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Master's Theses Theses and Dissertations

1980

The Use of Immunoperoxidase Method for theIdentification and Distribution of I G, I A, I M andC3 in Human Inflamed GingivaCesar Ruiz MoralesLoyola University Chicago

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THE USE OF IMMUNOPEROXIDASE METHOD

FOR THE IDENTIFICATION AND DISTRIBUTION

OF IgG, IgA, IgM AND C3 IN

HUMAN INFLAMED GINGIVA

By

Cesar Ruiz Morales

A Thesis Submitted to the Faculty of the Graduate School

of Loyola University in Partial Fulfillment of the

Requirements for the Degree of

Master of Science

May 1980

DEDICATION

To my beloved wife, Ginny, for having been patient when things

were difficult during the course of finishing this thesis and for her

love.

To my mother, Margarita, from whom I learned to be perseverant

and who has given me love and support.

To Doctor Toto, the teacher who has most affected my academic

growth.

i

VITA

Cesar Ruiz Morales, the fifth of six children from Leopolda Ruiz

Navarro and Margarita Morales. de Ruiz, was born on March 29, 1950, in

Mexico City, Mexico.

He obtained his Bachelor Degree from La Academia Militar Mexico,

Mexico City, in 1969. He entered the La Universidad Nacional Autonoma

de Mexico, in Mexico City, in January, 1970, in order to obtain the

degree of Business Administration, in 1973. In the same year, he

ingressed to La Facultad Nacional de Odontologia, in Mexico City, to

receive the degree of Cirujano Dentista, 1976.

In September of 1977, he began a two year program leading to a

certificate in Fixed Prosthodontics, at Loyola University, Maywood,

Illinois.

In 1979, he started a research to fulfill the requirements to

obtain the degree of Master of Science in Oral Biology at Loyola

University.

ii

ACKNOWLEDGFMENfS

To Doctor Patrick D. Toto, Professor and Chairman, of the Depart-

ment of General and Oral Pathology, at Loyola University, who suggested

this investigation and who supervised and guided me to its completion.

I want to offer my sincere gratitude and appreciation for his advise,

his kind assistance, and time spent on my behalf throughout my research

training.

To Doctor P~thony W. Gargiulo, Professor and Chairman, of the

Department of Periodontics, at Loyola University. I would like to

thank him for his assistance and valuable instructions in periodontology.

Lastly, I extend my sincere thanks to Doctor John P. Wortel,

Professor of Pathology, Department of General and Oral Pathology, who

taught me Oral Pathology, and for his valuable guidance to finish this

thesis.

iii

TABLE OF CONTENTS

I. INTRODUCTION ........................................ 1

II. LITERATURE REVIEW ................................... 3

A. The Inflamatory Process in Inflamed Gingiva ..... 4

1. Bacteria and Endotoxins ..................... 4 2. Host Reactive Cells ......................... 7

a. Mast Cell ................................ 8 b. Macrophages ............................ 10 c. Polymorphonuclear Leukocytes ........... 11 d. Lymphocytes and Plasma Cells ........... 13

B. Plasma Cells and Related Immunoglobulins Fluorrescine Isothiocyanate Studies ............ 17

C. The Role of Complement, Activation in Gingival Inflamation .................................... 20

D. Collagenolitic Activity During Inflamation of the Gingiva ................................. 23

E. Immunoperoxidase Method to Study Immuno-globulins, Plasma Cells and Complement 3 ••••••• 24

III. MATERIALS AND :ME'IBODS .............................. 26

IV. RESULTS ............................................ 29

A. Histological Findings of the Control Group ..... 29

B. Histological Findings of the Experimental Group .......................................... 29

C. Immunoperoxidase Conjugated Antibody · Findings; Control Group ........................ 30

D. Immunoperoxidase Conjugated Antibody · Findings; Experimental Group ................... 31

V. DISCUSSION ......................................... 33

iv

VI. S~~y AND CONCLUSIONS •.•.•...••..•..•.•.•.....•.•. 42

VII. BIBLIOGRAPHY .....•.•..•.•..............•.•.....•.•... 44

VI I I. TABLES ..•..•••••.•.•••....••...••......••..•..•..•... 51

IX. FIQJRES ........•••....•...•...•.....•..••.••.•••.••.. 63

v

LIST OF TABLES

TABLE PAGE

I Hematoxilin and Eosin Staining- Experiment Group .... 52

II Hematoxilin and Eosin Basement Membrane -Experiment Group ............... 53

III Hematoxilin and Eosin Connective Tissue -Experiment Group ............... 54

IV Hematoxilin and Eosin Epithelium- Control Group •.... 55

V. Hematoxilin and Eosin Basement Membrane -Control Group .................. 56

VI. Hematoxilin and Eosin Connective Tissue -Control Group .................. 57

VII. Irnrnunoperoxidase Staining -Fxperirnent Group ............... 58

VIII. Irnrnunoperoxidase Staining -Experiment Group ............... 59

IX. Irnrntmoperoxidase Staining -Experiment Group ............... 60

X. Irnrnunoperoxidase Staining -Control Group .................. 61

XI. CHI-Square Statistical Analysis ....................... 62

vi

LIST OF FIGURES

Figure 1 .............................. 64

Figure 2 ••.•..•••••••.••••.•••••.••.•• 65

Figure 3 ...•.........•................ 66

Figure 4 ••••••••••.••••.•••••••••••••. 67

Figure 5 .............................. 68

vii

CHAPTER I

INTRODUCTION

Thirty samples of clinically inflamed human gingival tissue were

stained with the immunoperoxidase method, in order to detect the presence

of IgG, IgA, IgM, and C3. Ten samples of clinically normal human gingi-

val tissue were used as control.

Both clinically normal and inflamed gingival tissue have shown to

be positive though the peroxidase conjugated anti-human Ig's and C3 in

the epithelial intercellular spaces, intracellular epithelial cells,

basement membrane, lamina propria, the cytoplasm of plasma cells, and

in the perivascular spaces.

The presence of Ig's and C3 in the clinically normal gingival tissue

may be interpreted as an immunologically state of defense of the host

against any antigenic substance that may gain access to the gingival

tissue, in order to maintain a state of homeostasis. Infiltration of

phagocytic cells in the epithelial intercellular spaces of the normal

gingival tissue was commonly observed. This infiltration can be expected

since C3, Ig's and supposedly antigens, which all are chemotactic agents,

are constantly present in the epithelium.

In the inflamed gingival tissue, a greater number of phagocytic

cells were identified infiltrating the epithelium, as compared to the

non-inflamed group. The concentration of Ig's and C3 in the inflamed

1

group was also stronger than that of the control group. This suggests

that once the homeostatic state is interrupted by the ingress of

greater gradient of antigens, to the gingival tissues, the immune

system starts to produce larger amounts of Ig's and C3.

2

The presence of larger amounts of Ig's, C3 and antigens means that

many chemotactic agents are present. This chemotactic agent attracts

neutrophils and macrophages cells that, in their effort to phagocyte

antigenic material, they destroy the surrounding gingival tissues.

CHAPTER II

LITERATIJRE REVIEW

A. The Inflamatory Process in Inflamed Gingiva

1. Bacteria and Endotoxins 2. Host Reactive Cells

a. Mast Cells b. Macrophages c. Polymorphonuclear Leukocytes d. Lymphocytes and Plasma Cells

B. Plasma Cells and Related Immunoglobulins -Immunofluorescent Studies

C. The Role of Complement 3, Activation in Gingivitis

D. Collagenase Study During Inflarnation of the Gingiva

E. Imrnunoperoxidase Method to Study Immunoglobulins, Plasma Cells and Complement 3

3

4

A. The Inflamatory Process in Inflamed Gingiva

The morphologic changes of human gingiva as seen in gingivitis

and periodontitis is characteristic of a chronic inflamatory process.

There is evidence that this change is attributed to the dental

plaque bacteria and the response of the host to such bacteria.

1. Bacteria and Endotoxins

The sulcular epithelium is not keratinized, lacks of

epithelial ridges and is thinner than the gingival epithelium.

For this reason, the sulcular epithelium is thought to be

more easily subject to inflamation, as bacterial toxins may

pass through it into the connective tissue. (l,Z, 3) Inversely,

fluids from the connective tissue may pass through the sulcular

epithelium into the gingival sulcus.C4)

As early as (1959), Gargiulo reported the permeability of

the mucosa by H2o2CS).

Brill and Krasse (1958), showed that after the systemic

administration of sodium fluorecein, it is possible to be

recovered in the gingival sulcus fluid of dogs. This obser-

vation demonstrated the permeability of the gingival sulcus

epithelium to fluids originating in the lamina propria derived

from the ·circulating blood stream. (6)

Mergenhagen (1960), showed that soluble endotoxins of oral

bacteria could elicit hemorrhagic and necrotic lesions in the

rabbit when'injected intracutaneously. He felt that endotoxic

properties of certain oral bacteria in the gingival sulcus

may be an important factor in periodontal disease. (7)

However, direct invasion of the gingival sulcus tissue

by the bacteria has not been reported.(8) Although List-

garten in 1965 reported bacteria in the gingival tissue of

humans with acute necrotizing ulcerative gingivitis.C9)

Brandtzaeg stated in 1966 that gingival sulcus fluid

exerted a rinsing effect sufficient for the removal of small

5

particles, exogenous pathogenic bacteria and bacterial plaque.

This finding suggests a function of cleansing by fluids

permeating from the gingival sulcus epithelium.ClO)

Rizzo and Berglund in 1969 demonstrated that endotoxins

in concentration as low as one hundred ug/ml could produce

an inflamatory response in gingival tissue of rabbits after

it penetrates the epithelium. (ll)

According to Tolo (1971), the basement membrane consti-

tutes a barrier to the passage of molecules with a molecular

weight greater than 50000 daltons. Therefore, one would

expect the macromolecules derived from dental plaque to "back

up" at the basement membrane. (lZ)

According to Loe and Holm-Pederson (1965), the gingival

flow rate increased during inflamation, even several days

prior to clinical detection of the inflamatory process. (l3)

6

Later on in 1967, Loe, Theilade, Jensen, and Schiatt

reported that the flow rate of the gingival fluid increased . (14)

concurrently with the accumulat1on of dental plaque.

According to Alfano's theory (1974), macromolecular

bacterial by-products accumulate at the basememt membrane,

and a standing osmotic gradient is generated in the gingival

sulcus and initiates the flow of gingival fluid. Since these

macromolecular by-products are composed of enzymes such as

hyaluronidase and collagenase, among others; their diffusion

through the sulcus epithelium could degrade the basement

membrane components of the gingival sulcus, allowing the

penetration of other bacteria into the connective tissue.

This would generate more exudate due to the resulting infla-

mation. (lS)

Schwartz, Stinson, and Parker (1970), showed the pene-

tration of tritiated endotoxins obtained from E. coli through

the intact sulcular epithelium in dogs, indicating it's

permeability to an injurious agent.Cl6)

Gerencser, Laudfried, and Slack (1971), identified

twelve species of filamentous or diphtheroid bacteria in

dental calculus by specific antisera using the fluorescent

antibody technique. Among those found were A. israelii,

A. viscosus, A. naeslundii, Arachnea propionica, R. dento-

cariosa and C. acnes.Cl7)

7

Caffesse and Nasjleti (1976), demonstrated that hyaluroni-

dase has the capability to penetrate through nonkeratinized

sulcular epithelium spaces and disorganizing the connective

lamina propria tissue in marmosets. They have shown further

that collagenase could then penetrate into the intercellular

epithelial spaces, but only after hyaluronidase first was

directly applied in the gingival sulcus. (lS)

2. Host Reactive Cells

The evidence that the gingival sulcus epithelium is

permeable to plasma originating in the lamina propria and to

the possible macromolecules originating in the sulcus con-

taining bacteria suggests that the homeostatic mechanism of

gingiva may be disturbed. Accordingly, should the macro-

molecules produced by bacteria located in the gingival sulcus

gain access through the epithelium, it may be expected that

this would result in the inflamation observed in gingivitis

and periodontitis. The histopathology of gingivitis and

periodontitis clearly shows a morphologic disturbance in the

gingiva characterized by changes in the epithelium and the

supporting connective tissues.

Inflamatory Cells Associated with Gingivitis and Periodontitis

The morphologic changes in the gingiva are always asso-

ciated by the evidence of polymorphonuclear leukocytes,

macrophage, small and medium sized lymphocytes, plasma cells

and mast cells infiltration.Cl9)

a. Mast Cells

8

Mast cells are supposed to derive from undifferentiated

mesenchYffial cells. They tend to congregate along small

blood vessels. Cytologically, mast cells are large round

cells with a pale nuclei frequently obscured by the

abundance of granules, which contain heparin eosinophilic

chemotactic factor, serotonin, and histamine. With basic

dye the granules stain intensely. Serotonin and histamine

are vasoactive and cause smooth muscle contraction and

vasodilation of venules in increasing their permeability.

Heparine is related to the coagulation process by its

inhibition of fibrin clot formation. (20)

According to Zachrisson (1967), clinically normal

human gingiva contains mast cells in larger numbers than

chronically inflamed human gingiva. (2l)

Zachrisson (1968), using different methods of staining,

found that mast cells disappeared during inflamation due

to the degranulation of the cells, with release of histamine

and serotonin, inflamatory mediators. He compared healthy

gingival tissue from 16 individuals with that of 20 indi-

viduals who were induced to gingivitis.C22)

9

Sheltcnand Hall (1968), compared mast cell populations

of gingival tissue from twenty individuals, ten with

clinically normal gingiva and ten with periodontitis.

They reported that the mast cell count decreased in gin-

givitis; at the same time the count of granules was also

decreased. However, after routine therapy the mast cell

count returned to "normal".(23)

Shapiro, Ulmansky and Schever in 1969, in contrast,

reported that mast cells were statistically, significantly

increased in inflamed human gingiva of thirty-five patients

as compared to healthy human gingiva of thirteen patients.

They suggest that chronic destructive periodontal disease

may be related to the increased number of mast cells,

since heparin has been related to bone resorption.C24)

Barnett (1973), found mast cells also in the epithelium

of chronically inflamed human gingiva. He suggested that

epithelial mast cells may have the function of phagocy-

tosis in the sulcular lesion of periodontal disease. The

liberation of trypsin by mast cells enzymatically affect

keratinocytes attachments, thereby increasing the size and

permeability of the sulcus epithelial intercellular

spaces. Barnett also suggested that an immune complex

effects mast cell degranulation in gingivitis.C25)

According to Ward (1972), complement 3 and 5

mediators of inflarnation have the potential to activate

mast cells to degranulate. (26)

b. Macrophages

The macrophages derived from mononuclear cells which

originate in the bone marrow. These cells circulate in

the body through the peripheral blood, where they are in

a form of monocytes. When they are released to the

tissues they become macrophages. Their primary function

10

is to phagocytize host tissue debris and any microorganism

and their by-products. (27)

Macrophages respond to chemotactic factors produced

by lymphocyte products, lyrnphokines, immune complexes,

and also from activated complement.C28)

Macrophages - derived substances may also effect cell

proliferation. (30) Activated macrophages have the capacity

to synthesize and secrete lysosomal acid hydrolases and

lysozyme. (3l)

The proteases released by macrophages in the site of

inflarnation cleave C3 into active fragments C3a and C3b,

which further activate C5-C9. Fragments C3a and CSa are

chemotactic and cause further accumulation of neutrophils

and macrophages thereby increasing the cellular infiltrate.C32)

According to Schluger, Yuodelis, and Page in 1977,

when antigens enter to the body, they become associated

with macrophages. Once these antigens are taken up by

macrophages, these cells present the antigens to the

11

B and T lymphocytes in the lymph nodes or spleen. B and T

lymphocytes then undergo blastogenesis which transform

in a large population of antigenic sensitive cells. The

small lymphocytes carry the information of the antigens

and leave the lymph nodes via the blood stream to localize

or approach the site of the injury. Finally the Bart

lymphocytes undergoes transformation to plasma cells or

lymphocytes respectively. The sensitized plasma cells

start to produce specific antibodies.C33)

c. Polymorphonuclear Leukocytes

In the blood the neutrophils comprise about 60% of

the total circulating leukocytes. Of these cells, 75%

are used and replaced every day. Neutrophils are the

first line of defense against any form of injury of the

tissues. Usually, these cells are present in all

inflamatory lesions.C34,35,36) Neutrophils have the

primary function of phagocytosis of tissue debris in-

cluding any microorganism or noxious substance in the

tissues. Their cytoplasm granules are both specific and

lysosomal. The granules contain many enzymes including

hyaluronidase, lactoferrin, alkaline phosphatase

myeloperoxidase, phospholipase, carbohydrases, pro-

teases, DNAse and RNAse. The neutrophils have the

capacity to respond to chemotactic factors directed

by movement in an ameboid fashion.C37,38,39)

According to Temple (1970), the P.M.N. 'sand

macrophages respond to two classified types of chemo-

tactic factors. The first includes bacterial chemo-

12

tactic factors that directly exert attraction to P.M.N. 's

and macrophage. The second chemotatic factor is produced

by interaction of bacteria or bacterial products with a

host factor.C40)

According to Kraal and Loesche, in 1974, complement

is one major source of endogenous leucotaxins, where

the complement system interacts with a variety of acti-

vating agents. C4l)

According also to Kraal and Loesche, in 1974, the

P.M.N. 's are also related to tissue destruction by the

release of their lysosomal enzymes. C42) Thilander in

1963 showed that when lysosomal enzymes were applied

into the gingival sulci of human volunteers, there re-

suited in a widening of intercellular spaces in the

sulcular epithelium.C43)

Taichman, Freedman, and Uriuhara in 1966, reported

that after injection of gingival plaque containing live

bacteria intradermally into the abdomen of albino

rabbits resulted in a formation of an abscess and massive

tissue destruction at the site of the injection; while

in leukopenic rabbits the injection of bacteria resulted

in dissiminating and caused death, but without tissue

destruction in the site of injection.C44)

d. Lymphocytes and Plasma Cells

Monocytic lymphocytes and histiocytes comprise the

principal cells of tissues immune system. (45)

Nesengard, in an excellent review of the immune

response (1977), explained that the lymphocytes derive

from a pleuripotent stem cells that can be primarily

found in the embryonic yolk sac. The lymphocyte migrates

first to the fetal liver and then to the bone marrow.

The lymphocytes travel to one of the two primary lymphoid

organs, the thymus gland and the bursa of the Fabricius

in chickens or the gut in humans. Thus, lymphocytes

influenced by the thymus gland are termed T cells or

thymus derived cells, while those which differentiated

in the gut are termed B cells, or bursa derived cells.

He also pointed out that antigen stimulation causes

blast transformation of both types of cells. B cells

13

differentiate further to plasma cells while T cells pro-

duce more lymphocytes and lymphokines. The plasma cells

produce immunoglobulins responsible for the antibody

activity (Humoral immunity), while the T lymphocytes are

responsible for the cellular immunity or cell mediated

immunity (CMI). C46)

He also explains that T cell lymphokines are released

14

as soluble mediators with a range of biological activities

as follows:

1. Inhibition of macrophage migration (MIF). 2. Chemotaxis for macrophage. 3. Activation of macrophage. 4. Inhibition of PMN migration. 5. Chemotaxis for PMN's. 6. Mitogens inducing blast formation of

nonsensitized lymphocytes. 7. Transfer factor cell-mediated immunity. 8. Cytotoxic for fibroblast. 9. Osteoclast activating factor (OAF). C47)

Horton, Raisz, Simmons, Appenheim, and Mergenhagen,

in 1972, reported that human peripheral blood leukocytes

produced a supernatant fluid under stimulation with

dental human plaque. This soluble factor they called

OSTEOCLAST ACTIVATING FACTOR (O.A.F.), which produces

bone resorption in organ cultures of fetal rat bone by

increasing the number of active osteoclast. (48)

15

Plasma Cells

Concerning the function of plasma cells in periodontal

disease, Nisengard states that they elaborate five classes

of immunoglobulins: IgG, IgA, IgM, IgE, IgD, of which

only IgG, IgA and IgM have been identified in inflamed

. . 1 . (49) gingiva tlssue.

Plasma cells as well as lymphocytes are known to be

present in inflamed gingiva. (50)

Toto (1961), described plasma cells arising directly

from perivascular undifferentiated connective tissue

reserve cells and in the absence of lymphocytes in

inflamed oral mucosa. The perivascular cells showed

increased synthesis of RNA prior to differentiation into

plasma cells. (5l)

According to Schneider, Toto, and Gargiulo (1966),

plasma cells are found in "normal" gingiva. They also

suggest that a specific antibody may always be present

in tissue in response to the presence of bacteria, thus

maintaining a relatively constant defense in healthy

individuals. (52)

Wittwer, Dickler, and Toto (1969), in a histologic

study of gingival tissue from fifty patients with

gingivitis, found a preponderance number of plasma

16

cells as compared to lymphocytes. They suggested that

these plasma cells were locally produced in response

to some antigenic plasma substance. The authors explained

that since plasma cells were associated with antigen-

antibody complex, an immunologic reaction was present

in gingivitis.C53)

Platt, Crosby, and Dalbrow (1970), found IgG and IgM

containing plasma cells in the gingival tissue of patients

with acute gingivitis. However, the presence of IgA

was inconclusive in their results. The tissue obtained

from patients with severe periodontal disease showed many

IgM positive plasma cells, moderate IgG positive plasma

cells but only few IgA positive plasma cells. They also

conclude that most of the bacteria present in the gingival

crevice are inactivated by immunoglobulins.C54)

Byers, Toto, and Gargiulo (1975), demonstrated IgG,

IgA, and IgM in the inflamed human gingiva by saline

extractions and assay by low level immunodiffusion plates.

IgG, IgA and IgM levels were increased over normal tissue

levels. It was demonstrated also that IgM was not

measurable in normal gingiva. They concluded that the

local immune response is operative in chronic perio-

dontitis. (55)

Schenkein and Genco (1977), using the immunoelectro-

phoretic technique, found that IgG showed higher concen-

tration as compared to IgA and IgM, in gingival fluid

taken from patients with chronically inflamed gingiva.

They suggest that this is due partially to the fact that

70 to 80% of the plasma cells of chronically inflamed

gingiva are producing IgG. (56)

Van Swol et.al. (1980), compared quantitively the

17

concentration of IgG, IgA, and IgM, between patients with

advanced periodontitis and periodontosis, using the

immunodiffusion technique. They found that IgG was

statistically significantly increased in the periodontosis

group as compared to the periodontitis group.C57)

B. Plasma Cells and Related Immunoglobulins

The major biological activity of the immunoglobulin molecules

is to bind to specific antigens. When IgM or IgG react with cell

surface antigens, complement activation occurs. The anterior

process causes profound changes to the cell membrane which often

includes lysis and death of that cell. (58)

In 1960 Brill and Bronnestam, using immuno-electrophoretic

analysis, found at least seven different serum proteins in the

gingival fluid, including gamma-globulin, transferrin and albumin.C59)

Brill and Bronnestam in 1960 fmmd gamma-globulin to be

present in fluid recovered from human gingival pocket. They were

able to demonstrate this by electrophoretic examination of the

fluid. They also specl1lated that antibodies may be present in

the fluid as well. (60)

Staffer, Kraus, and Holmes, in 1962, found seven salivary

proteins similar to plasma proteins by the irnrnunochemical method.

Among those proteins, IgG and IgM were included.(61)

18

According to Brandtzaeg, in 1965, IgG, IgA, and IgM are present

in serum in the approximate ratios 12:4:1 respectively. Similarly

the normal gingival sulcus fluid contained the same immunoglobulines

in proportions and concentrations of those seen in serum. (62)

Furthermore, Brandtzaeg, in 1965, also reported that IgG, IgA,

and IgM were present in saliva but in much lower proportions and

concentrations as compared to those found in serum. (63)

Saito et.al., in 1969, found increased levels of IgG in the

serum in eighteen of thirty patients with periodontitis as compared

to normal controls. Serum IgA was found to be increased only in

eight patients. They suggested that the increase levels of IgG

and IgA may be related to the development of periodontal disease,

by augmenting the inflamatory process. (64)

Berglund in 1971 noted that immunoglobulins in inflamed gingival

tissue originated both from the cells in the inflamed tissue as

well as from the serum. He also stated that immune complexes in

the gingiva may be important factors in the mediation of perio-

dontal inflamation, since he noted that such complexes activated

the complement system. (65)

Fluorescine Isothiocyanate Studies

Brandtzaeg and Kraus, in 1965, using the immunofluorensce method

reported that the most striking difference between clinically healthy

and inflamed gingival tissues was the frequent marked increase in the

number of plasma cells containing IgA. (66)

Thonard et.al., in 1966, using the direct and indirect immuno-

fluorescent technique detected the presence of IgM and IgA in gingival

tissue excised from patients with periodontal disease. However, they

were unable to detect IgG in those tissues.C67)

Cochrane and Dixon, in 1967, using fluorescent staining technique,

described tissue damage which may result after the antigen-antibody

interaction. Following local injection, antigen diffuses through the

tissue until it meets the antibody, at which point an antigen-antibody

complex occurs. Plasma complement is bound and activated, PMN's are

attracted and severe vasculitis occurs. Upon lysis, the PMN's release

lysosomes 1vhich contain many enzymes such as acid and alkaline phos-

phatase, ribonuclease and acid proteases. All these enzymes may

produce cellular injury and the local destruction of tissue. (68)

Crowley, in 1969, could not demonstrate the presence of IgA, IgM

and IgG in plasma cells using the immunofluorescent technique with

19

inflamed gingival tissue of dogs. He was able to demonstrate

large quantities of acid hydrolases in the plasma cells, in which

he suspected to be of major importance in tissue destruction of

. . 1 . (69) gingiva tissue.

Toto, Lin, and Gargiulo, using FITC, demonstrated IgG, IgM,

IgA and C3 in the basement membrane and blood vessels in the

gingiva in 25 patients with periodontitis. They suggested that

immune complexex may be important in the pathogenesis of the

inflamatory process.C 70)

C. The Role of Complement, Activation in Gingival Inflamation

Complement is made up by a system of eleven serum proteins

which often activation serves primarily to amplify the effect

of an interaction between specific antigen and its corresponding

antibody. There are two ways complement can be activated: one

is called the classical pathway and the other is called the

alternative or properdine pathway. Activation of the classical

pathway of complement usually requires an antigen-antibody

reaction. However, only IgM, and IgGl, IgG2 and IgG3 subclasses

20

of IgG are known to activate the complement system by the classical

pathway. The activation sequence is Cl, C4, C2, C3, CS, C6, C7, C8,

and C9. The alternative pathway of complement activation is

immunologically activated by IgA, IgG and IgE, and nonimrnunolo-

gically by endotoxins, properdin factors A and B, cobra venom and

zymosan. The alternative pathway differs from the classical path-

way as it begins with cleavage of C3. The sequence after C3 acti-

vation is the same as that of the classical pathway.

The third component of complement system (C3) plays a key

role in the amplification of the immune response. C7l)

Upon activation, C3 is cleaved into four fragments; C3a, C3b,

C3c, and C3d. Each of these fragments has a specific biological

function. (7Z)

Nisengard explains that one of the biological properties both

21

of C3a and CSa is the stimulation of mast cells to release histamine

resulting in vasoactivity and for this reason C3a and CSa are

called anaphylatoxins. He explains that C3a is also chemotactic

to Pfv1N' s and macrophages. C73)

Bokisch et.al., in 1969, found that C3a had a molecular weight

of 7000. They stated that C3 had both an anaphylatoxin activity

and chemotactic effect of leukocytes.C74)

Spiegelberg, Miescher and Benacerraf, in 1962, stressed the

importance of C3 when in an in vivo study they found the inability

of macrophages to phagocytize gram-negative bacteria in guinea

pigs depleted of C3.C74)

Ward, in 1972, stated that anaphylatoxins have the ability to

contract smooth muscle and to increase vascular permeability either

by a direct effect on vessels or by the activation on mast cells

to release histamine. He also explained that activated C3 not only

has a chemotactic effect for PMN's, but also monocytes and, to

a lesser extent, to eosinophils. He reported that C3a is shed

into the fluid where its anaphylatoxic and leukotactic effect

induces inflamation.C76)

According to Attstrom et.al., in 1975, neutrophils and mono-

cytes continuously migrate from the dento-gingival vessels into

the gingival crevice in both healthy and inflamed gingiva. Also,

in their study with electrophoretic technique, they found that

22

oral mixed saliva from healthy individuals contained a small amount

of C3, while that from patients with periodontitis contained larger

amounts of C3. They suggested that the C3 may enter the oral

saliva through fluid leakage from the marginal gingival. C77)

Koopman et.al., in 1976, studying C3 fragments in periodontal

disease, found that the generation of C3b during an inflamatory

reaction could augment the inflamatory response by stimulating

B cells to elaborate chemotactic and, presumably, other lympho-

kines. C7B)

Rizzo and Mergenhagen (1977), stated that C3b can interact

with specific receptors on the cell membrane of lymphocytes to

induce lymphokines production and release. C79)

Schenkein and Genco (1977), showed that C3 is significantly

decreased in gingival fluid from patients with severe periodontitis

as compared to normal gingival fluid, due to consumption upon

activation. They also were able to find that the alternative

23

pathway activation of complement occured in periodontal pocket. (8

0)

D. Collagenolitic Activity During Inflamation of the Gingiva

From and Schultz-Haudt in 1963 found collagen to comprise 50%

on a dry weight basis in a microchemical evaluation of the human

gingiva.C8l)

Muller and Martin in 1968 reported that the organic fraction of

bone is composed by 90% of collagen.C82)

Collagen is a unique protein consisting of three polypeptide

chains arranged in a triple helix. The majority of collagen in

bone, skin, and ligament, is comprised of two identical chains (al)

which differ in amino acid composition from the third chain ~2).

Native collagen can be degraded at its physiological pH and

temperature by the enzyme collagenase.C83)

Fulmer et.al. in 1969, by separating and culturing epithelial

cells from connective tissue, could identify which cells were

prodllcing collagenase. They incubated small pieces of gingiva in

elastase until epithelium could be mechanically dislodged from

the connective tissue. They reported that collagenase was readily

detectable in culture fluids from both epithelial cells and con-

nective tissue cells by viscometry technique and electronmicro-(84)

scopy.

Fulmer, in 1971, explained that collagenase acts at a neutral

to alkaline pH, and cleaves the collagen molecule in a highly

specific site, approximately three-quarters of the distance from

the N-terminal end. Then the resulting fragments of collagen are

denatured at a temperature lower than that for native collagen.

After initial cleavage by specific collagenase, the fragments of

collagen are assumed to be susceptible to nonspecific proteases

degradation. He resumed stating that collagenase is produced in

gingiva in the following increased order.

1. 2. 3.

Clinically normal gingiva. Chronically inflamed gingiva. Acute inflamed gingiva.(85)

Lazarus et.al. could extract collagenase directly from the

PIY!N' s granules .. C86)

24

Senior, Bielefeld and Jeffrey (1972), have shown that pulmonary

macrophage of humans and dogs can produce collagenase, also. (87)

E. Irrununoperoxidase Method to Study Irrununoglobulins, Plasma Cells and Complement 3

In 1966 Nakane and Pierce, for the first time, proposed the use

of enzymes such as peroxidase coupled antibodies for the detection

f . . b . "b d . (88) o tlssue antlgens y antlgen-antl o y reactlon.

In 1970 Davey and Busch, by using the peroxidase conjugated

antibody could demonstrate tissue antoantibody such as anti-basement

membrane and immune complexes in the kidney.C89)

The peroxidase labeled antibody has been used by Taylor and

Burns, in 1974, to demonstrate specific immunoglobulins and plasma

cells in sections taken from regular surgical pathology specimens

which have been previously fixed in formalin and processed as

paraffin sections.C90)

According to Nakamura and Cawley in 1978, the peroxidase

conjugated antibody technique has several advantages over

fluorescine isothiocyanate technique (FITC). These advantages

are as follows:

1. The slide can be examined by ordinary microscope.

2. The preparations are permanent and can be reexamined.

3. The preparations can be studied in more detail by staining with a radio-labeled antibody.

4. The peroxidase labeled antibody is able to penetrate

25

to ultracellular antigen more effectively than ferritin (9l)

labeled antibody because of its smaller molecular weight. ·

The purpose of this thesis is to corroborate the presence of

the Immunoglobulins and Complement in human gingival inflamation.

Whereas, the forgoing studies have shown Immunoglobulins and

Complement to be present in gingivitis and periodontitis by immuno-

chemical, histologic assays and the use of FITC, the employment of

the immunoperoxidase method has not been reported.

In view of the reported usefulness of the immunoperoxidase

method to detect Immunoglobulins and Complement, and because of

some of its advantages, this method will be employed in a study

of periodontitis.

CHAPTER III

MATERIALS AND METHODS

Thirty samples of clinically inflamed human gingival tissue were

collected during the routine prescribed gingivectomy procedure per-

formed in the dental school. Ten samples of clinically normal gingiva

tissue were also collected as control from patients who needed a

distal wedge gingivectomy extention. The procedure was performed

under local anesthesia employing regional blocks. All patients in-

cluded in the study were in good general health and were not at that

time taking any medication.

The specimens were immediately embedded in 10% buffered neutral

formalin and processed by routine paraffin embedding. The sections

were cut at six microns with a regular microtome (A0-820)* and dried

at 56°C in an oven for thirty minutes to one hour.

The peroxidase staining procedures was as follows:

1. Deparaffinize to 70% ethanol.

2. 1% a-naphthol in 40% ethanol with 0.2cc of 3% H2o2 added just before use, treat for five minutes.

3. Wash in tap water for fifteen minutes.

4. Stain one minute in 0.1% pyronin in 40% ethanol with 4cc added.

* Cappel Laboratories; Cochranville, PA 19330

26

5. Rinse in tap water and 3 changes of 0.01 Molar phosphate buffered saline (PBS), pH 7.4-7.6, ten minutes each.

6. Apply O.Sml of peroxidase conjugated goat antihuman* IgG, IgA, IgM or C3 to one each of the sections, incubate in a moist chamger for one hour.

7. Repeat step #5.

8. Apply O.lSml of Diaminobenzidine** (DAB) solution consisting of O.Smg DAB in lOml PBS, to which 0.2ml of 50% H2o2 diluted 1/50 in PBS has been added (final concentration of 0.01% H2o2), immediately before use. Develop for 1-5 minutes.

9. Rinse in three changes PBS, then distilled water. Blot the rinse with filter paper to prevent distortion or loss of the section.

10. Counterstain with 10% methyl green (MC/B, C.I. 42590, Norwood, Ohio) in 0.2M phosphate buffered, pH 4.0, five minutes.

11. Dehydrate in 70%, two changes 95%, uvo changes 100% ethanol, and 2 changes xylene.

The sections were examined under the light microscope for the

detection of inflamation and presence of the Ig's and C3 in the

epithelium and connective tissue. The cytochemical reaction of the

peroxidase enzyme can be detected by different substrates. In this

case 3-3' diaminobenzidine was used as a substrate, which produces a

brown-blue color.

One slide of every sample was stained with Hematoxilin and Eosin

(H & E) for morphologic examination in order to corroborate the presence

27

of inflamation in the experimental tissues as compared to that in control

tissue. The criteria used to score inflamation in the specimens was as

* Cappel Laboratories; Cochranville, PA 19330 ** Polyscience Inc., Warrington, PA 18976

positive or negative in this order:

Epithelium: 1. Widening of epithelial spaces. 2. Presence of PMN' s. 3. Presence of Macrophages. 4. Presence of Lymphocytes. 5. Presence of Micro-ulcerations.

Basement Membrane: 1. Basement membrane widening. 2. Basement membrane Lysis.

Connective Tissue: 1. Plasma Cells . 2. Lymphocytes. 3. PMN's. 4. Macrophages . 5. Proliferation of Blood Vessels. 6. Collagen Destruction. 7. Loss of Rete Pegs.

28

The criteria for the detection of Ig's and C3 was also as positive

or negative in this order:

Epithelium: 1. Intercellular spaces. 2. Intercellularly.

Basement Membrane

Connective Tissue: 1. Lamina. 2. Blood Vessels. 3. Plasma Cells containing IgG, IgA,

or IgM.

CHI-square analysis was used to see if there were any statistical

differences in the frequence of Ig's and C3 being present 1n the

experimental group sites, as compared to the control group.

CHAPTER IV

RESULTS

The examination of the gingival tissues both in the clinically

normal and chronic periodontitis patients shows both evidence of

chronic inflamation and the presence of immunoglobulins and complement

fragment 3. This suggests that immunoglobulins and C3 always are

present in human gingiva. Moreover, clinically normal gingiva may

change to gingivitis as a function, in part, of an increase in immuno-

globulins and C3.

A. Histological Findings of the Control Group

The histological findings of the normal gingival tissue, used

as control, showed few PMN's and macrophages infiltrating the

normal stratified squamous epithelium (TABLE IV). The connective

tissue contained fibroblast disposed between bundles of collagen

fibers. Infiltrating the connective tissue were PMN's, macro-

phages, plasma cells, lymphocytes, and mast cells (TABLE VI).

Blood and lymphatics were disposed as fine capillaries in the

lamina propria, and in the dense fibrous tissues (TABLE VI).

B. Histological Findings of the Experimental Group

The experimental group showed larger infiltration of PMN's and

macrophages in the epithelium as compared to the control group

(TABLE I). The epithelial spaces were widening and contain fluid,

29

30

indicating edema (FIGURES 1 and 4). The basement membrane appeared

thickened with loosely arranged fibriles and sometimes the basement

membrane was destroyed and infiltrated by inflamatory cells (TABLE II).

The collagen in the lamina propria was disorganized and lost (FIGURES

1 and 4). Many plasma cells and lymphocytes were found in close

proximity to capillaries and occupying these areas of lost collagen

(FIGURE 1). Macrophages and P.MN's disposed perivascularly and in

the lamina propria were more frequently observed than in the control

sections (FIGtffiE 4). Mast cells were present among the course of

capillaries and in the epithelium. Many blood vessels were found to

be dilated, partially filled with blood cells (FIGURE 5). Micro-

ulcerations were frequently observed in the epithelitrn where a

fibrino purulent exudate only covered the underlying dilated capi-

laries (TABLE III).

Two samples of the experimental group were observed to present

the histological features of the nonnal control group.

C. Immunopeioxidase Conjugated Antibody Findings; Control Group

The control group was positive to the peroxidase conjugated Anti

IgG, IgA, IgM and C3. The epithelial intercellular spaces were

positive and occasionally, few epithelial cells contained Ig's and

C3 in their cytoplasm._ The basement membrane was positive to Ig's

and C3. In the lamina propria, few plasma cells were seen to con-

tain IgA, IgG and IgM in their cytoplasm (TABLE X).

D. Immunoperoxidase Conjugated Antibody Findings; Experimental Group

The experimental group showed the same localizations of Ig's

and C3 but evidently in higher concentrations as compared to the

control group. The wider intercellular spaces of the epithelium

were strongly positive to the Ig's and C3 (FIGURES 1 and 4).

Also, the cytoplasm of the epithelial cells were more frequently

positive to the Ig's and C3 than the controls. The wider base-

ment membrane, the remaining collagen fibers, the blood and

lymphatic vessels in the lamina propria, were all strongly

positive to the Ig's and C3 as compared to the control group

(FIGURES 1 and 4). The numerous plasma cells were readily

identified (FIGURE 2). IgG, IgA, and IgM containing plasma cells

were more frequently found occupying the spaces where collagen

was lost in the lamina propria (FIGURE 3). In all cases, while

the plasma cells cytoplasm contained Ig's, it was clear that

other cells were negative. However, it was evident that IgG

containing plasma cells were most numerous in all sections examined

(TABLES VII, VIII, XIV, and FIGURE 3).

The distribution of IgG, IgA and IgM appeared to be signifi-

cantly greater in the sections of inflamed gingiva as compared to

clinical normal gingiva. The deposition of such immunoglobulins

31

in the basement membrane and lamina propria was significant at P 0.05.

Additionally, deposits were found in the perivascular spaces in sig-

nificantly greater quantities in the inflamed as compared to the

32

control gingiva (TABLE II).

The deposition of IgG, IgA and IgM in the intercellular epithelium

spaces both of inflamed and clinically normal gingiva appear equiva-

lent.

The distribution of C3, like IgG, IgA, and IgM, is significantly

greater in the basement membrane, lamina propria, and in the peri-

vascular spaces of the inflamed gingiva, as compared to the clinically

normal gingiva at the P 0.05. As in the case of Ig's, C3 deposits

intercellularly in the epithelium were equivalent in both the inflamed

and clinically normal gingiva (TABLE III).

CHAPTER V

DISCUSSION

Normal Non-Inflamed Gingival Tissue

The clinically healthy human gingiva contains immunoglobulins and

complement fragment 3 in the epithelium, basement membrane, lamina

propria, as detected by the immunoperoxidase method. In particular,

the deposition of the reduced diamenobenzidine appears to show a well

defined localization intercellularly on the epithelium, basement

membrane, and the periovascular spaces. Also, plasma cells, always

seen in small numbers in the lamina propria, contain in their cytoplasm

one of two specific classes of immunoglobulins.

The distribution of immunoglobulins and C3 is comparable to that

reported with the use of fluorescine isothi~cyanate conjugated antibody

by Brandtzaeg (1966)(10) and Schneider, Toto and Gargiulo (1966)fSZ)

Furthermore, the substantiating provided by the brown-blue deposits

produced by the irnmunoperoxidase reduction of Ig's and C3. The sensi-

tivity of this method and the facility afforded by the ability to store

such slides for later studies offers an advantage not provided by FITC.

The distribution of Ig's and C3 in the clinically normal gingiva

suggests that they derive from the blood circulation. However, while

only a few Ig contair1ing plasma cells are seen, no doubt they contribute

Ig to the gingival tissue as well. The Ig's and C3 appear to diffuse

33

throughout the gingival tissues as they are found perivascularly,

interstitially on collagen fibers, in the basement membrane and inter-

cellularly in the epithelium. Movement of Ig's and C3 from the

epithelium into the gingival sulcus could provide the source of anti-

bodies reported present in the gingival sulcus fluid.

The presence of Ig's and C3 in clinically normal gingiva suggests

that they may participate in the maintenance of homeostasis actively

serving in defense of the oral mucosa. In this view, both the presence

of Ig's, C3, and plasma cells in normal gingival sulcus mucosa suggests

that the gingiva is prepared to respond to the ingress of immunogenic

substances located in the gingival sulcus. This view supports that of

Scherder, Toto, and Gargiulo, who also suggest that the presence of

plasma cells in healthy gingiva serves as a constant state of defense

in order to maintain homeostasis.CSZ) Moreover, this view supports

that of Brandtzaeg (1966),ClZ) who proposed that Ig's arising in the

normal gingival tissues diffuse into the gingival sulcus where they

confront antigens. In this respect, the diffusion of Ig's has a

cleansing or "rinsing" effect in the human gingiva and the sulcus.

This view also has been taken by Attstrom, Lahsson and Sjoholm (1975),

who attributed the same activity to presence of C3 also found in the

gingival sulcus fluid. C77)

Inflamed Gingival Tissue

The inflamed gingival tissue showed the same distribution of Ig's

34

and C3 (FIGURE 4). The presence of widened intercellular spaces

occupied by edematous fluid may be interpreted as evidence of an

increase in the rate of diffusion of Ig's and C3 from the connective

tissue. The widened spaces could be created by squamous cell injury

and by liberation of lysosomal enzymes containing proteases and

carbohydrases. This could account for the loss of intercellular

"bridges" observed in widened intercellular spaces. Accordingly,

there is an increased intensity of immunoperoxidase staining produce

by Anti IgG, IgA, IgM, and C3 in periodontitis. This observation

also could explain the reported increase in Ig's and C3 found in the

sulcus fluid.

The widened intercellular spaces also could provide for the ingress

of the components of the sulcus fluid which presumably carry antigens

into such spaces. This possibility is supported by Schwartz, Stinson,

and Parker, in 1970, who demonstrated that endotoxins obtained from

E. coli could penetrate intact sulcular epithelium of dogs.Cl6)

PMN's and Macrophages Infiltrating the Epithelium

35

PMN's and occasionally macrophages are seen infiltrating the

epithelium intercellularly, in all samples of inflamed gingiva (FIGURE 4).

While such cells may be seen in clinically normal gingiva, they are in

relatively small numbers as compared to inflamed gingiva. In particular,

P~m's are seen at all levels of the epithelium, beginning at the basal

cell layer, and migrating through the widened, edematous, intercellular

spaces, they emerge on the surface to enter the gingival sulcus

(FIGURE 1). This evident migration of PMN's and macrophages must be

attributed to a gradient of chemotactic factors in the gingival

tissues.

36

The source of chemotactic factors may be provided by microorganisms

in the gingival sulcus and by the host gingiva. This view is supported

by the findings of Temple, Synderman, Jordan, and Mergenhagen (1970).

They have reported chemotactic activity both for PMN's and macrophages

by dental plaque bacteria as well as immune complexes found in the

host tissues. (40) As endotoxins may penetrate the gingival sulcus it

is possible for other antigenic substances to gain access to the

gingival tissue, according to Schwartz, Stinson, and Parker (1970).(16)

Should such immune complexes form and bind and activate complement,

chemotactic factors would be produced in the epithelium. Moreover,

lysosomal proteases liberated by P~W's, macrophages, and injured

epithelial cells, also can directly activate C3 in epithelium providing

demotactic factors.C73)

Intercellularly Positive Epithelial Cell

The cytoplasm of some epithelial cells were positive to Ig's and C3

in both experimental and control groups. The epithelial cells of the

experimental group were more frequently positive to Ig's and C3 as

compared to the control. This may be explained by a membrane disturbance

in some epithelial cells, resulting in an increase of cellular perme-

ability. This may be supported by the fact that peroxidase is an enzyme

of very small molecular weight and may penetrate cell membranes. (9l)

The Basement Membrane

The basement membrane varies morphologically, and appears thickened

and sometimes is destroyed in clinical inflamed gingiva (FIGURE 2).

When the basement membrane is widened it is strongly positive to Ig's

and C3 (FIGURES 1, 2, and 4). As macrophages and neutrophils infil-

trate the basement membrane (FIGURES 2 and 4), its observed changes

could be attributed to such cells as they produce botl1 collagenase and

hyaluronidase. Alteration in the basement membrane is associated with

an increase of tissue fluid diffusion into the epithelium. This

evident change in basement membrane permeability and increased inter-

cellular fluid is supported by the findings of Tolo, who in 1971,

reported that the basement membrane constitutes a barrier to the

passage of any molecule larger than SOOOOdaltons. (l2) Therefore, one

can expect that antigens and antibodies are concentrated in the base-

ment membrane.

Collagen in the Lamina Propria

The gingival collagen at the site of the lamina propria was found

disorganized, degraded, and lost. Residual collagen and spaces were

positive to Ig's and C3. The loss of collagen may be attributed to

collagenase and carbohydrases (FIGURE 3).

Fulmer, Gibson, Lazarus, Bladen, and Whedon, in 1969, detected

37

collagenase coming from both epithelial cells and connective tissue

cells in the gingiva. (84) Furthermore, Caffesse and Nasjleti, in

1976, demonstrated that collagenase was able to penetrate intact

38

sulcular epithelium of marmosets, and therefore contribute to collageno-

1 . . . . h 1 . . (l8) F 1m . 1971 1 . yt1c actl Vl ty 1n t e am1na-propr1a. u er, 1n , e:xp a1ns

that collagenase cleaves collagen and other proteases continue to

cleave collagen fragments.C 85) These suggest that when these fragments

are small enough, they may act as antigens to attract antibodies and

then be removed by phagocytic cells. Also, the diffusion of Ig and C3

through degrading collagen may adsorb into the surfaces. Thus, in the

morphologic absence of collagen, Ig's and C3 may be bound to the

collagen split products accounting for the intense immunoperoxidase

staining.

Plasma Cells

Plasma cells were readily identified with the immunoperoxidase

method (FIGURE 3). These cells were found in group form from five

to twelve approximately, and constitute the most numerous cells in

the chronic inflamed samples.

Wittwer, Dickler, and Toto, in 1969, found a preponderance number

of plasma cells as compared to lymphocytes in inflamed human gingival

tissue. Although this study was not a quantitiative, the use of

immunoperoxidase made evident that plasma cells containing IgG were

more frequently found as compared to IgA and IgM plasma cells. (S3)

39

Schenkein and Genco, in 1977, found that the gingival fluid contained

higher concentration of IgG as compared to IgA and IgM. They suggested

that this was due to the fact that 70 to 80 percent of plasma cells are

IgG producers. (S6)

The increased intensity of IgG, IgA, and IgM in the connective tis-

sue can be in part attributed to the plasma cells localized in the

gingival conne~tive tissue as they synthesize and secrete immuno-

globulins. Furthermore, they can contribute to the increased stain-

ability of both the basement membrane and epithelium, in addition to

the connective tissue.

Blood and Lymphatic Vessels

The vessels were found increased in number and dilated. The vessels

were positive to Ig's and C3 (FIGURE 5). According to Brandtzaeg, in

1966, Ig's are present in the serum and in the normal gingival sulcus

fluid. He reported the proportions of IgG, IgA, and IgM to be 12:4:1

respectively, in both the serum and the gingival sulcus fluid. (lO)

Berlund, in 1971, found that immunoglobulins of normal inflamed

gingival tissue originated from both plasma cells and the serum coming

from the gingival vessels.C65)

Alexander and Good, in 1977, explained that C3 is one of the eleven

serum proteins in the serum. Therefore, it can be expected that the

C3 seen in the gingiva has its source from the blood.

The presence of Ig's and C3 in the gingival tissue is very important

for the defense against antigenic agents that can cause periodontal

disease. Ig's and C3 together with the cellular immune system, may

maintain a stage of homeostasis in the gingival tissue. When larger

amounts of antigenic substances gain access into the gingival tissue,

they can alter this homeostasis state. Once the antigens gain access

to the gingival tissue, they form immune complexes with the Ig's that

are normally present. These immune complexes and antigens by them-

selves, activate the complement system either by the classical pathway

or by the alternative pathway. h~en C3 is activated it produces

anaphy1atoxins causing vasodilation. Vasodilation produces edema by

the easy scope of fluids from the blood vessels to the gingival tissue.

This may account for the larger amounts of Ig's and C3 present in the

gingival tissue. In gingivitis they are known to be found in the

serum fluid.

Antigens, immune complexes, and C3 are chemotactic agents to

neutrophils and macrophages, cells which easily escape from dilated

vessels together with sensitized T and B lymphocytes. Plasma cells

derived from B cells produce more Ig's. These Ig's form more immune

complexes with antigens. Lymphocytes release their lymphokines. Some

which are known to induce tissue destruction, like the osteoclast

activating factor, which induce osteoclastic activity, resulting in

bone absorption.

Neutrophils and macrophages, in an effort to phagocytize antigenic

40

substances and immune complexes, release lysosomal enzymes which are

known to produce tissue destruction.

41

GIAPTER VI

SUMMARY AND CONCLUSIONS

Forty samples of gingival tissue were obtained for the detection

and distribution of IgG, IgA, IgM, and C3 using the immuno-peroxidase

conjugated goat anti Ig's and C3 method. The patients from whom the

tissues were obtained all were in general good health. Thirty patients

showed clinical evidence of gingival inflamation and ten appeared

clinically without gingival inflamation.

The specimens were immediately embedded in neutral formalin and

processed by routine paraffin embedding. The sections were cut at

six microns and stained with immunoperoxidase goat antihuman Ig's and

C3, and Diaminobenzidine. One slide of every specimen was stained

with H & E for morphologic examination in order to corroborate the

presence of inflamation of the experimental group as compared to the

control group. Two samples of the experimental group were observed

to present the histological feature of the normal control group.

The slides were examined under the light microscope for the

detection of Ig's and C3. The peroxidase antibody method was proved

to be very useful for the detection of Ig's and C3 in gingival tissue.

Ig's and C3 were present in normal as well as in inflamed gingival

tissue. The presence of Ig's and C3 in the normal gingival tissue

may be interpreted as evidence to maintain a state of homeostasis.

42

43

Chronic antigenic stimulation of the gingiva may disturb such homeo-

stasis, resulting in an increase both in Ig's and C3 in the presence of

gingivitis.

The concentrations of Ig's and C3 in the inflamed gingival tissue

appeared to be greater as compared to the non-inflamed gingiva. This

may be explained by the fact that the antigens provided by plaque

bacteria cannot be cleared by the normal concentration of Ig's and

C3. The host response appears to be the production of an inflamatory

reaction accompanied by an increased production of Ig's by plasma

cells, and an ingress of blood Ig's and C3. The destructive effect

of the inflamatory reaction by neutrophils responding to chemotactic

factors of C3 may be the host reaction consequent to increases in

Ig's and C3.

CHAPTER VII

BIBLIOGRAPHY

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23. Shelton, L.E., and Hall, W.B.: "Human gingival mast cells". J. Perio. Res., 2:214, 1968.

24. Shapiro, S., Ulmansky, M., and Scheur, M.: ''Mast cells population in gingiva affected by chronic destruction periodontal disease". J. Periodontal, 40:276, 1969.

25. Barnett, M.L.: "The structure of human epithelial mast cells in periodontal disease". J. Periodontal, 8:371, 1973.

26. Ward, P .A.: "Biological activities of the complement system".

27. Schluger, S., Youdelis, R.A., and Page, R.A.: "Periodontal Disease". Lea and Febiger, 199-212, 1977.

28. Snyderman, R.H., Shin, H.W., and Hausmann, M.H.: "A chemotactic factor from mononuclear phagocytes". Proc. Soc. Exp. Biol. Med., 138:378, 1971.

29. Erb, P., and Feldman, M.: "Role of macrophage in vitro induction ofT-helper cell". Nature, 254:352, 1975.

30. Calderon, J., Williams, R.I., and Uname, E.R.: "An inhibitor of cell proliferation released by cultures of macrophages". Proc. Nat. Acad., Sec. 71:4273, 1974.

31. Pantalone, R.M., and Page, R.C.: "Lynphokine induced production and release of lysosomal enzymes by macrophages". Proc. Natl. Acad. Science, M.S.A., 72:2091, 1975.

32. Snyderman, R.H., Shin, H.W., and Darmemberg, A.M.: "Macrophage Proteinase and Inflamation: The production of chemotactic activity from the filter component of complement by macrophage proteinase. J. Immunol., 109:896, 1972.

33. Schluger, S., Youdelis, R.A., and Page, R.A.: "Periodontal Disease". Lea and Febiger, 1977.

34. Grand, D.A., Stern, I .B., and Evertt, F .B.: "Periodontics". Mosby, Fifth Edition, 1979.

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36. Schluger, S., Youdelis, R.A., and Page, R.A.: "Periodontal Disease". Lea and Febiger, 1977.

37. Grand, D.A., Stern, I.B., and Evertt, F.B.: "Periodontics". Mosby, Fifth Edition, 1979.

38. Ranfjord, S.P., and Ash, M.M.: "Periodontology and Periodontics". W.B. Saunders Company, 1979.

39. Schluger, S., Youdelis, R.A., and Page, R.A.: "Periodontal Disease". Lea and Febiger, 1977.

40. Temple, T.R., Snyderman, R., Jordan, H.J., and Mergenhagen, S.E.: "Factor from saliva and oral bacteria chemotactic for leukocytes: Their pass ible role in gingival inflama tion". J. Perio. , 41: 71, 1970.

46

41. Kraal, J.H., and Loesche, W.J.: "Rabbit polymorphonuclear leukocyte migration in vitro in response to dental plaque". J. Perio. Resch., 9:0, 1970.

42. IBID

43. Thilander, H.: "The effect of Leukocytes Enzymes activity on the structure of the gingival epithelium in man". Acta. Odont. Scand., 21:431, 1963.

44. Taichman, N.S., Freedman, H.L., and Uriahara, T.: "Inflamation and tissue injury. I. The response to ultradermal infection of human dentogingival plaque in normal and leukopenic rabbits". Arch. Oral Biol., 11:1385, 1966.

45. Schluger, S., Youdelis, R.A., and Page, R.A.: "Periodontal Disease". Lea and Febiger, 1977.

46. Nisengard, R.J.: "The role of immunology in periodontol disease". J. Periodontal., 48:507, 1977.

47. IBID

48. Horton, J.E., Raisz, L.G., Simmons, H.A., Oppenheim, J.J., and Mergenhagen, S.T.: "Bone resorption activity in supernatant fluid from culture human peripheral leukocytes". Science. 177:793, 1972.

49. Nisengard, R.J." "The role of immunology in periodontol disease". J. Periodontal., 48:507, 1977.

50. Thoma, K.H., and Goldman, H.M.: "Oral Pathology", Edition 5, St. Louis, C.V. Mosby Company, 449, 1960.

51. Toto, P. D. , : "Plasma cells in inflamed mucosa". Dent. Prog. 1: 199' 1961.

52. Schneider, T.F., Toto, P.D., Gargiulo, A.W., and Pollock, R.J.: "Specific bacterial antibodies in the inflamed human gingiva". Periodontics 4, 2:53, 1966.

53. Wittwer, J.W., Dickler, E.H., and Toto, P.D.: "Comparative frequencies of plasma cells and lymphocytes in gingivitis". J. Periodont. 40:274, 1969.

47

54. Platt, D., Crosby, R., and Dalbow, M.: "Evidence for the presence of immunoglobulins and antibody in inflamed periodontal tissu~'. J. Periodont., 41:215, 1978.

55. Byers, C.W., Toto, P.D., and Gargiulo, A.W.: "Levels of irmnuno-globulins IgG, IgA, and IgM in the human inflamed gingiva". J. Periodontal., 46:387, 1975 .

. 56. Schenkein, H.A., and Genco, R.J.: "Gingival fluid and serum in periodontal disease". J. Perio., 48:772, 1977.

57. Van Swol, R.L., Gross, A., Setterstrom, J.A., and D'Allesandro, S.M.: "Irmnunoglobulins in periodontal tissue: II concentrations of immunoglobulins in granlllation tissue pocket of periodontosis and periodontitis patients". J. Periodontal. 51: 20, 1980.

58. Alexander, J.W., and Good, R.A.: "Fundamental of clinical irmnunology". W.B. Saunders, 80-89, 1977.

59. Brill, N., and Bromestam, R.: "Irmnuno-electrophoretic study of tissue fluid from gingival pocket". Acta odont. scand. 18:95, 1960.

60. IBID

61. Stoffer, H.R., Kraus, F.W., and Holmes, A.C.: "IrmnunochemicaL identification of salivary proteins". Proc. Soc. Exp. Biol. and Med., III: 467, 1962.

48

62. Brandtzaeg, P.: "Immunochemical comparations of protein in human gingival pocket fluid, serum and saliva". Arch. Oral Biol. 10:795, 1965.

63. IBID

64. Saito, T.: "Serum protein analysis by immunophoresis in patients with periodontal disease". Arch. Oral Biol. 14:1113, 1969.

65. Berglund, S.E.: "Immunoglobulins in human gingiva with specificity for oral bacteria". J. Periodontal, 42:546, 1971.

66. Brandtzaeg, P., and Kraus, F. W.: "Autoimmunity and periodontal disease". Odont., 73:281, 1965.

67. Thonard, J. et.al.: "Detection of IgM and IgA immunoglobulins in diseased human periodontal tissue". I.A.D.R. Abst. 328, 1966.

68. Cochrane, C.G.: ''Mediators of the Arthus and related reaction". Prog. Allergy, 11:1, 1967.

69. Crowley, G.C.: "The initiation of gingival inflamation". J. Periodontal Res. , Sup. 4: 21, 1969.

70. Toto, P.D., Lin, L.M., and Gargiulo, A.W.: "Irrrrnunoglobulins and complement in human periodontitis". J. Perio. 49:631, 1978.

49

71. Alexander, J. W., and Good, R.A.: "Fundamentals of clinical irrrrnuno-logy". W.B. Saunders, 80-89, 1977.

72. IBID

73. Nisengard, R.L.: "The role of irrrrnunology in periodontal disease". J. Periodontal, 48:507, 1977.

74. Bokisch, V.A., Muller-Eberbrand, H.J., and Cochrane, C.G.: "Isolation of a fragment (C3a) of the third component of human complement containing anaphylatoxinand dumotactic activity and description of an anaphylatoxic inactivation of human sennn". J. Exp. Med., 129:1109, 1969.

7 5. Spiegelberg, H. , Miescher, P. , and Benacerraf, B. : "The role of complement in the irrrrnune clearance of rat and E coli by the R.E.S. of mice". J. Irrrrnunol., 90:751, 1963.

76. Ward, P.A.: "Biological activities of complement system". A. of Allergy, 30:307, 1972.

77. Attstrom, R., Laurel, A.B., Lahsson, V., and Sjoholm: "Complement factors in gingival crevice material from healthy and inflamed gingiva in humans". J. Perio. Res., 10:19, 1975.

78. Koopman, W.J., Sanberg, A.L., Wahl, S.M., and Mergenhagen, S.E.: Interaction of soluble C3 fragments with guinea pig lymphocytes. Comparison effect of C3a, C3b, C3c, and C3d on lymphokine production and lymphocyte proliferation". J. Irrrrnunol., 117:331, 1976.

79. Rizzo, A., and Mergenhagen, E.: "Host response in periodontal disease". International conference on research in the biology of periodontal disease. The College of Dentistry, University of Illinois, 200:203, 1977.

80. Schenkein, H.A., and Genco, R.J.: periodontal disease. II. Evidence C3 proactivator (Factor B) and C4 dontol, 48:778, 1977.

"Gingival fluid and sennn in for cleavage of complement C3, in gingival fluid. J. Perio-

81. From, S., and Schultz, Haudt, S.D.: "Comparative histological and microchemical evaluation of the collagen content of human gingiva". J. Periodont., 34:216, 1963.

82. Muller, E.J., and Martin, G.R.: "The collagen of bone". Clin. Orthop., 59:195, 1968.

83. Robertson, P .B., and Simpson, J.: "Collagenase: Current concept and relevance to periodontal disease". J. Periodontal, 42:20, 1976.

84. Fulmer, H.M., Gibson, W.A., Lazan1s, G.S., Bladen, H.A., and Whedon, K.A.: "The origin of collagenase in periodontal tissues of man". J. Dent. Res., 48:646, 1969.

85. Fulmer, H.M.: "Collagenase and periodontal disease. A Review". J. Dent. Res., 50:288, 1971.

86. Lazarus, G.S., Daniel, J.R., Brown, R.S., Bladen, H.A., and Fulmer, H.M.: "Degradation of collagen by a human granulocyte collagucolitic system". L Clin. Invest., 47:2622, 1968.

50

87. Senior, R.M., Bielefeld, D.R., and Jeffrey, J.J.: "Collagenolitic activity in alveolar macrophage". Clinical Res., 20:88, 1972.

88. Nakane, P.K., and Pierce, G.B.: "Enzyme-labeled antidose: Preparation and application for the localization of antigens". J. Hist. Cytochern., 19:929, 1966.

89. Davey, F .R., and Busch, G .J.: "Immunohistochemistry of glomerulonephritis using horseredish peroxidase and fluorescein-labeled antibody: A composition of two techniques". Am. J. Clin. Path., 53:531, 1970.

90. Taylor, C.R., and Burns, J.: "The demonstration of plasma cells and other immunoglobulins - containing cells in formalin -fixed paraffin - unbedded tissues using peroxidase - labeled antibody". J. Clinic. Path., 27:14, 1974.

91. Nakamura, R.M., and Cawley, C .P.: "Immunocroscopy in clinical immunology". American Soc. Clinic Path. National Meeting, 50, 1978.

CHAPTER VIII

TABLES

51

52

TABLE I

HEMATOXILIN AND EOSIN STAINING EXPERIMENT GROUP

Widening of Micro Epithel Spaces Th1N's Macrophages Lymphocytes Ulcerations

1 + + + + + + + + +

3 + + + + 4 + + + 5 + + 6 + + + + + 7 + + + 8 + + 9 + + +

10 + + + + + 11 + + + + 12 + + + 13 + + + 14 + + + + + 15 + + + + 16 + + + + 17 + + 18 + + + + + 19 + + + + 20 + + + + 21 + + + 22 + + + + 23 + + + + + 24 + + + + 25 + + + + 26 + + + + + 27 + + + + + 28 + + + +

53

TABLE II

HEMATOXILIN Ai'lD EOSIN BASEMENT MEMBRANE EXPERIMENT GROUP

Basement Basement Membrane Membrane Widening Lysis

1 + + 2 + + 3 + 4 + 5 + 6 + + 7 8 9 + +

10 + + 11 + + 12 + + 13 + 14 + 15 + 16 + + 1 + + 18 +

+ + +

+ + + + +

+ + + + + + +

+

54

TABLE III

HEMATOXILIN AND EOSIN CONNECTIVE TISSUE EXPERIMENT GROUP

Blood Plasma Lympho- Vessels Collagen Loss of Cells Cytes P.M~N. 's Macrophage Increase Lysis Rete Pegs

1 + + + + + + + 2 + + + + + + + 3 + + + + + + +

+ + + + + + 5 + + + + + +

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

1 2 3 4 5 6 7 8 9

10

Widening of Epith. Spaces

TABLE IV

HEMATOXILIN AND EOSIN EPITHELIUM CONTROL GROUP

55

PMN's Macrophages Lymphocytes Microu1cers ·.

+

+

+

1 2 3 4 5 6 7 8 9

10

TABLE V

HEMATOXILIN AND EOSIN BASEMENT MEMBRANE

CONTROL GROUP

Basement Membrane Widening·

Basement Membrane Lysis

56

57

TABLE VI

HEMATOXILIN AND EOSIN CONNECTIVE TISSUE

COI\TTROL GROUP

Blood Plasma Lympho- Vessels Collagen Loss of Cells Cytes PMN's Macrophage Increase Lysis Rete Pegs

1 + + + +

3 + + + + + 4 + 5 + + + + + 6 + 7 + + + + 8 +

+ + + 10 +

58

TABLE VII

I~MUNOPEROXIDASE STAINING EXPERIMENT GROUP

Inter- Intra-Cell Cell Basal La1nina Blood Plasma Spaces Spaces !vfemb. .Prop. Vessels Cells

IgG + + + + + + IgA + + + + IgM + + + + + +

1 ,..,-IJ.) + + + + + + + + + +

+ + + + + + + + +

2 + + + + + + + + + + + + + + + + + +

3 + + + + + + + + + + + + + + + +

4 + + + + + + + +

·+ + + + + +

5 + + + + + + + + + + + + + + + + + +

6 + + + + + + + + + + + + + +

7 + + + + + + + + +

8 + - + + + + + + +

+ 9 + + +

+ + + + + + +

+ + + 10 + + +

59

TABLE VIII

IM\IUNOPEROXIDASE STAINING EXPERIMENT GROUP

Inter- Intra-Cell Cell Basal Lamina Blood Plasma Spaces Spaces Memb. Propria Vessels Cells

I"G + + + + + + _ _]_gA + + + +

IgM + + + + ll C3 + + + +

IgG + + + + + + I:;-A + + + + IgM + + + + +

1?. C3 + + + + IgG + + + + + +

_ _l_?A + + + + + + IgM + + + + + +

13 C3 + + + + + _lgG + + + + +

IgA + + + + + I&\1 + + + +

14 C3 + + + + + I G + + + + +

+ + + + + I ·M + + + +

15 C3 + + + + Ir:rG + + + + JoA + + + + + + I[;M + + + + +

16 r"' -~J + + + + + + + + + + + + + + + + + + + +

17 + + + + + + + + + + + + + + + + + + + + +

18 + + + + + + + + + + + + + + + + + + + + +

19 + + + + + + + + + + + + + + + + + + + +

20 + + +

60

TABLE IX

I~~OPEROXIDASE STAINING EXPERIJ.VfENT GROUP

Inter- Intra-Cell Cell Basement Lamina Blood Plasma Spaces Spaces Membrane Propria Vessels Cells

IgG + + + + + IgA + + + + + + I M +

21 C3 + + + + I G + + + + + IgA + + + + + + IgM + + + + +

22 C3 + + + + I G + + + + + I A + + + + + IgM + + + + +

23 C3 + + + + I G + + + + + + IgA + + + + + + I M + + + + + +

24 C3 + + + + + IgG + + + + + + I A + + + + + + I M + + + + + +

25 C3 + + + + IgG + + + + + I A + + + + + IgM + + + + +

26 C3 + + + + -----IgG + + + + + + ----------I A + + + + + IgM + + + +

27 C3 + + + + IgG + + + + IgA + + + + IgM + + + +

28 C3 + + + + -------------

61

TABLE X

IMMUNOPEROXIDASE STAINING CONTROL GROUP

Inter- Intra-Cell Cell Basement Lamina Blood Plasma Spaces Spaces Membrane Propria Vessels Cells

+ +

1 +

+ +

2 + +

+ + 3

+ + +

+ 4

+ + +

5 + + +

6 + + + +

7 + + + +

8 + + + +

9 + + + +

+ + +

10 +

62

TABLE XI

CHI-SQUARE STATISTICAL ANALYSIS

Sites Corrected Chi-Square p at.05 Significant

IgG Intercellular Spaces .2.48 3.8 No IgG Intracellular Spaces 0.0 3.8 No IgG Basement Membrane 8.63 3.8 Yes IgG Lamina Propria 19.59 3.8 Yes IgG Vessels 0.77 3.8 No IgG Plasma Cells 1.7 3.8 No

IgA Intercellular Spaces 3.41 3.8 No IgA Intracellular Spaces 1. 69 3.8 No IgA Basement Membrane 15.69 3.8 Yes IgA Lamina Propria 23.76 3.8 Yes IgA Vessels 9.00 3.8 Yes IgA Plasma Cells 2.91 3.8 No

IgM Intercellular Spaces 1. 67 3.8 No IgM Intracellular Spaces 1.15 3.8 No IgM Basement Membrane 4.25 3.8 Yes IgM Lamina Propria 18.35 3.8 Yes Igt-.1 Vessels 5.65 3.8 Yes IgM Plasma Cells 5.26 3.8 Yes

C3 Intercellular Spaces 5.60 3.8 Yes C3 Intracellular Spaces 2.78 3.8 No C3 Basement Membrane 9. 72 3.8 Yes C3 Lamina Propria 25.08 3.8 Yes C3 Vessels 5.38 3.8 Yes

TABLE IX

FIGURES

63

Figure 1. Chronic gingival inflamation shows positive staining to Peroxidase conjugated anti-IgG, deposits ar e on the Periphery of the Blood Vess el (Bv) and the Basement Membrane (Bm).

64

Figure 2. Chronic gingival inflamation shows positive staining to Peroxidase conjugated anti-IgG, deposits are seen in the cytoplasm of Plasma Cell (pc) and Basement Membrane (bm).

65

Figure 3. Chronic gingival inflamation . The Peroxidase conjugated anti-IgG deposits are seen in the cytoplasm of the Plasma Cell (PC).

66

Figure 4. Chronic gingival inflamation shows positive staining to Peroxidase conjugated anti-C3, deposits are seen in the Basement Membrane (brn) and in the Epithelial Intercellular Spaces (is). Monocytic cells can be seen infiltrating the Epithelium (MI) .

67

Figure 5. Chronic gingival inflamation shows positive staining to Peroxidase conjugated anti-C3 , deposits are seen on the Periphery of the Blood Vessel (bv) .

68

APPROVAL SHEET

This thesis, submitted by Cesar Ruiz Morales, has been read

and approved by three members of the faculty of the Graduate School.

The final copies have beenexamined by the director of the

thesis and the signature which appears below verifies the fact that

any necessary changes have been incorporated and that the thesis is

now given final approval with reference to content, form, and mechanical

accuracy.

The thesis is therefore accepted in partial fulfillment of the

requirements for the Degree of Master of Science.

Patrick D. Toto, B.S., M.S., D.D.S., F.A.C.S.S.

/~~JJ·~·- &c ~ eo Adviso I

APPROVAL SHEET

The thesis submitted by Cesar Ruiz Morales has been read and

approved by the following committee:

Dr. Patrick D. Toto, Professor and Chairman Department of General and Oral Pathology, Loyola

Dr. Anthony W. Gargiulo, Professor and Chairman Department of Periodontics, Loyola

Dr. John P. Wortel, Professor of Pathology Department of General and Oral Pathology, Loyola

The final copies have been examined by the director of the

thesis and the signature which appears below verifies the fact that

any necessary changes have been incorporated and that the thesis is

now given final approval by the Committee with reference to content

and form.

The thesis is therefore accepted in partial fulfillment of the

requirements for the Degree of Master of Science.

Patrick D. Toto, B.S., M.S., D.D.S., F.A.C.S.S.

1 1/f-- /o/@ Date

~a:6-tuzs

Loyola University ChicagoLoyola eCommons1980

The Use of Immunoperoxidase Method for the Identification and Distribution of I G, I A, I M and C3 in Human Inflamed GingivaCesar Ruiz MoralesRecommended Citation

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