1. Periodontal Immunology
Dr. Aaron Weinberg DMD, PhD
Department of Biological Sciences
CASE School of Dental Medicine

2. Outline  Periodontal diseases (PDs) as opportunistic infections
Confusion in defining microbial pathogenesis
 Socransky’s criteria for periodontopathogens
 Specific bacteria-Specific disease
Specific plaque hypothesis
 Gingival crevicular fluid and PD
 Primary PMN abnormalities and PDs
 Subtle PMN defects and PDs
 Experimental neutropenia
 Chronic phase of PD
Role of lymphocytes/monocytes

3. Periodontal diseases
 A local “infection” by indiginous plaque bacteria
This is an opportunistic infection (my bias)
Infection by true pathogens???
 A “time dependent” local immune response that mimics the natural history of inflammation
This response includes remodeling of the subjacent connective tissues, including bone.
 An opportunistic infection reflects a failure of innate immune mechanisms
 The chronic immune response results in increase of specific defenses (ex., humoral immunity); remodeling of tissues
 Tissue remodeling involves cycles of destruction and reconstruction

4. There are some bacteria that cause a disease, but there are some diseases that bring about a condition that is ideal for the growth of some bacteria.
-Pasteur

5. Reasons for Uncertainty/Confusion in Defining Microbial Periopathogens
“Periodontal disease” may be periodontal diseases
Mixed infections
Large number of species present
Many species are difficult to grow
Time of sampling may be wrong
Different sites in same patient may have different bacteria
Opportunistic species grow as result of disease rather than as cause.
Association studies
Carrier states
Phenotypically “normal”, but infected with pathogen
Strains of putative pathogens may vary in virulence. Some may harbor phage or plasmids.

6. Criteria proposed by Dr. S
Criteria proposed by Dr. S. Socransky to implicate a microbe in the etiology of a form of periodontal disease
It must be found in high numbers in proximity to the periodontal lesion
It must be absent, or present in much smaller numbers in periodontally healthy subjects or in subjects with other forms of periodontal disease.
The organism must have high levels of serum, salivary and gingival crevicular fluid antibodies against it in periodontally diseased patients.
It must be found to produce virulence factors in-vitro which can be correlated with clinical histopathology
Experimental implantation of the organism into the gingival crevice of an appropriate animal model should lead to development of some characteristics of naturally occurring periodontal disease.
Clinical improvement following treatment must eliminate the putative pathogen from the periodontal lesion

7. Specific bacteria, specific diseases

8. Specific infections- the specific plaque hypothesis
Widely accepted; never rigorously proven; certain perio diseases result from infection by specific microbes
Remember:
We all possess these bacteria
Not everyone with elevated levels of these bacteria exhibit periodontal disease.
Host factors must be important in PD pathogenesis (Gemmell et al, 1996)

9. Gingival crevicular fluid (GCF) and periodontal disease
 Antimicrobial activities of the GCF
From neutrophil release and/or lysis
Lactoferrin, calprotectin, -defensins
Concentration range of ~0.5 g/ml to >1000 g/ml (Miyasaki et al, 1998)
Levels > 10 g/ml can be microbicidal
 Complement and PD
Found in high levels in GCF
C3, factor B, C4 can attain 25%, 62%, 85% of serum levels in GCF (Schenkein, 1991)
Certain periopathogens can cleave components of C’
P. gingivalis enzymes cleave C3, resulting in inactive C3a (Wingrove et al, 1992)
Are people with classical or alternative pathway deficiencies more susceptible to PD?
No compelling studies to date

10. PMNs get into the gingival crevice
Transepithelial migration
Requires a chemotactic gradient
IL-8, important chemokine
PMNs found routinely w/i junctional epithelium of healthy people (Yamasaki et al, 1979)
Junctional epithelium expresses IL-8 and ICAM-1 in normal states and form chemotactic gradient from basal layers toward gingival sulcus (Tonetti et al, 1998)
Hypoxia stimulates IL-8 mediated PMN epithelial transmigration (Colgan et al, 1996)
Bacterial attachment to epithelium, sufficient to initiate epithelial transmigration of PMNs (Savkovic et al, 1997)

15. Neutrophils and PD
 Evidence that PMNs are protective against periodontal destruction
Primary PMN abnormalities strongly associated with severe PD
Healthy persons with severe perio problems appear to have subtle defects in their PMNs (some in the field support this)
Exptal neutropenia in animals leads to rapid periodontal infection
 Primary PMN abnormalities
Neutropenia and agranulocytopenia
normal: /ml; neutropenia: <1500/ml; agranulo.: <500/ml
genetic (primary) or drug, infection, autoimmune (secondary)
major periodontal problems
Chediak-Higashi syndrome (CHS)
rare, autosomal recessive, localized to chromosome 1q43; megabodies form (structural defects involving azurophil and other granules); neutropenia; depressed inflammation; reduced oxygen metabolites; severe periodontitis, oral ulceration

16. Neutrophils and PD cont’d
Primary PMN abnormalities
G-CSF treated morbus Kostmann syndrome
MKS is a rare form of neutropenia
Treating with granulocyte colony stimulating factor doesn’t help even though numbers of PMNs increases
Found that there is a in -defensins and LL37 in the granules
Specific granule deficiency (SGD)
Probably autosomal recessive
Defective packaging of antimicrobial agents in granules
Severe periodontitis and oral ulceration
Papillon-LeFevre syndrome (PLS); Haim Munk syndrome (HMS); Non Syndromic Prepubertal Periodontitits (NS-PPP) (Hart et al, 2000)
PLS and HMS exhibit rapid generalized destruction of alveolar bone (primary and secondary dentition affected) and palmoplantar hyperkeratosis (PPK)
HMS also shows atrophic changes of nails, deformity of fingers
NS-PPP does not show PPK

17. Neutrophils and PD cont’d
PLS, HMS, NS-PPP all connected with allelic variants of cathepsin C (Hart et al, 2000)
PLS, 2126C T; HMS, 2127A G; NS-PPP, 1040A G substitution
Cathepsin C found in PMN and leukocyte granules, important in protein degradation in PMNs and proenzyme activation in T-cells
Chronic granulomatous disease (CGD)
Defects of the NADPH oxidase system so that normal respiratory burst and free radical production is diminished
Diagnosed by recurrent infections by catalase + bacteria
Bacteria gain access to connective tissues leading to formation of granulomas by chronic immune cells
CGD is not strongly associated with periodontitis, suggesting that phagocyte defense against facultative bacteria invading “normoxic” connective tissue is less important than defense against anaerobic bacteria in hypoxic gingival crevice
Because the host phagocytes are unable to mount a normal respiratory burst (ie. Reduce oxygen) they have difficulty controlling organism which do not release reduced oxygen metabolites themselves.
Catalase negative bacteria release enough hydrogen peroxide to assist PMNs to perform oxidative killing.

19. Severe periodontal diseases associated with subtle neutrophil defects
 Localized juvenile periodontitis (LJP)
Severe form of early onset periodontitis affecting children (4-20 yrs)
Frequency: Caucasians, 0.02%; Asians, %; African descent, 0.8% (Boughman et al, 1990)
Frequency in U.S., 0.53% (Löe, Brown, 1991)
Attachment loss ( mm loss/yr; AP loss is mm/yr)
1st molars and incisors affected
70-80% of cases, characterized by PMN chemotaxis defect
Some evidence of respiratory burst activity w/localized tissue destruction
75% of cases, massive tissue invasive infection by A.a.spreading apically along tooth and w/i adjacent gingival tissues (Waehaug, 1976; Saglie et al, 1982)
 Window of opportunity hypothesis (Shenker et al, 1990)
 window opens as general perio infection during mixed dentition period (ugly duckling phase; permanent molars/ incisors erupted);
 window closes with formation of protective Abs.
 Abs + C’ required to opsonize A.a. (Baker, Wilson, 1989)
 A.a. gets foothold before proper Ab isotype, specificity and avidity can be deployed

20. Poor production of effective Abs against periodontal pathogens
 R. Page group at U. Washington, late 80’s-90’s
 Many perio patients don’t develop the right Abs against P.g., A.a.
 Scaling and root planing stimulates Ab production (leads to seroconversion)
Argument for poor antibodies (ie, wrong isotype) in PD individuals
Debatable
May be good for certain PDs, but not LJP
Not all seropositive individuals are immune from periodontitis

21. LJP-1, LJP-2, LJP-3  LJP-1 - MAbs to GP110 inhibit chemotaxis  LJP-2
in PMN chemotaxis to C5a, fMLP, leukotriene B4, IL-8 (Offenbacher et al, 1987)
Pan (global)-receptor defect
79-80% of LJP cases are LJP-1
May be associated with expression of GP110 (~40%) involved somehow with chemotaxis receptors. PMNs from normal and LJP patient
analyzed by western blot with Ab to GP110 (Van Dyke et al, 1987)
- MAbs to GP110 inhibit chemotaxis
 LJP-2
Similar clinical lesions as LJP-1, but no decreased PMN
chemotaxis
 LJP-3
Specific defect in fMLP
chemotaxis (Perez et al, 1991)
fMLP= formyl-Methionyl-leucyl-phenylalanine; strong chemoattractant; from bacterial breakdown products
Leukotrine B4, an arachidonic acid metabolite

22. Generalized juvenile periodontitis (GJP)
 Synonymous with EOP
 Afflicts young adults/post –pubescent individuals
 Frequency, 0.13% in U.S. (Oliver et al, 1998)
 Associated with plaque/calculus; different from LJP
 PMN chemotaxis disorder may be present but no alteration in GP110
Rapidly progressing adult periodontitis (RAP)
 Form of severe periodontitis, not well defined
 Mean age 40 (30-62)
 16% present with PMN chemotaxis defects
 32% exhibit serum Ig factors that inhibit chemotaxis (Lavine et al, 1979)
 One case reported presence of chemotactic factor inactivator (CFI);inhibits attractant
 Serum from RAP patients does not support phagocytosis of A.a. (Sjöström et al, 1992)

23. Refractory periodontitis (RP)
 Resistance to resolution by conventional perio therapy
 Normal PMN chemotaxis; depressed PMN phagocytosis (MacFarlane et al, 1992)
 90% of persons with RP are smokers
Suggests tobacco adversely affects PMNs??
Studies support this (Haffajee et al, 1997; Kornman et al, 1997)
In vitro studies: tobacco smoke (unsaturated aldehydes) inhibits PMN chemotaxis (Bridges et al, 1977) and phagocytosis (Kenney et al, 1977)
 Other acquired PMN dysfunctions by:
Nutritional, hormonal, drug-induced, radiation-induced, viral, immune, autoimmune
Ex, estradiol inhibits PMN chemotaxis (Miyagi et al, 1992)
antihistamines (promethazine) and “tranquilizers” (trifluoperazine) impede PMN phagolysosome fusion (Meers et al, 1997)

24. Microbially induced PMN defects
Leukotoxin
A.a. expresses 116 kDa “RTX” toxin that binds to PMNs (CD18) resulting in lysis (Lally et al, 1997) or apoptosis (Yamaguchi et al, 2001)
A.a. shares this toxin with other species of the genus Pasteurellaceae (Jacques, Mikhael, 2002)
Immunosuppressive factor (ISF)
A.a. expresses an ISF that delays specific immune responses; ie, antigen presentation to T-cells (Shenker et al, 1994)
Inhibition of neutrophil migration into gingival crevice
In vitro studies; P.g. impedes transepithelial migration of PMNs in response to fMLP, IL-8 (Madianos et al, 1997)
E. coli factor (110 kDa) with similar outcome in intestines (Hofman et al 1998), suggesting a common strategy for mucosal pathogens
P.g. prevents gingival epithelium from secreting IL-8 (Darveau et al, 1998)
“RTX” toxins are exotoxins secreted by Gram negative bacteria. RTX refers to the repeated peptide sequences found in the toxin. RTX toxins are either hemolysins or leukotoxins.

26. Experimental neutropenia
Elimination of neutrophils in animals can lead to rapid apical extension of the bacterial plaque front into/beyond the junctional epithelium (Rylander et al, 1977)
Observed w/i 4 days
No bacterial invasion across
sulcular epithelium
Results show the importance of PMNs
in protecting periodontium
 Tendency of bacteria to spread in
apical manner may be due to inability
of host to defend junctional epithelium
Experimental neutropenia can be induced by either chemotherapy or injection of anti-neutrophil serum.

27. Role of lymphocytes and monoctyes
Experiment of Nature
Oral manifestations of AIDS
Both CD4+ T cells and monocyte activities are altered
Clinical findings: large areas of soft tissue undergo necrosis and expose bone
Results suggest:
Inadequate defense against
mucocutaneous infection
(2) Disorganization of the local
immune response

28. SCID Mice Lack mature T and B cells
Both SCID and normal mice develop bone loss when infected with P. gingivalis
Normal mice develop more bone loss than SCID mice (Baker et al, 1994)
Suggests importance of chronic immune system
Loss of bone by SCID mice indicates that B and T cells are not absolutely required for bone loss
Implicates myeloid cells as having some role in periodontal bone loss
SCID=severe combined immunodeficiency ; T and B cells missing, not myeloid cells

29. Hypofunction of lymphocytes and Periodontal Disease
Does not relate to severe forms of PD as observed in hypofunction of neutrophils
Experimental lymphosupression in animals w/ cyclosporin A or anti-thymocyte serum does not result in increased PD.
Lymphosuppressed humans (corticosteroids, prednisone)
are not predisposed to increased PD.
Hyporesponsiveness may be related to less gingival inflammation!!!
Cyclosporin A, immunosuppressive drug; interferes with T cell activity.

30. Increase in lymphocyte activity and susceptibility to PD
Levamisole (drug that enhances T cell activity), increases gingival inflammation in humans (Lehner et al, 1977)
Dinitrochlorobenzene (DNB, skin contact antigen) induces gingival cell mediated immune lesions in dogs.
Painting gingival margins with DNB in sensitized animals resulted in periodontal lesions.
No neutrophils participated in lesions (no microbial chemotaxis; confirmed histologically)
Lymphocyte and monocyte hyperfunction can induce PD without neutrophils and without a microbial challenge
Levamisole found to increase lymphocyte-monocyte interactions.
Dinitrochlorobenzene induces type IV hypersensitivity reaction. Macrophages release IL-12 that stimulates proliferation of CD4+ T cells that then secrete IL-2 and IFN gamma, further inducing Th1 cytokine release; i.e. cell mediated immunity.

31. How is this explained?
Increased monocyte/macrophage activity as predisposing factors to PD.
Peripheral blood monocytes from PD patients release 2-3 times more IL-1β (osteoclast activating factor) (McFarlane et al, 1990)
This increase was also observed when these monocytes were stimulated with LPS
collagen breakdown
⇗ ⇘ fibroblast  collagenase Tissue Breakdown
⇗ ⇗
M  IL bone resorption ⇘ ⇗
osteoblast  osteoclast

32. IL-1 polymorphism and PD
Genetic polymorphism = variation in a sequence of a gene
A single base pair transition, i.e., A-T to G-C can alter some aspect of the gene product.
Single nucleotide polymorphism (SNP) is a variation in the identity of a single nucleotide at a specific site in the gene that can result in an amino acid change.
If in promoter region, results in expression levels
If in exon region, results in conformational changes.
IL-1polymorphism (hyperinflammatory genotype) seen as a severity factor in adult PD (Kornman et al, 1997)
IL-1+ individuals express 2-4 times more IL-1 than “normals.” (Kornman et al, 1997)
Papillon- Lefèvre and cathepsin C (Toomes et al, 1999; Hart et al, 1999)
Cathepsin C, lysosomal protease; hydrolyzes proteins into polypeptides.

33. Risk factors associated with periodontal disease
Condition
Odds Ratio
Diabetes
2.3 (J Periodontol 65: , 1994)
Heavy Smoking
4.8 (J Periodontol 65: , 1994)
IL-1 genotype positive
7-19 (J Clin Periodontol 24:72-77, 1997)
Cholesterol
2.4

34. Systemic Lymphocyte Activity and PD
Serum antibodies
Exposure to bacterial antigens
Based on work by many, patients with AP have Abs to Pg and to a lesser degree to Aa, while patients with LJP have Abs to Aa and to a lesser degree to Pg
This is good immunological evidence that seroconversion is occurring; i.e., an infection occurs that is protracted enough to elicit regional lymph node response.

35. Systemic Lymphocyte Activity and PD
Serum antibodies and disease
Susceptibility to LJP can relate to some genetic variable controlling ability to respond to the antibodies one produces.
Certain people produce different isotype Abs to an Ag than other people
Dominant Ab isotype to Aa in LJP is IgG2 subclass against high mol wt LPS (Wilson and Bronson, 1997)

36. Serum response and the “Window of Opportunity”
In African Americans with LJP, IgG2 is predominant Ab against Aa (Zhang et al, 1996)
Mitogen stimulation of B cells from AA LJP patients results in a 2 fold higher output of IgG2 than B cells from healthy controls.
IgG is a secondary immunoglobulin class molecule; i.e. requires (1) correct T cell activators (gp39 [CD40L]; gp36 doesn’t work) and (2) correct T-cell switch cytokines
To kill Aa need good obsonizing Abs for effective phagocytosis; i.e., Aa is serum resistant (C’ or C’ + Ab don’t kill A.a.)

37. CD32 (FcRII)
Problem may be that phagocytes of LJP patients express a form of IgG Fc receptors that doesn’t bind IgG2 well. (Mike Wilson)
Fc receptor that binds IgG2 is CD32 (FcRII)
Allelic variation w/i Fc receptors can lead to a low affinity CD32 receptor predisposing the person to risk.
Genotyping A.A. LJP subjects is ongoing to determine if they possess the “wrong’ form of CD32.
So far, 73% of patients are R131/R131 homozygous; i.e., R131 allele encodes a CD32 low affinity receptor.
Stay tuned.

38. Summary for LJP
LJP may represent a complex of at least 3 problems related to immunologic responses to A.a.
(1) hyporesponsiveness of G protein coupled receptors as assessed by chemotaxis; i.e., phacocyte attraction.
(2) T-cell and B-cell communication “errors; ” i.e. nonideal isotype class switching.
(3) allelic variation w/i the Fc receptor; i.e., low affinity receptor for IgG2.
Don’t forget the A.a. leukotoxin!

39. Serum antibodies and correlation with PD bacteria
Serum Abs correlate with bugs 80% of the time when cultured from active sites
Serum Abs correlate with bugs 20% of the time when cultured from inactive sites. (Ebersole et al, 1987)
Seroconversion may occur after lesion has developed; seen in LJP
A.a. elaborates an immunosuppressive factor (Shenker et al, 1982)

40. Longitudinal evaluation of serum antibodies against P. gingivalis
Evaluation of serum Ab titers to Pg after scaling (SC) and aggressive treatment (ST)
 73% of patients showed titers to Pg
 titers began to peak at 200 days after scaling
 Reason for titers may relate to
inoculation of bugs into host or
to elimination of immuno-
suppresive organism.
 Surgery + antibiotics results in
gradual decline in Ab titer.
SC=scaling; ST=surgery+antibiotics; dots=appointments

41. Gingival changes observed in T and B cells
In children, gingivitis is a stable lesion which does not progress to periodontitis
 Dominant cell type w/i tissue is the T-cell (Seymour et al, 1981)
 In exptal gingivitis in adults that can lead to PD, dominant cell type is B-cell (Page and Schroeder, 1976)
Concept that periodontitis is a B-cell lesion
 Proportion of B cells in tissues of active periodontitis sites can be as high as 90%.
 T:B ratio drops significantly in
active lesions when compared
to health and stable conditions
 This is pronounced in the
sulcular region (S)
T:B ratio distribution in periodontal disease
(Reinhardt et al, 1988)

42. Gingival changes observed in T and B cells continued
The decrease in T:B ratio is not due to a decrease in T cells
 It is due to an increase in B cells
 B cells increase substantially as periodontal health declines
 T cell density in active periodontal lesions is similar to that seen in health
B-cell distribution in periodontal lesions T-cell distribution in periodontal lesions

43. Changes in intragingival T-cell subpopulations
Number of mononuclear cells recovered from periodontally diseased tissue is ~3 times greater than from normal tissue.
 The ratio of CD4+ to CD8+ T-cell is ~2:1 in normal peripheral blood and gingivitis of children (Armitt et al, 1986)
 The ratio is 1:1 in AP and JP
 The shift seems paradoxical since there is an increase in B-cells. Why?

44. Th1 vs Th2 in Periodontal Disease
Problem: When to sample?
Hypothesis: individuals susceptible to PD have a Th2 response
while those resistant have a Th1 cytokine profile (Gemmell and Seymour, 1994)
 Data is conflicting
 Some studies show predominantly Th2 with high mRNA levels of IL-5 and IL- 6 in gingival mononuclear cells. (Fujihashi e tal, 1994)
 Others show predominantly Th1
with IL-12 presence (Yamazaki et al, 1997)
 Still others show presence of both
(Taubman et al, 1994)
Polyclonal activation of B cells
leads to massive IL-1β production *