Immunology 1992 76 42-47

Detection of inflammatory cytokine messenger RNA (mRNA)-expressing cells in human inflamed gingiva by combined in situ hybridization and immunohistochemistry Y. MATSUKI, T. YAMAMOTO* & K. HARA Department of Periodontology, Niigata University School of Dentistry *Department of Pathology, Institute of Nephrology, Niigata University School of Medicine, Niigata, Japan

and

Accepted for publication 2 January 1992

SUMMARY Cells expressing messenger RNA (mRNA) for inflammatory cytokines [interleukin- 1 (IL-1), tumour necrosis factor-alpha (TNF-a), IL-6, IL-8] were demonstrated by in situ hybridization in human inflamed gingiva. When this technique was used in conjunction with immunohistochemistry, IL-la and/or ,B and IL-8 messages were observed predominantly on macrophages infiltrating the gingiva. TNF-a messages were abundant on macrophages and T cells. In contrast, the IL-6 mRNA were more widely distributed on many types of cells such as macrophages, T cells, fibroblasts, endothelial cells and B cells. This study clearly identified the cells which express mRNA for inflammatory cytokines in inflamed gingiva and suggested an involvement of cytokine network in the generation of human periodontitis.

stimulation of IL-I: and TNF-a by using a reverse transcription-polymerase chain reaction analysis.'4 GCF obtained from inflamed sites contains more than 90% polymorphonuclear leucocytes (PMN) of all cellular components;'5 furthermore, IL8 exerts a potent chemotactic attraction for neutrophils across endothelial monolayers, leading to the transmigration of up to 90% of neutrophils within 1 hr,'6 so IL-8 may contribute to a neutrophil-mediated process of periodontal disease. According to these reports, cytokines are thought to be important mediators in the generation of periodontitis. However, little is known about the cells producing these cytokines in inflamed gingiva or about their mechanism or sites of production in vivo. The present study was designed to detect and identify the cells expressing mRNA for the inflammatory cytokines (IL-1, TNF-a, IL-6 and IL-8) in human inflamed gingival tissues by combined immunohistochemistry and in situ hybridization. The main source of these cytokines and the pathway of the possible network with respect to gingival inflammation were sought.

INTRODUCTION Interleukin-l (IL-1), IL-6, IL-8 and tumour necrosis factoralpha (TNF-a) are typical examples of multifunctional cytokines involved in the regulation of the immune response and inflammation. 1-3 Periodontitis, an inflammatory disease in periodontal tissues, causes loss of connective tissues, formation of periodontal pockets and resorption of alveolar bone. More stages of the disease lead to loosening and finally loss of teeth. Higher IL- activity was found in gingival crevicular fluid (GCF) obtained from inflamed gingival sites than that from messenger RNA (mRNA) and non-inflamed sites.4-6 IL-l protein were detected in the inflamed gingival tissues of patients with periodontitis.7 In addition, TNF-a was found in GCF obtained from patients with periodontitis.8 TNF-a can stimulate fibroblasts, including those in gingiva, to produce collagenase,9 an enzyme implicated in the tissue destruction of periodontal disease, and to stimulate bone resorption as well as IL-1.° Mononuclear cells isolated from inflamed gingiva of patients with periodontitis spontaneously produce biologically active IL-6," a key cytokine for the terminal differentiation of B cells to become immunoglobulin-secreting cells.'2 IL-8 is a polypeptide chemotactic for neutrophils originally identified in the culture supernatant of lipopolysaccharide (LPS)-stimulated monocytes.'3 The expression of human gingival fibroblastderived IL-8 mRNA was detected at an early stage after

severe

MATERIALS AND METHODS

Gingival tissues We used samples from 19 adult patients (27 sites) with moderate to severe periodontitis who had been referred to Niigata University Dental Hospital, Japan. Control specimens (six sites) were taken from three clinically healthy patients undergoing orthodontic treatment at the time of tooth extraction. Pieces of gingival tissues were fixed in 10% neutral buffered formalin for 6-12 hr, embedded in paraffin, and sectioned at 6 ym thickness onto poly-L-lysine (0 01 %; Sigma Chemical Co., St Louis, MO) coated slides.

Abbreviations: GCF, gingival crevicular fluid; DEPC, diethyl pyrocarbonate; SSC, standard saline-citrate buffer. Correspondence: Dr K. Hara, Dept. of Periodontology, Niigata University School of Dentistry, Gakko-cho 2-5274, Niigata 951, Japan.

42

Cytokine mRNA-expressing cells in gingiva Immunohistochemistry The sections were deparaffinized through xylene and graded ethanol (100, 95, 90, 75, 50 and 30%) and immersed in distilled water. To inhibit endogenous peroxidase, the sections were placed in a bath of methanol containing 0 3% hydrogen peroxide for 20 min. After blocking with 5% normal rabbit serum or 5% normal goat serum for 30 min, monoclonal antibodies against human macrophages (CD68;KPI,'7 Dako Japan Co., Ltd, Tokyo, Japan), human T cells (OPD4,'8"9 CD45RO;UCHL-1,20 Dako), and human B cells (CD20;L26,2' Dako) were reacted with the sections for 60 min to label infiltrating leucocytes in the gingival tissues. Polyclonal rabbit antibodies against human CD322 (Dako) and von Willebrand factor (a gift from Dr Theodore S. Zimmerman, Scripps Clinic and Research Foundation, La Jolla, CA) were used to identify T cells and endothelial cells, respectively. They were incubated sequentially with rabbit anti-mouse antibody or goat anti-rabbit antibody for 30 min, peroxidase-anti-peroxidase complex (mouse or rabbit, Dako) for 60 min, and diaminobenzidine tetrahydrochloride (WAKO Pure Chemical Industries, Ltd, Osaka, Japan) in 0 05 M Tris-HCl buffer (pH 7 4) containing 0 01 % hydrogen peroxidase for 5 to 10 min. In situ hybridization In situ hybridization was performed as described previously.23 After immunoperoxidase staining, the sections were washed in phosphate-buffered saline (PBS) and immersed in 0 2 M HCI at room temperature for 10 min to remove basic proteins. The sections then were washed in distilled water supplemented with 0 1% diethyl pyrocarbonate (DEPC, Sigma) twice for 2 min, treated with I % Triton X- 100 for 90 seconds at room temperature, and washed again in 01 % DEPC. Then the sections were digested with proteinase K (1 0 [g/ml) in 20 mm Tris-HCl, pH 7 2, containing 2 mm CaCI2 for 15 min at 37°. As a negative control, slides were treated with a mixture of RNase Tl (200 U/ ml; Bethesda Research Laboratories, Gaithersburg, MD) and RNase A (100 ,ug/ml; Sigma) at 370 for 30 min to digest tissue mRNA. After brief rinsing in 0 1% DEPC, they were immersed in 0-2% glycine in PBS for 3 min to inhibit proteinase K, transferred to PBS supplemented with 0-1% DEPC, dehydrated through graded ethanol containing 0 3 M ammonium acetate, and then air dried. Synthetic oligonucleotides were purchased from British Biotechnology Limited (Oxford, U.K.). These were complementary to human cytokines as shown in Table 1. As a negative control probe, a synthetic oligonucleotide (GGCGACGCGCCG Table 1. Synthetic oligonucleotides complementary to human cytokines

IL-lo IL-1I, TNF-ar IL-6 IL-8 Control *

t

Length (mer)

GC (%)*

Tm (O)t

Exon

30 30 29 30 30 30

43 43 48 40 43 43

86 86 86 84 86 86

5, 6, 7 5,6,7 4A 2, 3, 4, 5 5', mid, 3' region

GC (%), percentage of 'guanine and cytosine' contents. Tm(0), melting temperature.

43

TATTTATAATTCATTATG) was prepared on a DNA synthesizer (Cyclone Plus DNA Synthesizer, MilliGen/Biosearch Division of Millipore, Tokyo, Japan). Extremely low homology of this sequence to known complementary DNA was verified by searching GenBank data base (June 1990) using the program DNASIS (Hitachi Software Engineering Co., Ltd, Yokohama, Japan). These oligonucleotides were radiolabelled with 35SdATP (New England Nuclear/Dupont, Boston, MA) by using terminal deoxynucleotidyl transferase (TdT) (Takara Shuzo, Co., Ltd, Kyoto, Japan) as follows. Oligonucleotides of 50 ng each were mixed with 35S-dATP (100 pCi) and TdT 20 U in a total volume of 20 pl tailing buffer and incubated for 90 min at 370. The labelled probes were purified by Sephadex G-25 gel filtration, and radioactivity was measured in a scintillation counter. The specific activities of these probes were essentially equal (approximately 1 x 108 d.p.m./pg). The concentrations of the probes were adjusted to approximately 1 x 105 d.p.m./pl in a hybridization mix consisting of 50% formamide, 5 x Denhardt's solution, 5 x hybridization salt, 10% dextran sulphate, and 500 pg/ml denatured salmon sperm DNA. Finally, dithiothreitol was added to the mixture at a final concentration of 10 mM. Sections were incubated with the probes overnight at 370 in a humid chamber. Slides were washed sequentially in 2 x standard saline-citrate buffer (SSC) at room temperature for 15 min twice, 0 1 x SSC at room temperature for 10 min twice, 0-1 x SSC at 60° for 10min, 0 1 x SSC at 37° for 15 min, and then 2 x SSC for 10 min at room temperature. After serial dehydration through graded ethanol (30-100%), the slides were

dipped in autoradiography emulsion (NR-M2, Konica Co., Tokyo, Japan), air dried, and maintained for 7 days in the dark at 4°. Finally the slides were developed in Konicadol-X (Konica) for 5 min, fixed in Konicafix (Konica) for 10 min, and counterstained with haematoxylin for 20 seconds. The number of these grain-positive cells per random area

(0 42 x 0 54 mm: 0 23 mm2, five areas chosen in each section) on the sections was quantitated under a light microscope. The gingival lesions were classified into two categories by histology and immunohistochemistry; one was the lesion which was infiltrated with macrophage predominantly (more than 10% of the infiltrating cells, M + lesion) and the other was characterized by less macrophage infiltration (less than 10% of the infiltrating cells, M - lesion). The percentages of the mRNA-positive cells among the immunohistochemically identified cells were separately evaluated in these lesions. RESULTS By histology and immunohistochemistry, M+ lesion was characterized by conspicuous infiltration of macrophage (10-30% of the infiltrating cells) identified by immunostaining for CD68. On the other hand, macrophages were few (5-10% of the infiltrating cells) in the M- lesion. The M- lesion was composed mainly (40-60%) of B cells and plasma cells in the tissue. The expression of mRNA for the inflammatory cytokines was more remarkable in the M + lesion than M - lesion. The predominant cells expressing cytokine mRNA on the human inflamed gingiva were macrophages identified by staining for CD68 as shown on Fig. 1. IL- 1 and IL-8 mRNA expressions were frequently observed on the macrophages. Interestingly, many CD45RO+ cells accumulated around IL-1 mRNA-expressing cells (Fig. 2a). TNF-a was expressed on both CD68+ macrophages and

44

Y. Matsuki, T. Yamamoto & K. Hara

Figure 1. Inflammatory cytokines mRNA-expressing macrophages are demonstrated by in situ hybridization and immunohistochemistry. Original magnification x 500. Macrophages express inflammatory cytokines mRNA: IL-la (a); TNF-a (b); IL-6 (c); IL-8 (d).

CD3 + T cells. In contrast, IL-6 mRNA was expressed on various types of cell such as macrophages, T cells (Fig. 2b), CD20+ B cells (Fig. 2c), endothelial cells (Fig. 2d) and fibroblasts. In addition, IL-6 mRNA-expressing cells were frequently located in the B-cell- and plasma cell-rich region (Fig. 2b) and were observed both in the M + lesion and the M - lesion. IL-8 mRNA was observed frequently on macrophages (Fig. Id) and occasionally on endothelial cells in the M+ lesion, but the M- lesion contained very little IL-8 mRNA (Fig. 2e). The frequency of these cytokine mRNA-expressing cells in the gingival tissue examined is summarized in Table 2. The expression of mRNA for these cytokines in the healthy gingival tissues was hardly (less than one cell per mm2 area) observed by in situ hybridization (not shown). The control probe did not hybridize to any cells in the inflamed gingiva (Fig. 2f). In addition, RNase pretreatment eliminated the hybridization of these probes to any cells in the inflamed gingiva (not

shown). DISCUSSION This study identified cell types which express cytokine mRNA in human gingival tissue. The number of infiltrating macrophages was different in each tissue. The expression of mRNA for cytokines was, therefore, separately evaluated in the M + lesion

and M- lesion. The cytokines have been disclosed as important mediators of inflammatory response. Although the in vitro production of cytokines by monocytes stimulated with bacterial products such as LPS and muramyl dipeptide has been shown,'3 in vivo production has not yet been clearly demonstrated. The present study revealed that macrophages were predominantly expressing cytokine mRNA in the human inflamed gingiva and some of the T cells, B cells, fibroblasts and endothelial cells were also cells expressing cytokine messages in periodontitis. These findings suggested a complicated involvement of inflammatory cytokines in the induction and development of periodontitis. An extraordinarily large number of pathways might lead to periodontitis, but for many or most routes, the cytokine network probably involves regulating the related disease activity and tissue destruction. Both IL-I and TNF-a have been found to stimulate collagenase, prostaglandin E2 (PGE2) or IL-6 production by fibroblasts,924 and to induce bone resorption.2526 In addition, these cytokines also stimulate mononuclear cells or endothelial cells to produce IL-8, which induces neutrophil infiltrates.2728 Since CD45RO has been shown mainly on T cells but also on minor populations of macrophages or B cells, our findings that close association of IL- 1 mRNA-expressing cells with CD45RO+ cells indicate a probable role of IL- I in the activation and proliferation of T cells in the inflamed tissue (Fig. 2a).

Cytokine mRNA-expressing cells in gingiva

45

Figure 2. (a) IL-l: mRNA-expressing cells are located around accumulating CD45RO+ cells. (b) OPD4+ cells expressing IL-6 mRNA are shown in plasma-like cells lesion (arrow). (c) Although very seldom, IL-6 mRNA-expressing CD20+ cells are observed (arrows). (d) Endothelial cells identified by anti von Willebrand factor express IL-6 mRNA. (e) Very few IL-8 mRNA-expressing macrophages are shown in plasma cell-rich lesion (arrow). (f) Even if many macrophages are observed, any positive grains are not demonstrated by using control probe. Original magnification x 500.

Although IL-I mRNA-expressing cells were mostly macrophages in the inflamed gingival tissues, they were assumed to play various roles in the generation of the lesions. IL-6 had the widest range of producer cell types: macrophages, endothelial cells, fibroblasts, T cells and B cells. B cells and plasma cells are the predominant inflammatory cells in advanced periodontal lesions.29 It is reasonable to assume,

therefore, that factors with the ability to differentiate B cells and plasma cells occupy these lesions. IL-6 is a well-established cytokine that induces maturation of B cells into immunoglobulin-secreting cells.'2 Judging from the evidence, the IL-6 mRNAexpressing cells might induce maturation of surrounding B cells into plasma cells and lead to an advanced periodontal lesion. Stimulation with a combination of CD2, PMA, and CD28 was

Y. Matsuki, T. Yamamoto & K. Hara

46

Table 2. Summary of the cytokine mRNA-expressing cells in human inflamed gingiva (27 sites) IL-6

IL-8

%*

IL-la

IL-If

M+20-30 M- 5-10

+ - ++ +

+ - ++ +

M+20-50 M-40-50

-

-

± - +

+

-

+

+

-

B cell (CD20) +plasma cell

M+20-30 M-40-60

-

-

-

+ +

-

Endothelial cell (vWF)

M+

± - + +

± - + +

-

- + +

+ +

+ +

-

± - + +

Cell type

Macrophage(CD68) Tcell(CD3,OPD4,CD45RO)

MM+

Fibroblast

M-

TNF-a - + +

- + +

+ - ++ +

± - + +

+ +

The incidence (%) of grain-positive cells in a special cell type was arbitrarily graded from + + to -: + + = >40%, + =10-40%, + = < -0%,-=negative. * Approximate composition (%) among the infiltrating cells. M +, M + lesion, n = nine sites; M -, M - lesion, n = 18 sites.

shown to induce IL-6 production by memory (CD4+CD45RO+) subsets of T cells.30 Our finding that OPD4+ and/or CD45RO+ cells occasionally expressed IL-6 mRNA suggested IL-6 production by T-cell subsets in vivo. These T-cell subsets could be involved in activation or maturation of B cells. IL-8 mRNA-expressing cells were hardly observed in the M- lesion but they were demonstrated frequently in the M+ lesion. IL-8 could play a role as an initial mediator of the disease in attraction of neutrophils as supposed in vitro."3 This study elucidated the main source of inflammatory cytokines in inflamed gingiva. However, the biological function of a particular cytokine may vary, depending on the phase of inflammation and the target cells. Over-production of the cytokines studied here leads to tissue destruction such as loss of attachment, pocket formation and bone resorption through stimulation of collagenase or PGE2 production. It is, therefore, important to identify all factors in the cytokine network so as to clarify the role of cytokines and to inhibit or to break the pathway of their activities in periodontal disease.

ACKNOWLEDGMENTS We thank Dr H. Yoshie (Dept. of Periodontology, Niigata University School of Dentistry) for access to patients under his care. We are grateful to Professor I. Kihara, Dr K. Kawasaki, E. Yaoita (Dept. of Pathology, Institute of Nephrology, Niigata University School of Medicine) for their helpful advice. We also thank Ms P. Minick for her skilful editing of the manuscript and Mr K. Yoshida for sectioning the specimens. This study was supported in part by a Grant-in-Aid for Fellowships of the Japanese Society for the Promotion of Science for Japanese Junior Scientist, Japan.

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