is devoid of epidermal Langerhans cells. J Exp Med. 1996;184:2417-2422.

[15.] Rosenstein ED, Eskow RN, Lederman DA, Kramer N. Case report: Langerhans cell histiocytosis associated with elevation of angiotensin-converting enzyme levels. Am J Med Sci. 1995;310:65-67.

[16.] Vuk-Pavlovic Z, Kreofsky TJ, Rohrbach MS. Characteristics of monocyte angiotensin-converting enzyme (ACE) induced by dexamethasone. J leukocyte Biol. 1989;45:503-509.

[17.] Falkenhahn M, Franke F, Bohle RM, et al. Cellular distribution of angiotensin-converting enzyme after myocardial infarction. Hypertension. 1995;25: 219-226.

[18.] Kurihara N, Bertolini D, Suda T, Akiyama Y, Roodman GD. IL-6 stimulates osteoclast-like multinucleated cell formation in long term human marrow cultures by inducing IL-1 release, J Immunol. 1990;144:4226-4230.

[19.] Feldman RS, Krieger NS, Tashjian AH Jr. Effects of parathyroid hormone and calcitonin on osteoclast formation in vitro. Endocrinology. 1980;107:1137-1143.

[20.] Foss HD, Herbst H, Araujo I, et al. Monokine expression in Langerhans' cell histiocytosis and sinus histiocytosis with massive lymphadenopathy (Rosai-Dorfman disease) [see comments]. J Pathol 1996;179:60-65.

[21.] Elomaa I, Blomqvist C, Porkka L, Holmstrom T. Experiences of clodronate treatment of multifocal eosinophilic granuloma of bone. J Intern Med. 1989;225: 59-61.

[22.] Berenson JR, Lipton A. Pharmacology and clinical efficacy of bisphosphonates. Curr Opin Oncol. 1998;10:566-571.

Accepted for publication March 1, 2000.

From the Divisions of Laboratory Medicine and Pediatrics, Penn State Geisinger Health System, Danville, Pa, and Pennsylvania State University, College of Medicine, Hershey, Pa.

Reprints: Robert E. Brown, MD, Division of Laboratory Medicine, Geisinger Medical Center, Danville, PA 17822-0131 (e-mail:
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Author:Brown, Robert E.
Publication:Archives of Pathology & Laboratory Medicine
Geographic Code:1USA
Date:Sep 1, 2000
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