Periodontal Immunology

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Periodontal Immunology

• Dr. Aaron Weinberg DMD, PhD
• Department of Biological Sciences
• CASE School of Dental Medicine

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Outline

• ? Periodontal diseases (PDs) as opportunistic
  infections
• Confusion in defining microbial pathogenesis
• ? Socranskys criteria for periodontopathogens
• ? Specific bacteria-Specific disease
• Specific plaque hypothesis
• ? Gingival crevicular fluid and PD
• ? Primary PMN abonormalities and PDs
• ? Subtle PMN defects and PDs
• ? Experimental neutropenia
• ? Chronic phase of PD
• Role of lymphocytes/monocytes

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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

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• 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

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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.

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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

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Specific bacteria, specific diseases
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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)

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Gingival crevicular fluid (GCF) and periodontal
disease

• ? Antimicrobial activities of the GCF
• From neutrophil secretions and/or lysis
• Lactoferrin, calprotectin, ?-defensins
• Concentration range of 0.5 ?g/ml to gt1000 ?g/ml
  (Miyasaki et al, 1998)
• Levels gt 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

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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 and germfree animals (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)

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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
• Exptal neutropenia in animals leads to rapid
  periodontal infection
• ? Primary PMN abnormalities
• Neutropenia and agranulocytopenia
• normal 4000-8000/ml neutropenia lt1500/ml
  agranulo. lt500/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

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Neutrophils and PD contd

• Primary PMN abnormalities
• G-CSF treated morbus Kostmann syndrome
• MKS is a rare form of neutropenia
• Treating with granulocyte colony stimulating
  factor doesnt 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

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Neutrophils and PD contd

• 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 and proenzyme
  activation in PMNs and 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

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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, 0.2-0.47
  African descent, 0.8 (Boughman et al, 1990)
• Frequency in U.S., 0.53 (Löe, Brown, 1991)
• Attachment loss (1.08-1.8 mm loss/yr AP loss is
  0.072-0.36 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

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Poor production of effective Abs against
periodontal pathogens

• ? R. Page group at U. Washington, late 80s-90s
• ? Many perio patients dont 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 PDs individuals
• Debatable
• May be good for certain PDs, but not LJP
• Not all seropositive individuals are immune from
  periodontitis

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LJP-1, LJP-2, LJP-3

• ? LJP-1
• 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)

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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)
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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)

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Microbially induced PMN defects

• Leukotoxin
• A.a. expresses 116 kDa RTX toxin that binds to
  PMN 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)

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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

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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

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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 monocytes as having some role in
  periodontal bone loss

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Hypofunction of lymphocytes and monocytes and PD

• 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!!!

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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

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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-1ß (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-1
bone resorption ?
?
osteoblast ? osteoclast
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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)

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Risk factors associated with periodontal disease
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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.

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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)

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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 doesnt 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 dont kill A.a.)

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CD32 (Fc?RII)

• Problem may be that phagocytes of LJP patients
  express a form of IgG Fc receptors that doesnt
  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.

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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.
• Dont forget the A.a. leukotoxin!

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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)

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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.

SCscaling STsurgeryantibiotics
dotsappointments
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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.
• ? TB ratio drops significantly in
• active lesions when compared
• to health and stable conditions
• ? This is pronounced in the
• sulcular region (S)

TB ratio distribution in periodontal disease
(Reinhardt et al, 1988)
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Gingival changes observed in T and B
cellscontinued

• The decrease in TB 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
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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 21 in
  normal peripheral blood and gingivitis of
  children (Armitt et al, 1986)
• ? The ratio is 11 in AP and JP
• ? The shift seems paradoxical since there is an
  increase in B-cells. Why?

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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-1ß production