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Heat shock proteins in middle ear cholesteatoma
EXPERIMENTAL STUDY ILSA SCHWARTZ, PhD Experimental Study Editor
Heat shock proteins in middle ear cholesteatoma HIDEO SHINODA, MD, and CHENG-CHUN HUANG, PhO,New York, New York
Heat shock proteins [HSPs], particularly HSP60 and HSP70, may have integral roles in several aspects of the pathogenesis of acquired cholesteatoma. Cholesteatoma is usually associated with an inflammatory reaction occurring in the middle ear cavity, leading to proliferation, differentiation, and programmed cell death of keratinocytes. The presence of HSP60 and HSP70 in cholesteatoma was demonstrated by the immunobiotting assay with specific anti-HSP60 and anti-HSP70 antibodies after protein extraction. The distribution of HSP60 and HSP70 in the cholesteatoma tissues was studied immunohistochemically with paraffin-embedded sections. HSP60 and HSP70 appeared to be localized in the cytoplasm of keratinocytes in all layers of the epithelium of cholesteatoma. Moreover, HSP70 was found in the nucleus of keratinocytes. Both HSPswere not labeled in the epidermis of the external ear canal skin or normal facial skin, except for significant staining on keratinocytes of hair follicles in facial skin. High density of HSF'60 and HSP70 may be induced by the inflammatory reaction and immune responses in the middle ear cavity during the clinical development of cholesteatoma. Induction of HSP60 and HSP70 in cholesteatoma epithelium may also be related to hyperproliferation and active differentiation of basal keratinocytes, which cause the accumulation of keratin debris, a characteristic feature of cholesteatoma. In the nucleus of keratinocytes, HSP70 may act to stabilize p53 protein, which functions as a negative regulator of cellular proliferation and is crucial to apoptosis of keratinocytes. This study demonstrates HSPs in cholesteatoma and suggests important roles for HSPs in the clinical development of cholesteatoma. [OTOLARYNGOL HEAD NECK SURG 1996;t 14:77-83.]
C e l l u l a r expression of heat shock proteins (HSPs) was first recognized as a response to elevated temperatures.E Later, it was also found that production of HSPs can be induced by other environmental and pathophysiologic stresses. HSPs are named according to their apparent molecular weights and classified into families, for example, HSP60 and HSP70, with molecular weights of 60 and 70 kD. These proteins enable the cell to survive and recover from stressful conditions by as yet incompletely understood mechanisms. However, the role of the HSPs is not likely to be confined to cellular stress. Many of these proteins are also synthesized under normal From the Department of Otolaryngology-Headand Neck Surgery,Collegeof Physiciansand Surgeons, ColumbiaUniversity. Received for publication Nov. 10, 1994; revision received April 27, 1995; accepted May 5, 1995. Reprint requests: C-C. Huang, PhD, Department of Otolaryngology-Headand Neck Surgery,ColumbiaUniversity,630 West 168th St., New York, NY 10032. Copyright © 1996by the American Academyof OtolaryngologyHead and Neck Surgery Foundation, Inc. 0194-5998/96/$5,00 + 0 23/10/66124
conditions and fulfill many central functions in the normal cells. The HSP60 and HSP70 cognates are essential for the folding and unfolding, the assembly and disassembly, and the sorting and transaction of many other proteins. Accordingly, they have been termed chaperons. 2-5 DeNagel and Pierce ~ have shown that HSPs are the dominant antigens of a variety of pathogens and serve as agents in antigen processing and/or presentation. These findings revealed that in a wide variety of organisms, including human beings, HSPs play a critical role both in normal functions and in the response to stress/ Cholesteatoma is a chronic middle ear disease characterized by the accumulation of keratinizing epithelium resulting from proliferation, differentiation, and programmed cell death of epithelial cells. In response to inflammation in the middle ear cavity, keratinocytes in the epidermis of the ear canal skin migrate, proliferate, differentiate, and cornify to cause the accumulation of keratin debris to form a cholesteatoma. It is interesting to investigate the presence of HSPs in cholesteatoma during its development because HSPs may be involved in the 77
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inflammatory response; therefore it is important to understand the function of HSPs in the pathogenesis of cholesteatoma. This investigation focused on studying the presence and localization of HSP60 and HSP70 in cholesteatoma tissues. Of the HSPs known, HSP60 and HSP70 have been most associated with inflammatory and immune responses. METHODS AND MATERIAL Antibodies
Antibodies against HSPs were obtained from Stress Gen (Victoria, British Columbia, Canada). Polyclonal rabbit antibody (SPA-805) was used for studying HSP60 by immunoblotting assay and immunohistochemistry. This antibody recognizes HSP60 constitutively synthesized in some types of cells. This protein is also stress inducible. Both the constitutive (HSCT0) and the inducible (HSP70) types are highly homologous and can be recognized by either polyclonal or monoclonal anti-HSP70 (SPA-820). Tissues
Specimens were obtained from patients during middle ear operations. Specimens included 15 middle ear cholesteatomas and 3 normal external ear canal skins. Three specimens of normal human facial skins obtained from patients undergoing facial plastic surgery were also used for study. The ages of the patients ranged from 21 to 56 years, with a median age of 42 years. The protocols for using specimens for this study were approved by the Columbia Unigersity Institutional Human Use Committee Review Board, and the studies were in compliance with all federal, state, and local regulations concerning the use of human subjects and materials in research. One portion of cholesteatoma specimens was fixed in 10% formalin in 10 mmol/L phosphate-buffered saline (PBS), embedded in paraffin, and sectioned at 6-txm thicknesses. These sections were used for the immunohistochemical studies. The other portion of the tissue specimens was used to prepare a protein extraction for the immunoblot assay. Extraction of Proteins
The cholesteatoma tissues (six specimens) without fixation with 10% formalin were homogenized with a glass homogenizer and then sonicated for 5 seconds three times at 4° C in 50 mmol/L Tris-HC1 (pH 7.6) containing 100 txg/ml phenylmethylsulfonyl chloride protease inhibitor. The tissue homogenate was centrifuged at 3000 g for 5 minutes at 4° C, the supernatant was collected,
Otolaryngology Head and Neck Surgery January 1996
and the pellet was further subjected to extraction once more with 50 mmol/L Tris/HC1 (pH 7.6) containing 100 Ixg/ml phenylmethylsulfonyl chloride. The supernatants were combined and used for immunoblot assay. Gel Electrophoresis and Immunobloffing
The protein extracts of cholesteatoma tissues (80 Ixg protein/well) were subjected to electrophoresis on a 10% sodium dodecylsulfate polyacrylamide slab gel (SDS-PAGE) with MINI-PROTEAN II (BIO-RAD, Richmond, Calif.) according to the procedure of Laemmli? The proteins separated on the gel by SDS-PAGE were electrophoretically transferred onto a nitrocellulose membrane with MINI-TRANS-BLOT (BIO-RAD). HSPs in the nitrocellulose membrane cut into strips were identified by immunoblot analysis. For identification of HSP60, the following steps were used: (1) the nitrocellulose membrane strips were incubated with 0.2% glutaraldehyde for 30 minutes at room temperature, (2) strips were incubated with 3% nonfat milk-l% normal goat serum for 30 minutes, (3) strips were incubated with polyclonal rabbit anti-HSP60 protein antibody (1:1000 dilution) at 4 ° C overnight, (4) strips were incubated with peroxidase-c0njugated goat antirabbit IgG (Kirkegaard & Perry Laboratories, Gaithersburg, Md.) (0.5 Ixg/ml) for 30 minutes at room temperature, and (5) color development was assessed with TMB membrane peroxidase substrate (Kirkegaard & Perry Laboratories) for 30 seconds at room temperature. For control staining, normal rabbit IgG was used to replace the primary antibody. For identification of HSP70, the following steps were used: (1) the nitrocellulose membrane strips were incubated with 0.2% glutaraldehyde for 30 minutes at room temperature, (2) strips were incubated with 3% nonfat milk-l% normal horse serum (Oncogene Science, Uniondale, N.Y.) for 30 minutes, (3) strips were incubated with monoclonal mouse anti-HSP70 antibody (1:1000 dilution) at 4° C overnight, (4) strips were incubated with biotinconjugated horse antimouse IgG (Oncogene Science) (1:200 dilution) for 30 minutes at room temperature, (5) strips were incubated with premixed avidin and biotin peroxidase (Oncogene Science) for 30 minutes, and (6) color development was assessed with TMB membrane peroxidase substrate for 30 seconds at room temperature. For control staining, normal mouse IgG was used to replace the primary antibody.
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Immunohistochemical Staining For immunolocalization of HSP60 and HSP70, the tissue' sections were stained by the indirect immunoperoxidase method with polyclonal rabbit anti-HSP60 antibody or anti-HSP70 antibody. ~ Immunoperoxidase staining included the following incubation steps after deparaffinization of tissue sections: (1) 1.0% hydrogen peroxide for 30 minutes, (2) 3% normal goat serum for 30 minutes, (3) polyclonal rabbit anti-HSP60 protein antibody (4 i~g/ml) for 1.5 hours or polyclonal rabbit anti-HSP70 protein antiserum (1:500) for 1.5 hours, (4) peroxidaseconjugated goat antirabbit IgG, 2.5 Ixg/ml for 30 minutes, and (5) 0.01% hydrogen peroxide-0.05% 3,3'-diaminobenzidine-HC1 in PBS for 3 minutes. As a control, rabbit anti-HSP60 or anti-HSP70 antibody was substituted by rabbit nonimmune IgG. The sections were then mounted with glycerol in PBS and observed under an Olympus BH-2 microscope (Olympus Optical Co., Tokyo, Japan). RESULTS Immunoblot Analysis The immunoblot analyses were performed to probe the presence of HSP60 and HSP70 in cholesteatoma tissues. Figure 1A shows a specific band exhibiting molecular weight 60 kD, indicating that polyclonal rabbit anti-HSP60 antibody recognized HSP60 in the cholesteatoma extract. The two light protein bands with molecular weights larger than 60 kD were immunoreactive bands and were caused by the nonspecific binding of other protein in the extracts to the rabbit IgG, which are also shown in the membrane strip with nonimmune rabbit IgG used as a control. In Fig. 1B, the immunoblot assay with monoclonal anti-HSP70 shows a specific band exhibiting molecular weight 70 kD. A control study with nonimmune mouse IgG used to replace the primary antibody showed no staining of protein bands. In our study, all cholesteatoma specimens examined showed the presence of HSP60 and HSPT0. Immunohistochemical Staining Polyclonal rabbit antibodies against HSP60 and HSP70 were used to localize HSPs in cholesteatoma tissue sections. HSP60 was detected as a brownish precipitate in keratinocytes of all layers of epithelium and confined to the cytoplasm in cholesteatoma sections (Fig. 2). In the control study using nonimmune IgG, no staining of HSP60 was seen in cholesteatoma (not shown). Also, no HSP60 staining was seen on the epidermis of both external
,~---70 60--~
1
2 A
1
2 B
Fig. 1. Immunoblot analysis of HSP60 and HSP70 in cholesteatoma tissue. Tissue extracts were separated by $DS-PAGE, and proteins were transferred to nitrocellulose and probed with polyclonal anti-HSP60 and monoclonal anti-HSP70 antibody. A, Lane I indicates the presence of HSP60 at a molecular weight of 60 kD. Two light proteins with molecular weights larger than 60 kD were caused by nonspecific binding to the rabbit IgG and are also seen in the control in lane 2. Other bands with molecular weights less than 60 kD may be the d e g r a d e d products of HSP60. Lane 2 indicates no HSP60 staining for a control study with normal rabbit IgG used to replace the primary antibody. B, Lane I indicates no staining for a control study with normal mouse IgG used to replace the primary antibody. Lane 2 indicates the presence at HSP70 at a molecular weight of 70 kD.
ear canal skin and normal facial skin (not shown). The immunoperoxidase staining showed that HSP70 appears to be localized throughout all the layers of epithelium and confined to both the cytoplasm and the nucleus of keratinocytes in the epithelium of cholesteatoma (Fig. 3). In the control study using nonimmune IgG, no staining of HSP70 was seen in cholesteatoma (not shown). Normal external ear canal skins and epidermis of normal human skins were not significantly stained for HSP70 (Fig. 4A). However, an intense immunoreactivity of HSP70 was seen in keratinocytes of the
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Fig. 2. Immunoperoxidase staining of cholesteatoma with polyclonal anti-HSP60 antibody and 3,3'-diaminobenzidine reaction. Positive brownish staining can be seen in all layers of cholestearoma epithelium (E) and confined to the cytoplasm of keratinocytes (arrows). K, Keratin debris; C, inflammatory connective tissue, [Original magnification x 132.)
hair follicle area of normal human skin and confined in the cytoplasm (Fig. 4B). In this study, 13 of 15 cholesteatoma specimens showed localization of HSP60 and HSP70 in keratinocytes in the cholesteatoma epithelium. DISCUSSION
In this study we investigated the presence and distribution of HSP60 and HSP70 in cholesteatoma tissues by immunoblot assay and immunohistochemical study. With anti-HSP60 and anti-HSP70 antibodies that recognize both constitutive and induced HSPs, our immunoblot assay indicated the presence of both constitutive and induced HSP60 and HSP70 in the protein extracts of cholesteatoma tissues. Our immunolocalization studies revealed that HSP60 and HSP70 are localized in the cytoplasm of keratinocytes in cholesteatoma epithelium. These findings are in accord with those of other studies, ~°'I1 which have suggested that HSPs, particularly HSP60 and HSP70, are associated with an inflammatory response of diseases. It has been suggested that HSP60 and HSP70, particularly the constitutive HSPs, are molecular chaperons in cytoplasm, having roles in the acquisition of native monomeric and oligomeric proteins after their synthesis on ribosomes or after transfer across membranes. Several investigators'210 have suggested that a function of HSPs may be to stabilize protein conformations
distinct from the stably folded, native structure, to mediate targeting and transportation of polypeptides across membranes, to assemble oligomeric structures, and to facilitate interactions with other proteins. Shielding of certain sequences by HSP60 and HSP70 might reduce the tendency of proteins to fold and aggregate improperly. With elevated rates of protein synthesis resulting from the proliferation and differentiation of keratinocytes in cholesteatoma, both HSP60 and HSP70 of both induced and constitutive HSPs may play a role in the cytoplasm of keratinocytes to prevent premature folding and aggregation of proteins in the cytoplasm. Production of HSP60 and HSP70 may also be associated with immune responses during the development of cholesteatoma, as has been found with several other diseases.6 '12 Our previous studies 13,14 and those of others ~5have found Langerhans' cells-antigen-presenting cells in direct communication with T lymphocytes- in cholesteatoma, and we have demonstrated the presence of intercellular adhesion molecule-1 in the epithelium and granulation tissue of cholesteatoma, ~6to suggest that cholesteatoma is associated with immune responses. With our present studies demonstrating the presence of HSP60 and HSP70 in cholesteatoma, these HSPs may cooperate with Langerhans' cells and intercellular adhesion molecule-1 in the immune responses during the development of cholesteatoma.
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Fig. 3. Immunoperoxidase staining of cholesteatoma with polyclonal anti-HSP70 antibody and 3,3'-diaminobenzidine reaction. Positive brownish staining can be seen in all layers of cholestearoma epithelium (E) and confined to both cytoplasm and nucleus of keratinocytes [arrows). I(, Keratin debris; C, inflammatory connective tissue. [Original magnification × 132.]
The heat-inducible HSP70 protein of mammalian and Drosophila cells is a major protein product of heat-shocked cells. On heat shock, HSP70 accumulates in the nucleus of these cells. 1.7LaThangue ~8has also reported that the nuclear accumulation of HSP70 is enhanced in proliferating cells. Our immunohistochemical staining of HSP70 showed that keratinocytes in the cholesteatoma epithelium exhibited an intense nuclear staining besides the cytoplasm staining. This suggests that the induced HSP70 may be associated particularly with cellular proliferation of keratinocytes in cholesteatoma epithelium. Also, in our present studies, normal skin showed HSP70 present only in the cytoplasm of keratinocytes of normal hair follicles, where keratinocytes arc: in active proliferation. Gatto et al. ~ have found that normal human skin stored at 4° C (a kind of thermal shock) for several hours exhibited the presence of HSP70 predominantly in the basal layer, confined to the nucleus of keratinocytes, whereas no immunolabeling was seen in conventional skin biopsy specimens frozen immediately in liquid nitrogen. Our findings suggest that it is the hyperproliferative state of cholesteatoma epithelium that is associated with the strong presence of HSP70, particularly the inducible HSP70 in the nucleus of keratinocytes in all layers of epithelium. Our previous studies indicated the hyperproliferation of keratinocytes in cholesteatoma by demonstrating the
presence of cytokeratin 16, a marker for hyperproliferation, and high levels of epidermal growth factor, transforming growth factor-u, and their receptors in th e epithelium of cholesteatoma. 2° The accumulation of keratin debris, the unique pathologic feature of cholesteatoma, is derived from an increased rate of programmed cell death, known as apoptosis, driven by the differentiation of keratinocytes. Keratinocyte cell death involves keratinization and cornification, the last step of the differentiation process, which may involve the activities of p53. The p53 protein, a nuclear phosphoprotein, has been identified as a product of a tumor-suppressor gene and functions as a negative regulator of cellular proliferation and is crucial to the apoptosis pathway inducing DNA damage. 2t Our recent study indicated that p53 protein was densely present in the nucleus of keratinocytes in the granular layer of cholesteatoma epithelium. = Generally, the cellular content of p53 is limited under normal conditions and has a short half-life, approximately 20 minutes. The presence of a detectable amount of p53 protein in keratinocytes in the granular layer of cholesteatoma epithelium suggests that accumulation of the protein may be caused by the stable complex formation of p53 protein with other proteins, such as HSP70 present in the nucleus. 23 Growth of cholesteatoma is possibly mediated by such inflammatory cytokines as interleukin-1 and
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Fig. 4. Immunoperoxidase staining of normal external ear canal skin and normal facial skin with polyclonal anti-HSP70 antibody and 3,3'-diaminobenzidine reaction. A, In external ear canal skin, no significant staining for HSP70 is seen in the cytoplasm of keratinocytes {arrows) of the epidermis {El. D, Dermis. B, Positive brownish staining can be seen in the cytoplasm of keratinocytes {arrows) of the hair follicle area {H) but not in any other area of epidermis {E} of facial skin. [Original magnification x 132.]
tumor necrosis factor-co. 2<2~Both HSP60 and HSP70 might be induced by these cytokines, which have been shown to stimulate various cell types to produce these HSPs. 1~ However, the mechanisms of induction of these HSPs in keratinocytes in epithelium of cholesteatoma are still not known. Further investigations on the mechanisms of induction of these HSPs by cytokines such as interleukin-1 and tumor necrosis factor-c~ is necessary.
REFERENCES
1. Ritossa F. A new puffing pattern induced by temperature shock and DNP in Drosophila. Experentia 1962;18: 571-3, 2. Langer T, Neupert W. Heat shock proteins hsp60 and 70. Curr Top Microbiol Immunol 1991;167:3-30. 3. Hartl FU. Heat shock proteins in protein folding and membrane translocation. Semin Immunol 1991;3:5-16. 4. Welch JW. How cells respond to stress. Scientific Am 1993; 268:56-64. 5. Burel C, Mezger V, Pinto M, Rallu M, Trigon S, Morange M.
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Mammalian heat shock protein families. Expression and functions. Experientia 1992;48:629-34. DeNagel DC, Pierce SK. Heat shock proteins in immune responses: Crit Rev Immunol 1993;13:71-81. Subjeck JR, Shyy TT. Stress protein systems of mammalian cells. Am J Physiol 1986;250:C1-17. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227: 680-5. Welch W, Feramisco J. Nuclear and nucleolar localization of the 72,000-dalton heat shock protein in heat-shocked mammalian cells. J Biol Chem 1984;259:4501-13. Kantengwa S, Donati YRA, Clerget M, et at. Heat shock proteins: an autoprotective mechanism for inflammatory cells? Semin Immunol 1991;3:49-56. Polla BS, Kantengwa S. Heat shock proteins and inflammation. Curr Top Microbiol Immunol 1991;167:93-105. DeNagel DC, Pierce SK. Heat shock proteins implicated in antigen processing and presentation. Semin Immunol 1991; 3:65-71. Chao WY, Jin YT, Huang CC. Langerhans' cells in human middle ear cholesteatoma. Eur Arch Otorhinolaryngol 1992; 249:380-4. Kamide Y, Sasaki H, Abramson M, Huang CC. Effects of epidermal Langerhans cell's conditioned medium on keratinocytes: a role of Langerhans cells in cholesteatoma. Am J Otolaryngol 1991;12:307-15. Takahashi S, Nakano Y. Immunohistochemical demonstration of Langerhans' cell in cholesteatoma using an antiserum against S-100 protein. Arch Otorhinolaryngol 1989;246:4852. ShinodaH, HuangCC. Localization of intercellular adhesion
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molecule-1 in middle ear cholesteatoma. Eur Arch Otolaryngol 1995 (in press). Lewis MJ, Pelham HRB. Involvement of ATP in the nuclear and nucleolar functions of the 70 kd heat shock protein. EMBO J 1985;4:3137-42. LaThangue NB. A major heat-shock protein defined by a monoclonal antibody. EMBO J 1984;32:1871-9. Gatto H, Charveron M, Schmitt D. Effects of thermal shocks on interleukin-1 levels and heat shock protein 72 (HSP72) expression in normal human keratinocytes. Arch Dermatol Res 1992;284:414-7. Huang CC, Sasaki H, Chao WY, Huang TY, Yabe T, Huang E. Evidence that cholesteatoma is a hyperproliferative epidermal disease: expression of epidermal growth factor receptors and abnormal cytokeratin 16 [Abstract]. Association for Research in Otolaryngology 1994;17:147. Lovine A, Moman J, Finlay CA. The p53 tumor suppressor gene. Nature 1991;351:453-6. Shinoda H, Huang CC. Expression of c-jun and p53 proteins in human middle ear cholesteatoma: related to keratinocyte proliferation, differentiation and programmed cell death. Laryngoscope (in press). Matsumoto H, Shimura M, Omatsu T, Okaichi K, Majima H, Ohnishi T. p53 Proteins accumulated by heat stress associate with heat shock proteins HSP72/HSC73 in human glioblastoma cell lines. Cancer Lett 1994;87:39-46. Ahn JM, Huang CC, Abromson M. Localization of interleukin-1 in human cholesteatoma. Am J Otolaryngol 1990;11: 71-7. Yan SD, Huang CC. Tumor necrosis factor alpha in middle ear cholesteatoma and its effect on keratinocytes in vitro. Ann Otol Rhinol Laryngol 1991;100:157-61.
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Heat shock proteins in middle ear cholesteatoma HIDEO SHINODA, MD, and CHENG-CHUN HUANG, PhO,New York, New York
Heat shock proteins [HSPs], particularly HSP60 and HSP70, may have integral roles in several aspects of the pathogenesis of acquired cholesteatoma. Cholesteatoma is usually associated with an inflammatory reaction occurring in the middle ear cavity, leading to proliferation, differentiation, and programmed cell death of keratinocytes. The presence of HSP60 and HSP70 in cholesteatoma was demonstrated by the immunobiotting assay with specific anti-HSP60 and anti-HSP70 antibodies after protein extraction. The distribution of HSP60 and HSP70 in the cholesteatoma tissues was studied immunohistochemically with paraffin-embedded sections. HSP60 and HSP70 appeared to be localized in the cytoplasm of keratinocytes in all layers of the epithelium of cholesteatoma. Moreover, HSP70 was found in the nucleus of keratinocytes. Both HSPswere not labeled in the epidermis of the external ear canal skin or normal facial skin, except for significant staining on keratinocytes of hair follicles in facial skin. High density of HSF'60 and HSP70 may be induced by the inflammatory reaction and immune responses in the middle ear cavity during the clinical development of cholesteatoma. Induction of HSP60 and HSP70 in cholesteatoma epithelium may also be related to hyperproliferation and active differentiation of basal keratinocytes, which cause the accumulation of keratin debris, a characteristic feature of cholesteatoma. In the nucleus of keratinocytes, HSP70 may act to stabilize p53 protein, which functions as a negative regulator of cellular proliferation and is crucial to apoptosis of keratinocytes. This study demonstrates HSPs in cholesteatoma and suggests important roles for HSPs in the clinical development of cholesteatoma. [OTOLARYNGOL HEAD NECK SURG 1996;t 14:77-83.]
C e l l u l a r expression of heat shock proteins (HSPs) was first recognized as a response to elevated temperatures.E Later, it was also found that production of HSPs can be induced by other environmental and pathophysiologic stresses. HSPs are named according to their apparent molecular weights and classified into families, for example, HSP60 and HSP70, with molecular weights of 60 and 70 kD. These proteins enable the cell to survive and recover from stressful conditions by as yet incompletely understood mechanisms. However, the role of the HSPs is not likely to be confined to cellular stress. Many of these proteins are also synthesized under normal From the Department of Otolaryngology-Headand Neck Surgery,Collegeof Physiciansand Surgeons, ColumbiaUniversity. Received for publication Nov. 10, 1994; revision received April 27, 1995; accepted May 5, 1995. Reprint requests: C-C. Huang, PhD, Department of Otolaryngology-Headand Neck Surgery,ColumbiaUniversity,630 West 168th St., New York, NY 10032. Copyright © 1996by the American Academyof OtolaryngologyHead and Neck Surgery Foundation, Inc. 0194-5998/96/$5,00 + 0 23/10/66124
conditions and fulfill many central functions in the normal cells. The HSP60 and HSP70 cognates are essential for the folding and unfolding, the assembly and disassembly, and the sorting and transaction of many other proteins. Accordingly, they have been termed chaperons. 2-5 DeNagel and Pierce ~ have shown that HSPs are the dominant antigens of a variety of pathogens and serve as agents in antigen processing and/or presentation. These findings revealed that in a wide variety of organisms, including human beings, HSPs play a critical role both in normal functions and in the response to stress/ Cholesteatoma is a chronic middle ear disease characterized by the accumulation of keratinizing epithelium resulting from proliferation, differentiation, and programmed cell death of epithelial cells. In response to inflammation in the middle ear cavity, keratinocytes in the epidermis of the ear canal skin migrate, proliferate, differentiate, and cornify to cause the accumulation of keratin debris to form a cholesteatoma. It is interesting to investigate the presence of HSPs in cholesteatoma during its development because HSPs may be involved in the 77
78
SHINODA and HUANG
inflammatory response; therefore it is important to understand the function of HSPs in the pathogenesis of cholesteatoma. This investigation focused on studying the presence and localization of HSP60 and HSP70 in cholesteatoma tissues. Of the HSPs known, HSP60 and HSP70 have been most associated with inflammatory and immune responses. METHODS AND MATERIAL Antibodies
Antibodies against HSPs were obtained from Stress Gen (Victoria, British Columbia, Canada). Polyclonal rabbit antibody (SPA-805) was used for studying HSP60 by immunoblotting assay and immunohistochemistry. This antibody recognizes HSP60 constitutively synthesized in some types of cells. This protein is also stress inducible. Both the constitutive (HSCT0) and the inducible (HSP70) types are highly homologous and can be recognized by either polyclonal or monoclonal anti-HSP70 (SPA-820). Tissues
Specimens were obtained from patients during middle ear operations. Specimens included 15 middle ear cholesteatomas and 3 normal external ear canal skins. Three specimens of normal human facial skins obtained from patients undergoing facial plastic surgery were also used for study. The ages of the patients ranged from 21 to 56 years, with a median age of 42 years. The protocols for using specimens for this study were approved by the Columbia Unigersity Institutional Human Use Committee Review Board, and the studies were in compliance with all federal, state, and local regulations concerning the use of human subjects and materials in research. One portion of cholesteatoma specimens was fixed in 10% formalin in 10 mmol/L phosphate-buffered saline (PBS), embedded in paraffin, and sectioned at 6-txm thicknesses. These sections were used for the immunohistochemical studies. The other portion of the tissue specimens was used to prepare a protein extraction for the immunoblot assay. Extraction of Proteins
The cholesteatoma tissues (six specimens) without fixation with 10% formalin were homogenized with a glass homogenizer and then sonicated for 5 seconds three times at 4° C in 50 mmol/L Tris-HC1 (pH 7.6) containing 100 txg/ml phenylmethylsulfonyl chloride protease inhibitor. The tissue homogenate was centrifuged at 3000 g for 5 minutes at 4° C, the supernatant was collected,
Otolaryngology Head and Neck Surgery January 1996
and the pellet was further subjected to extraction once more with 50 mmol/L Tris/HC1 (pH 7.6) containing 100 Ixg/ml phenylmethylsulfonyl chloride. The supernatants were combined and used for immunoblot assay. Gel Electrophoresis and Immunobloffing
The protein extracts of cholesteatoma tissues (80 Ixg protein/well) were subjected to electrophoresis on a 10% sodium dodecylsulfate polyacrylamide slab gel (SDS-PAGE) with MINI-PROTEAN II (BIO-RAD, Richmond, Calif.) according to the procedure of Laemmli? The proteins separated on the gel by SDS-PAGE were electrophoretically transferred onto a nitrocellulose membrane with MINI-TRANS-BLOT (BIO-RAD). HSPs in the nitrocellulose membrane cut into strips were identified by immunoblot analysis. For identification of HSP60, the following steps were used: (1) the nitrocellulose membrane strips were incubated with 0.2% glutaraldehyde for 30 minutes at room temperature, (2) strips were incubated with 3% nonfat milk-l% normal goat serum for 30 minutes, (3) strips were incubated with polyclonal rabbit anti-HSP60 protein antibody (1:1000 dilution) at 4 ° C overnight, (4) strips were incubated with peroxidase-c0njugated goat antirabbit IgG (Kirkegaard & Perry Laboratories, Gaithersburg, Md.) (0.5 Ixg/ml) for 30 minutes at room temperature, and (5) color development was assessed with TMB membrane peroxidase substrate (Kirkegaard & Perry Laboratories) for 30 seconds at room temperature. For control staining, normal rabbit IgG was used to replace the primary antibody. For identification of HSP70, the following steps were used: (1) the nitrocellulose membrane strips were incubated with 0.2% glutaraldehyde for 30 minutes at room temperature, (2) strips were incubated with 3% nonfat milk-l% normal horse serum (Oncogene Science, Uniondale, N.Y.) for 30 minutes, (3) strips were incubated with monoclonal mouse anti-HSP70 antibody (1:1000 dilution) at 4° C overnight, (4) strips were incubated with biotinconjugated horse antimouse IgG (Oncogene Science) (1:200 dilution) for 30 minutes at room temperature, (5) strips were incubated with premixed avidin and biotin peroxidase (Oncogene Science) for 30 minutes, and (6) color development was assessed with TMB membrane peroxidase substrate for 30 seconds at room temperature. For control staining, normal mouse IgG was used to replace the primary antibody.
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Immunohistochemical Staining For immunolocalization of HSP60 and HSP70, the tissue' sections were stained by the indirect immunoperoxidase method with polyclonal rabbit anti-HSP60 antibody or anti-HSP70 antibody. ~ Immunoperoxidase staining included the following incubation steps after deparaffinization of tissue sections: (1) 1.0% hydrogen peroxide for 30 minutes, (2) 3% normal goat serum for 30 minutes, (3) polyclonal rabbit anti-HSP60 protein antibody (4 i~g/ml) for 1.5 hours or polyclonal rabbit anti-HSP70 protein antiserum (1:500) for 1.5 hours, (4) peroxidaseconjugated goat antirabbit IgG, 2.5 Ixg/ml for 30 minutes, and (5) 0.01% hydrogen peroxide-0.05% 3,3'-diaminobenzidine-HC1 in PBS for 3 minutes. As a control, rabbit anti-HSP60 or anti-HSP70 antibody was substituted by rabbit nonimmune IgG. The sections were then mounted with glycerol in PBS and observed under an Olympus BH-2 microscope (Olympus Optical Co., Tokyo, Japan). RESULTS Immunoblot Analysis The immunoblot analyses were performed to probe the presence of HSP60 and HSP70 in cholesteatoma tissues. Figure 1A shows a specific band exhibiting molecular weight 60 kD, indicating that polyclonal rabbit anti-HSP60 antibody recognized HSP60 in the cholesteatoma extract. The two light protein bands with molecular weights larger than 60 kD were immunoreactive bands and were caused by the nonspecific binding of other protein in the extracts to the rabbit IgG, which are also shown in the membrane strip with nonimmune rabbit IgG used as a control. In Fig. 1B, the immunoblot assay with monoclonal anti-HSP70 shows a specific band exhibiting molecular weight 70 kD. A control study with nonimmune mouse IgG used to replace the primary antibody showed no staining of protein bands. In our study, all cholesteatoma specimens examined showed the presence of HSP60 and HSPT0. Immunohistochemical Staining Polyclonal rabbit antibodies against HSP60 and HSP70 were used to localize HSPs in cholesteatoma tissue sections. HSP60 was detected as a brownish precipitate in keratinocytes of all layers of epithelium and confined to the cytoplasm in cholesteatoma sections (Fig. 2). In the control study using nonimmune IgG, no staining of HSP60 was seen in cholesteatoma (not shown). Also, no HSP60 staining was seen on the epidermis of both external
,~---70 60--~
1
2 A
1
2 B
Fig. 1. Immunoblot analysis of HSP60 and HSP70 in cholesteatoma tissue. Tissue extracts were separated by $DS-PAGE, and proteins were transferred to nitrocellulose and probed with polyclonal anti-HSP60 and monoclonal anti-HSP70 antibody. A, Lane I indicates the presence of HSP60 at a molecular weight of 60 kD. Two light proteins with molecular weights larger than 60 kD were caused by nonspecific binding to the rabbit IgG and are also seen in the control in lane 2. Other bands with molecular weights less than 60 kD may be the d e g r a d e d products of HSP60. Lane 2 indicates no HSP60 staining for a control study with normal rabbit IgG used to replace the primary antibody. B, Lane I indicates no staining for a control study with normal mouse IgG used to replace the primary antibody. Lane 2 indicates the presence at HSP70 at a molecular weight of 70 kD.
ear canal skin and normal facial skin (not shown). The immunoperoxidase staining showed that HSP70 appears to be localized throughout all the layers of epithelium and confined to both the cytoplasm and the nucleus of keratinocytes in the epithelium of cholesteatoma (Fig. 3). In the control study using nonimmune IgG, no staining of HSP70 was seen in cholesteatoma (not shown). Normal external ear canal skins and epidermis of normal human skins were not significantly stained for HSP70 (Fig. 4A). However, an intense immunoreactivity of HSP70 was seen in keratinocytes of the
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Fig. 2. Immunoperoxidase staining of cholesteatoma with polyclonal anti-HSP60 antibody and 3,3'-diaminobenzidine reaction. Positive brownish staining can be seen in all layers of cholestearoma epithelium (E) and confined to the cytoplasm of keratinocytes (arrows). K, Keratin debris; C, inflammatory connective tissue, [Original magnification x 132.)
hair follicle area of normal human skin and confined in the cytoplasm (Fig. 4B). In this study, 13 of 15 cholesteatoma specimens showed localization of HSP60 and HSP70 in keratinocytes in the cholesteatoma epithelium. DISCUSSION
In this study we investigated the presence and distribution of HSP60 and HSP70 in cholesteatoma tissues by immunoblot assay and immunohistochemical study. With anti-HSP60 and anti-HSP70 antibodies that recognize both constitutive and induced HSPs, our immunoblot assay indicated the presence of both constitutive and induced HSP60 and HSP70 in the protein extracts of cholesteatoma tissues. Our immunolocalization studies revealed that HSP60 and HSP70 are localized in the cytoplasm of keratinocytes in cholesteatoma epithelium. These findings are in accord with those of other studies, ~°'I1 which have suggested that HSPs, particularly HSP60 and HSP70, are associated with an inflammatory response of diseases. It has been suggested that HSP60 and HSP70, particularly the constitutive HSPs, are molecular chaperons in cytoplasm, having roles in the acquisition of native monomeric and oligomeric proteins after their synthesis on ribosomes or after transfer across membranes. Several investigators'210 have suggested that a function of HSPs may be to stabilize protein conformations
distinct from the stably folded, native structure, to mediate targeting and transportation of polypeptides across membranes, to assemble oligomeric structures, and to facilitate interactions with other proteins. Shielding of certain sequences by HSP60 and HSP70 might reduce the tendency of proteins to fold and aggregate improperly. With elevated rates of protein synthesis resulting from the proliferation and differentiation of keratinocytes in cholesteatoma, both HSP60 and HSP70 of both induced and constitutive HSPs may play a role in the cytoplasm of keratinocytes to prevent premature folding and aggregation of proteins in the cytoplasm. Production of HSP60 and HSP70 may also be associated with immune responses during the development of cholesteatoma, as has been found with several other diseases.6 '12 Our previous studies 13,14 and those of others ~5have found Langerhans' cells-antigen-presenting cells in direct communication with T lymphocytes- in cholesteatoma, and we have demonstrated the presence of intercellular adhesion molecule-1 in the epithelium and granulation tissue of cholesteatoma, ~6to suggest that cholesteatoma is associated with immune responses. With our present studies demonstrating the presence of HSP60 and HSP70 in cholesteatoma, these HSPs may cooperate with Langerhans' cells and intercellular adhesion molecule-1 in the immune responses during the development of cholesteatoma.
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Fig. 3. Immunoperoxidase staining of cholesteatoma with polyclonal anti-HSP70 antibody and 3,3'-diaminobenzidine reaction. Positive brownish staining can be seen in all layers of cholestearoma epithelium (E) and confined to both cytoplasm and nucleus of keratinocytes [arrows). I(, Keratin debris; C, inflammatory connective tissue. [Original magnification × 132.]
The heat-inducible HSP70 protein of mammalian and Drosophila cells is a major protein product of heat-shocked cells. On heat shock, HSP70 accumulates in the nucleus of these cells. 1.7LaThangue ~8has also reported that the nuclear accumulation of HSP70 is enhanced in proliferating cells. Our immunohistochemical staining of HSP70 showed that keratinocytes in the cholesteatoma epithelium exhibited an intense nuclear staining besides the cytoplasm staining. This suggests that the induced HSP70 may be associated particularly with cellular proliferation of keratinocytes in cholesteatoma epithelium. Also, in our present studies, normal skin showed HSP70 present only in the cytoplasm of keratinocytes of normal hair follicles, where keratinocytes arc: in active proliferation. Gatto et al. ~ have found that normal human skin stored at 4° C (a kind of thermal shock) for several hours exhibited the presence of HSP70 predominantly in the basal layer, confined to the nucleus of keratinocytes, whereas no immunolabeling was seen in conventional skin biopsy specimens frozen immediately in liquid nitrogen. Our findings suggest that it is the hyperproliferative state of cholesteatoma epithelium that is associated with the strong presence of HSP70, particularly the inducible HSP70 in the nucleus of keratinocytes in all layers of epithelium. Our previous studies indicated the hyperproliferation of keratinocytes in cholesteatoma by demonstrating the
presence of cytokeratin 16, a marker for hyperproliferation, and high levels of epidermal growth factor, transforming growth factor-u, and their receptors in th e epithelium of cholesteatoma. 2° The accumulation of keratin debris, the unique pathologic feature of cholesteatoma, is derived from an increased rate of programmed cell death, known as apoptosis, driven by the differentiation of keratinocytes. Keratinocyte cell death involves keratinization and cornification, the last step of the differentiation process, which may involve the activities of p53. The p53 protein, a nuclear phosphoprotein, has been identified as a product of a tumor-suppressor gene and functions as a negative regulator of cellular proliferation and is crucial to the apoptosis pathway inducing DNA damage. 2t Our recent study indicated that p53 protein was densely present in the nucleus of keratinocytes in the granular layer of cholesteatoma epithelium. = Generally, the cellular content of p53 is limited under normal conditions and has a short half-life, approximately 20 minutes. The presence of a detectable amount of p53 protein in keratinocytes in the granular layer of cholesteatoma epithelium suggests that accumulation of the protein may be caused by the stable complex formation of p53 protein with other proteins, such as HSP70 present in the nucleus. 23 Growth of cholesteatoma is possibly mediated by such inflammatory cytokines as interleukin-1 and
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Fig. 4. Immunoperoxidase staining of normal external ear canal skin and normal facial skin with polyclonal anti-HSP70 antibody and 3,3'-diaminobenzidine reaction. A, In external ear canal skin, no significant staining for HSP70 is seen in the cytoplasm of keratinocytes {arrows) of the epidermis {El. D, Dermis. B, Positive brownish staining can be seen in the cytoplasm of keratinocytes {arrows) of the hair follicle area {H) but not in any other area of epidermis {E} of facial skin. [Original magnification x 132.]
tumor necrosis factor-co. 2<2~Both HSP60 and HSP70 might be induced by these cytokines, which have been shown to stimulate various cell types to produce these HSPs. 1~ However, the mechanisms of induction of these HSPs in keratinocytes in epithelium of cholesteatoma are still not known. Further investigations on the mechanisms of induction of these HSPs by cytokines such as interleukin-1 and tumor necrosis factor-c~ is necessary.
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