Phenotypic characterization of mononuclear inflammatory cells in salivary glands of bio-breeding rats

Archs oral Biol. Vol. 42, No. 9, pp. 649-655, 1997 © 1997 ElsevierScienceLtd. All rights reserved Printed in Great Britain PII: S0003-9969(97)00056-3 0003-9969/97 $17.00 + 0.00

Pergamon

P H E N O T Y P I C C H A R A C T E R I Z A T I O N OF M O N O N U C L E A R I N F L A M M A T O R Y CELLS IN S A L I V A R Y G L A N D S OF BIOB R E E D I N G RATS R. E. C O H E N , ~'* G. T A L A R I C O : and B. N O B L E 3 ~Department:; of Periodontology and Oral Biology, School of Dental Medicine, University at Buffalo, Buffalo, NY 14214, U.S.A., 2Department of Periodontology, School of Medicine, University at Buffalo, Buffalo, NY 14214, U.S.A. and 3Department of Microbiology, School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY 14214, U.S.A. (Accepted 7 May 1997)

Summary--The purpose of this study was to assess whether mononuclear cell abnormalities exist in salivary glands fiom autoimmune Bio-Breeding (BB) rats. Frozen sections of gland tissues were prepared from five diabetes-resistant BB rats (BB-DR), from five BB rats with diabetes (BB-DP) and from five Wistar rats. P~ panel of six monoclonal antibodies was used to identify membrane antigens associated primarily with monocytes (ED1), mature tissue macrophages (ED2), lymphoid macrophages (ED3), MHC class II (Ia) antigen (OX6), CD5 + T lymphocytes (OX19), and rat B lymphocytes (OX33). Normal submandibular, sublingual and parotid glands contained few EDl-positive cells, usually two or fewer per field. Tissue macrophages identified by clone ED2 comprised a major mononuclear cell subset in both Wistar and BB rats. However, the number of ED2-positive mononuclear cells was significantly depressed in the submandibular and parotid glands from BB-DR and BB-DP animals, being present in quantities 25 50% of those observed in glands from normal Wistar rats (p < 0.001). In contrast, 25- to 30-fold greater numbers of ED3-positive macrophages were observed in submandibular glands from BB rats (p < 0.001). MHC class II (Ia) antigen expression also was 4- to 6-fold greater in BB rat submandibular glands, compared to Wistar rats (p < 0.001). CD5 + T-lymphocytes were rare or entirely absent in BB sublingaal glands (0 to 1 cell per 0.87 mm 2 field), compared to 47 cells per field from Wistar sublingual glands. No B lymphocytes were identified with antibody OX33 in any of the rat strains. These findings indicate that BB rat salivary glands differ significantly from Wistar salivary glands. In BB rats there is a rich population of ED3-positive macrophages and T lymphocytes in submandibular gland, low quantities of T lymphocytes in sublingual gland, and fewer ED2-positive macrophages in all three major salivary glands. These differences in mononuclear cell subpopulations may also influence salivary gland function in mucosal immunity. © 1997 Elsevier Science Ltd Key words: lymphocytes, macrophages, T cells, immunohistochemistry, inflammation, salivary glands.

INTRODUCTION

et al., 1988). At 6 months of age, twice-daily insulin administration typically is required for survival of Bio-Breeding (BB) rats exhibit a profound C D 4 + diabetic BB rats. and C D 8 + T-lymphopenia (Bellgrau et al., 1992; Diabetes in diabetes-prone BB rats is characterGreiner et al., 1986:; W o d a et al., 1986; van Rees et ized by an Ia + lymphocytic and monocytic infilal., 1988). A high percentage of BB rats also trate that destroys pancreatic/~-cells (Weringer and develop spontaneous insulin-dependent diabetes Like, 1988; Kastern et al., 1990). As in human insumellitus ( N a k h o o d a et al., 1977). Functional T-lymlin-dependent diabetes, diabetes-prone BB rats exhiphocyte abnormalities include delayed skin allograft bit a heightened autoreactivity, reflected in an rejection and depre,;sed lymphocyte proliferative reincrease in serum IgG (Scott, 1990). Other BB rats sponses in vitro. In neonatal and young adult anifail to develop autoimmune disease and diabetes, mals, responses to thymus-dependent antigens are and are clinically normal (diabetes-resistant). delayed and decrea:sed (Bellgrau et al., 1982; Elder In humans, Sj6gren's syndrome is associated with and MacLaren 1983; Jackson et al., 1983; van Rees a C D 4 + and CD8 + T-lymphocytic infiltrate that affects salivary and lacrimal glands, ultimately lead*To whom all corre,;pondence should be addressed at: ing to tissue damage. Salivary IgG autoantibodies State University o1' New York at Buffalo, Department also are elevated (Fox and Saito, 1994). of Periodontolog~, 250 Squire Hall, Buffalo, New Unfortunately, animal models of human salivary York 14214, USA [Tel: (716) 829-3845; Fax: (716) 837gland disease have so far proved to be relatively 7623; E-mail: [email protected]]. u n s a t i s f a c t o r y . Although some such models do exist Abbreviations: TBS, Tfis-buffered saline.

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(e.g. NOD mice; Kikutani and Makino, 1992), the degree of inflammation is usually variable, and well-characterized panels of monoclonal antibodies to mouse mononuclear-cell epitopes are not yet available. As diabetes in BB rats is seconda~y~to the autoimmune defect associated with those animals, it is reasonable to expect that other organs also may exhibit mononuclear-cell abnormalities. Although BB rats serve as a laboratory model for human insulin-dependent diabetes, potential autoimmune abnormalities of the salivary glands have not been reported. Consequently, we wished to assess whether BB rat salivary glands are associated with inflammation and aberrant mononuclear-cell expression analogous to that observed in other autoimmune rodents such as NOD mice. We also wanted to assess whether any such mononuclearcell abnormalities of BB rats may be useful for the study of salivary gland immunoregulation. As a result, we now measured the expression of lymphocyte and macrophage subsets in salivary glands from diabetes-prone and -resistant BB rats, and compared the expression of salivary mononuclear cell phenotype in BB rats to that of Wistar rats.

MATERIALS A N D M E T H O D S

Study design

Immunocytochemical techniques, with monospecific, well-characterized monoclonal antibodies, were used to identify and quantitate resident macrophage, lymphocyte and dendritic cell subsets from major salivary glands of BB rats. Salivary gland tissues from five diabetes-prone and five diabetes-resistant BB rats were assessed and compared to salivary glands from six Wistar rats. Rats

Our experimental procedures were reviewed and approved by the Laboratory Animal Care Committee of the Schools of Medicine and Dental Medicine, State University of New York at Buffalo. Male diabetic and diabetic-resistant Bio-Breeding rats were obtained from the University of Massachusetts Medical School (NIH contract colony), Worcester, M A at 12-15 weeks of age. They were housed in individual metabolism cages and were maintained on a 24% protein pellet diet (Teklad, Madison, WI). Diabetic rats received a daily subcutaneous injection'of protamine zinc insulin (0.5-0.8 units/100 g body wt in the late morning; Eli Lilly) in the tissues overlying the pectoralis muscles. The insulin dose was adjusted so that diabetic rats routinely exhibited glycosuria (4+ by Testape; Eli Lilly) with no evidence of ketoacidosis (Ketodiastix; Ames). This protocol was selected on the basis of previously published studies that examined renal glomerular function in spontaneously

diabetic BB rats (Zamlauski-Tucker et al., 1992). The duration of diabetes was 8-12 weeks, and the rats were used in our studies at 20-23 weeks of age. Wistar rats (150-200 g; Harlan Sprague-Dawley, Indianapolis, IN, U.S.A.) were maintained on food pellets (R-M-H 2000; Agway, Inc., Syracuse NY) and water ad libitum, and housed in air-conditioned, humidity-controlled facilities, as described by Cohen et al. (1992, 1994). Animals were anaesthetized intraperitoneally with ether and killed by exsanguination within 1 min of induction of anaesthesia. The salivary glands were removed, cut into 2 3 mm 3 pieces, and immediately frozen in liquid nitrogen-cooled isopentane. The tissues were stored in liquid nitrogen until frozen sections were prepared (within 4-6 weeks). Tissue specimens

Sublingual, submandibular and parotid glands were obtained from 6-12-month-old, mate diabetesprone and diabetes-resistant BB rats. Frozen tissue specimens were prepared from five animals of each type. Salivary gland tissues from normal Wistar rats also were examined and compared to the BB tissues. Animals were anaesthetized by an overdose of inhaled ether, and then killed by exsanguination as above. Immunochemical reagents

Immunoperoxidase methods were essentially as reported by Cohen et al. (1992, 1994). Optimal dilutions of primary and secondary antibodies were determined in preliminary trials by checkerboard titration of tissue sections using varying concentrations of primary and secondary reagents, and were chosen to maximize specific staining with minimal non-specific reaction. All primary antibodies were obtained as ascites fluids from Bioproducts For Science, Inc., Cambridge, MA. Normal rabbit serum was obtained from GIBCO (Grand Island, NY), and normal rat serum was obtained by cardiac puncture of anaesthetized, untreated, 350 400 g female Wistar rats. Rabbit anti-mouse immunoglobulins and mouse peroxidase-antiperoxidase complex were obtained from Dako Corporation (Carpinteria, CA). The mouse monoclonal antibodies used to localize cellular antigens within rat salivary glands, their specificities, immunoglobulin subclass, and dilutions used for immunocytochemistry, are described in Table 1. Immunocytochemical procedures

Cryostat sections were cut at 5 /~m from frozen tissue specimens. Tissue sections were stored in liquid nitrogen, and used for immunocytochemistry within 1 week of sectioning, as described by Cohen et al. (1990, 1992, 1995). All sections were thawed at 23°C for 20 min, and then fixed for 2 min in 95% ethanol (with clones ED1, ED2 and ED3) or with acetone:chloroform (1:1; with clones OX6,

Rat salivary gland mononuclear cells

651

Table 1. Description of antibodies used for immunocytochemistry Monoclonal antibody ED1 ED2 ED3 OX6 OX19 OX33

Specificity Cytoplasmic antigen monocyte and most macrophage and granulocyte subpopulations Membrane antigen on IgG2a tissue macrophage of lymphoid and nonlymphoid organs Membrane antigen on macrophages predominantly confined to lymphoid organs Rat M HC Class II (Ia) antigen (rnonomorphic) Rat CD5 antigen (thymocytes and peripheral T cells) Rat B cells

OX19, and OX33). After fixation, the sections were washed twice for 10 min in TBS (20 m M Tris-HC1, 500 m M NaC1, p H 7.5), followed by incubation for 20 min in normal rabbit serum diluted 1:20 in TBS. U p o n removal of 1:he normal serum, the sections were treated for 45 min with primary monoclonal antibody appropriately diluted in TBS, as described in Table 1. The sections were subsequently washed three times for 10 rain in TBS, then treated for 45 min with rabbit ant:i-mouse immunoglobulin diluted 1:50 in TBS containing 5% rat serum, in order to eliminate cross-reactivity of the anti-mouse reagent with rat tissues. After washing in TBS as described above, mouse peroxidase-antiperoxidase complex was applied to each section for 30 min, followed by further washing in TBS. Tissue localization of antigen was achieved by development with a solution, prepared by mixing 3-amino-9-ethylcarbazole (4 mg/ml in N,N-dimethyl formamide/0.1 M acetate buffer, pH 5.2) for 40 min at 23°C, which produced a red precipitate in the presence of peroxidase. Mayer's haematoxylin was used as a counterstain, and the sections were examined by light microscopy. Negative controls included salivary gland tissues from rats that were incubated with non-specific ascites fluid obtained from the same parental myel-

Dilution

Immunoglobulin subclass

1:1000

IgG1

Damoiseaux et al. (1994)

1:500

IgG2a

Dijkstra et al. (1985)

1:100

IgG2a

Dijkstra et al. (1985)

1:100

IgG1

1:1000

IgG1

McMaster and Williams (1979) Williams (1985)

1:100

IgG1

Gillian et al. (1985)

Reference

oma cell line as the primary monoclonal antibody. Sections of normal rat spleen served as positive controls. Quantitation o f mononuclear phagocyte subsets

Semiquantitative analysis was as described by H o n d a et al. (1968), and as modified by us (Cohen et al., 1992, 1994). Ten randomly selected sites were analysed by counting the number of cells labelled with each monoclonal antibody at ×200 magnification (0.87 m m 2 per field) from each of two duplicate submandibular gland sections. The data from each duplicate were combined and the average number of cells per field positive for each antibody was evaluated for each animal. The data for each major salivary gland from rats in the two BB and the Wistar groups were combined to obtain the mean (±SD). Significant differences among groups were determined by analysis of variance (F) tests corrected for multiple comparisons; the sample size (n) was the number of animals per group.

RESULTS

Immunoperoxidase staining of salivary glands from the BB and Wistar rats with monoclonal antibodies to ED1 (monocytes), ED2 (tissue macro-

Table 2. Distribution of mononuclear inflammatory cells in submandibular glands from Wistar and Bio-Breeding rats

Monoclonal antibody

Bio-Breeding diabetes-prone Bio-Breeding diabetesWistar (BB-DP) resistant (BB-DR) (positive cells per 0.87 mm 2 microscopic field; mean + SD)

ED1 ED2 ED3 OX6 OX19 OX33

2_+1 165 _+28 2+ 2 18 + 14 4 ___2 <1

*Significantly different from Wistar (p < 0.001). **Significantly different from Wistar (p < 0.01). tSignificant differences between BB-DP and BB-DR (p < 0.001). ttSignificant differences between BB-DP and BB-DR (p < 0.01).

<1 75 ___25* 70 _+ 14*,t'j" 112 _+ 15*,f 1 + 1"* <1

<1 62 -L-_10" 50 + 7*,t'j" 80 _+ 3"£t < 1"* <1

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R.E. Cohen et al. Table 3. Distribution of mononuclear inflammatory cells in sublingual glands from Wistar and Bio-Breeding rats

Monoclonal antibody

Bio-Breeding diabetes-prone Bio-Breeding diabetesWistar (BB-DP) resistant (BB-DR) (positive cells per 0.87 mm 2 microscopic field; mean + SD)

ED1 ED2 ED3 OX6 OX19 OX33

3+ 1 113 + 37 16+8 35 + 14 47 + 18 <1

<1 67 + 14 23+7 64 + 10*,t 1 + 1" <1

<1 67 + 12 11 +5 33 _+ 7t <1 <1

*Significantly different from Wistar (p < 0.01). tSignificant differences between BB-DP and BB-DR (p < 0.01) phages) and ED3 (lymphoid macrophages) is summarized in Tables 2, 3 and 4. Positive cells were dendritic, with relatively small nuclei, sparse cytoplasm and numerous slender, elongated processes. Figures 1 and 2 illustrate their typical morphology, which was similar for both Wistar and BB salivary glands, and consistent with our previous reports of mononuclear cell subsets in rat salivary tissues (Cohen et al., 1995). N o r m a l submandibular, sublingual and parotid glands exhibited few ED1positive cells, usually two or fewer per field. Those cells were usually located within connective tissue near blood vessels, although a few were found at the periphery of the acini. Tissue macrophages identified by ED2 comprised a major mononuclear cell subset both in Wistar and BB rats; these data are consistent with our previous work (Cohen et al., 1992, 1994, 1995). However, the number of ED2-positive mononuclear cells was significantly fewer in the submandibular and parotid glands from both diabetes-resistant and diabetes-prone BB animals, with ED2-positive cells being present in quantities 25-50% of those observed in the corresponding salivary glands from normal Wistar rats (p < 0.001; Tables 2 and4). Significant differences in ED2 expression were not observed between BB animals with or without diabetes in any of the salivary glands examined. In contrast, 25- to 30-fold greater numbers of ED3-positive macrophages were detected in submandibular glands from both types of BB rat (70 and 50 cells per field in diabetes-prone and diabetes-resistant submandibular glands, respectively, vs 2 cells per field in Wistar submandibular glands;

p < 0.001). The difference in expression of ED3 markers between each of the BB rat groups was also significant in submandibular glands, although at a lower confidence interval (p < 0.01; Table 2). Similarly, M H C class II (Ia) antigen expression also was 4- to 6-fold greater in submandibular glands from BB rats, compared to Wistar rats (p < 0.001). Ia antigen expression was also elevated in sublingual glands from BB diabetes-prone rats, compared to BB diabetes-resistant and Wistar rats (p < 0.01; Table 3). Substantially fewer C D 5 + T lymphocytes were observed in BB rat sublingual glands (0-1 cell per 0.87 mm 2 field) compared to Wistar sublingual glands (47 cells per field) (p < 0.001). N o B lymphocytes were identified with antibody OX33 in any of the rat strains. Finally, controls incubated without the application of the primary antibody did not result in staining in any of the salivary gland sections.

DISCUSSION

Our findings indicate that cells of the mononuclear lymphocyte and phagocyte lineage are present within the interstitial submandibular, sublingual and parotid gland tissues of diabetesprone and diabetes-resistant rats. The differences in resident mononuclear cell subsets among each of the major salivary glands further support our previous results indicating that immunoregulatory mechanisms may operate differently in these glands (Cohen et al., 1992, 1995).

Table 4. Distribution of mononuclear inflammatory cells in parotid glands from Wistar and Bio-Breeding rats

Monoclonal antibody

Bio-Breeding diabetes-prone Bio-Breeding diabetesWistar (BB-DP) resistant (BB-DR) (positive cells per 0.87 mm 2 microscopic field; mean + SD)

EDI ED2 ED3 OX6 OX19 OX33

2+1 96 ___4 4+ 1 14 + 14 1+ 1 <1

*Significantly different from Wistar (p < 0.001). tSigniflcantly different from Wistar (p < 0.01)

<1 20 _+9* 10 + 6 17___9 <1 <1

<1 29 + 8* 17 + 6** 33 + 12 <1 <1

Rat salivary gland mononuclear cells

Fig. 1. Photomicrograph of a frozen section of parotid gland from a BB diabetes-prone rat. The section was processed by the indirect peroxidase-antiperoxidase technique, developed wiLh aminoethylcarbazole, and lightly counterstained with Mayer's haematoxylin. A decrease in the number of ED2-positive resident tissue macrophages was observed in parotid glands from BB rats, compared with normal Wistar rats. Magnification x400.

Mononuclear cell distribution in BB rat salivary glands is substanti~Llly different from that of normal Wistar rats. Although ED2-positive macrophages are the predominant cell subpopulation in all three major salivary glands from Wistar rats, markedly fewer ED2-positive cells are present in BB rats, approaching approx. 50% of levels observed in Wistar glands. The observed depletion of ED2-positive mononuclear c,ells is consistent with the results of van Rees et al. (1988), who described a similar paucity of ED2-positive cells in the thymus, spleen and pancreas of BB rats. In BB submandibular glands, highly significant elevations in the ~aumber of ED3-positive macrophages and in MHC class II antigen expression were detected, compared to Wistar submandibular

Fig. 2. Photomicrograph of a frozen section of sublingual gland from a BB diabetes-prone rat. The section was processed by the indiiect peroxidase-antiperoxidase technique, detected with monoclonal antibody OX6, developed with aminoethylcarbazole and lightly counterstained with Mayer's haematoxyLLn. Cells expressing MHC class II determinants are st~.ined positively. Magnification x400.

653

glands. However, the number of CD5 + T lymphocytes in sublingual glands detected with clone OX19 decreased substantially. Collectively, these data suggest that the immune defect inherent in BB rats reduces the number of ED2-positive macrophages in all glands, and increases submandibular gland ED3-positive cells and Ia antigen expression. This is accompanied by near total elimination of CD5 + T lymphocytes in sublingual gland. We have previously proposed (Cohen et al., 1995) that the rich population of ED3-positive macrophages and T lymphocytes, and the marked Ia antigen expression, in Wistar sublingual glands all indicate that the sublingual gland may have important antigen-presenting and immunoregulatory functions, and that it may be the best adapted of the major salivary glands to play a part in mucosal immunity. Consequently, the immune defect associated with BB rats may compromise the animals' ability to perform those functions. Our data also indicate that the diabetic status of the BB rats may influence mononuclear cell phenotype of salivary glands. For example, larger numbers of ED3-positive cells and greater MHC class II antigen expression were found in diabetes-prone than diabetes-resistant BB submandibular glands. Similarly, class II antigen expression was elevated in sublingual glands of diabetes-prone BB rats compared to diabetes-resistant. However, no other differences between these two groups were detected with any of the monoclonal antibody probes in any of the three salivary glands. These data suggest that the primary determinant of abnormal phenotype in BB rats is the genetic immune defect characteristic of those animals, rather than diabetic status. Dendritic cells in lymphoid and non-lymphoid tissues are thought to have important antigen-presenting functions in cellular immunity. Dendritic cells occur within non-lymphoid tissues, and are responsible for both antigen recognition and processing. Inflammatory mediators and cytokines also contribute to the development of dendritic leucocyte precursors by promoting the migration of immature cells into secondary lymphoid tissues. The maturation of these cells leads to antigen presentation in the form of peptide-MHC complexes and T-lymphocyte activation (Austyn, 1992). Our data therefore support the presence of defects in oral mucosal immunity in BB rats, and suggest that further studies are indicated to determine whether defects exist in cytokine expression and periodontal inflammatory mediators. BB rats are associated with a profound T-cell lymphocytopenia, with suppression of both CD4 + and CD8 + (helper/suppressor) subsets (Walker et al., 1988). It has been proposed that the T-lymphocyte defect may be associated with alterations in Tcell maturation or differentiation (Elder and MacLaren, 1983; Greiner et al., 1986), and T-cell mediated immunoresponses have been shown to be

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impaired in vitro (Bellgrau et al., 1982; Elder and MacLaren, 1983). F r o m birth, a profound reduction in macrophage subpopulations in thymic cortex occurs, but not in spleen and lymph nodes. The population of CD8 + cells fails to expand from 10 days after birth in the thymic cortex, and 14 days after birth in the spleen and lymph nodes, supporting an intrathymic maturational defect of the C D 8 + cells in BB rats (Van Rees et al., 1988). In humans, Sj6gren's syndrome is characterized by keratoconjunctivitis sicca, rheumatoid arthritis and xerostomia, resulting in part from the destruction of the lacrimal and salivary glands. The exact aetiology of the disease is unknown, but lymphocytic infiltration consists primarily of activated CD4 + T lymphocytes (Fox and Saito, 1994). Although the BB rat does not possess many of those disease indicators, it nevertheless may serve as a potential model of human salivary gland inflammation, such as chronic obstructive sialadenitis or sialolithiasis. However, further characterization of the immune defect in BB rats in general, as well as salivary gland effects in particular, is required. We demonstrate that, in addition to the known diabetic, renal and pancreatic abnormalities inherent in the Bio-Breeding rat model, there exist previously unappreciated salivary gland effects. These findings indicate that, compared to normal Wistar rats, BB rats exhibit a rich population of ED3-positive submandibular gland macrophages and T lymphocytes, low quantities of sublingualgland T lymphocytes, and a generalized decline in the number of ED2-positive macrophages in all three major salivary glands. The resulting alterations in mononuclear cell subpopulations may also have pronounced effects on oral mucosal immunity. Consequently, the BB rat model may be valuable for the study of periodontal immunobiology, as well as complementing previous and ongoing studies of salivary gland immune regulation in murine models. Acknowledgements--This study was supported in part by

a grant from the Sjogren's Syndrome Foundation, Inc., and by USPHS Training Grant No. DE07106. REFERENCES

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