inducible nitric oxide synthase in chronic periodontiits

1. PRESENTED BY:
SONAL GOYAL
2ND YR POSTGRADUATE
DEPARTMENT OF PERIODONTOLOGY
Inducible Nitric Oxide
Synthase (iNOS) In Chronic
Periodontitis
GUIDED BY:
DR. MANORANJAN
DR. RACHANA

2. Contents
1. Introduction
2. Nitric oxide
3. Synthesis of nitric oxide
4. Nitric oxide synthase
5. Inducible nitric oxide synthase
6. Role in periodontitis
7. Clinical implications
8. Conclusion
9. Reference

3. Introduction
• The pathogenesis of periodontitis consists of a cascade of inflammatory
and immunological reactions, which have not yet been fully elucidated.
• Recently, NO has been shown to be a vital molecule in inflammatory
processes.
• Since its discovery as a biologically active molecule in the late 1980’s,
nitric oxide (NO) has been found to play an important role as signal
molecule in many parts of the organism as well as cytotoxic or
regulatory effector molecule of the innate immune response.
Lappin DF, Kjeldsen M, Sander L, Kinane DF: Inducible nitric oxide synthase expression in
periodontitis. J Periodont Res 2000; 35: 369-373.

4. Nitric oxide
• Nitric oxide, also known as nitrogen monoxide
• Highly reactive radical produced from the amino acid arginine by the
enzyme nitric oxide synthase (NOS).
• Implicated in a wide variety of regulatory mechanisms ranging from
vasodilatation and blood pressure control to neurotransmission.
Jackson MJ, Papa S, Bolanos J, et al. Antioxidants, reactive oxygen and nitrogen species, gene
induction and mitochondrial function. Mol Aspects Med 2002;23:209-285.

5. Synthesis of nitric oxide
• NO is formed by the :
Oxidative deamination of the amino acid l-arginine
Conversion of l-arginine to l-citrulline plus nitric oxide.
Catalysed by nitric oxide synthases.
Jackson MJ, Papa S, Bolanos J, et al. Antioxidants, reactive oxygen and nitrogen species, gene
induction and mitochondrial function. Mol Aspects Med 2002;23:209-285.

6. The second step is a three electron oxidation, again requiring
molecular oxygen and NADPH to perform an electron removal,
oxygen insertion, and carbon-nitrogen bond scission to form L-
citrulline and the free radical nitric oxide.
Two primary steps have been identified. The first step, a two
electron oxidation, is a hydroxylation of one of the guanidino
nitrogens of L-arginine requiring molecular oxygen and
nicotinamide adenine diphosphate (NADPH) to form NG-hydroxy-
Larginine.
Jackson MJ, Papa S, Bolanos J, et al. Antioxidants, reactive oxygen and nitrogen species, gene
induction and mitochondrial function. Mol Aspects Med 2002;23:209-285.

7. • Thus the five electron oxidation of the terminal guanido nitrogen
of the amino acid l -arginine to form NO plus l-citrulline, in a
complex reaction involving....
• Molecular oxygen
• NADPH
• Enzyme bound heme, FAD, FMN
• Reduced thiols, and tetrahydrobiopterin.
cosubstrates
Redox factors
Jackson MJ, Papa S, Bolanos J, et al. Antioxidants, reactive oxygen and nitrogen species, gene
induction and mitochondrial function. Mol Aspects Med 2002;23:209-285.

8. • For all three NOS isoforms, NO synthesis depends upon the :
• [Ca2+ i = resting intracellular] are required for their binding
calmodulin and, consequently, for their becoming fully activated.
• For eNOS and bNOS, increase in resting intracellular [Ca2+ i]
concentrations required.
Jackson MJ, Papa S, Bolanos J, et al. Antioxidants, reactive oxygen and nitrogen species, gene
induction and mitochondrial function. Mol Aspects Med 2002;23:209-285.

9. • By contrast, iNOS appears able to bind calmodulin with extremely
high affinity even at the low [Ca2+ i] characteristic of resting cells.
• Hence, the activity of iNOS in immunoactivated cells is no longer
temporally regulated by intracellular calcium transients.
Lappin DF, Kjeldsen M, Sander L, Kinane DF: Inducible nitric oxide synthase expression in
periodontitis. J Periodont Res 2000; 35: 369-373.

10. Since NO may be either stabilized or degraded through its
interactions with diverse intracellular or extracellular chemical
moieties, the localization of NOS within the cell might be
expected to influence the biological role and chemical fate of the
NO produced by the enzyme.
Almost every conceivable intracellular organelle has been
postulated as a possible site for NO synthesis, from the plasma
membrane to the cell nucleus.
Lappin DF, Kjeldsen M, Sander L, Kinane DF: Inducible nitric oxide synthase expression in
periodontitis. J Periodont Res 2000; 35: 369-373.

11. NITRIC OXIDE SYNTHASES
Lappin DF, Kjeldsen M, Sander L, Kinane DF: Inducible nitric oxide synthase expression in
periodontitis. J Periodont Res 2000; 35: 369-373.
Isoforms of
NOS
Constitutively
produced
(cnos)
Neuronal
Endothelial
Maintains
normal
physiology
Inducible (inos)
Involved
principally in
inflammatory
processes.

13. Human genes for the NOS isoforms are officially categorized in the order of
their isolation and characterization; thus, the human genes encoding nNOS,
iNOS, and eNOS are termed NOS1, NOS2 , and NOS3 , respectively.
Jackson MJ, Papa S, Bolanos J, et al. Antioxidants, reactive oxygen and nitrogen species, gene
induction and mitochondrial function. Mol Aspects Med 2002;23:209-285.

14. Localization of
iNOS:
Macrophages
Lymphocytes
PMN in
connective
tissue
Endothelial cells
of blood vessel
Fibroblasts
Basal layer of
epithelium

15. • Absent in resting cells, but the gene is rapidly expressed in
response to stimuli such as proinflammatory cytokines.
• Once present, iNOS synthesize 100–1000 times more NO than the
constitutive enzymes and does so for prolonged periods.
•This high concentration of NO may inhibit a large variety of
microbes, but may also potentially damage the host, thereby
contributing to pathology.
Inducible nitric oxide synthases
Lappin DF, Kjeldsen M, Sander L, Kinane DF: Inducible nitric oxide synthase expression in
periodontitis. J Periodont Res 2000; 35: 369-373.

16. • Induced in macrophages.
• Major cytotoxicity.
• Because of its affinity to protein-bound iron, NO can inhibit a
number of key enzymes that contain iron in their catalytic centres.
• Down-regulates Th1 cytokine responses.
Lappin DF, Kjeldsen M, Sander L, Kinane DF: Inducible nitric oxide synthase expression in
periodontitis. J Periodont Res 2000; 35: 369-373.

17. • It seems reasonable to assume that the increased levels of NO are
produced by iNOS expressing cells during inflammation of the
periodontal tissue.
• This is analogous to other phlogistic molecules produced during the
inflammatory process, such as prostaglandin E2, IL-1β and TNF-α.
• The presence of iNOS and conceivably NO release at the
inflammatory sites may have a role in selecting the type of T-cell
response.
Lappin DF, Kjeldsen M, Sander L, Kinane DF: Inducible nitric oxide synthase expression in
periodontitis. J Periodont Res 2000; 35: 369-373.

18. • Proinflammatory cytokines (e.g. IL-1β) and Th1 cytokines (e.g. IL-2
& IFN-γ) have been shown to have a stimulatory effect on iNOS
expression,
• Whereas Th2 cytokines (e.g. IL-4) have been shown to have an
inhibitory effect in vivo,
• Thus indicating an important reciprocal role of Th1 and Th2 T-cell
subsets in iNOS synthesis.
Lappin DF, Kjeldsen M, Sander L, Kinane DF: Inducible nitric oxide synthase expression in
periodontitis. J Periodont Res 2000; 35: 369-373.

19. • The presence of iNOS expressing macrophages in an environment
rich in Th2 cytokines is not altogether surprising because iNOS
expression by IFN-γ and/ or TNF-γ primed macrophages is
augmented by IL-10 stimulation.
Lappin DF, Kjeldsen M, Sander L, Kinane DF: Inducible nitric oxide synthase expression in
periodontitis. J Periodont Res 2000; 35: 369-373.

21. Role in periodontitis

22. • Increased NO production is seen with macrophage stimulation by
Porphyromonas gingivalis LPS, Actinobacillus
actinomycetemcomitans, P. intermedia, P. nigrescens, and F.
nucleatum.
• Conflicting results regarding its role in periodontitis exists:
An increase in local production of nitric oxide via iNOS can be
seen in periodontal diseases, however,
Batista AC, Silva TA, Chun JH, Lara VS. Nitric oxide synthesis and severity of human
periodontal disease. Oral diseases 2002;8:254-260.

23. The presence of NO is
increased in alveolar
bone resorption
This molecule may have
an effect on the growth
and survival of the
bacteria implicated in
periodontal disease
May function as a
mediator for the control
of clastic activity and
avoiding excessive bone
resorption

24. Inducible production of NO in periodontitis
Increased number of iNOS immunoreactive
inflammatory cells in tunica propria and in
immunoreactivity of basal layer and the uppermost
layer of epithelial lining of the gingiva.
Induce iNOS production
Gingivomucosal immune and epithelial cells
Toxins, enzymes, metabolites of gram negative
bacteria
Lohinai ZM, Szabo C. Role of nitric oxide in physiology and patophysiology of periodontal
tissues. Med sci monit 1998;4:1089-1095.

25. Pro-inflammatory cytokines produced by
inflammatory cells
Trigger resident and/or immigrant cell
population
iNOS
Non-specific immunity.
Acts as cytotoxic and cytostatic agents.
Lohinai ZM, Szabo C. Role of nitric oxide in physiology and patophysiology of periodontal
tissues. Med sci monit 1998;4:1089-1095.

26. Immunoregulation + low concentration of cellular l-arginine
iNOS (in macrophages)
NO Superoxide
Forms toxic oxidant peroxynitrite
Lohinai ZM, Szabo C. Role of nitric oxide in physiology and patophysiology of periodontal
tissues. Med sci monit 1998;4:1089-1095.

27. Killing or stasis of microorganism
Bacterial wall produce more NO
Positive feedback cycle
Wall component of gram negative bacteria killed by NO
Lappin DF, Kjeldsen M, Sander L, Kinane DF: Inducible nitric oxide synthase expression in
periodontitis. J Periodont Res 2000; 35: 369-373.

28. DNA injury
Tissue breakdown via
Oxidation reaction Nitration reaction
Inhibition of
energy generating
enzyme
Activation of
proinflammatory
enzyme
Cytotoxicity towards the host tissue
Excessive NO production
Cyclooxygenase
+
MMP
Periodontal
tissue damage

29. • In periodontitis, stimulatory and inhibitory actions of nitric oxide
have been found, according to differences in concentration level.
• It is difficult to predict whether increased production of NO during
inflammation is likely to increase bone loss or prevent it.
Role Of iNOS In Bone Destruction
Batista AC, Silva TA, Chun JH, Lara VS. Nitric oxide synthesis and severity of human
periodontal disease. Oral diseases 2002;8:254-260.

30. • Batista et al and Kroncke et al concluded that:
• Since, PMNs is the major source of NO and taking into account
that these cells do not present an innermost contact with bone, NO
originating from PMNs may be related to beneficial effects rather
than bone loss in periodontal disease.

31. The presence of constitutive and
inducible forms of NOS have been
demonstrated in both osteoblasts and
osteoclasts.
Osteoblasts have been shown to
produce a low basal level of NO
which is important for normal
function.
Significant increases or decreases in
NO production via inflammatory
stimuli or NOS inhibitors have been
found to inhibit osteoblast
proliferation and differentiation, and
in some cases, result in apoptosis.

32. • Low NO levels also appear to be important for the normal function
of osteoclasts.
• If NO production is abolished or significantly increased, then bone
resorption ceases under normal conditions.
• However, NO produced by iNOS also serves as an autocrine
negative feedback signal that regulates osteoclastogenesis.
Jackson MJ, Papa S, Bolanos J, et al. Antioxidants, reactive oxygen and nitrogen species, gene
induction and mitochondrial function. Mol Aspects Med 2002;23:209-285.

33. High NO levels appear to
antagonize the effects of PGE2 on
bone resorption, whereas low
levels appear to act in synergy in
enhancing bone resorption.
Therefore, in the presence of
inflammatory or pro-osteoclastic
mediators, the inhibition of iNOS may
result in significantly increased bone
resorption.
Jackson MJ, Papa S, Bolanos J, et al. Antioxidants, reactive oxygen and nitrogen species, gene
induction and mitochondrial function. Mol Aspects Med 2002;23:209-285.

34. Beneficial effects Detrimental effects
•Antimicrobial activity
•Immune modulation
•Inhibition of microvascular
thrombosis
•Increased tissue perfusion
•Cytotoxic action towards host tissue
including alveolar bone
•Gingival redness- due to vasodilatory
effect of NO
•Gingival swelling – vascular
permeability
•Increasing effect of NO
•BOP due to inhibitory effect of NO
on platelet aggregation and adhesion
• Increased alveolar resorption – due
to stimulatory effect of NO on the
activity of the osteoclasts.

35. • NO and other free radicals participate in the activation of
neutrophil pro-collagenase and they also suppress proteoglycan
and collagen synthesis.
• This may be one of the mechanisms by which NO contributes to
the profound early loss of collagen in gingival lesions.
• The induction of iNOS expression may also inhibit fibroblast
proliferation and induce apoptosis, contributing to the imbalance
of tissue destruction with tissue repair that is characteristic of
periodontitis.
Batista AC, Silva TA, Chun JH, Lara VS. Nitric oxide synthesis and severity of human
periodontal disease. Oral diseases 2002;8:254-260.

36. Batista AC, Silva TA, Chun JH, Lara VS. Nitric oxide synthesis and severity of human
periodontal disease. Oral diseases 2002;8:254-260.
NO may also activate the recruitment of
phagocytes and,
Conversely, modulate the degree of
inflammation,
Inducing a strong and specific inhibition
of T cell proliferation in both Th subsets.

37. • As B cells and plasma cells appear to play a more important role in
the progressive lesion of chronic inflammation in periodontal
disease than T cells,
• It is possible that NO plays a role in the progression of periodontal
disease through the suppression of the T- cell immune response.
Batista AC, Silva TA, Chun JH, Lara VS. Nitric oxide synthesis and severity of human
periodontal disease. Oral diseases 2002;8:254-260.

38. The lack of significant differences in the number of iNOS+ cells
between gingivitis and periodontitis may suggest that it is a
normal part of the inflammatory process, and the larger number
of cells in gingivitis would indicate that it is not related to tissue
destruction or bone loss.
Nevertheless, taking into account that NO production is
kept remarkably elevated throughout the continuing
increase in disease severity, the killer properties of NO may
not be related to the concentration of NO generated per
enzyme, but rather to the duration of NO produced.
Number or duration??
Batista AC, Silva TA, Chun JH, Lara VS. Nitric oxide synthesis and severity of human
periodontal disease. Oral diseases 2002;8:254-260.

42. • Akopov and Kankanian
Activated PMN
Inhibits NO- induced
stimulation of cGMP
accumulation in cultured
fibroblasts
More in periodontitis
patients than in healthy
Deactivation of NO by
activated PMNs maybe one
of the pathophysiological
mechanism of periodontitis.
Release superoxide
NO
peroxynitrite
Prevents activation
of guanylate cyclase

43. Relationship between ROS and RNS
During acute
inflammation, the NO
generated by NOS is a
mediator of the non
specific defense, the NO,
together with ROS,
having a cytotoxic and
cytostatic effect on the
bacterial agents.
In the inflammatory cells
it is stimulated the
generation of NO and of
superoxide anion (O2•–).
NO reacts with O2•– and
other ROS, leading to
the generation of RNS.
Camelia A, Alb S, Suciu S, et al. Oxygen and nitrogen reactive species implications in the
etiopathogenesis of the periodontal disease. Bulletin USAMV-CN 2007;64:1-5.

45. Paradoxically, ROS and
RNS take part in the
regulation of some
physiological processes,
but, on the same time,
they destroy non-self and
self cells.
The impact of their
chronic synthesis on the
periodontium has
consequences unrelated
with their synthesis rate
and with the composition
of the environment in
which this process takes
place
Camelia A, Alb S, Suciu S, et al. Oxygen and nitrogen reactive species implications in the
etiopathogenesis of the periodontal disease. Bulletin USAMV-CN 2007;64:1-5.

46. Healthy periodontium
Superoxide dismutase
Decreased catalyzes dismutation of superoxide
Prevents the formation of peroxynitrite
Anti-oxidant ascorbic acid
Scavenger of reactive nitrogen species.
Lohinai ZM, Szabo C. Role of nitric oxide in physiology and patophysiology of periodontal
tissues. Med sci monit 1998;4:1089-1095.

47. Clinical implications
• Selective iNOS inhibition-
1. Mercaptoethylguanidine (MEG):
 Selective inhibitor
 Scavenger of peroxynitrite
 Significantly reduces the plasma extravasation in gingivomucosal
tissue
 Decreased degree of alveolar bone destruction.
Lohinai ZM, Szabo C. Role of nitric oxide in physiology and patophysiology of periodontal
tissues. Med sci monit 1998;4:1089-1095.

48. 2. Glucocorticoids:
 iNOS inhibiting ability.
3. Tetracyclines
Used in treatment of juvenile periodontitis
Eliminates bacteria
Prevent bone loss
Inhibits peroxynitrite- dependent oxidative process.
Lappin DF, Kjeldsen M, Sander L, Kinane DF: Inducible nitric oxide synthase expression in
periodontitis. J Periodont Res 2000; 35: 369-373.

49. AGGRESSION

50. Studies
Author Study Conclusion
Lappin et al,
2000
Examined the localisation of
iNOS in biopsies from
patients with periodontitis.
The periodontitis diseased tissue
demonstrated a greater level of iNOS
expression than the healthy tissue.
The source of iNOS in the periodontal
tissues was determined to be the
macrophage, with the endothelial cells
also contributing.
A role for NO in the inflammatory
response of periodontal tissues is
suggested, but the precise role was not
elucidated.

51. Authors Study Conclusion
Kendall et al,
2001
Described the role of nitric
oxide in periodontal tissue
destruction.
iNOS, identified in several cell
types such as macrophages and
polymorphonuclear cells, is
expressed in response to
inflammatory stimuli, such as IL-1,
TNF-a, IFN-c and LPS, preferably
in a synergic manner, yielding high
amounts of NO for a long time
period.
Michiko et al,
2001
Expression of cytokine and
inducible nitric oxide
synthase in inflamed
gingival tissue.
iNOS expression in gingiva with
periodontitis was significantly
higher than that in the healthy
gingiva. NO production by
macrophages and PMNL via iNOS
was enhanced in periodontal
lesions and resulted in the
progression of periodontitis.

52. Authors Study Conclusion
Camelia et al,
2007
Oxygen and nitrogen
reactive species
implications in the
etiopathogenesis of the
periodontal disease
Found a statistic significant
increase of the nitric oxide in
mixed saliva of patients with
periodontal disease, which is the
expression of the involvement of
the nitro-oxidative stress.
Batista et al,
2002
To identify and quantify the
expression of iNOS in
samples of plaque induced
gingivitis and localized
chronic periodontitis, to
suggest the possible
relationship between this
molecule and bone
resorption activation and
tissue destruction during the
progression of periodontal
disease.
iNOS increases in the presence of
periodontal disease. Also,
polymorphonuclear cells present
an additional activation pathway
in periodontal disease, expressing
significant iNOS and probably
representing an important source
of NO in human periodontal
disease.

53. Conclusion
Low concentrations of basally produced NO maintain normal
homeostasis, and are protective under physiological conditions
in circumdental tissues.
However, NO may be detrimental when produced in excess
in inflammation, may destroy the host tissues as well not
only the invading microorganisms.
Hence, selective inhibition of the inducible isoform of
nitric oxide synthase and maintenance of constitutive NO
production may be of therapeutic utility in periodontitis.

54. Reference
1. Lappin DF, Kjeldsen M, Sander L, Kinane DF: Inducible nitric oxide
synthase expression in periodontitis. J Periodont Res 2000; 35: 369-
373.
2. Lohinai ZM, Szabo C. Role of nitric oxide in physiology and
patophysiology of periodontal tissues. Med Sci Monit 1998;4:1089-
1095.
3. Batista AC, Silva TA, Chun JH, Lara VS. Nitric oxide synthesis and
severity of human periodontal disease. Oral diseases 2002;8:254-
260.

55. 4. Camelia A, Alb S, Suciu S, et al. Oxygen and nitrogen reactive species
implications in the etiopathogenesis of the periodontal disease. Bulletin
USAMV-CN 2007;64:1-5.
5. Jackson MJ, Papa S, Bolanos J, et al. Antioxidants, reactive oxygen
and nitrogen species, gene induction and mitochondrial function. Mol
Aspects Med 2002;23:209-285.
6. Hirose M, Ishihara K, Saito A, et al. expression of cytokines and
inducible nitric oxide synthase in inflamed gingival tissue. J
Periodontol 2001;72:590-597.