American Journal of Pathology, Vol. 138, No. 3, March 1991 Copyrght C) American Association of Pathologists

S-100 Protein Antibodies Do Not Label Normal Salivary Gland Myoepithelium Histogenetic Implications for Salivary Gland Tumors

Neoplastically modified myoepithelial cells have a key role in developing the histologic characteristics of some salivary gland tumors. S-100 protein expressed in certain of these tumors is suggested to support this role, as the principal component in the human salivary gland reported to be S-100 proteinpositive is myoepithelium. Confirmation of such an important aspect is required. Immunoperoxidase staining of parotid salivary gland shows considerably different patterns obtained with antibodies to S-00protein, neuron-specific enolase, and neurofilaments compared with those for muscle-specific actin and cytokeratin 14; many more cells and their processes associated with acini and ducts are evident with the latter two antibodies. Double immunofluorescent staining with antibodies to either S-100 protein or neuron-specific enolase combined with muscle-specific actin does not reveal colocalization of these antigens in myoepithelial cells. The former localize only to nerve fibers adjacent to, but separate from, acini, and the latter only to myoepithelial cells. It is apparent that S-100 protein staining of the rich network of unmyelinated nerves in the interstitial tissues, evident ultrastructurally, has been misinterpreted as myoepithelium. This result has important implications for histogenetic classifications of salivary gland tumors. (AmJPathol 1991, 138:619-628)

protein composed of a,B, gp, and aa, respectively.4 Interesting variations in the staining patterns of tissues or their component cell types are observed, depending on the use of polyclonal or monoclonal antibodies to S-1 00 protein or its various subunits.'9 Indeed this applies to salivary gland, where S-100,B is stated to preferentially localize to the myoepithelial cell, while antibodies to S-1OO0 more often label ducts or acini.410 Detection of S-100 protein in certain tissues or cells has made antibodies to this protein of use diagnostically in general11 1 and in the salivary gland in particular.7 12-l4 Because of the essential role of the myoepithelial cell in salivary gland pathology,1-19 the expression of S-100 protein in these tumors is claimed to have major histogenetic implications. 14 To draw conclusions from immunohistochemical results, particularly with respect to histogenetic aspects of tumors, the specificity of staining patterns in normal tissues must be determined with certainty. Is S-100 protein definitely present in the myoepithelium of normal salivary gland? Immunohistochemical studies give conflicting results. Myoepithelial cells are recorded as being positively stained in some studies,2,3,10,11,20,21 but were reportedly negative in other investigations of salivary gland,9'1921,22 sweat gland,823 and breast.24 Certain statements by Zarbo et al,25 as part of an extensive study of salivary gland neoplasms, perhaps best illustrate the staining pattern observed in normal salivary gland: 'The S-100 protein immunostaining in normal salivary glands showed considerable variability. In most glands, myoepithelium stained, but at varying intensities.' Haimoto and associates4 also noted that antibodies to S-1 00b stained some myoepithelial cells in the parotid gland. Our experience has been similar. More specific markers for salivary gland myoepithelial

S-100 protein, although originally isolated from brain tissue, has since been shown to be present in a wide variety of tissues and cell types using immunohistochemical techniques.1" Three forms constitute this protein, S100a, S-100b, and S-100a0, each of which is a dimeric

Supported by a grant from the Conn Smythe Foundation, Toronto, Ontario, and by the Charlie Conacher Cancer Research Fund, Toronto General Hospital Foundation, Toronto, Ontario, Canada. Accepted for publication October 22, 1990. Address reprint requests to Dr. I. Dardick, Department of Pathology, Banting Institute, Room 072, 100 College St., Toronto, Ontario, M5G 1 L5, Canada.

Irving Dardick,* Michael Stratis,* William R. Parks,t Franco G. DeNardi,t and Harriette J. Kahnt From the Departments of Pathology, Toronto General Hospital,* Toronto; Ottawa Civtic Hospital,t Ottawa; and Women's College Hospital,t Toronto, Ontario, Canada

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cells, such as muscle-type actin26 30 and cytokeratin 14,31 34 are currently available. We have taken advantage of this specificity to investigate the location of S-1 00 polypeptides in human parotid gland using the technique of double immunofluorescence labeling.

Materials and Methods The superficial lobe of nine adult human parotid salivary gland was obtained at the time of surgery for benign pleomorphic adenomas. Some portions of this tissue remote from the tumor were fixed both in methanol/acetic acid (95:5 by volume) and 10% buffered formaldehyde for 18 to 24 hours before embedding in paraffin, while other pieces were placed in Beem capsules containing OCT medium (Lab-Tek, Naperville, IL) and snap frozen in liquid nitrogen. Additional salivary gland tissue was diced and immediately fixed in 2.5% glutaraldehyde in 0.1 mol/l (molar) sodium cacodylate buffer for 24 hours before embedding in epon-araldite resin. Human control tissues consisted of sural nerve biopsies, cortical areas of brain (obtained at autopsy), and fetal cartilage, all of which were snap frozen and fixed in both methanol/acetic acid and formalin.

(1:50) and anti-cytokeratin 14 (1:50), ant[-S-1 00 protein (1:50) and anti-neurofilament (1:20) and antineuron-specific enolase (1:50) and HHF35 (1:2500) were applied to sections for 30 minutes before washing with phosphate-buffered saline (PBS). This was followed by a solution containing anti-rabbit IgG tagged with rhodamine (1:50: supplied by Biocan, Mississauga, Ontario) and an anti-mouse IgG/fluorescein combination (1:50; Bio/Can, Mississauga, Ontario) for 30 minutes before washing in PBS and coverslipping using an aqueous 50% glycerol/PBS solution containing 0.1% p-phenylenediamine. Sections were viewed and photographed using a Leitz epifluorescent microscope equipped with appropriate filters.

Electron Microscopy Epon-araldite blocks containing acini and ducts from three of the normal salivary glands were sectioned on an ultramicrotome and mounted on copper grids. After staining with uranyl acetate and lead citrate, the sections were surveyed using a Philips EM400 at 60 kV. Areas in which small nerve fibers were seen adjacent to acini were

photographed.

Immunohistochemistry An indirect immunoperoxidase technique was used to immunostain both alcohol- and formalin-fixed salivary gland tissue using the following antibodies: 1) anti-rabbit S-100 protein (Dako Corporation, Santa Barbara, CA; 1:200); 2) anti-rabbit neuron-specific enolase (Dako Corporation; 1:300); 3), anti-mouse neurofilament (Dako Corporation; clone 2F1 1, 1:20 to 1:50); 4), anti-mouse cytokeratin 14 (CK 14, supplied by Sigma Chemical Co., Mississauga, Ontario; 1 :100); and 5), HHF35, an anti-mouse muscle-specific actin (Enzo Biochemical, New York; 1:2500). Primary antibodies were applied for 1 hour at room temperature, followed by an incubation of either anti-mouse or anti-rabbit immunoglobulin fractions conjugated with horseradish peroxidase (Bio/Can, Mississauga, Ontario) for 45 minutes. The chromagen was 3,3'-diaminobenzidine.

Immunofluorescent Microscopy Deparaffinized sections of methanol/acetic acid-fixed salivary gland tissue were simultaneously double-labeled with combinations of polyclonal and monoclonal antibodies as previously described.336 Mixtures of antiKS-100 protein (1:50) and HHF35 (1:2500), anti-S-100 protein

Control Procedures Portions from the same specimens of normal salivary gland, peripheral nerve, and brain were fixed both in

methanol/acetic acid and in formalin and then all six samples were embedded in the same paraffin block. Sections from this block were stained using a polyclonal antibody to S-100 protein (Dako Corporation) and an indirect immunoperoxidase procedure. In addition, frozen sections were cut from normal parotid, normal nerve, and skin containing nevus or melanoma cells and mounted on the same slide. To assess if methanol/acetic acid fixation resulted in extraction of S-100 protein, some slides were fixed for 10 minutes in either acetone, methanol/ acetic acid (95:5), or buffered formalin, while other slides were fixed in methanol/acetic acid for 30 minutes, before immunostaining. Indirect immunoperoxidase (IMP) and immunofluorescent (IMF) staining of additional sections of methanol/acetic acid-fixed normal parotid gland used monoclonal antibodies to the subunits of S-100 protein (anti-S-100a from Dako Corporation [IMP at 1:200 and IMF at 1:20] and anti-S100 protein antibody predominantly recognizing the p-subunit from the Hospital for Sick Children, Toronto, Ontario [IMP at 1:200 and IMF at 1:20]).

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Results

Immunohistochemistry Figures 1 and 2 allow a comparison of the staining patterns obtained in the parotid salivary gland with antibodies to S-100 protein (Figure 1), neuron-specific enolase (Figure 2A), muscle-specific actin (HHF35) (Figure 2B), and cytokeratin 14 (Figure 2C). Using ant4-100 protein antisera, major nerve bundles, as well as moderate to small-sized branches, adjacent to large excretory ducts and blood vessels, were strongly stained (Figure 1 A). Within the lobules of the salivary gland parenchyma, there were a few small, round to elongated patches of positively stained tissue between but closely applied to the acini and striated ducts (Figure 1 B). An identical pattern of parenchymal staining was

Figure 1. Normal parotid gland. A: AntiS-100 protein. This antibody strongly stains the major nerves in the interlobular tissues, but at this magnification, the location of staining within the gland parendrnyma is difficult to appreciate. B: Anti-S-400 protein. At higher magnification, there is staining of linear- to triangular-shaped structures (arrows) adjacent to acini. Immunoperoxidase with hematoxylin counterstain (A, x 130; B, X325).

obtained with the antibody to neuron-specific enolase (Figure 2A), and the interlobular nerves (Table 1) were also positive. The antibody to neurofilament polypeptides strongly stained nerve fibers associated with interlobular and larger intralobular ducts, but it was difficult in most cases to detect any staining in the tissues associated with the acini and smaller ducts; only focal staining could be appreciated in the occasional sample of parotid tissue (Table 1). However the staining patterns obtained with both anti-muscle-specific actin (Figure 2B) and anticytokeratin 14 (Figure 2C) were completely different. Not only were there more crescent-shaped cells associated with acini and intercalated ducts, but a considerable number of fine cytoplasmic processes of myoepithelial cells were readily apparent. By comparison, the features and distribution of these cells were different from the staining patterns evident for S-100 protein and neuronspecific enolase (Figures 1 and 2A), and the impression

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Figure 2. Normal parotid gland. A: Antineuron-specific enolase. Mainly linear structures (arrows) are stained in a distribution pattern similar to that obtained with S-100 protein. B: Anti-muscle-specific actin. Acini are encircled partially by many more crescent-shaped structures then are evident with either S-100 protein or neuron-specific enolase. In addition, linear and triangularly shaped cells (arrowheads) are stained on the outer aspect of striated ducts. C: Anticytokeratin 14. The pattern and distribution of staining is that evident with muscle-specific actin antibody. Immunoperoxidase with hematoxylin counterstain (A, B, and C, X325).

gained was that the latter peptides were localized to small nerve fibers in the interstitial tissue. The results of staining with the series of antibodies used in this part of the study are summarized in Table 1.

Immunofluorescence Microscopy Figure 3 displays portions of typical fields of human parotid salivary gland immunostained with a combination of

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Table 1. Immunostaining Patterns of Myoepithelial Cells and Nerves in Normal Parotid Salivary Glands (n = 9) Tissue Component Minor Major Myoepithelial Antibody nerves nerves cells to* + to ++ + to ++ NSE + -to +t S-100 + -$ NF ++ _ CK14 ++ MSA * NSE, neuron-specific enolase (monoclonal); S-100, S-100 protein (polyclonal); NF, neurofilament (monoclonal); CK 14, cytokeratin 14 (monoclonal); MSA, muscle-specific actin (monoclonal).

t S-100 protein staining of nerves associated with acini and small ducts was readily seen using immunofluorescent microscopy, but difficult to appreciate in immunoperoxidase-stained sections in most cases. t Nerves associated with acini and intercalated ducts were difficult to visualize by either immunoperoxidase or immunofluorescent techniques.

fluorescein isothiocyanate (FITC)-tagged musclespecific actin antibody (yellow fluorescence) and rhodamine-labeled S-100 protein antibody (red fluorescence). Interlobular blood vessels and nerves served as independent positive controls and disclosed no colocalization of these antigens in either structure (a lack of green fluorescence) (Figure 3A). The many cytoplasmic processes of myoepithelial cells ringing each salivary gland acinus were strongly decorated by the antimuscle-specific actin (Figure 3B). The identical fluorescent coloration of myoepithelium and the smooth muscle cells of blood vessels again indicated the absence of S-100 protein in the former. When compared with the staining of the interlobular nerves, the red fluorescence associated with the S-1 00 protein antibody was confined to small nerves closely associated with the periphery of some acini and was distinct from the alternatively labeled myoepithelial cells (Figure 3B). The specificity of S-100 protein antibody for nerves (Figure 3C) was shown by double immunofluorescence labeling with an antibody to neurofilament polypeptides (Figure 3D). A higher magnification of parotid acini cophotographed with FITC-labeled muscle-specific actin antibody and rhodamine-tagged S-100 protein antibody verified the independent localization of each of these antigens (Figure 3E); fine terminal branches of autonomic nerves were closely applied to the periphery of acini (Figure 3E). The actin-rich myoepithelial cell processes were not only located to one side of these nerves, but they definitely did not express any S-100 protein (Figure 3E). Similar results were obtained with the combination of muscle-specific actin and neuron-specific enolase antibodies (Figure 3F), whereas the mixture of musclespecific actin and cytokeratin 14 antibodies did show colocalization to myoepithelial cells associated with acini

and intercalated and striated ducts, as reported

previously.35 36

Electron Microscopy Based on the information obtained from both the immunoperoxidase and immunofluorescent studies, three of the normal parotid glands were surveyed ultrastructurally. In each of these, unmyelinated nerves were readily detected both in cross (Figure 4A) and longitudinal (Figure 4B) section. Such nerves were located in the interstitial tissues associated with both blood vessels and acini. The nerves, however, were definitely separated from both structures and external to the basal lamina of the acini and their bordering myoepithelial cell processes (Figure 4). It should be noted that the distance separating the nerves and acini can be as little as 300 nm (Figure 4B).

Control Tissues S-100 protein antibodies stained glial and Schwann cells in normal brain and nerve, respectively, in both alcoholand formalin-fixed tissues. Only the major nerves were consistently stained in normal salivary gland (included with the above tissues) using both fixatives: in $ome cases, a small amount of staining was seen associated with acini in a linear, crescentic, or round pattern, using anti-S-100 protein antisera especially with formalin-fixed tissue, but no staining reminiscent of myoepithelial cells was evident. To further assess the effect of methanol fixation on S-100 protein staining, frozen sections of salivary gland, nerve, and skin containing nevus or melanoma cells were fixed in either formalin, methanoVacetic acid, or acetone and stained for immunofluorescent microscopy with both monoclonal and polyclonal anti-S-i 00 protein antibodies. In all cases, staining of nerve or nevus and melanoma cells was evident, although the intensity was always somewhat greater with formalin-fixed tissues compared with both methanol/acetic acid and acetone fixation. In small nerve fibers, the nuclei of Schwann cells were particularly intensely fluorescent.

Discussion Immunohistochemical staining patterns for a wide variety of antigens found in the duct and acinar cells of salivary gland tissue are often used to imply histogenetic bases for salivary gland tumors. S-1 00 protein is a case in point, particularly because of the key role that the myoepithelial cell has in developing the characteristic histologic appearances of these tumors 145,1 17,18,37

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Some reports have emphasized the histogenetic and prognostic role for myoepithelial cells, in terms of the classification of salivary gland tumors, based on the suggested staining of these cells by antibodies to S-100 protein.14'15'25 It would appear that such conclusions are unwarranted. As we have demonstrated, autonomic nerves are the only S-100 protein-containing structure in the parotid gland. Not only were the immunofluorescent microscopy results obtained by staining normal parotid gland with neuron-specific enolase antibody remarkably similar to those obtained with all S-100 protein antibodies, but just as with neuron-specific enolase, no colocalization of S-100 protein occurred to the muscle-specific actincontaining myoepithelial cells. It is apparent that the proximity of terminal nerve fibers to and their alignment with the periphery of acini led to confusion with myoepithelium. In reviewing the results that we11'20 and others,2'4'10'21 have reported, it is evident that the figures detailing S-100 protein immunoperoxidase staining in these studies are similar to those in the current illustrations. The original misinterpretation has thus been perpetuated. Although S-100 protein staining patterns were originally interpreted as myoepithelial cells, the results with more definitive markers, muscle-specific actin and cytokeratin 14, demonstrate that this is incorrect. Surveying the parotid gland in the current series does not reveal even a subpopulation of myoepithelial cells with a combined expression of S-100 protein and muscle-specific actin. In contrast, the myoepithelial/basal cells of striated ducts are a mixed population; some are actin positive and others are not.-'31Basal cells associated with excretory ducts are also heterogeneous in terms of interme0 In both locations, diate filament expression.3 however, S-100 protein-positive cells are not detected. The absence of S-100 protein in myoepithelial cells and the complexity of myoepithelial/basal cells in the duct system in salivary gland,' 3441 including the presence of actin-positive myoepithelium in striated ducts,33.42 means that many aspects of histogentic concepts for salivary gland tumors must be reevaluated. Histologic assessment of nerve fibers in the salivary gland does not appear to have been well documented, although the gland is well endowed with sympathetic and -4

parasympathetic nerves.435 Electron microscopy of parotid gland confirmed the relatively rich network of nonmyelinated nerves associated with acini and ducts, a feature also demonstrated by immunostaining for S-1 00 protein, neuron-specific enolase, and neurofilament polypeptides. Of these proteins, neuron-specific enolase gave the most consistent and reliable staining of these small nerves, but all three strongly stained larger, particularly interlobular, nerves. S-100 protein was more reliably demonstrated in small nerves using immunofluorescent techniques, probably because of its better sensitivity along with increased concentrations of the antibody. Nevertheless, staining was detected at least focally in a proportion of samples whether fixed in methanol or formalin, but particularly in the latter. Perhaps the failure of both neurofilament and S-100 protein antibodies to consistently stain the small, peripheral branches is due to the limited complement of intermediate filaments and other polypeptides in the few axons comprising these nerves. Indeed S-100 protein is preferentially localized to the nuclei of Schwann cells in terminal nerve fibers. Frozen and fixed tissues, various fixatives, and a variety of polyclonal antibodies to S-100 protein and its a and ,B subunits were used as controls to ensure that S-100 protein was not extracted by methanol/acetic acid or that certain components of S-100 protein were not specifically localized to the myoepithelium. Rather than supporting a role for myoepithelial cells in certain human salivary gland tumors, the lack of S-100 protein in the normal secretory and ductular tissues reflects an equally important aspect of neoplasms, ie, the acquisition of ectopic proteins. Pleomorphic adenoma and myoepithelioma are lesions in which S-100 protein7,9,13.14,19-22.25.46.47 and glial fibrillary acidic protein222536.48 have been demonstrated immunohistochemically and even by other immunologic techniques.21 2549 It is essential to note that not all tumor cells in these lesions express such proteins. As well, the myoepithelial cell component of such tumors show many structural and functional alterations when compared with its normal counterpart.36'50'51 Emphasis needs to be placed on understanding the genetic mechanisms involved in producing the morphology of these neoplasms rather than concern for a particular cell of origin. There is

Figure 3. Normalparotidgland, double immunofluorescence labeling. A: Double-exposure micrograph of interlobular tissues showing that with a combination of anti-muscle-specific actin (FITC) and anti-S-100 protein (rhodamine), the former is confined to smooth muscle cells of blood vessels (open arrow) and the latter to nerves (arrow). B: Double exposure of gland parenchyma in which the rich network of autonomic nerves is evident at low magnification. The actin-containing myoepithelial cells (FITC) are separated clearlyfrom the rhodaminelabeled small nerves (anti-S-100 protein antiserum) bordering the acini. A glancing section of one acinus (arrow) reveals a myoepithelial cell, with its cytoplasmic extensions, only decorated by the muscle-specific actin antibody. Note the branching of one nervefiber (open arrow). C: Double immunolabelingfor S-100 protein (rbodamine) and neurofilament proteins (FITC). Single exposure with S-100 protein localized to three interlobular nerves (arrows). D: Single exposure of the same area as in C. The FITC-tagged neurofilament protein antibody has exactly the same localization as the antibody to S-1OO protein. E: Double exposure for neuron-specific enolase (rhodamine) staining of interlobular nerves and muscle-specific actin (FITC) labeling a large interlobular artery. F: Double exposure for neuron-specific enolase (rhodamine) and muscle-specific actin (FITC). The pattern for neuron-specific enolase is identical to thatfor S-1OO protein. The antibody to muscle-specific actin produces the same coloration and distribution as detected when this antibody is combined wih the anti-S-100 protein antiserum as in A and B (A, C, D, and E, x600; B, x350; F, xi100).

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Figure 4. Normalparotid gland. A: A cross section of an unmryelinated nerve (N) ispresent in the angleformed by two acinar cells (A) and a capillay (C). One axon of the nerve contains membrane-bound secretory granules (arrow); these are enkarged in the inset. Thefilamentladenedprocesses of a myoepithelial cell (M) border the acinar cells, both of which are lined by a narrow basal lamina (arrowbeads). B: An unmyelinated nerve (N) runs parallel to the surface of adjacent, zymogen-containing (Z) acinar cell. The nerve comes within 300 nanometers of the acinus (arrowhead), which again displays extensions of myoepithelial cells (arrows) (A, x 14,800 (inset, X20,000); B, x 10,000).

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support for a morphologic connection between various lesions,15'17'18 but no evidence that the specific cells involved in the induction of these tumors92 has any value in classification. Thus the absence of S-100 protein in normal myoepithelial cells does not negate a major role for neoplastic myoepithelium, variably modified both cytologically and functionally with no set pattern even within any one subtype, in salivary gland tumors. Whatever diagnostic role the expression of S-100 protein may have in these tumors needs further investigation, but the presence or absence of this particular peptide cannot be used to evoke a histogenetic classification for salivary gland tumors.

Acknowledgments The authors thank Ken Ekem, Sayoko Yamada, Luitgard Weyer, and Laura Boutilier for technical assistance.

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