Am J Otolaryngol 12:s349.
1991
The Role of Tumor Necrosis Factor-Alpha in Bone Resorption of Cholesteatoma SHI-DU YAN, MD, AND CHENG-CHUN HUANG, PHD Recent investigations have indicated that cytokines such as tumor necrosis factor-alpha (TNF-a) play a potential role in the bone resorption associated with inflammatory diseases. In this immunoperoxidase study, TNF-a was localized in mononuclear cells, macrophages, fibroblasts, osteoblasts, and osteoclasts adjacent to bone resorption areas in both human and experimental middle ear cholesteatomas. In vitro, TNF-a stimulated monocytes to form multinucleated cells that demonstrate tartrate-resistant acid phosphatase activity, a marker enzyme for osteoclasts. These multinucleated osteoclast-like cells induce resorption of devitalized bone. The extent of bone resorption was increased by the co-cultures of osteoblasts and osteoclasts in the presence of TNF-a, suggesting that cell to cell interaction plays a significant role in bone resorption. Moreover, TNF-a was capable of stimulating macrophages to produce acid phosphatase and collagenase, and osteoblasts to produce prostaglandin E, and collagenase. These chemical mediators have been known to lead to bone resorption. Our findings suggest that TNF-a may play an important clinical role in the destructive process of cholesteatoma. AM J OTOLARYNGOL 12:83-89. Copyright 0 1991 by W.B. Saunders Company Key words: tumor necrosis factor-alpha, bone resorption, cholesteatoma.
enhance production of prostaglandin E, (PGE,) and collagenase by human synovial cells and derma1 fibroblasts,4 to increase the number of osteoclasts, and to stimulate bone resorption.5-7 In a previous study, we observed the localization of TNF-a in the epithelium of human middle ear cholesteatoma (unpublished data). This study demonstrates that TNF-(w is involved in the bone resorption process associated with middle ear cholesteatoma.
Bone resorption is an important feature of cholesteatoma. However, the exact mechanisms underlying it have been elusive. Recently, more attention has been paid to the role of cytokines, such as tumor necrosis factor (TNF), interleukin 1, and granulocyte-macrophage colony-stimulating factor (GM-CSF), in studies of bone resorption associated with inflammatory diseases. Cytokines are known to regulate both cell growth and cell differentiation Tumor necrosis factor-alpha (TNF-o) is one of the major inflammatory cytokines produced by activated macrophages.’ Recent investigations have indicated that it may be responsible for the tissue degeneration seen in acute and chronic infections.2’3 Tumor necrosis factor-alpha may play a significant role in bone resorption via several different mechanisms. It has been shown to
MATERIALS AND METHODS /mmunohistochemica/ Staining. Five experimental cholesteatomas and five human middle ear cholesteatoma specimens were studied. Human middle ear cholesteatoma specimens were taken from patients during ear surgery. Experimental cholesteatomas were induced in rats by instilling propylene glycol into the middle ear cavity.’ All specimens were fixed in 10% formalin, decalcified, embedded in paraffin, and sectioned to a 5-pm thickness. Tissue sections were stained by the indirect immunoperoxidase method using rabbit anti-human or anti-mouse TNF-ok immunoglobulin G (IgG). Briefly, the procedure of immunoperoxidase staining included the following incubation steps:
Received August 9, 1990. from the Department of Otolaryngology, College of Physicians and Surgeons, Columbia University, New York, NY. Accepted for publication January 15, 1991.
Supported by grants from the Deafness Research Foundation and the National Institute on Deafness and Other Communication Disorders (grant DC-00225). Address correspondence and reprint requests to ChengChun Huang, PhD, Department of Otolaryngology, Columbia University, 630 W 168th St, New York, NY 10032. Copyright 0 1991 by W.B. Saunders Company 0196-0709/91/1202-0005$5.00/O
83
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TNF-ALPHA IN BONE RESORPTION OF CHOLESTEATOMA
(a) 0.3% H,O,, 5 minutes: (b) 3% normal goat serum (Cappel, Cochranville, PA), 30 minutes; (c) rabbit anti-human or anti-mouse TNF-a IgG (Genzyme Boston, MA) diluted 1:40,30 minutes; (d) peroxidase goat anti-rabbit or anti-mouse (Cappel) diluted 1:400or l:lOO, 30 minutes; and (e) 0.03% H,O, to 0.6% &$diaminobenzidine-HCL in phosphate-buffered saline (PBS), 10 minutes. In each step, tissue sections were washed twice with PBS. The whole process was performed in a humidified chamber at room temperature. We completed three control studies. One study was performed by the absorption of rabbit antihuman or anti-mouse TNF-a IgG (step c) with excess antigen: recombinant human or mouse TNF-a (Genzyme) to determine specificity. Rabbit anti-human or anti-mouse TNF-a IgG antibody was preincubated for 2 hours at 37% with an excess of recombinant human or mouse TNF-a, and placed in refrigeration at 4“C overnight prior to application on sections. The other two studies involved substituting PBS, or nonimmune normal rabbit serum (1:4O), for primary antibody (step c) to determine nonspecific staining sites. The sections were then mounted with permount and observed under a microscope.
of adhering tissues. The marrow cavity was flushed with DMEM. The marrow cells were collected by centrifugation, and then cultured in DMEM with 10% calf serum at 5 X lo5 cells/well in four-chamber plates. Bone marrow mononuclear cells were cultured in the presence of recombinant mouse TNF-a (100 ng/mL; Genzyme) for 10 days. All cultures were maintained at 37°C in a humidified atmosphere of 5% CO, in air. After designated periods of time in culture, the cells were fixed with acetone and stained for tartrateresistant acid phosphatase (TRACP) using a commercially available kit (Sigma). The cells containing three or more nuclei were counted as multinucleated cells in 10 random fields at 200 X magnification.
Preparation of Osfeoblask Osteoblasts were prepared from the calvaria of neonatal rats.g Calvaria were dissected free of periosteum and associated soft tissues and subjected to four sequential 20-minute collagenase (2 mg/mL) (Sigma, St Louis, MO) digestions at 37°C. Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Grand Island, NY) with 10% calf serum. Alkaline phosphatase histochemistry revealed that the 95% cells were alkaline phosphatase positive. Alkaline phosphatase is a marker for the osteoblast. After reaching confluence, the cells were cultured by serum-free DMEM in the presence of TNF-a (2 to 100 ng/mL) for 72 hours. After 72 hours, the medium was harvested and the levels of collagenase and PGE, were determined. Preparation of Macrophages. Macrophages were prepared by the intraperitoneal injection of mineral oil into rats 2 days prior to death.*’ Macrophages were collected from rat peritoneal cavities. The cells (1 x 106)were then cultured in a 24-well cultureplate with serum-free DMEM in the presence of TNF-a (2 to 100 ng/mL). After a 96-hour incubation, the medium was harvested and the levels of collagenase and acid phosphatase activities were measured. Bone Marrow Cell Culture. The adult rats were killed by an overdose of pentobarbital. Femurs and tibiae were removed and dissected free
Bone Resorption Assay With Bone Slices. Rat bone marrow cell suspension (1 x 10~ cells/ml) was added to bone slices (ICN Biomedicals, Inc, Lisle, IL) in a well of 24-well plates. Cells were incubated with bone slices overnight, after which they were lifted out of the wells and incubated in fresh DMEM with 10% calf serum for 14 days in the presence of TNF-a (100 ng/mL). In some wells, osteoblasts (1 x lo5 cells/ml), together with bone marrow cells (1 x lo6 cells/ml), were added to bone slices as described above. For the controls, (1) cells were not added, but the slices were otherwise treated in the same manner, (2) bone marrow cells or calvaria osteoblasts were incubated without TNF-a, and (3) calvaria osteoblastmarrow cell co-cultures were incubated without TNF-a. At the end of the culture period, bone slices were fixed with 10% formalin, stained with hematoxylin-eosin, and examined under a light microscope. Assays of Acid Phosphatase, Collagenase, and Prostaglandln E2. Acid phosphatase activity in macrophages was determined using a commercially available kit (Sigma) after solubilization of cells with 0.5% Triton X-100. Collagenase activity was assayed by using i4Ccollagen as a substrate.” Prostaglandin E, was measured by radioimmunoassay using an antibody specific to PGE, (Sigma). RESULTS Histologic and lmmunohistochemical Studies. Human and experimental cholesteatoma tissues, which included the subepithelial granulation tissues with adjacent bone, demonstrated various degrees of inflammatory reaction characterized by infiltration of mononuclear cells (lymphocytes and macrophages) with capillary proliferation. The bony defects were filled with granulation tissues. The immunohistochemical study showed
85
YAN AND HUANG
tissue. The bone erosion area was filled with granulation tissue. Figure 1. Human middle ear cholesteatoma factor-alpha was localized in mononuclear cells, macrophages (M), and osteoblasts (Ob). B, Bone. (Magnification
that TNF-a was localized in granulation tissue and appeared in mononuclear cells, macrophages, fibroblasts, osteoclasts, and osteoblasts near bone surfaces (Figs I and 2). Osteoclasts were not found
Tumor necrosis X 330.)
in the human samples. This might be due to the relatively short life of the osteoclast or to the timing of the surgery, which is usually done after inflammation has been controlled.12
Figure 2. Rat middle ear cholesteatoma tissue. Tumor necrosis factor-alpha appeared to be in the granulation in mononuclear cells, macrophages (M), fibroblasts (F), and osteoclasts (0~). (Magnification x 330.)
tissue,
specifically
86
TNF-ALPHA
IN BONE RESORPTION
OF CHOLESTEATOMA
Figure 3. Control immunoperoxidase staining of TNF-a in human cholesteatoma tissue. No staining of TNF-a granulation tissue near bone resorption regions. GT, Granulation tissue; B, bone. [Magnification x 330.)
In the control study, no staining of TNF-a was seen in the subepithelial granulation tissue adjacent to the bone resorption area when the rabbit anti-human TNF-a or anti-mouse TNF-a antibody was absorbed with antigen, human TNF-a (Fig 3), or mouse TNF-a, or replaced by PBS or normal rabbit serum (data not shown). The fact that immunohistochemical staining can be blocked by the absorption of anti-human or anti-mouse TNF-a antibody with its antigen human or mouse TNF-a suggests that the reaction is indeed specific.13 Effect of Tumor Necrosis Factor-Alpha on Multinucleated Cell Formation. The addition of recombinant mouse TNF-a to bone marrow cultures significantly stimulated multinucleated cell formation (Table 1). These multinucleated cells demonstrated TRAGP activity (Fig 4), a marker for the osteoclast. Resorption on Devitalized Bovine Bone Slices. When marrow mononuclear cells were cultured with TNF-a for 14 days, resorption lacunae were formed on the surfaces of the bone slices (Fig 5, top left). Moreover, TNF-a markedly stimulated bone resorption in osteoblast-marrow cell co-
was seen in the
cultures. Some multinucleated cells could be seen on the resorption lacunae (Fig 5, top right). Neither bone marrow mononuclear cells, calvaria cells, nor a co-culture of both cell types (data not shown) without the addition of TNF-a showed any morphologic evidence of bone resorption (Fig 5, bottom). The absence of resorption was also noted in the bone slices with TNF-a alone without cells (data not shown). Effect of Tumor Necrosis Factor-Alpha on Collagenase, Prostaglandin E2, and Acid Phosphatase Production. The addition of recombinant TNF-a to the culture of calvaria osteoblasts stimulated both collagenase and PGE, production in a dose-related manner (Figs 6 and 7). In addition, TNF-a enhanced the production of collageTABLE
Effect of Tumor Necrosis Factor-Alpha 1. Multinucleated Cell Formation in Bone Marrow Culture ADDITION
MNC (%)
None TNF-a
1.08 +- 1.02 9.50 + 1.10
on
NOTE. Bone marrow mononuclear cells were cultured with TNF-a for 10 days. The extent of multinucleated cells (MNC) is expressed as the percentage mean f SD for 10 random fields (P < .05).
87
YANANDHUANG
Figure 4. Enzyme histochemistry for TRACP of bone marrow cells treated with TNF-a. Tartrat-resistant acid phosphatase-positive multinucleated cells (MNC) and TRACP-positive mononuclear cells [MC) were seen. [Magnification x 330.)
Figure 5. Micrographs of bovine bone slices. (Top left) Resorption lacunae (arrow) were formed on the surfaces of bone slices on which bone marrow cells were cultured with TNF-u for 14 days. (Magnification x 132.) (Top right] The extent of resorption (arrow) was significantly increased in bone slices in which bone marrow cells plus osteoblasts were cultured in the presence of TNF-a. (Magnification x 132.) (Bottom) The absence of resorption lacunae was noted on the control bone slice with bone marrow cells but without TNF-c(. MC, Mononuclear cells. (Magnification x 132.)
88
TNF-ALPHA
nase (Fig 8) and acid phosphatase by macrophages (Table 2).
=
IN BONE RESORPTION
OF CHOLESTEATOMA
800 5 3 a600 _a
DISCUSSION Although the causative factors of bone erosion in cholesteatoma have not been completely elucidated, a local inflammatory reaction seems to play a major role. This study showed the presence of TNF-a in subepithelial granulation tissue in the vicinity of bone erosion areas, suggesting that TNF-a was involved in the disease process of cholesteatoma. According to our in vitro study, TNF-a stimulated the formation of the multinucleated osteoclast-like cells derived from bone marrow cells. Evidence indicates that osteoclasts are formed by the fusion of mononuclear precursors derived from hematopoietic progenitor cells.14p15 These multinucleated osteoclasts are the major cells responsible for bone resorption in cholesteatomal’ and exhibit typical morphologic features of osteoclasts, including TEACP, a marker enzyme for osteoclasts.‘” Tumor necrosis factor-alpha may increase multinucleated cell formation through the direct stimulation of multinucleated cell precursor proliferation and fusion, or through the indirect stimulation of GM-CSF production,17 which, in turn, stimulates multinucleated cell formation.” Tumor necrosis factor-alpha induced bone resorption when bone marrow cells were cultured on bone slices. However, the extent of bone resorption was significantly increased by the addition of osteoblasts to bone slices in the presence of osteoclasts. Without the addition of TNF-cq resorption was not seen, even in the presence of both osteoblasts and marrow cells. This suggests that TNF-cx enhances osteoclastic bone resorption through a primary effect on osteoblasts, which
t-l
p
oLn_LL 0
2
TW-alpha
20
100
(ng/ml)
Figure 7. Effect of TNF-a on PGE, production by osteoblasts. Tumor necrosis factor-alpha stimulated PGE, production by osteoblasts. Values are means of four wells of cultures f SD (P < .05).
produces a factor that stimulates osteoclastic resorption.*Q-21 Our study and others have shown that TNF-a stimulated osteoblasts to produce PGE,.” However, its action of osteoclastic bone resorption appears not to be completely dependent on prostaglandins.6s’Q Increased release of CSF by osteoblasts in response to TNF-cx may be a mechanism whereby the osteoblasts could influence osteoclast recruitment, thus enhancing bone resorption.20 In vitro, TNF-(w stimulates osteoblasts to produce collagenase that exposes the mineral of the bone surface to osteoclastic resorptionzl This might be the other mechanism in which osteoblasts are involved in osteoclastic bone resorption. These findings further suggest that cell to cell interaction between osteoblasts and osteoclasts plays an important role in bone resorption. Bone resorption includes demineralization and dissolution of the organic component of bone. It has been reported that chemical mediators (for example, collagenase, PGE,, acid phosphatase, and neutral proteases) are needed to decalcify bone 10 r
L
20
TW-alpha
kg/ml)
Figure 6. Effect of TNF-a on collagenase production by osteoblasts. Tumor necrosis factor-alpha enhanced production of collagenase. Values are means of four wells of cultures + SD (P < .05).
TNF-alpha
(ng/ml)
Figure 8. Effect of TNF-u on collagenase production by macrophages. Tumor necrosis factor-alpha stimulated collagenase production in a dose-related manner (P < .05).
89
YANANDHUANG TABLE
Tumor Necrosis Factor-Alpha-Stimulated 2. Acid Phosphatase Activity in Macrophage Cultures ADDITION
NMOLtWELL
None TNF-o 2 ngimL 20 ng/mL 100 ng/mL
11.8 * 0.9 22.0 2 0.9 22.0 + 0.9 19.0 * 1.0
NOTE. Mouse peritoneal macrophages were cultured with TNF-a for 4 days. Data are the mean 2 SD of four culture wells (P < .Ol).
and to dissolve bone protein. In this study, TNF-(w was found to be localized in mononuclear cells, fibroblasts, macrophages, osteoblasts, and osteoclasts adjacent to bone resorption areas. These cells have been implicated as active agents of bone resorption by releasing the extracellular agents mentioned above.22-25 Based on our in vitro studies, TNF-cx may also affect bone resorption indirectly through the release of bone-resorbing agents such as prostaglandin, collagenase, and acid phosphatase. In conclusion, this study demonstrates that TNF-a is present in granulation tissue adjacent to bone resorption areas in cholesteatoma. Tumor necrosis factor-alpha induces bone erosion directly by stimulating osteoclast formation through the fusion of osteoclast precursors, and indirectly by releasing PGE,, collagenase, and acid phosphatase. Our results provide evidence that TNF-CY is an important mediator in the bone destruction associated with cholesteatoma. Acknowledgment. The authors thank Drs Maxwell Abramson and Jack Wazen for providing the specimens for this study, and Drs Robert Parks and Meredith FernStrom for their review and criticism of this manuscript. References 1. Carswell EA, Old LJ, Kassel RL, et al: An endotoxininduced serum factor that causes necrosis of tumor. Proc Nat1 Acad Sci USA 1975; 72:3666-3670 2. Old LJ: Tumor necrosis factor (TNF). Science 1985; 230:630-632 3. Clark IA, Cowden WB, Butcher GA, et al: Possible roles of tumor necrosis factor in the pathology of malaria. Am J Path01 1987; 129:192-199 4. Dayer JM. Beutler B, Cerami A: Cachectinltumor necrosis factor stimulates collagenase and prostaglandin E, production by human synovial cells and dermal fibroblasts. J Exp Med 1985; 162:2163-2168 5. Johnson RA, Boyce BF, Mundy GR, et al: Tumor produc-
ing human tumor necrosis factor induce hypercalcemia and osteoclastic bone resorption in nude mice. Endocrinology 1989; 124:1424-1427 6. Pfeilschifter J, Chenu C, Bird A, et al: Interleukin-1 and tumor necrosis factors stimulate the formation of human osteoclastlike cells in vitro. J Bone Miner Res 1989; 4:113-118 7. Bertolini DR, Nedwin GE, Bringman TS, et al: Stimulation of bone resorption and inhibition of bone formation in vitro by human tumor necrosis factor. Nature 1986; 319:516518 8. Huang CC, Shi GS, Yi ZX: Experimental induction of middle ear cholesteatoma in rats. Am J Otolaryngol 1988; 9:165-172 9. Tatakis DN, Schneeberger G, Dziak R: Recombinant interleukin-1 stimulates prostaglandin E, production by osteoblastic cells: Synergy with parathyroid hormone. Calcif Tissue Int 1988; 42:358-362 10. Wahl LM, Wahl SM, Mergenhagen SE, et al: Collagenase production by endotoxin-activated macrophages. Proc Nat1 Acad Sci USA 1974; 71:3598-3601 11. Terato K, Nagai Y, Kawanishi K, et al: A rapid assay method of collagenase activity using %-labelled soluble collagen as substrate. Biochim Biophys Acta 1976; 445:753-762 12. Chole RA: Osteoclasts in chronic otitis media, cholesteatoma and otosclerosis. Ann Otol Rhino1 Laryngol 1988; 97:661-666 13. Petrusz P, Sar M, Ordronneau P, et al: Specificity in immunocytochemical staining. J Histochem Cytochem 1976; 24:1110-1112 14. Mundy GR, Roodman GD: Ontogeny and function of the osteoclast. Bone Miner Res 1987; 5:209-281 15. Chambers TJ: The origin of the osteoclast. Bone Miner Res 1989; 6:1-25 16. Minkin C: Bone acid phosphatase: Tartrate-resistant acid phosphatase as a marker of osteoclast function. Calcif Tissue Int 1982; 34:285-290 17. Munker R, Gasson J, Ogawa M, et al: Recombinant human TNF induces production of granulocvte-monocvte col_ ony-stimulating factor. Nature 1986; 323:79-82 18. MacDonald BR. Mundv GR. Clark S. et al: Effects of human recombinant CSF-GM and highly purified CSF-1 on the formation of multinucleated cells with osteoclast characteristics in long-term bone marrow cultures. J Bone Miner Res 1986; 1:227-233 19. Thomson BM, Mundy GR, Chambers TJ: Tumor necrosis factors a and p induce osteoblastic cells to stimulate osteoclastic bone resorption. J Immunol 1987; 138:775-779 20. Felix R, Fleisch H, Elford PR: Bone-resorbing cytokines enhance release of macrophage colony-stimulating activity by the osteoblastic cell MC3T3-El. Calcif Tissue Int 1989; 44:356360 21. Kahn AJ, Partridge NC: New concepts in bone remodeling, an expanding role for the osteoblasi. Am J Otolaryngol 1987; 8:258-264 22. Tashjian AH, Voelkel EF, Lazzaro M, et al: Tumor necrosis factor-a (cachectin) stimulates bone resorption in mouse calvaria via a prostaglandin-mediated mechanism. Endocrinology 1987; 120:2029-2036 23. Moriyama H, Huang CC, Abramson M: Cell cooperation on bone resorption in chronic otitis media. Arch Otorhinolaryngol 1984; 241:89-93 24. Abramson M, Huang CC: Localization of collagenase in middle ear cholesteatoma. Laryngoscope 1977; 87:771-791 25. Robinson DR, Tashjian AH, Levine L: Prostaglandin stimulated bone resorption by rheumatoid synovia. A possible mechanism for bone destruction in rheumatoid arthritis. J Clin Invest 1975; 56:1181-1188
1991
The Role of Tumor Necrosis Factor-Alpha in Bone Resorption of Cholesteatoma SHI-DU YAN, MD, AND CHENG-CHUN HUANG, PHD Recent investigations have indicated that cytokines such as tumor necrosis factor-alpha (TNF-a) play a potential role in the bone resorption associated with inflammatory diseases. In this immunoperoxidase study, TNF-a was localized in mononuclear cells, macrophages, fibroblasts, osteoblasts, and osteoclasts adjacent to bone resorption areas in both human and experimental middle ear cholesteatomas. In vitro, TNF-a stimulated monocytes to form multinucleated cells that demonstrate tartrate-resistant acid phosphatase activity, a marker enzyme for osteoclasts. These multinucleated osteoclast-like cells induce resorption of devitalized bone. The extent of bone resorption was increased by the co-cultures of osteoblasts and osteoclasts in the presence of TNF-a, suggesting that cell to cell interaction plays a significant role in bone resorption. Moreover, TNF-a was capable of stimulating macrophages to produce acid phosphatase and collagenase, and osteoblasts to produce prostaglandin E, and collagenase. These chemical mediators have been known to lead to bone resorption. Our findings suggest that TNF-a may play an important clinical role in the destructive process of cholesteatoma. AM J OTOLARYNGOL 12:83-89. Copyright 0 1991 by W.B. Saunders Company Key words: tumor necrosis factor-alpha, bone resorption, cholesteatoma.
enhance production of prostaglandin E, (PGE,) and collagenase by human synovial cells and derma1 fibroblasts,4 to increase the number of osteoclasts, and to stimulate bone resorption.5-7 In a previous study, we observed the localization of TNF-a in the epithelium of human middle ear cholesteatoma (unpublished data). This study demonstrates that TNF-(w is involved in the bone resorption process associated with middle ear cholesteatoma.
Bone resorption is an important feature of cholesteatoma. However, the exact mechanisms underlying it have been elusive. Recently, more attention has been paid to the role of cytokines, such as tumor necrosis factor (TNF), interleukin 1, and granulocyte-macrophage colony-stimulating factor (GM-CSF), in studies of bone resorption associated with inflammatory diseases. Cytokines are known to regulate both cell growth and cell differentiation Tumor necrosis factor-alpha (TNF-o) is one of the major inflammatory cytokines produced by activated macrophages.’ Recent investigations have indicated that it may be responsible for the tissue degeneration seen in acute and chronic infections.2’3 Tumor necrosis factor-alpha may play a significant role in bone resorption via several different mechanisms. It has been shown to
MATERIALS AND METHODS /mmunohistochemica/ Staining. Five experimental cholesteatomas and five human middle ear cholesteatoma specimens were studied. Human middle ear cholesteatoma specimens were taken from patients during ear surgery. Experimental cholesteatomas were induced in rats by instilling propylene glycol into the middle ear cavity.’ All specimens were fixed in 10% formalin, decalcified, embedded in paraffin, and sectioned to a 5-pm thickness. Tissue sections were stained by the indirect immunoperoxidase method using rabbit anti-human or anti-mouse TNF-ok immunoglobulin G (IgG). Briefly, the procedure of immunoperoxidase staining included the following incubation steps:
Received August 9, 1990. from the Department of Otolaryngology, College of Physicians and Surgeons, Columbia University, New York, NY. Accepted for publication January 15, 1991.
Supported by grants from the Deafness Research Foundation and the National Institute on Deafness and Other Communication Disorders (grant DC-00225). Address correspondence and reprint requests to ChengChun Huang, PhD, Department of Otolaryngology, Columbia University, 630 W 168th St, New York, NY 10032. Copyright 0 1991 by W.B. Saunders Company 0196-0709/91/1202-0005$5.00/O
83
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TNF-ALPHA IN BONE RESORPTION OF CHOLESTEATOMA
(a) 0.3% H,O,, 5 minutes: (b) 3% normal goat serum (Cappel, Cochranville, PA), 30 minutes; (c) rabbit anti-human or anti-mouse TNF-a IgG (Genzyme Boston, MA) diluted 1:40,30 minutes; (d) peroxidase goat anti-rabbit or anti-mouse (Cappel) diluted 1:400or l:lOO, 30 minutes; and (e) 0.03% H,O, to 0.6% &$diaminobenzidine-HCL in phosphate-buffered saline (PBS), 10 minutes. In each step, tissue sections were washed twice with PBS. The whole process was performed in a humidified chamber at room temperature. We completed three control studies. One study was performed by the absorption of rabbit antihuman or anti-mouse TNF-a IgG (step c) with excess antigen: recombinant human or mouse TNF-a (Genzyme) to determine specificity. Rabbit anti-human or anti-mouse TNF-a IgG antibody was preincubated for 2 hours at 37% with an excess of recombinant human or mouse TNF-a, and placed in refrigeration at 4“C overnight prior to application on sections. The other two studies involved substituting PBS, or nonimmune normal rabbit serum (1:4O), for primary antibody (step c) to determine nonspecific staining sites. The sections were then mounted with permount and observed under a microscope.
of adhering tissues. The marrow cavity was flushed with DMEM. The marrow cells were collected by centrifugation, and then cultured in DMEM with 10% calf serum at 5 X lo5 cells/well in four-chamber plates. Bone marrow mononuclear cells were cultured in the presence of recombinant mouse TNF-a (100 ng/mL; Genzyme) for 10 days. All cultures were maintained at 37°C in a humidified atmosphere of 5% CO, in air. After designated periods of time in culture, the cells were fixed with acetone and stained for tartrateresistant acid phosphatase (TRACP) using a commercially available kit (Sigma). The cells containing three or more nuclei were counted as multinucleated cells in 10 random fields at 200 X magnification.
Preparation of Osfeoblask Osteoblasts were prepared from the calvaria of neonatal rats.g Calvaria were dissected free of periosteum and associated soft tissues and subjected to four sequential 20-minute collagenase (2 mg/mL) (Sigma, St Louis, MO) digestions at 37°C. Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Grand Island, NY) with 10% calf serum. Alkaline phosphatase histochemistry revealed that the 95% cells were alkaline phosphatase positive. Alkaline phosphatase is a marker for the osteoblast. After reaching confluence, the cells were cultured by serum-free DMEM in the presence of TNF-a (2 to 100 ng/mL) for 72 hours. After 72 hours, the medium was harvested and the levels of collagenase and PGE, were determined. Preparation of Macrophages. Macrophages were prepared by the intraperitoneal injection of mineral oil into rats 2 days prior to death.*’ Macrophages were collected from rat peritoneal cavities. The cells (1 x 106)were then cultured in a 24-well cultureplate with serum-free DMEM in the presence of TNF-a (2 to 100 ng/mL). After a 96-hour incubation, the medium was harvested and the levels of collagenase and acid phosphatase activities were measured. Bone Marrow Cell Culture. The adult rats were killed by an overdose of pentobarbital. Femurs and tibiae were removed and dissected free
Bone Resorption Assay With Bone Slices. Rat bone marrow cell suspension (1 x 10~ cells/ml) was added to bone slices (ICN Biomedicals, Inc, Lisle, IL) in a well of 24-well plates. Cells were incubated with bone slices overnight, after which they were lifted out of the wells and incubated in fresh DMEM with 10% calf serum for 14 days in the presence of TNF-a (100 ng/mL). In some wells, osteoblasts (1 x lo5 cells/ml), together with bone marrow cells (1 x lo6 cells/ml), were added to bone slices as described above. For the controls, (1) cells were not added, but the slices were otherwise treated in the same manner, (2) bone marrow cells or calvaria osteoblasts were incubated without TNF-a, and (3) calvaria osteoblastmarrow cell co-cultures were incubated without TNF-a. At the end of the culture period, bone slices were fixed with 10% formalin, stained with hematoxylin-eosin, and examined under a light microscope. Assays of Acid Phosphatase, Collagenase, and Prostaglandln E2. Acid phosphatase activity in macrophages was determined using a commercially available kit (Sigma) after solubilization of cells with 0.5% Triton X-100. Collagenase activity was assayed by using i4Ccollagen as a substrate.” Prostaglandin E, was measured by radioimmunoassay using an antibody specific to PGE, (Sigma). RESULTS Histologic and lmmunohistochemical Studies. Human and experimental cholesteatoma tissues, which included the subepithelial granulation tissues with adjacent bone, demonstrated various degrees of inflammatory reaction characterized by infiltration of mononuclear cells (lymphocytes and macrophages) with capillary proliferation. The bony defects were filled with granulation tissues. The immunohistochemical study showed
85
YAN AND HUANG
tissue. The bone erosion area was filled with granulation tissue. Figure 1. Human middle ear cholesteatoma factor-alpha was localized in mononuclear cells, macrophages (M), and osteoblasts (Ob). B, Bone. (Magnification
that TNF-a was localized in granulation tissue and appeared in mononuclear cells, macrophages, fibroblasts, osteoclasts, and osteoblasts near bone surfaces (Figs I and 2). Osteoclasts were not found
Tumor necrosis X 330.)
in the human samples. This might be due to the relatively short life of the osteoclast or to the timing of the surgery, which is usually done after inflammation has been controlled.12
Figure 2. Rat middle ear cholesteatoma tissue. Tumor necrosis factor-alpha appeared to be in the granulation in mononuclear cells, macrophages (M), fibroblasts (F), and osteoclasts (0~). (Magnification x 330.)
tissue,
specifically
86
TNF-ALPHA
IN BONE RESORPTION
OF CHOLESTEATOMA
Figure 3. Control immunoperoxidase staining of TNF-a in human cholesteatoma tissue. No staining of TNF-a granulation tissue near bone resorption regions. GT, Granulation tissue; B, bone. [Magnification x 330.)
In the control study, no staining of TNF-a was seen in the subepithelial granulation tissue adjacent to the bone resorption area when the rabbit anti-human TNF-a or anti-mouse TNF-a antibody was absorbed with antigen, human TNF-a (Fig 3), or mouse TNF-a, or replaced by PBS or normal rabbit serum (data not shown). The fact that immunohistochemical staining can be blocked by the absorption of anti-human or anti-mouse TNF-a antibody with its antigen human or mouse TNF-a suggests that the reaction is indeed specific.13 Effect of Tumor Necrosis Factor-Alpha on Multinucleated Cell Formation. The addition of recombinant mouse TNF-a to bone marrow cultures significantly stimulated multinucleated cell formation (Table 1). These multinucleated cells demonstrated TRAGP activity (Fig 4), a marker for the osteoclast. Resorption on Devitalized Bovine Bone Slices. When marrow mononuclear cells were cultured with TNF-a for 14 days, resorption lacunae were formed on the surfaces of the bone slices (Fig 5, top left). Moreover, TNF-a markedly stimulated bone resorption in osteoblast-marrow cell co-
was seen in the
cultures. Some multinucleated cells could be seen on the resorption lacunae (Fig 5, top right). Neither bone marrow mononuclear cells, calvaria cells, nor a co-culture of both cell types (data not shown) without the addition of TNF-a showed any morphologic evidence of bone resorption (Fig 5, bottom). The absence of resorption was also noted in the bone slices with TNF-a alone without cells (data not shown). Effect of Tumor Necrosis Factor-Alpha on Collagenase, Prostaglandin E2, and Acid Phosphatase Production. The addition of recombinant TNF-a to the culture of calvaria osteoblasts stimulated both collagenase and PGE, production in a dose-related manner (Figs 6 and 7). In addition, TNF-a enhanced the production of collageTABLE
Effect of Tumor Necrosis Factor-Alpha 1. Multinucleated Cell Formation in Bone Marrow Culture ADDITION
MNC (%)
None TNF-a
1.08 +- 1.02 9.50 + 1.10
on
NOTE. Bone marrow mononuclear cells were cultured with TNF-a for 10 days. The extent of multinucleated cells (MNC) is expressed as the percentage mean f SD for 10 random fields (P < .05).
87
YANANDHUANG
Figure 4. Enzyme histochemistry for TRACP of bone marrow cells treated with TNF-a. Tartrat-resistant acid phosphatase-positive multinucleated cells (MNC) and TRACP-positive mononuclear cells [MC) were seen. [Magnification x 330.)
Figure 5. Micrographs of bovine bone slices. (Top left) Resorption lacunae (arrow) were formed on the surfaces of bone slices on which bone marrow cells were cultured with TNF-u for 14 days. (Magnification x 132.) (Top right] The extent of resorption (arrow) was significantly increased in bone slices in which bone marrow cells plus osteoblasts were cultured in the presence of TNF-a. (Magnification x 132.) (Bottom) The absence of resorption lacunae was noted on the control bone slice with bone marrow cells but without TNF-c(. MC, Mononuclear cells. (Magnification x 132.)
88
TNF-ALPHA
nase (Fig 8) and acid phosphatase by macrophages (Table 2).
=
IN BONE RESORPTION
OF CHOLESTEATOMA
800 5 3 a600 _a
DISCUSSION Although the causative factors of bone erosion in cholesteatoma have not been completely elucidated, a local inflammatory reaction seems to play a major role. This study showed the presence of TNF-a in subepithelial granulation tissue in the vicinity of bone erosion areas, suggesting that TNF-a was involved in the disease process of cholesteatoma. According to our in vitro study, TNF-a stimulated the formation of the multinucleated osteoclast-like cells derived from bone marrow cells. Evidence indicates that osteoclasts are formed by the fusion of mononuclear precursors derived from hematopoietic progenitor cells.14p15 These multinucleated osteoclasts are the major cells responsible for bone resorption in cholesteatomal’ and exhibit typical morphologic features of osteoclasts, including TEACP, a marker enzyme for osteoclasts.‘” Tumor necrosis factor-alpha may increase multinucleated cell formation through the direct stimulation of multinucleated cell precursor proliferation and fusion, or through the indirect stimulation of GM-CSF production,17 which, in turn, stimulates multinucleated cell formation.” Tumor necrosis factor-alpha induced bone resorption when bone marrow cells were cultured on bone slices. However, the extent of bone resorption was significantly increased by the addition of osteoblasts to bone slices in the presence of osteoclasts. Without the addition of TNF-cq resorption was not seen, even in the presence of both osteoblasts and marrow cells. This suggests that TNF-cx enhances osteoclastic bone resorption through a primary effect on osteoblasts, which
t-l
p
oLn_LL 0
2
TW-alpha
20
100
(ng/ml)
Figure 7. Effect of TNF-a on PGE, production by osteoblasts. Tumor necrosis factor-alpha stimulated PGE, production by osteoblasts. Values are means of four wells of cultures f SD (P < .05).
produces a factor that stimulates osteoclastic resorption.*Q-21 Our study and others have shown that TNF-a stimulated osteoblasts to produce PGE,.” However, its action of osteoclastic bone resorption appears not to be completely dependent on prostaglandins.6s’Q Increased release of CSF by osteoblasts in response to TNF-cx may be a mechanism whereby the osteoblasts could influence osteoclast recruitment, thus enhancing bone resorption.20 In vitro, TNF-(w stimulates osteoblasts to produce collagenase that exposes the mineral of the bone surface to osteoclastic resorptionzl This might be the other mechanism in which osteoblasts are involved in osteoclastic bone resorption. These findings further suggest that cell to cell interaction between osteoblasts and osteoclasts plays an important role in bone resorption. Bone resorption includes demineralization and dissolution of the organic component of bone. It has been reported that chemical mediators (for example, collagenase, PGE,, acid phosphatase, and neutral proteases) are needed to decalcify bone 10 r
L
20
TW-alpha
kg/ml)
Figure 6. Effect of TNF-a on collagenase production by osteoblasts. Tumor necrosis factor-alpha enhanced production of collagenase. Values are means of four wells of cultures + SD (P < .05).
TNF-alpha
(ng/ml)
Figure 8. Effect of TNF-u on collagenase production by macrophages. Tumor necrosis factor-alpha stimulated collagenase production in a dose-related manner (P < .05).
89
YANANDHUANG TABLE
Tumor Necrosis Factor-Alpha-Stimulated 2. Acid Phosphatase Activity in Macrophage Cultures ADDITION
NMOLtWELL
None TNF-o 2 ngimL 20 ng/mL 100 ng/mL
11.8 * 0.9 22.0 2 0.9 22.0 + 0.9 19.0 * 1.0
NOTE. Mouse peritoneal macrophages were cultured with TNF-a for 4 days. Data are the mean 2 SD of four culture wells (P < .Ol).
and to dissolve bone protein. In this study, TNF-(w was found to be localized in mononuclear cells, fibroblasts, macrophages, osteoblasts, and osteoclasts adjacent to bone resorption areas. These cells have been implicated as active agents of bone resorption by releasing the extracellular agents mentioned above.22-25 Based on our in vitro studies, TNF-cx may also affect bone resorption indirectly through the release of bone-resorbing agents such as prostaglandin, collagenase, and acid phosphatase. In conclusion, this study demonstrates that TNF-a is present in granulation tissue adjacent to bone resorption areas in cholesteatoma. Tumor necrosis factor-alpha induces bone erosion directly by stimulating osteoclast formation through the fusion of osteoclast precursors, and indirectly by releasing PGE,, collagenase, and acid phosphatase. Our results provide evidence that TNF-CY is an important mediator in the bone destruction associated with cholesteatoma. Acknowledgment. The authors thank Drs Maxwell Abramson and Jack Wazen for providing the specimens for this study, and Drs Robert Parks and Meredith FernStrom for their review and criticism of this manuscript. References 1. Carswell EA, Old LJ, Kassel RL, et al: An endotoxininduced serum factor that causes necrosis of tumor. Proc Nat1 Acad Sci USA 1975; 72:3666-3670 2. Old LJ: Tumor necrosis factor (TNF). Science 1985; 230:630-632 3. Clark IA, Cowden WB, Butcher GA, et al: Possible roles of tumor necrosis factor in the pathology of malaria. Am J Path01 1987; 129:192-199 4. Dayer JM. Beutler B, Cerami A: Cachectinltumor necrosis factor stimulates collagenase and prostaglandin E, production by human synovial cells and dermal fibroblasts. J Exp Med 1985; 162:2163-2168 5. Johnson RA, Boyce BF, Mundy GR, et al: Tumor produc-
ing human tumor necrosis factor induce hypercalcemia and osteoclastic bone resorption in nude mice. Endocrinology 1989; 124:1424-1427 6. Pfeilschifter J, Chenu C, Bird A, et al: Interleukin-1 and tumor necrosis factors stimulate the formation of human osteoclastlike cells in vitro. J Bone Miner Res 1989; 4:113-118 7. Bertolini DR, Nedwin GE, Bringman TS, et al: Stimulation of bone resorption and inhibition of bone formation in vitro by human tumor necrosis factor. Nature 1986; 319:516518 8. Huang CC, Shi GS, Yi ZX: Experimental induction of middle ear cholesteatoma in rats. Am J Otolaryngol 1988; 9:165-172 9. Tatakis DN, Schneeberger G, Dziak R: Recombinant interleukin-1 stimulates prostaglandin E, production by osteoblastic cells: Synergy with parathyroid hormone. Calcif Tissue Int 1988; 42:358-362 10. Wahl LM, Wahl SM, Mergenhagen SE, et al: Collagenase production by endotoxin-activated macrophages. Proc Nat1 Acad Sci USA 1974; 71:3598-3601 11. Terato K, Nagai Y, Kawanishi K, et al: A rapid assay method of collagenase activity using %-labelled soluble collagen as substrate. Biochim Biophys Acta 1976; 445:753-762 12. Chole RA: Osteoclasts in chronic otitis media, cholesteatoma and otosclerosis. Ann Otol Rhino1 Laryngol 1988; 97:661-666 13. Petrusz P, Sar M, Ordronneau P, et al: Specificity in immunocytochemical staining. J Histochem Cytochem 1976; 24:1110-1112 14. Mundy GR, Roodman GD: Ontogeny and function of the osteoclast. Bone Miner Res 1987; 5:209-281 15. Chambers TJ: The origin of the osteoclast. Bone Miner Res 1989; 6:1-25 16. Minkin C: Bone acid phosphatase: Tartrate-resistant acid phosphatase as a marker of osteoclast function. Calcif Tissue Int 1982; 34:285-290 17. Munker R, Gasson J, Ogawa M, et al: Recombinant human TNF induces production of granulocvte-monocvte col_ ony-stimulating factor. Nature 1986; 323:79-82 18. MacDonald BR. Mundv GR. Clark S. et al: Effects of human recombinant CSF-GM and highly purified CSF-1 on the formation of multinucleated cells with osteoclast characteristics in long-term bone marrow cultures. J Bone Miner Res 1986; 1:227-233 19. Thomson BM, Mundy GR, Chambers TJ: Tumor necrosis factors a and p induce osteoblastic cells to stimulate osteoclastic bone resorption. J Immunol 1987; 138:775-779 20. Felix R, Fleisch H, Elford PR: Bone-resorbing cytokines enhance release of macrophage colony-stimulating activity by the osteoblastic cell MC3T3-El. Calcif Tissue Int 1989; 44:356360 21. Kahn AJ, Partridge NC: New concepts in bone remodeling, an expanding role for the osteoblasi. Am J Otolaryngol 1987; 8:258-264 22. Tashjian AH, Voelkel EF, Lazzaro M, et al: Tumor necrosis factor-a (cachectin) stimulates bone resorption in mouse calvaria via a prostaglandin-mediated mechanism. Endocrinology 1987; 120:2029-2036 23. Moriyama H, Huang CC, Abramson M: Cell cooperation on bone resorption in chronic otitis media. Arch Otorhinolaryngol 1984; 241:89-93 24. Abramson M, Huang CC: Localization of collagenase in middle ear cholesteatoma. Laryngoscope 1977; 87:771-791 25. Robinson DR, Tashjian AH, Levine L: Prostaglandin stimulated bone resorption by rheumatoid synovia. A possible mechanism for bone destruction in rheumatoid arthritis. J Clin Invest 1975; 56:1181-1188
