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Loyola University ChicagoLoyola eCommons
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
This Thesis is brought to you for free and open access by the Theses and Dissertations at Loyola eCommons. It has been accepted for inclusion inMaster's Theses by an authorized administrator of Loyola eCommons. For more information, please contact [email protected].
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.Copyright © 1980 Cesar Ruiz Morales
Recommended CitationMorales, Cesar Ruiz, "The Use of Immunoperoxidase Method for the Identification and Distribution of I G, I A, I M and C3 in HumanInflamed Gingiva" (1980). Master's Theses. Paper 3071.http://ecommons.luc.edu/luc_theses/3071
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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
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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
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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
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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
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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
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VI. S~~y AND CONCLUSIONS •.•.•...••..•..•.•.•.....•.•. 42
VII. BIBLIOGRAPHY .....•.•..•.•..............•.•.....•.•... 44
VI I I. TABLES ..•..•••••.•.•••....••...••......••..•..•..•... 51
IX. FIQJRES ........•••....•...•...•.....•..••.••.•••.••.. 63
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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
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LIST OF FIGURES
Figure 1 .............................. 64
Figure 2 ••.•..•••••••.••••.•••••.••.•• 65
Figure 3 ...•.........•................ 66
Figure 4 ••••••••••.••••.•••••••••••••. 67
Figure 5 .............................. 68
vii
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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
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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.
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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
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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
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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)
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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)
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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)
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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)
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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)
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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
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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)
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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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2. Ranfjord, S.P. and Ash, M.M.: "Periodontology and Periodontics". W.B. Saunders Company, 1979.
3. Schluger, S., Youdelis, R.A. and Page, R.A.: "Periodontal Disease". Lea and Febiger, 1977.
4. Ranfjord, S.P. and Ash, M.M.: "Periodontology and Periodontics". W.B. Saunders Company, 1979.
5. Gargiulo, A.W.: "Mitotic Activity". Thesis. Loyola University, 1959.
6. Brill, N. and Krasse, B.: "The Passage of fluid into the clinically healthy gingival pocket". Acta Odont. Scand. 16:233-245, 1958.
7. Mergenhagen, S.E.: "Exotoxic Properties of Oral Bacteria as Revealed by the local Schwartzman Reaction". J. Dent. Res. 39:267, 1960.
8. Caffesse, R.U., Nasjleti, C.E.: "Enzimatic penetration through intact sulcular epithelium". J. Periodontal. 47:391-397, 1976.
9. Listgarten, M.: "Electromicroscopy observations of the bacterial flora of acute necrotizing ulcerative gingivitis". J. Periodont. 36:328, 1965.
10. Brandtzaeg, P.: "Local factors of resistance in the gingival area''. J. Perio. Res. , 1: 19, 1966.
11. Rizzo, A., and Berglund, S.E.: "Response of Rabbit Gingiva and Regional Lymph Nodes After Prolonged Contact Exposure to Bacteria Endotoxins". I.A.D.R. Abts. #69, 1969.
12. Tolo, K.: "Transport across stratified nonkeratinized epithelium". J. Perio. Res., 6:237, 1971.
44
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13. Loe, H. and Holm-Pedersen, P.: "Absence and Presence of Fluid from Normal and Inflamed Gingiva". Periodontics 3:171, 1965.
14. Loe, H., Theilade, E., Jensen, S.B. ~ and Schiott, C.R.: "Experi-mental Gingivitis in Man". J. Peno. Res., 2:282, 1976.
15. Alfano, M. C. : "The origin of gingival fluid". J. Theor. Biol. 47:127, 1974.
16. Schwartz, J., Stinson, F., and Parker, R.: "The passage of tritiatic bacteria endotoxin across intact gingival crevicular epithelium". J. Periodontal, 43:270, 1970.
17. Gerenesu, M., Laudfried, S., and Slack, J .M.: "Identification of Actimonyces and related bacteria in dental calculus by fluorescent antibody technique". J. Dent. Res., 50:78, 1971.
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45
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46
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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
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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.
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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.
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60. IBID
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48
62. Brandtzaeg, P.: "Immunochemical comparations of protein in human gingival pocket fluid, serum and saliva". Arch. Oral Biol. 10:795, 1965.
63. IBID
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68. Cochrane, C.G.: ''Mediators of the Arthus and related reaction". Prog. Allergy, 11:1, 1967.
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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.
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"Gingival fluid and sennn in for cleavage of complement C3, in gingival fluid. J. Perio-
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50
87. Senior, R.M., Bielefeld, D.R., and Jeffrey, J.J.: "Collagenolitic activity in alveolar macrophage". Clinical Res., 20:88, 1972.
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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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