Histochem Cell Biol (2000) 113:241–245
© Springer-Verlag 2000
O R I G I N A L PA P E R
S. Heilmann · T. Hummel · F.L. Margolis · M. Kasper M. Witt
Immunohistochemical distribution of galectin-1, galectin-3, and olfactory marker protein in human olfactory epithelium
Accepted: 20 December 1999
Abstract The expression pattern of galectin-1 and galectin-3 in the human olfactory epithelium was investigated in relation to olfactory marker protein (OMP) using confocal laser immunofluorescence in human specimens and postmortem biopsies. OMP expression was found in olfactory receptor neurons (ORNs) in the olfactory mucosa and in fibers of the olfactory nerve crossing the submucous connective tissue. Galectin-1 was expressed in both the connective tissue of the nasal cavity and in the basal layer of the olfactory epithelium. In contrast, galectin-3 expression was limited to cells of the upper one-third of the olfactory epithelium. Expression of galectin-3 occurred in a subset of OMP-positive cells. However, between areas of galectin-1 and galectin-3 expression in the lower and upper portion of the epithelium, OMP-positive ORNs did not stain for both galectins. Considering the potential role of galectin-1 and galectin3 in cell differentiation and maturation, the differential localization of galectins in the olfactory epithelium appears to be consistent with a significant role of these molecules in the physiological turnover of ORNs.
Introduction The cellular characteristics of the olfactory epithelium have already been described in great detail, especially with the extensive use of electron microscopy. However, S. Heilmann · T. Hummel (✉) Department of Otorhinolaryngology, University of Dresden Medical School, Fetscherstrasse 74, 01307 Dresden, Germany e-mail:
[email protected] Tel.: +49-351-4584189, Fax: +49-351-4584326 F.L. Margolis Department of Anatomy and Neurobiology, University of Maryland at Baltimore School of Medicine, 685 West Baltimore Street, Baltimore MD 21201, USA M. Kasper · M. Witt Department of Anatomy, University of Dresden Medical School, Fetscherstrasse 74, 01307 Dresden, Germany
although the cellular subpopulations of this particular epithelium are identified, their functional significance is not yet fully understood and the nature of their interactions is only described to a certain extent, for example, with regard to the regenerative capacities of the olfactory epithelium (Farbman et al. 1998). To characterize intraepithelial cell populations, olfactory marker protein (OMP) was used to identify olfactory receptor cells. This protein has been shown to be a reliable marker for olfactory cells (Buiakova et al. 1996; Getchell et al. 1993; Lee and Pixley 1994; Pixley 1996; Ring et al. 1997). It is regarded as a molecule being solely expressed by cells involved in the perception of odorants, and it allows the discrimination between olfactory receptors and basal/supporting cells. Recent studies indicate that OMP may play a role in neurogenesis in olfactory tissue (Farbman et al. 1998). There is evidence for OMP playing a physiological role in olfactory processing (Buiakova et al. 1994; Youngentob and Margolis 1999), the exact mechanism yet being unknown. Due to their regulatory functions and modulating effects in various tissues (Perillo et al. 1998), galectins, a family of endogenous lectins, have been the subject of intensive research in recent years. Their appearance in a wide range of organisms has led to insights into mechanisms of cell regulation (Zanetta et al. 1994). Effects of the members of this lectin family range from cell maturation to apoptosis. They are involved in the mediation of cell adhesion and, seemingly, in the regulation of celldependent cytotoxicity (Truong et al. 1993). Effects of lectins and, in particular, galectins are highly differentiated. Some of their effects even appear to be designed in an antagonistic manner. For example, galectin-1 appears to play a role in differentiation and maturation. It is expressed in developing brain tissue, but not in mature brain (Joubert et al. 1989). In contrast, galectin-3 seems to mark a mature cell population (Gillenwater et al. 1996). Reduced galectin-3 expression was found in carcinomas with little differentiation, i.e., greater aggressive potential and higher malignancy (Bresalier et al. 1997; Castronovo et al. 1996; Lotz et al. 1993). Thus, it has
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been hypothesized that both galectin- 1 and galectin-3 are associated with cell differentiation. Since galectin-3 has been reported in non-human material to promote: (1) cell proliferation, (2) neural cell adhesion, and (3) neurite growth, as well as being an antagonist to galectin-1 in certain cell models and thus (4) protecting cells from apoptosis, we were interested in the expression of both molecules in the human olfactory epithelium. This epithelium is known to regenerate constantly which is exceptional for neuronal tissue (compare Cagrassi and Graziadei 1995).
plied for 1 h at RT, followed by an incubation with corresponding FITC or Texas red-labeled secondary antibodies (1:100; Dianova, Hamburg, Germany). The sections were mounted with PBSgelatin and analyzed in a confocal laser scanning microscope (Leica, Bensheim, Germany). The following controls were carried out: (1) omission of the primary antibody in order to rule out non-specific binding of the secondary antibody and (2) parallel incubation of tissue, usually rat olfactory epithelium, previously reported to be immunopositive to the markers tested (Mahanthappa et al. 1994; Tenne-Brown et al. 1998).
Results Materials and methods
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We examined biopsies taken from human olfactory mucosa of ten healthy volunteers (six males, four females, age range 18–90 years, mean age 46 years) who underwent nasal surgery under general anesthesia after written consent was given. In addition, material from six cadavers (volunteer donors from the Department of Anatomy; one male, five females, age range 68–89 years, mean age 82 years). The study design was approved by the Ethics Committee of the Dresden University Medical School. In both cases, tissue was fixed in 4% buffered formaldehyde, dehydrated, and embedded in paraffin. Sections were cut (5–10 µm) and mounted on silane-coated slides. Immunohistochemistry was performed using: (a) the avidin-biotin-peroxidase technique (Hsu et al. 1981) and (b) indirect immunofluorescence. Deparaffinized sections were pretreated with microwaves (800 W) and exposed to 0.3% H2O2 for 30 min in order to block endogenous peroxidases. Then the sections were incubated with goat antiserum against OMP (Keller and Margolis 1975), diluted 1:5000 in phosphate-buffered saline (PBS), pH 7.2, containing 0.7% bovine serum albumin for 1 h at room temperature (RT). The antibody reacts against rodent OMP and crossreacts with human OMP (Buiakova et al. 1996). After washing in PBS, the sections were exposed to a rabbit anti-goat biotinylated IgG (Vectastain Elite kit; Vector, Burlingame, Calif., USA) for 1 h at RT. Polyclonal antibodies against protein gene product 9.5 (PGP 9.5; Biotrend, Cologne, Germany; 1:2000) and galectin-1 (Mahanthappa et al. 1994; 1:800) were detected with a swine anti-rabbit IgG (Dako, Copenhagen, Denmark; 1:400). The reaction product was visualized with an avidin-biotin-peroxidase complex (Vector) followed by incubation with 0.3% diaminobenzidine/H2O2. The sections were counterstained with hematoxylin. For colocalization studies, a double immunofluorescence technique was applied as follows. The sections were incubated overnight with OMP (1:400). After rinsing, antibodies against galectin-1 (1:80), galectin-3 (Novocastra, Newcastle upon Tyne, UK; 1:20), or PGP 9.5 (1:200) were each apFig. 1 a Galectin-1 in basally situated cells of the human olfactory epithelium (arrows). Scale bar 15 µm. b Double immunoreaction with antibodies against olfactory marker protein (OMP; Texas red fluorescence) and galectin-3 (FITC). Olfactory receptor neurons reactive to OMP are situated predominantly in the middle layer of the epithelium. Some cells are immunopositive for both markers (arrows, yellow). Section adjacent to a. Scale bar 10 µm Fig. 2 a Galectin-1 is expressed in basal cells of the olfactory epithelium on the basement membrane (arrows) as well as in a variety of subepithelial cells, for example, fibrocytes and endothelial cells. b Galectin-3 is expressed in slender cells with nuclei in the middle layer of the epithelium (as shown in Fig. 1b). Scale bar 10 µm. c OMP is detected mainly in perikarya of the lower onethird of the epithelium. Individual perikarya migrate immediately underneath the apical boundary of the epithelium. d The control (omission of primary antibody) showed no reaction. Counterstain with hematoxylin. Scale bar 10 µm
Olfactory receptor neurons (ORNs) and non-olfactory nerve fibers were identified by positive immunohistochemical reaction with PGP 9.5. As expected, OMP expression was located in intraepithelial ORNs. Immunoreactivity (IR) occurred in cells situated near the basal layer, in clearly marked ORN perikarya in the middle part of the epithelium located between the nuclei of basal and supporting cells. In addition, the dendrites of ORNs were found to be OMP positive. While ORNs were clearly identified by their OMP reactivity, striking differences were seen for the galectins examined. Galectin-1 was expressed in the connective tissue underlying the olfactory mucosa and in the basal layer of the olfactory epithelium (Figs. 1, 2). Galectin-3 expression was limited to nuclei and cytoplasm of both the middle and the upper one-third of the olfactory epithelium. OMP-positive cells in the area beyond the nuclei of the supporting cells were clearly reactive for galectin-3, while ORNs in the basal section of the mucosa showed no galectin-3 expression (Figs. 1, 2). Between both areas of galectin expression, OMP-positive ORNs were devoid of either one of the investigated galectins (Fig. 1). Controls omitting the secondary antibody rendered negative results (Fig. 2d).
Discussion Galectin-1 has been shown to be involved in the development of the rodent olfactory system (Puche and Key 1995; Puche et al. 1996; Raabe et al. 1997; Tenne-Brown et al. 1998); so far, no corresponding data have been reported for humans. Galectin-1 was reported to support fasciculation and adhesion in the mouse olfactory system (Tenne-Brown et al. 1998). In fact, it was the first lectin demonstrated to have an outgrowth-promoting effect on neurons. It is now thought to play a role in neural pathfinding, as shown in the developing mouse olfactory system (Puche et al. 1996). Primary ORNs in galectin-1 null-mutant animals failed to make contact with their target, the olfactory bulb (Puche et al. 1996; Tenne-Brown et al. 1998). In addition, in T-cells galectin-1 was shown to be a mediator for apoptosis (Perillo et al. 1995), and in malignant tumors galectin-1 expression was found to be associated with less-differentiated cell types (Skrincosky et al. 1993).
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Galectin-3 has been shown to stimulate neural cell growth and to promote neuronal adhesion (Pesheva et al. 1998). It is part of the advanced glycation endoproduct (AGE)-receptor complex which plays a role in the elimination of AGEs (Vlassara et al. 1995). Its expression is modulated in malignant tumors (Bresalier et al. 1997). In some carcinomas galectin-3 expression is associated with a lower degree of metastatic potential, i.e., less malignancy and better differentiation (Perillo et al. 1998). In keratinocytes galectin-3 expression has been shown to be dependent on the degree of cellular differentiation (Gillenwater et al. 1996). As it protected cells from apoptosis, effects of galectin-3 were found to be opposite to those of galectin-1 in a system of T-cell apoptosis (Yang et al. 1996). Neural cells within the olfactory epithelium display unique cellular features, being constantly replaced with cells from undifferentiated stem cells located in the basal layer of the epithelium. Earlier investigations in the rodent olfactory system have shown an apoptosis-like character of the observed cell death (Magrassi and Graziadei 1995; Mahalik 1996). Regulatory mechanisms of this cell cycle are still widely unknown. Although an age-dependent expression of both galectins cannot be deduced from our data, the results obtained might suggest a modulated expression of both molecules during development and death of ORNs. Expression of galectin-1 in basal cells, i.e., in still immature ORNs, supports reports of its role in the stimulation of neural growth and neural pathfinding, which allows freshly differentiated cells to establish contact with higher-order olfactory relays in the olfactory bulb. Although expression of galectin-1 in rodent primary ORNs has not been investigated, galectin-1 was found to be localized in glial cells in the olfactory nerve pathway (Tenne-Brown et al. 1998). Our results in human tissue show galectin-1 expression in cells of the basal epithelial layer. Thus, galectin-1 expression of basal cells might be a sign of their low degree of maturation, at the same time stimulating neural growth of the young ORNs and their adhesion to the olfactory bulb. In contrast, galectin-3 IR of the more superficial perikarya of ORNs may be viewed as a sign of differentiation and maturity, while a protective effect of galectin-3 with regard to apoptosis remains speculative since it has only been shown in the T-cell model. Taken together, our preliminary results indicate a role of galectins in ORN turnover. Their expression patterns were found to correspond to their function. Specifically, galectin-1 may stimulate neural cell growth and neural pathfinding in developing cells and is found in basal layers of the olfactory epithelium, while expression of galectin-3 has been shown to depend on the degree of cellular differentiation and to protect from apoptosis in certain cell models. Accordingly, galectin-3 IR was found in the upper one-third of the epithelium. Acknowledgements We are indebted to Dr. Douglas Cooper, Department of Anatomy, University of California, San Francisco, Parnassus Avenue, San Francisco, Calif., USA, for providing the antibody against galectin-1. We appreciate the technical assistance of Mrs. B. Georgieva and Mrs. C. Nipproschke.
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