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Immunolocalization of interleukin-6 in salivary gland tumors
Immunolocalization of Interleukin-6 in Salivary Gland Tumors REGINA GANDOUR-EDWARDS, MD, SILLOO B. KAPADIA, MD, PAUL H. GUMERLOCK, PHD, AND LEON BARNES, MD Interleukin-6 (IL-6) is a multifunctional cytokine that regulates immune responses and acute phase reactions. It has demonstrated a growth factor function in several tumors, including those of salivary, plasma cell, and renal origin. We performed immunohistochemical staining for IL-6 localization on 57 salivary tumors. Reactivity was scored by intensity (0 to 4 + ) and percentage of cells staining, and the tumors were classified into three groups representing low (0 to 1+, 0% to 30%), moderate (2 to 3+, 31% to 75%), or high ( > 3 to 4 + , 76% to 100%) reactors. High reactivity was found in all primary pleomorphic adenomas (N = 10), five of eight recurrent pleomorphic adenomas, and all polymorphous low grade adenocarcinomas (N = 4 ). Moderate reactivity was observed in four of seven basal cell adenomas and three of five myoepitheliomas. Low reactivity characterized all acinic cell carcinomas (N = 3) and mucoepidermoid carcinomas
(N : 3) as well as six of nine primary adenoid cystic carcinomas and all metastatic adenoid cystic carcinomas (N = 3). Carcinoma ex pleomorphic adenoma (N = 5) had three low and two moderate reactors. A pattern emerged in which the benign and low grade malignant tumors showed stronger reactivity than the metastatic or high grade malignant tumors. This suggests an inverse relationship between the presence of ID6 and the biological aggressiveness of salivary gland tumors. The function of ID6 in salivary gland neoplasia awaits further study and elucidation. HUM PATHOL 26:501503. Copyright © 1995 by W.B. Saunders Company Key words: salivary tumors; interleukin-6; pleomorphic adenoma; salivary gland; adenoid cystic carcinoma. Abbreviations: IL-6, interleukin-6.
Interleukin-6 (IL-6) is a multifunctional cytokine produced by a wide variety of cells that mediates inflammatory and immune responses. It is postulated to be the major regulator of the hepatic secretion of acute phase reactants, eg, C-reactive protein, and to modulate B-cell lymphocyte development, v4 Significantly, IL-6 also has been shown to function as a growth factor in several h u m a n tumors, including plasmacytoma, multiple myeloma, melanoma, and renal cell carcinoma. TM The precise mechanisms of growth regulation are the subject of ongoing investigation; however, both growth inhibition and growth stimulation have been observed in in vitro models. 4-7 Gallo et al 7 described IL-6 autocrine growth regulation of a cell line derived from a pleomorphic adenoma of the parotid gland. Interleukin-6 was detected by immunocytochemical staining of light microscopic sections. Immunoelectron cytochemistry demonstrated IL6 in the vicinity of the nucleus as well as scattered in the cytoplasm. A joilot study of 26 salivary tumors by Gandour-Edwards°demonstrated strong IL-6 reactivity in routinely processed tissue sections of resected pleoinorphic adenoma and other tumors. We elected to study a wide variety of benign and malignant salivary tumors to determine the range and extent of IL-6 expression by standard immunohistochemical methodology.
MATERIALS AND METHODS Resection specimens of salivary tumors were obtained f r o m the files of the D e p a r t m e n t of Pathology at the University of Pittsburgh. A total of 57 benign and malignant salivary tumors was studied, including p l e o m o r p h i c a d e n o m a (10), r e c u r r e n t p l e o m o r p h i c a d e n o m a (8), basal cell a d e n o m a (7), c a r c i n o m a ex p l e o m o r p h i c a d e n o m a (5), acinic cell carcin o m a (3), primary a d e n o i d cystic c a r c i n o m a (9), metastatic a d e n o i d cystic c a r c i n o m a (3), m y o e p i t h e l i o m a (5), mucoepide r m o i d carcinoma (3), and p o l y m o r p h o u s low grade adenoc a r c i n o m a (4). Adjacent sections of n o r m a l salivary gland were present in most cases. Paraffin sections were e x a m i n e d by an i m m u n o h i s t o c h e m i c a l t e c h n i q u e using a m o n o c l o n a l i m m u n o g l o b u l i n (IgG) antibody to h u m a n IL-6 (Genzyme, Cambridge, MA) at a dilution of 1:20. Formalin-fixed, paraffin-embedded cell blocks from an IL-6-secreting prostate cancer cell line (PC-3) were used as positive controls. Briefly, 5micron-thick sections were deparaffinized and rehydrated, and e n d o g e n o u s peroxidase was blocked with 3% hydrogen p e r o x i d e in absolute methanol. Sections then were heated for 10 minutes in distilled water in a microwave oven to optimize antigen exposure. After blocking with 10% normal e q u i n e serum, a 1-hour incubation at r o o m temperature with the primary m o n o c l o n a l antibody was p e r f o r m e d followed by a phosphate-buffered saline (PBS) rinse. A biotin-conjugated e q u i n e antimouse secondary antibody (Vector, Burlingame, CA) at a dilution of 1:1000 followed, and after PBS rinsing an avidin-biotin c o m p l e x (ABC Elite, Vector) was applied. After PBS rinsing diaminobenzidene-peroxidase (ScyTek
TABLE 1. From the Department of Pathology and the Division of Hematology and 0ncology, Department of Internal Medicine, University of California Davis, Sacramento, CA; and the Department of Pathology, University of Pittsburgh, Pittsburgh, PA. Accepted for publication August 29, 1994. Address correspondence and reprint requests to Regina Gandour-Edwards, MD, Department of Pathology, University of California Davis Medical Center, 2315 Stockton Blvd, Sacramento, CA 95817. Copyright © 1995 by W.B. Saunders Company 0046-8177/95/2605-000755.00/0
501
Interleukin-6 Immunohistochemical Staining in Benign Salivary Tumors Diagnosis
Low
Moderate
High
Pleomorphic adenoma (n = 10) Recurrent pleomorphic adenoma (n = 8) Basal cell adenoma (n = 7) Myoepithelioma (n = 5) Total (N = 30) (%)
--
--
10
1 1 2 4 (13)
2 4 3 9 (30)
5 2 -17 (57)
HUMAN PATHOLOGY TABLE 2.
Volume 26, No. 5 (May 1995)
Interleukin-6 I m m u n o h i s t o c h e m i c a l Staining in M a l i g n a n t Salivary Tumors
Diagnosis Carcinoma ex pleomorphic adenoma (n = 5) Adenoid cystic carcinoma (n = 9) Adenoid cystic carcinoma, metastatic (n = 3) Mucoepidermoid carcinoma (n = 3) Acinic cell carcinoma (n = 3) Polymorphous low grade adenocarcinoma (n = 4) Total (N = 27) (%)
Low
Moderate
High
3
2
--
6
2
1
3
--
--
3 3
---
---
-18 (67)
-4 (15)
4 5 (19)
Labs, Logan, UT) was applied to achieve a permanent color change in the reactive cells. Duplicate sections subjected to all phases of staining except that of the primary antibody served as negative controls. Sections were stained with hematoxylin and evaluated by light microscopy for reactivity.
FIGURE 2. Immunolocalization of IL-6 comparing the absence of staining in a carcinoma ex pleomorphic adenoma (A) with the cytoplasmic and nuclear staining of a pleomorphic adenoma (B). (Hematoxylin with DAB, original magnification × 100.)
I n t e r p r e t a t i o n o f i m m u n o h i s t o c h e m i c a l staining was p e r f o r m e d by all three pathologists (R.G.E., S.B.K., a n d L.B.). T h e reactivity was primarily cytoplasmic a n d g r a n u l a r in nature. In several t u m o r s the n u c l e u s app e a r e d stained, which we i n t e r p r e t e d to r e p r e s e n t overlying cytoplasm r a t h e r t h a n intrinsic n u c l e a r staining. T h e t u m o r s were evaluated as to the d e g r e e o f staining intensity using a 0 to 4 + scale a n d by the p e r c e n t a g e o f cells stained. T h e t u m o r s t h e n were classified into t h r e e g r o u p s r e p r e s e n t i n g low (0 to 1+, 0% to 30%), m o d e r a t e (2 to 3 + , 31% to 75%), o r high ( > 3 to 4 + , 76% to 100%) reactors (Tables 1 a n d 2). Strong cytoplasmic staining was observed in intercalated a n d striated ducts o f a d j a c e n t n o r m a l salivary tissue (Fig 1). All 10 o f the p r i m a r y p l e o m o r p h i c a d e n o -
mas d e m o n s t r a t e d strong granular, cytoplasmic, a n d n u c l e a r reactivity in b o t h the epithelial a n d myoepithelial cells, whereas the s t r o m a was unstained. In comparison, five o f eight r e c u r r e n t p l e o m o r p h i c a d e n o m a s were strong reactors, whereas two were m o d e r a t e a n d o n e was low. A contrast was n o t e d in the three specim e n s o f c a r c i n o m a ex p l e o m o r p h i c a d e n o m a in that three showed a low d e g r e e a n d two s h o w e d a m o d e r a t e d e g r e e o f cytoplasmic staining (Fig 2). Two o t h e r benign tumors, basal cell a d e n o m a a n d m y o e p i t h e l i o m a , s h o w e d p r e d o m i n a t e l y m o d e r a t e cytoplasmic staining. Six o f nine p r i m a r y a d e n o i d cystic carcinomas were low reactors as were all three o f the metastatic a d e n o i d cystic lesions. A c o m p a r i s o n between the p r i m a r y a n d metastatic lesions was available for only o n e o f the patients a n d virtually n o difference in staining was noted. In contrast, however, all f o u r p o l y m o r p h o u s low grade a d e n o c a r c i n o m a s were strongly reactive (Fig 3). T h e high g r a d e m u c o e p i d e r m o i d c a r c i n o m a s (N = 3) a n d
FIGURE 1. Immunolocalization of IL-6 in normal salivary tissue. Intense granular cytoplasmic staining is present in the ducts, whereas the acinar cells and adipose cells are negative. (Hematoxylin with DAB, original magnification x 100.)
FIGURE 3. Immunolocalization of IL-6 comparing the strong cytoplasmic staining of a polymorphous low grade adenocarcinoma (A) with the absence of staining in an adenoid cystic carcinoma (B). (Hematoxylin with DAB, original magnification x 100.)
RESULTS
502
IL-6 IN SALIVARY TUMORS (Gandour-Edwards et al)
the low grade acinic cell carcinomas (N = 3) were all low reactors. In summary, four (13%) of the benign tumors were low reactors, nine (30%) were intermediate, and 17 (57%) were highly reactive with the monoclonal antibody to IL-6. O f the malignant tumors, 18 (67%) reacted weakly, four (15%) reacted moderately, and five (19%) reacted strongly to anti-IL-6.
DISCUSSION Interleukin-6 is a multifunctional cytokine that is p r o d u c e d by a wide variety of cells and by a growing n u m b e r of h u m a n tumors. Tabibzadeh et al 9 observed IL-6 staining in 30 different h u m a n tumor types and proposed that the cytokine plays a major role in regulating local and systemic host-tumor interaction. Interleukin-6 has been shown to induce acute-phase protein production by hepatocytes, to p r o m o t e the growth and differentiation of B and T lymphocytes, and to enhance the activation of natural killer cells? An examination of the role of IL-6 in neoplastic growth control has shown a diverse range of behaviors. In plasmacytoma, multiple myeloma, and renal cell carcinoma, IL-6 functions as an autocrine growth stimulator. 1'4'1°-n In contrast, in lung and breast carcinoma its behavior is inhibitory to growth, lz'l~ A recent report by Lu and Kerbel 5 describes the interesting finding that exogenous IL-6 inhibited the growth of early stage (metastatically incompetent) melanoma cells, but that advanced stage (metastatically competent) cells were not inhibited. Furthermore, several advanced stage melan o m a cell lines p r o d u c e d IL-6, which functioned as a growth stimulator. This suggests that cells can make the transition from responding to IL-6 as a paracrine growth inhibitor to responding to IL-6 as an autocrine growth stimulator during carcinogenesis. We recently examined IL-6 staining in 10 invasive pituitary adenomas involving the sphenoid sinus and c o m p a r e d the results with those of 10 noninvasive pituitary adenomas and five normal pituitary glands. 14 Our study demonstrated strong expression of IL-6 in normal glands with cytoplasmic reactivity in all cell types. Interleukin-6 staining was diminished in the noninvasive adenomas with only two showing moderate cytoplasmic reactivity. However, five (50%) of the invasive adenomas demonstrated strong granular cytoplasmic staining, suggesting a change in cellular function. We propose that in invasive pituitary adenomas IL-6 may u n d e r g o a transition from a normal cell regulator of secretion to an autocrine growth stimulator. T h e results o f this study of salivary gland tumors d e m o n s t r a t e d m o d e r a t e to strong IL-6 staining in n o r m a l salivary gland ducts and several b e n i g n tumors, including primary and r e c u r r e n t p l e o m o r p h i c a d e n o m a , basal cell a d e n o m a , and myoepithelioma.
503
Strong reactivity also was observed in p o l y m o r p h o u s low grade a d e n o c a r c i n o m a . Low reacting tumors inc l u d e d primary and metastatic a d e n o i d cystic carcinoma, acinic cell carcinoma, m u c o e p i d e r m o i d carcinoma, and c a r c i n o m a ex p l e o m o r p h i c a d e n o m a . T h e s e results show a pattern o f stronger reactivity in the b e n i g n and low grade malignant tumors than in metastatic or high grade malignant tumors. Thus, this evidence suggests an inverse relationship between the p r e s e n c e o f IL-6 and the biological aggressiveness o f salivary gland tumors. This decrease o f IL-6 c o n t e n t in malignant salivary gland tumors may provide insight into the f u n c t i o n o f IL-6 in n o r m a l salivary glands and neoplastic change. These questions will guide o u r future investigations.
Acknowledgment. We thank Pauline Yau at the University of California Davis and Karen Mule at the University of Pittsburgh for their superb technical assistance. REFERENCES 1. Akira S, Taga T, Kishimoto T: Interleukin-6 in biology and medicine, in Dixon FJ (ed): Advances in Immunology, vol 54. San Diego, CA, Academic, 1993, pp 1-78 2. Hirano T, Akira S, Taga T, et al: Biological and clinical aspects of interleukin 6. Immunol Today 11:443449, 1990 3. Cox G, Gauldie J: Structure and function of interleukin-6, in Kunkel S, Remick DG (eds): Cytokines in Health and Disease. New York, NY, Dekker, 1992, pp 97-120 4. Scibienski RJ, Paglieroni T, Caggiano V, et al: Factors affecting the in vitro evolution of a myeloma cell line. Leukemia 6:940-947, 1991 5. Lu C, Kerbel RS: Interleukin-6 undergoes transition from paracrine growth inhibitor to autocrine stimulator during human melanoma progression. J Cell Biol 120:1281-1288, 1993 6. Takizawa H, Ohtoshi T, Ohta K, et al: Growth inhibition of human lung cancer cell lines by interleukin 6 in vitro: A possible role in tumor growth via an autocrine mechanism. Cancer Res 53:41754181, 1993 7. Gallo O, Bani D, Toccafondi G, et al: Characterization of a novel cell line from pleomorphic adenoma of the parotid gland with myoepithelial phenotype and producing interleukin-6 as an autncrine growth factor. Cancer 70:559-568, 1992 8. Gandour-Edwards R: Immunolocalization of interleukin-6 (IL-6) in salivary tumors. Otolargyngol Head Neck Surg 109:338, 1993 9. Tabibzadeh SS, Poubouris D, May LT, et al: Interleukin-6 immunoreactivity in human tumors. Am J Pathol 135:427433, 1989 10. Kawano M, Hirano T, Matsuda P, et al: Autocrine generation and essential requirement of BSF-2/IL-6 for human multiple myelomas. Nature 332:83-85, 1988 11. Miki S, Iwano M, Miki Y, et al: Interleukin-6 (IL-6) functions as an in vitro autocrine growth factor in renal cell carcinoma. FEBS Lett 250:607-610, 1989 12. Takizawa H, Ohtoshi T, Ohta K, et al: Growth inhibition of human lung cancer cell lines by interleukin 6 in vitro: A possible role in tumor growth via an autocrine mechanism. Cancer Res 53:41754181, 1993 13. Chen L, Mory Y, Zilberstein A, et al: Growth inhibition of human breast carcinoma and leukemia/lymphoma cell lines by recombinant interferon-ft. Proc Natl Acad Sci U S A 85:8037-8041, 1988 14. Gandour-Edwards R, Kapadia SB, Janecka IP, et al: Biologic markers of invasive pituitary adenomas involving the sphenoid sinus. Mod Pathol 8:160-164, 1995
(N : 3) as well as six of nine primary adenoid cystic carcinomas and all metastatic adenoid cystic carcinomas (N = 3). Carcinoma ex pleomorphic adenoma (N = 5) had three low and two moderate reactors. A pattern emerged in which the benign and low grade malignant tumors showed stronger reactivity than the metastatic or high grade malignant tumors. This suggests an inverse relationship between the presence of ID6 and the biological aggressiveness of salivary gland tumors. The function of ID6 in salivary gland neoplasia awaits further study and elucidation. HUM PATHOL 26:501503. Copyright © 1995 by W.B. Saunders Company Key words: salivary tumors; interleukin-6; pleomorphic adenoma; salivary gland; adenoid cystic carcinoma. Abbreviations: IL-6, interleukin-6.
Interleukin-6 (IL-6) is a multifunctional cytokine produced by a wide variety of cells that mediates inflammatory and immune responses. It is postulated to be the major regulator of the hepatic secretion of acute phase reactants, eg, C-reactive protein, and to modulate B-cell lymphocyte development, v4 Significantly, IL-6 also has been shown to function as a growth factor in several h u m a n tumors, including plasmacytoma, multiple myeloma, melanoma, and renal cell carcinoma. TM The precise mechanisms of growth regulation are the subject of ongoing investigation; however, both growth inhibition and growth stimulation have been observed in in vitro models. 4-7 Gallo et al 7 described IL-6 autocrine growth regulation of a cell line derived from a pleomorphic adenoma of the parotid gland. Interleukin-6 was detected by immunocytochemical staining of light microscopic sections. Immunoelectron cytochemistry demonstrated IL6 in the vicinity of the nucleus as well as scattered in the cytoplasm. A joilot study of 26 salivary tumors by Gandour-Edwards°demonstrated strong IL-6 reactivity in routinely processed tissue sections of resected pleoinorphic adenoma and other tumors. We elected to study a wide variety of benign and malignant salivary tumors to determine the range and extent of IL-6 expression by standard immunohistochemical methodology.
MATERIALS AND METHODS Resection specimens of salivary tumors were obtained f r o m the files of the D e p a r t m e n t of Pathology at the University of Pittsburgh. A total of 57 benign and malignant salivary tumors was studied, including p l e o m o r p h i c a d e n o m a (10), r e c u r r e n t p l e o m o r p h i c a d e n o m a (8), basal cell a d e n o m a (7), c a r c i n o m a ex p l e o m o r p h i c a d e n o m a (5), acinic cell carcin o m a (3), primary a d e n o i d cystic c a r c i n o m a (9), metastatic a d e n o i d cystic c a r c i n o m a (3), m y o e p i t h e l i o m a (5), mucoepide r m o i d carcinoma (3), and p o l y m o r p h o u s low grade adenoc a r c i n o m a (4). Adjacent sections of n o r m a l salivary gland were present in most cases. Paraffin sections were e x a m i n e d by an i m m u n o h i s t o c h e m i c a l t e c h n i q u e using a m o n o c l o n a l i m m u n o g l o b u l i n (IgG) antibody to h u m a n IL-6 (Genzyme, Cambridge, MA) at a dilution of 1:20. Formalin-fixed, paraffin-embedded cell blocks from an IL-6-secreting prostate cancer cell line (PC-3) were used as positive controls. Briefly, 5micron-thick sections were deparaffinized and rehydrated, and e n d o g e n o u s peroxidase was blocked with 3% hydrogen p e r o x i d e in absolute methanol. Sections then were heated for 10 minutes in distilled water in a microwave oven to optimize antigen exposure. After blocking with 10% normal e q u i n e serum, a 1-hour incubation at r o o m temperature with the primary m o n o c l o n a l antibody was p e r f o r m e d followed by a phosphate-buffered saline (PBS) rinse. A biotin-conjugated e q u i n e antimouse secondary antibody (Vector, Burlingame, CA) at a dilution of 1:1000 followed, and after PBS rinsing an avidin-biotin c o m p l e x (ABC Elite, Vector) was applied. After PBS rinsing diaminobenzidene-peroxidase (ScyTek
TABLE 1. From the Department of Pathology and the Division of Hematology and 0ncology, Department of Internal Medicine, University of California Davis, Sacramento, CA; and the Department of Pathology, University of Pittsburgh, Pittsburgh, PA. Accepted for publication August 29, 1994. Address correspondence and reprint requests to Regina Gandour-Edwards, MD, Department of Pathology, University of California Davis Medical Center, 2315 Stockton Blvd, Sacramento, CA 95817. Copyright © 1995 by W.B. Saunders Company 0046-8177/95/2605-000755.00/0
501
Interleukin-6 Immunohistochemical Staining in Benign Salivary Tumors Diagnosis
Low
Moderate
High
Pleomorphic adenoma (n = 10) Recurrent pleomorphic adenoma (n = 8) Basal cell adenoma (n = 7) Myoepithelioma (n = 5) Total (N = 30) (%)
--
--
10
1 1 2 4 (13)
2 4 3 9 (30)
5 2 -17 (57)
HUMAN PATHOLOGY TABLE 2.
Volume 26, No. 5 (May 1995)
Interleukin-6 I m m u n o h i s t o c h e m i c a l Staining in M a l i g n a n t Salivary Tumors
Diagnosis Carcinoma ex pleomorphic adenoma (n = 5) Adenoid cystic carcinoma (n = 9) Adenoid cystic carcinoma, metastatic (n = 3) Mucoepidermoid carcinoma (n = 3) Acinic cell carcinoma (n = 3) Polymorphous low grade adenocarcinoma (n = 4) Total (N = 27) (%)
Low
Moderate
High
3
2
--
6
2
1
3
--
--
3 3
---
---
-18 (67)
-4 (15)
4 5 (19)
Labs, Logan, UT) was applied to achieve a permanent color change in the reactive cells. Duplicate sections subjected to all phases of staining except that of the primary antibody served as negative controls. Sections were stained with hematoxylin and evaluated by light microscopy for reactivity.
FIGURE 2. Immunolocalization of IL-6 comparing the absence of staining in a carcinoma ex pleomorphic adenoma (A) with the cytoplasmic and nuclear staining of a pleomorphic adenoma (B). (Hematoxylin with DAB, original magnification × 100.)
I n t e r p r e t a t i o n o f i m m u n o h i s t o c h e m i c a l staining was p e r f o r m e d by all three pathologists (R.G.E., S.B.K., a n d L.B.). T h e reactivity was primarily cytoplasmic a n d g r a n u l a r in nature. In several t u m o r s the n u c l e u s app e a r e d stained, which we i n t e r p r e t e d to r e p r e s e n t overlying cytoplasm r a t h e r t h a n intrinsic n u c l e a r staining. T h e t u m o r s were evaluated as to the d e g r e e o f staining intensity using a 0 to 4 + scale a n d by the p e r c e n t a g e o f cells stained. T h e t u m o r s t h e n were classified into t h r e e g r o u p s r e p r e s e n t i n g low (0 to 1+, 0% to 30%), m o d e r a t e (2 to 3 + , 31% to 75%), o r high ( > 3 to 4 + , 76% to 100%) reactors (Tables 1 a n d 2). Strong cytoplasmic staining was observed in intercalated a n d striated ducts o f a d j a c e n t n o r m a l salivary tissue (Fig 1). All 10 o f the p r i m a r y p l e o m o r p h i c a d e n o -
mas d e m o n s t r a t e d strong granular, cytoplasmic, a n d n u c l e a r reactivity in b o t h the epithelial a n d myoepithelial cells, whereas the s t r o m a was unstained. In comparison, five o f eight r e c u r r e n t p l e o m o r p h i c a d e n o m a s were strong reactors, whereas two were m o d e r a t e a n d o n e was low. A contrast was n o t e d in the three specim e n s o f c a r c i n o m a ex p l e o m o r p h i c a d e n o m a in that three showed a low d e g r e e a n d two s h o w e d a m o d e r a t e d e g r e e o f cytoplasmic staining (Fig 2). Two o t h e r benign tumors, basal cell a d e n o m a a n d m y o e p i t h e l i o m a , s h o w e d p r e d o m i n a t e l y m o d e r a t e cytoplasmic staining. Six o f nine p r i m a r y a d e n o i d cystic carcinomas were low reactors as were all three o f the metastatic a d e n o i d cystic lesions. A c o m p a r i s o n between the p r i m a r y a n d metastatic lesions was available for only o n e o f the patients a n d virtually n o difference in staining was noted. In contrast, however, all f o u r p o l y m o r p h o u s low grade a d e n o c a r c i n o m a s were strongly reactive (Fig 3). T h e high g r a d e m u c o e p i d e r m o i d c a r c i n o m a s (N = 3) a n d
FIGURE 1. Immunolocalization of IL-6 in normal salivary tissue. Intense granular cytoplasmic staining is present in the ducts, whereas the acinar cells and adipose cells are negative. (Hematoxylin with DAB, original magnification x 100.)
FIGURE 3. Immunolocalization of IL-6 comparing the strong cytoplasmic staining of a polymorphous low grade adenocarcinoma (A) with the absence of staining in an adenoid cystic carcinoma (B). (Hematoxylin with DAB, original magnification x 100.)
RESULTS
502
IL-6 IN SALIVARY TUMORS (Gandour-Edwards et al)
the low grade acinic cell carcinomas (N = 3) were all low reactors. In summary, four (13%) of the benign tumors were low reactors, nine (30%) were intermediate, and 17 (57%) were highly reactive with the monoclonal antibody to IL-6. O f the malignant tumors, 18 (67%) reacted weakly, four (15%) reacted moderately, and five (19%) reacted strongly to anti-IL-6.
DISCUSSION Interleukin-6 is a multifunctional cytokine that is p r o d u c e d by a wide variety of cells and by a growing n u m b e r of h u m a n tumors. Tabibzadeh et al 9 observed IL-6 staining in 30 different h u m a n tumor types and proposed that the cytokine plays a major role in regulating local and systemic host-tumor interaction. Interleukin-6 has been shown to induce acute-phase protein production by hepatocytes, to p r o m o t e the growth and differentiation of B and T lymphocytes, and to enhance the activation of natural killer cells? An examination of the role of IL-6 in neoplastic growth control has shown a diverse range of behaviors. In plasmacytoma, multiple myeloma, and renal cell carcinoma, IL-6 functions as an autocrine growth stimulator. 1'4'1°-n In contrast, in lung and breast carcinoma its behavior is inhibitory to growth, lz'l~ A recent report by Lu and Kerbel 5 describes the interesting finding that exogenous IL-6 inhibited the growth of early stage (metastatically incompetent) melanoma cells, but that advanced stage (metastatically competent) cells were not inhibited. Furthermore, several advanced stage melan o m a cell lines p r o d u c e d IL-6, which functioned as a growth stimulator. This suggests that cells can make the transition from responding to IL-6 as a paracrine growth inhibitor to responding to IL-6 as an autocrine growth stimulator during carcinogenesis. We recently examined IL-6 staining in 10 invasive pituitary adenomas involving the sphenoid sinus and c o m p a r e d the results with those of 10 noninvasive pituitary adenomas and five normal pituitary glands. 14 Our study demonstrated strong expression of IL-6 in normal glands with cytoplasmic reactivity in all cell types. Interleukin-6 staining was diminished in the noninvasive adenomas with only two showing moderate cytoplasmic reactivity. However, five (50%) of the invasive adenomas demonstrated strong granular cytoplasmic staining, suggesting a change in cellular function. We propose that in invasive pituitary adenomas IL-6 may u n d e r g o a transition from a normal cell regulator of secretion to an autocrine growth stimulator. T h e results o f this study of salivary gland tumors d e m o n s t r a t e d m o d e r a t e to strong IL-6 staining in n o r m a l salivary gland ducts and several b e n i g n tumors, including primary and r e c u r r e n t p l e o m o r p h i c a d e n o m a , basal cell a d e n o m a , and myoepithelioma.
503
Strong reactivity also was observed in p o l y m o r p h o u s low grade a d e n o c a r c i n o m a . Low reacting tumors inc l u d e d primary and metastatic a d e n o i d cystic carcinoma, acinic cell carcinoma, m u c o e p i d e r m o i d carcinoma, and c a r c i n o m a ex p l e o m o r p h i c a d e n o m a . T h e s e results show a pattern o f stronger reactivity in the b e n i g n and low grade malignant tumors than in metastatic or high grade malignant tumors. Thus, this evidence suggests an inverse relationship between the p r e s e n c e o f IL-6 and the biological aggressiveness o f salivary gland tumors. This decrease o f IL-6 c o n t e n t in malignant salivary gland tumors may provide insight into the f u n c t i o n o f IL-6 in n o r m a l salivary glands and neoplastic change. These questions will guide o u r future investigations.
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