Localization of IgE immunoglobulin in human dental periapical lesions by the peroxidase-antiperoxidase method

Arch\ orul llinl. Vol. 26. pp. 677 to 681. 1981 Pnnwd in Great Britam

ooO3-9969/81/080677-05602.00!0 Pergamon Pm, Lfd

LOCALIZATION OF IgE IMMUNOGLOBULIN IN HUMAN DENTAL PERIAPICAL LESIONS BY THE PEROXIDASE-ANTIPEROXIDASE METHOD M. TORABINEJAD’, J. D. KETTERING’and L. K. BAKLAND~ ‘Associate Professor of Endodontics, School of Dentistry, Loma Linda University, Loma Linda, California, U.S.A. 24ssociate Professor of Microbiology, School of Medicine, Loma Linda University, Loma Linda, California, U.S.A 3Associate

Professor

and Chairman,

Department of Endodontics, Loma Linda, California,

School U.S.A.

of Dentistry,

Loma

Linda University,

Summary-Twenty-eight dental periapical lesions were examined for the presence of light chains of human immunoglobulins, IgG, and IgE by the peroxidase-antiperoxidase method; 27 stained positively for light chains and IgG and 20 for the presence of IgE. One tissue specimen, diagnosed histologically as periapical scar tissue, contained no immunoglobulins. The presence of IgE immunoglobulin in 74 per cent of periapical tissue specimens suggests that IgE-mediated reactions participate in pathogenesis of human dental periapical lesions.

MATERIALSAND

INTRODUCTION Toller and Holborow (1969), using the immunofluorescent antibody technique, found numerous IgAand some IgG- and IgM-containing cells in the walls of human periapical cysts. Skaug (1974) measured the levels of IgG, IgA and IgM, a- and b-globulins and albumin in non-keratinizing jaw cysts by single radio immunodiffusion; his findings indicate a local synthesis of immunoglobulins, mainly IgG and IgA, in the cyst walls. Naidorf (1975) studied the extracted immunoglobulins from three human dental periapical lesions by immunoelectrophoresis and single radioimmunodiffusion. In two lesions, diagnosed as granulomas, IgG, IgA, and IgM were found; the third lesion, diagnosed as scar tissue, contained no immunoglobulins. Morse, Lasater and White (1975), utilizing the pyronin-methyl green stain, found many positively stained plasma cells in periapical granulomas and cysts. They suggested that the positive cells contained immunoglobulins. Malmstrom (1975), Kuntz er al. (1977) and Morton, Clagett and Ya.vorsky (1977) examined periapical biopsy specimens by immunofluorescence techniques (IFT) and found numerous positive cells for IgG, IgM and IgA immunoglobulins. Jones and Lally (1980) examined five biopsy specimens of human periapical lesions by autoradiography and found that IgG and IgA were synthesized in ail five of the lesions. However, no trace of IgM biosynthesis was found. By contrast, the literature concerning IgE in these lesions is sparse. Pulver, Taubman and Smith (1978), using IFT, found that 5-10 per cent of immunoglobulin-containing cells in periapical granulomas and cysts were positive for IgE. Levy (1978) reported that 59 per cent of his periapical biopsy samples had cells positive for IgE. Our aim was to confirm the presence of IgE immunoglobulin in human periapical lesions using the peroxidase-antiperoxidase (PAP) method. 617

METHODS

Collection, fixation and sectioning of tissue samples Twenty-eight biopsy samples were obtained from patients undergoing periapical surgery in the Department of Endodontics at Loma Linda University, School of Dentistry. The indications for surgery were in general those recommended by Ingle and Beveridge (1976). Medical histories of all patients contained nothing relevant. The biopsy samples were immediately fixed in a mixture of 1 part Zenker fixative and 9 parts 10 per cent buffered formalin at 4°C for 4 h and then soaked in 30 per cent sucrose solution overnight according to Eidelman and Berchauer (1969). The specimens were later embedded in drops of mounting medium (O.C.T., Ames Co., Elkhart, Ind.) on a small wood block and stored at -70°C. The biopsies were in due course embedded in paraffin wax and sectioned at 6pm. Tissue staining For histopathologic diagnosis of the periapical lesions, at least one section of each specimen was stained with haematoxylin and eosin. Five to ten adjacent sections were then stained for the presence of light chains (K or A)of immunoglobulins, IgG and IgE by the PAP method of Taylor, Russel and Chandor (1978). The PAP method consisted of the following steps: slides were de-paraffinized and hydrated by passage through alcohol to tris-saline buffer. While still wet, they were placed in a humidity chamber and incubated with normal swine serum for 15 min at room temperature. Each slide was briefly rinsed with trissaline and wiped dry, except for the tissue section area. A 1:500 or 1: 1000 dilution of rabbit anti-human IgG, IgE, or K or I light chains of human immunoglobulins (DAK0 Corp., Santa Barbara, Calif.) was placed upon the appropriate section and incubated for 45 min. Appropriate dilutions of all immuno-

M. Torabinejad, J. D. Kettering and L. K. Bakland

678

Table 1. Frequency of light chains (ICand 1) of immunoglobulins IgG and IgE in formalin-fixed and paraffin-embedded human periapical lesions* Substance localized

No. positive

No. tested

Percentage

K

27 27 27 20

27 27 27 27

100 100 100 74

1 IgG IgE

* Periapical scar tissue sample was not included.

globulin reagents used were determined by a previous pilot study. After incubation, the slides were rinsed and placed in a tris-saline bath for 30 min, rinsed again and dried except for the tissue section area, and returned to the humidity chamber. A 1: 30 dilution of swine anti-rabbit serum was added to all sections, which were then incubated for another 30min. The slides were rinsed as described above and placed in the humidity chamber. A 1:200 dilution of peroxidase-antiperoxidase complex (DAK0 Corp., Santa Barbara, Calif.) was added to the sections, incubated for 30 min and then rinsed as described. After the last tris-saline bath, the slides were placed on a staining rack and treated with diaminobenzidine reagent plus hydrogen peroxide for 3-5 min. They were then thoroughly rinsed with tap water, counterstained with haematoxylin, dehydrated and mounted with Permount. Positive controls consisted of 6 pm thick sections of formalin-fixed, paraffin-embedded human adenoid tissue from patients with symptoms of inhalant allergy and augmented IgE levels. Although the monospecificity of the anti-IgG and anti-IgE used here had been tested by the manufacturer, some representative sections of adenoid tissue and the periapical lesions were also stained by the PAP method except for the application of the primary antisera. Negative staining after by-passing the first step in the PAP method indicated the monospecificity of the eliminated reagent. Microscopy and data collection

The sections were examined with a light microscope and photographs of selected sections taken. The presence of K or I light chains, IgG and IgE in histologic sections was manifested by a brown cytoplasmic colour in cells with the classic morphologic configuration of human plasma cells. Samples were regarded as positive when at least 5 sections had one or more peroxidase-positive plasma cell in each high-power field ( x 400 magnification). RESULTS Examination of the haematoxylin and eosin-stained specimens showed that, of the 28 periapical lesions, 17 were granulomas, 10 were apical periodontal cysts and 1 was periapical scar tissue. The sections of formalin-fixed and paraffin-embedded adenoid tissue displayed positive IgE-containing plasma cells beneath the surface epithelium and

within and around the germinal centres (Fig. 1). Tissue sections from these patients also had positive IgG-stainable cells. Of the 28 periapical samples, 27 showed numerous K and 1 light chains and IgG-positive plasma cells (Figs 2 and 3). The periapical scar tissue was negative for IgG and for the immunoglobulin light chains (Fig. 4). Of the 27 granulomas and cysts, 20 had IgEpositive plasma cells (Fig. 5). Of the 7 periapical lesions without positive staining for IgE, 4 were cysts and 3 were granulomas. The periapical scar tissue specimen contained no cells positive for IgE. The frequency of K and i, light chains, IgG and IgE in the periapical specimens are reported in Table 1. Although the number of IgG- or IgE-containing cells were not quantitated for each specimen, IgG-cells appeared 5-10 times more numerous than IgE-cells. The specificity of the antisera was confirmed when control sections did not stain for IgG or IgE after elimination of rabbit anti-human IgG or IgE immunoglobulins (Fig. 6). DISCUSSION

Although the techniques of direct or indirect immunofluorescence are highly specific, they have several drawbacks (Taylor, 1978): (1) The specimens stained by IFT are impermanent and the fluorophores fade upon excitation by light. (2) In IFT, the specimens usually require immediate freezing resulting in some loss of morphologic detail. (3) Examination of specimens prepared by IFT requires specialized microscopy. Alternatives include various procedures using horseradish peroxidase as a labelling enzyme and diaminobenzidine plus hydrogen peroxide as chromogenic substrate for visual observations. Peroxidase, either conjugated (direct and indirect) or non-conjugated (hybrid antibody method, enzyme bridge method and the PAP method), are various modifications of immunoperoxidase methods (Taylor, 1978). The PAP method, initially introduced by Sternberger (1974), not only circumvents the shortcomings of other immunoperoxidase methods and IFT, but is also 1~1000 times more sensitive than IFT. We therefore used the PAP method. The positive cytoplasmic staining of plasma cells for IgG and IgE we found in control tissues and the absence of staining when the primary antiserum was eliminated indicate the monospecificity of the antisera used and the reliability of our findings. Further evi-

IgE in human

dental

dence for this is the simultaneous presence of positive and negative plasma cells in a given microscopic field. Absence of positive IgG-, IgE-, K- and i-containing cells in one periapical lesion which was histologically scar tissue indicates that the PAP method specifically stains cells containing immunoglobulins. The presence of positive light-chain containing plasma cells in all the granulomas and cysts indicates that local production of immunoglobulins occurs in periapical lesions. Our results confirm findings by Naidorf (1975). The observation of numerous plasma cel,s stained positively for IgG in every granuloma and cyst indicates that the primary antibody response to ;he antigens egressing from the root canal system is IgG. Our results confirm earlier reports of IgG in human periapical lesions (Toller and Holborow, 1969; SkiLug, 1974; Naidorf, 1975; Kuntz et al., 1977; Mortar, et a/., 1977; Jones and Lally, 1980). l’ulver et al. (1978) found an average of 15 positive I#-stainable cells in a x 40 field in 13 periapical lesions. The one or more positive cells in a x 400 field in our study is almost equal to the average number obtained by Pulver et al. Because the data in the two studies were collected and treated differently, the findine,s cannot be directly compared. However, both imestigations show the presence of IgE-containing plasma cells in periapical lesions. The presence of Igl-forming plasma cells in 74 per cent of our periap.cal lesions and 59 per cent in those of T. A. Levy (unpublished) also indicates a potential involvement of tgE-mediated reactions in the pathogenesis of periap cal lesions. Plasma cells in periapical lesions may produce IgE im:nunoglobulin against various antigens egressing from the root canal system or, as Stanworth (1973) speculated, IgE may be produced as a by-product of Ig!\ biosynthesis. Studies with known antigens leading to production of specific antibodies in the periapical tissues of experimental animals are needed to identify the sequence in the development of various classes of immunoglobulins. ‘Ne made no special attempt to identify mast cells, bu: Mathiesen (1973) found numerous mast cells in human periapical lesions. This and the apparent produ:tion of IgE molecules in response to the egress of anrigens from the root make it reasonable to speculat: that, in addition to immune-complex mediated and delayed hypersensitivity reactions (Torabinejad an.1 Kettering, 1979; Stabholz and McArthur, 1978), IgE-mediated reactions may participate in the pathogenesis of human dental periapical lesions. A(.inowledgements-We would like to recognize and thank MI Ray Russell, University of Southern California, Los Angeles County Medical Center, for his valuable contribu-

Plate

periapical

679

lesions

tions and his technical

assistance

throughout

this experi-

ment. REFERENCES

Eidelman S. and Berschauer J. 1969. A method for Immunocytochemical study of human gastrointestinal section biopsies. Stain Techno/. 44, 43-44 Ingle J. I. and Beveridge E. E. 1976. Endodontic,s. 2nd edn. pp. 595-611. Lea & Febiger, Philadelphia. Jones 0. J. and Lally E. T. 1980. Biosynthesis of immunoglobulin isotypes in human periapical lesion. J. Endodont. 6, 672-677. Kuntz D. D., Genco R. J., Guttuso J. and Natiella .I. R. 1977. Localization of immunoglobulins and the third component of complement in dental periapical lesions. J. Endodont. 3, 68-73. Malmstrom M. 1975. Immunoglobulin classes of IgG, IgM, IgA and complement components C3 in dental periapical lesions of patients with rheumatoid disease. Stand. J. Rheumat. 4, 57-64. Mathiesen A. 1973. Preservation and demonstration of mast cells in human apical granulomas and radicular cysts. Stand. J. dent. Res. 81, 218-299. Morse D. R., Lasater D. R. and White D. C. 1975. Presence of immunoglobulin-producing cells in periapical lesions. J. Endodont. 1, 338-343. Morton T. H., Clagett J. A. and Yavorsky D. J. 1977. Role of immune complexes in human periapical periodontitis. J. Endodonr. 3, 261-268. Naidorf I. 1975. Immunoglobulins in the periapical granulomas: a preliminary report. J. Endodok. 1, -15-l < Pulver W. H., Taubman M. A. and Smith D. J. 1978. Immune components in human dental periapical lesions. Archs oral Biol. 23. 435-443. Skaug N. 1974. Proteins in fluid from non-keratinizing jaw cysts. 4. Concentrations of immunoglobulins IIeG. IeA ,and IgM) and some non-immunoglobulin proteins: relevance to concepts of cyst wall permeability and clearance of cyst proteins. J. oral Path. 3, 47-61. Stabholz A. and McArthur W. P. 1978. Cellular immune response of patients with periapical pathosis to necrotic dental pulp antigens determined by release of LIF. J. Endodont. 4, 282-287. Stanworth D. R. 1973. Immediate Hypersensiril-if!. pp. 275--289. North-Holland, Amsterdam. Sternberger L. A. 1974. Immunocytochemisrr!*. PrenticeHall, Englewood Cliffs, N.J. Taylor C. R. 1978. Immunoperoxidase techniques. Archs Path. lab. Med. 102, 113-121. Taylor C. R.. Russel R. and Chandor S. 1978. An immunohistologic study of multiple myeloma and related conditions using an immunoperoxidase method. Am. J. chin. Path. IO, 612-622. Toller P. A. and Holborow E. J. 1969. Immunoglobulins and immunoglobulin containing cells in cysts of the jaws. Lancet 2, 178-181. Torabinejad M. and Kettering J. D. 1979. Detection of immune complexes in human dental periapical lesions by anticomplement immunofluorescence technique. Ornl Surg. 48, 256261.

1 overleaf.

Y

680

M. Torabinejad, J. D. Kettering and L. K. Bakland

Plate 1. Fig. 1. Immunoperoxidase staining of &E-containing cells (immunoglobulin stained brown) in the germinal centers of human adenoid tissue with inhalant allergy. Formaldehyde-parat% section; counterstained with haematoxylin. x 400 Fig. 2. Immunoperoxidase staining of K light-chain containing cells in human periapical Formaldehyde-paraffin section; counterstained with haematoxylin. x 400

lesions.

Fig. 3. Immunoperoxidase staining of IgG containing cells in human periapical lesion. Formaldehydeparaffin section; counterstained with haematoxyhn. x 400 Fig. 4. Immunoperoxidase staining of K light-chain containing cells in human periapical scar tissue. Formaldehyde-paraffin section; counterstained with haematoxylin. x 400 Fig. 5. Immunoperoxidase staining of IgE containing cells in human periapical lesion. Formaldehydeparaffin section; counterstained with haematoxylin. x 400 Fig. 6. Blocking of immunoperoxidase staining of IgE-containing cells in human periapical lesion. Formaldehyde-paraffin section; counterstained with haematoxylin. x 400

IgE in human

dental

periapical

lesions

681