••

•• Mucosal &SystemicMucosal &Systemicimmunity of oral cavityimmunity of oral cavity

•• Lecture (10)Lecture (10)

•• Dr.Dr. BahaBaha, H.AL, H.AL--AmiediAmiedi

•• Ph.D.MicrobiologyPh.D.Microbiology

•• It is clear, therefore, that the tooth surface isIt is clear, therefore, that the tooth surface isinfluenced by both local salivary immuneinfluenced by both local salivary immunemechanisms, mediated largely through secretarymechanisms, mediated largely through secretaryIgAIgA, and by systemic immunity involving all the, and by systemic immunity involving all thevaried immune components present in blood. Thevaried immune components present in blood. Theway in which these contributing factors interact toway in which these contributing factors interact toprovide immunity within the oral cavityprovide immunity within the oral cavity

•• mucosal component MALT:mucosal component MALT:

•• The mucosal surfaces of the body are secretary inThe mucosal surfaces of the body are secretary innature and have their own immune system, thenature and have their own immune system, themucosamucosa--associated lymphoid tissue, or MALT.associated lymphoid tissue, or MALT.The main mucosal surfaces are theThe main mucosal surfaces are thegastrointestinal tract (including oral mucosa andgastrointestinal tract (including oral mucosa andthe salivary glands), the genitourinary tract, andthe salivary glands), the genitourinary tract, andthe respiratory tract. The secretion of thethe respiratory tract. The secretion of themammary glands (mammary glands (colostrumcolostrum) is also part of the) is also part of theMALT. This branch of the immune system isMALT. This branch of the immune system isstimulated through mucosastimulated through mucosa--associated lymphoidassociated lymphoidtissue, which is organized into discrete areas intissue, which is organized into discrete areas inthe gut known asthe gut known as Peyer'sPeyer's patches,patches,

•• just below the epithelial cells in the laminajust below the epithelial cells in the laminapropriapropria.. Antigen travels through specializedAntigen travels through specializedepitheliumepithelium ((M cellsM cells)) to theto the subepithelialsubepitheliallymphoid tissuelymphoid tissue.. The main antibody produced atThe main antibody produced atthese sites isthese sites is secretorysecretory IgAIgA, a, a dimericdimericimmunoglobulin which acquires itsimmunoglobulin which acquires its secretorysecretorycomponent during transport across thecomponent during transport across theepitheliumepithelium

•• Oral immunity:Oral immunity:

•• Oral health is dependent on the integrity ofOral health is dependent on the integrity ofthe oral mucosa, which normally functionsthe oral mucosa, which normally functionsas an effective barrier againstas an effective barrier againstmicroorganisms. In addition, the oralmicroorganisms. In addition, the oralmucosa is in continuity with a number ofmucosa is in continuity with a number ofanatomical structures, if the oral defensesanatomical structures, if the oral defensesbreak down.break down. amajoramajor area of risk is thearea of risk is thejunction between the gingival and the toothjunction between the gingival and the toothand the various forms of periodontaland the various forms of periodontaldisease.disease.

•• There are several factors which may preventThere are several factors which may preventpenetration of intact oral mucosa by micropenetration of intact oral mucosa by micro--organismsorganisms ((Fig.1).Fig.1).

•• Protective barrier:Protective barrier:

•• 11--SalivaSaliva

•• 22--KeratinKeratin

•• 33--Granular layerGranular layer

•• 44--Basement membraneBasement membrane

•• 55--leucocytesleucocytes

•• 66--AntibodyAntibody

•• SalivaSaliva

•• Saliva is a very important component of the oralSaliva is a very important component of the oraldefenses, both by its mechanical washing activity anddefenses, both by its mechanical washing activity andby means of the antimicrobial factors that it contains.by means of the antimicrobial factors that it contains.The important antimicrobial activities andThe important antimicrobial activities andcomponents of saliva are summarized incomponents of saliva are summarized in ((TabelTabel.)..).

•• 11--Mechanical cleaningMechanical cleaning

•• 22--LysozymeLysozyme

•• 33--peroxidaseperoxidase

•• 44--lactoferrinlactoferrin

•• 55--lecocyteslecocytes

•• 66--SecratorySecratory IgAIgA

•• Role of SecretaryRole of Secretary IgAIgA in oralin oral GavityGavity::

•• SecretarySecretary IgAIgA is by far the most importantis by far the most importantimmunoglobulin in saliva.immunoglobulin in saliva. IgAIgA is secreted by salivaryis secreted by salivarygland plasma cells, two molecules of which aregland plasma cells, two molecules of which arecombined by means of a J chain (Fig.2), which is alsocombined by means of a J chain (Fig.2), which is alsosecreted by local plasma cells. The resultantsecreted by local plasma cells. The resultant dimericdimericIgAIgA is then complexes to the secretary component,is then complexes to the secretary component,synthesized by epithelial cells of the salivary acing, andsynthesized by epithelial cells of the salivary acing, andthe complete secretarythe complete secretary IgAIgA is transported into theis transported into theduct lumen and thence into the mouth. Secretaryduct lumen and thence into the mouth. Secretary IgAIgAis more resistant to proteolysis degradation than otheris more resistant to proteolysis degradation than otherimmunoglobulin. It probably functions by combiningimmunoglobulin. It probably functions by combiningwith microwith micro--organisms and preventing their adherenceorganisms and preventing their adherenceto host surfaces.to host surfaces.

•• Role of GingivalRole of Gingival crevicularcrevicular fluidfluid

•• Blood components, including leucocytes, are able toBlood components, including leucocytes, are able toreach the oral cavity via the flow of fluid through thereach the oral cavity via the flow of fluid through thejunctionaljunctional epithelium of theepithelium of the gingivagingiva.. The flow of thisThe flow of thissoso--called gingivalcalled gingival crevicularcrevicular fluidfluid ((GCFGCF)) increasesincreasesgreatly with the inflammation accompanyinggreatly with the inflammation accompanyingperiodontal disease. Experiments usingperiodontal disease. Experiments using radiolabelledradiolabelledIgGIgG,, IgMIgM,, IgAIgA andand neutrophilsneutrophils have shown that bothhave shown that bothhumoralhumoral and cellular components from blood canand cellular components from blood canreach the oral cavity in GCFreach the oral cavity in GCF

•• In addition to immunoglobulin, complementIn addition to immunoglobulin, complementcomponents have been detected in GCF, suggesting thatcomponents have been detected in GCF, suggesting thatboth the classical and alternative complement pathwaysboth the classical and alternative complement pathwaysmay be activated in the gingival crevice. Othermay be activated in the gingival crevice. Othercomponents include enzymes such ascomponents include enzymes such as lysozymelysozyme,,proteases andproteases and collagenasescollagenases released by cells of both thereleased by cells of both thehost and bacteria. Specific proteases which inactivatehost and bacteria. Specific proteases which inactivateIgAIgA have been described.have been described.

•• The cellular component of GCF comprises mainlyThe cellular component of GCF comprises mainlyneutrophilsneutrophils, with small numbers of macrophages and B, with small numbers of macrophages and B--and Tand T--lymphocytes. These cells migrate continuouslylymphocytes. These cells migrate continuouslyfrom the blood through thefrom the blood through the junctionaljunctional epithelium intoepithelium intothe gingival crevice. Over 80% ofthe gingival crevice. Over 80% of neutrophilsneutrophils in thein thegingival crevice are functional and cangingival crevice are functional and can phagocytosephagocytosemicromicro--organisms.organisms.

•• It is clear, therefore, that the tooth surface isIt is clear, therefore, that the tooth surface isinfluenced by both local salivary immuneinfluenced by both local salivary immunemechanisms, mediated largely through secretarymechanisms, mediated largely through secretaryIgAIgA, and by systemic immunity involving all the, and by systemic immunity involving all thevaried immune components present in blood. Thevaried immune components present in blood. Theway in which these contributing factors interact toway in which these contributing factors interact toprovide immunity within the oral cavity isprovide immunity within the oral cavity isillustrated in Figillustrated in Fig..

Immunological techniquesImmunological techniques

•• the study of biological reactions relevant to thethe study of biological reactions relevant to thepathogenesis of disease, epidemiology, andpathogenesis of disease, epidemiology, anddiagnosisdiagnosis.. Serology, the measurement of specificSerology, the measurement of specificantibodies in serum to diagnose indirectly aantibodies in serum to diagnose indirectly aspecific infection, still has a major rook inspecific infection, still has a major rook inclinical microbiologyclinical microbiology.. Some of the mostSome of the mostcommonly used techniques involve the use ofcommonly used techniques involve the use oftagged, or labeled antibodies, where the label cantagged, or labeled antibodies, where the label canbe a fluorescent dye,be a fluorescent dye,

•• An enzyme, or a radioactive isotopeAn enzyme, or a radioactive isotope.. Two ofTwo ofthe most commonly used techniques arethe most commonly used techniques areenzymeenzyme--linkedlinked immunosorbenimmunosorben assayassay((ELISAELISA)), and, and immunobiotcingimmunobiotcing ((WesternWesternblottingblotting).). Both techniques involve theBoth techniques involve theultimate detection of antibodyultimate detection of antibody--antigenantigencomplexes, where either the antigen or thecomplexes, where either the antigen or theantibody is being assayedantibody is being assayed.. The thirdThe thirdtechnique, latex agglutinationtechnique, latex agglutination ((or a variationor a variationcalledcalled coagglutinationcoagglutination)) is used as anis used as anextremely rapid technique for the detectionextremely rapid technique for the detectionof antigen or toxin in clinical specimensof antigen or toxin in clinical specimens..

•• ELISA Principle :ELISA Principle :

•• . It can be used to detect antibodies in serum or. It can be used to detect antibodies in serum orother body fluids, by binding them to an antigenother body fluids, by binding them to an antigencoated to a solid phase (a well in a polystyrene,coated to a solid phase (a well in a polystyrene,multimulti--well microwell micro titretitre plate). The bound antibodyplate). The bound antibodycan then be detected by adding a tagged secondcan then be detected by adding a tagged secondantibody, which recognizes the first as an antigen.antibody, which recognizes the first as an antigen.The second antibody covalently labeled with theThe second antibody covalently labeled with thean enzymes.an enzymes.

•• The complex of antigen, first antibody, and secondThe complex of antigen, first antibody, and second

•• antibodyantibody--enzyme conjugate is detected by adding aenzyme conjugate is detected by adding achromogenkchromogenk substrate for the enzyme, isubstrate for the enzyme, i..ee.. the productthe productof the enzyme is colored, or a different color from theof the enzyme is colored, or a different color from thesubstratesubstrate.. The intensity of the colored produced,The intensity of the colored produced,which is proportional to the amount of antibodywhich is proportional to the amount of antibodybound by the antigen, can be read quantitatively in abound by the antigen, can be read quantitatively in apurposepurpose--designed micro plate spectrophotometerdesigned micro plate spectrophotometer

ELISA ProcedureELISA Procedure

•• *The Antigen adsorbed on plastic well*The Antigen adsorbed on plastic well•• * then washed* then washed•• *Dilution of the test antibody solution are added to Antigen*Dilution of the test antibody solution are added to Antigen

adsorbed onto plastic wells,adsorbed onto plastic wells,•• * the complex is washed,* the complex is washed,•• *and the an enzyme*and the an enzyme--conjugated ,anticonjugated ,anti--isoiso-- type antibody istype antibody is

added.added.•• *After that washing,*After that washing,•• * the enzyme substrate is added* the enzyme substrate is addedThe resulting color is measure using spectrophotometer ,The resulting color is measure using spectrophotometer ,

The simplest form of this technique is summarized inThe simplest form of this technique is summarized inFigarFigar..

•• Latex agglutinationLatex agglutination

•• This is performed with microscopic latex (reallyThis is performed with microscopic latex (reallypolystyrene) beads which have been coated with apolystyrene) beads which have been coated with aspecific antibody. When these beads are mixed with aspecific antibody. When these beads are mixed with afluid containing the specific antigen they bind to thefluid containing the specific antigen they bind to theantigen and agglutinate, forming a visible precipitateantigen and agglutinate, forming a visible precipitate

Antigen-bindingsites

Antibody A

Antigen

Antibody B

Antibody C

Epitopes(antigenicdeterminants)