Role of phagocytic cells in periodontal health

Presented by: Dr. Rajendra Kumar

The phagocytic cells of the innate or nonspecific immune system, especially neutrophils, monocytes and macrophages, maintain health by preventing and controlling infection of the host by bacteria. The nonspecific defense systems constitute the first line of defense, or acute reaction to insult, by invading bacteria or other foreign material.

This contrasts with acquired immunity: the development of immune recognition by lymphoid cells and specific reaction to defined antigens. Phagocytic cells are able to detect invaders through a variety of mechanisms. The predominant mechanism is opsonization, which is mediated by complement proteins in the acute phase and antibody after the development of acquired immunity. The neutrophil plays a pivotal role in host defense against infectious periodontal disease.

Understanding the role of phagocytic cells in protecting the host from periodontal disease requires and awareness of normal neutrophil function. Phagocytic cells are derived from the lymphoid and myeloid arms of hemopoietic system. In the bone marrow, the myeloid arm give rise to phagocytes.
Phagocytes
Mononuclear macrophages ( monocytes) Polymorphonuclear Microphages (Neutrophils Eosinophils Basophils)

Neutrophils and Monocytes/Macrophages

Neutrophils and monocytes are closely related phagocytic leukocytes. The fundamental difference between these two cells is that neutrophils differentiate almost completely within the bone marrow (14 days), whereas monocytes exit the bone marrow after 2 days in a relatively immature state and may differentiate in the tissues. Neutrophils and monocytes are the same size (9-10m diameter) in the blood.

They possess many lysosomes within their cytoplasm. Because neutrophils do not need to differentiate substantially to function, they are suited for rapid responses. When neutrophils leave the blood, they always retain their small size and hence were once called microphages. Neutrophils possess receptors for metabolites of the complement molecule C3, designated complement receptor 1, 3, and 4 (CR1, CR3, CR4); and C5aR. They also possess receptors for IgG antibody (FcyR). These receptors enable neutrophils to participate in the inflammatory response and to ingest foreign molecules and cells in the process of phagocytosis.

 Monocytes are referred to as macrophages when they leave the blood. They complete their differentiation in the local tissues and may become greater than 22 m diameter. Because macrophages differentiate and live in the local tissues, they are suited for communicating with lymphocytes and other surrounding cells. Together, macrophages and lymphocytes coordinate the chronic immune response. Monocytes/macrophages possess CRl, CR3, CR4, C5aR receptors, several classes of Fcy receptors, and molecules important in antigen presentation (MHC Class II receptor, CD1).

Role of Neutrophils in Phagocytosis

Neutrophils are the primary phagocytic cells in the acute response. The normal function of neutrophils can be broken down into a defined series of molecular events useful in describing how neutrophils respond to bacterial invasion. Normal neutrophil function can be discussed as quantifiable events:

1.Stimulation of the acute phase (generation of the signal) 2. Recognition of the signal 3.Migration to the source of the signal 4. Recognition of the invader 5. Phagocytosis 6. Microbicidal activity

Stimulation of the acute phase (generation of the signal) or complement pathway

The complement cascade comprises 30 heat-labile plasma proteins that autoassemble after initiation of inflammation, forming a series of enzymes that catalyze each subsequent step. The net effect of complement activation is to augment opsonization of bacteria by antibodies, to allow some antibodies to kill bacteria, to recruit phagocytes to the site of complement activation and to attack the membrane of pathogens forming pores in the cell and lysis.

Complement is activated through three different pathways:

The classical pathway is activated by an antigen-antibody complex, requiring an acquired humoral immune response. The lectin pathway is initiated by binding of a serum lectin, the mannose binding protein, to mannose-containing proteins or to carbohydrates on bacteria. The alternative pathway is initiated by lipopolysaccharide or other bacterial products, resulting in the direct cleavage of the third component of complement, C3, which initiates activation of the terminal proteins of the cascade.

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Although multiple pathways exist for the initial activation of complement, all pathways ultimately result in the production of a protease called C3 convertase that is covalently bound to the surface of the pathogen.

Biological effects of complement components

C3a, C5a and C5b are mediators of inflammation that recruit fluid, cells and proteins to the site of infection. In all three pro-inflammatory complement components, C5a is the most stable and has the highest specific biological activity. All three mediators induce smooth muscle contraction and increase vascular permeability; C3a and C5a can activate mast cells to release mediators that cause similar effects.

The increased vascular permeability ensures that antibody, complement and phagocytic cells are readily exposed to the site of the infection, accelerating clearance of the pathogen through opsonization and phagocytosis or clearance to the local lymph nodes. Importantly, C5a is a potent chemotactic factor, or chemical attractant, for neutrophils and monocytes and macrophages.

Recognition of the signal

Chemotactic factor receptors are a distinct group of molecules with a unique structure found on the surface of neutrophils and other cells. The neutrophil is a fully differentiated, dedicated phagocyte constituting the primary cellular defense against bacterial insult. Neutrophils are selectively recruited into distressed tissues by a sequence of pro-inflammatory events, promoted at the site of insult by secretion of soluble mediators by both indigenous host cells and the bacteria themselves

Neutrophils are initially the predominant host defense cell found in bacterial infections, including periodontal lesions. Neutrophils have several selective mechanisms for controlling bacteria, including both intracellular and extracellular oxidative and nonoxidative killing mechanisms , which can be triggered by endogenous signals, such as antibody and complement component binding (C3b), or exogenous bacterial factors.

These signals profoundly affect chemotaxis, anaphylaxis and phagocytosis. In the neutrophil, C5a and neutrophil granule enzyme breakdown of C5 serves as a positive feedback loop for neutrophil chemotaxis, phagocytosis and granule release . Both C5a and C3a have been reported to be chemotactic for monocytes and macrophages.

Migration to the site of infection

Neutrophils and monocytes appear to move into the tissues through similar mechanisms. The initial adherence of cells to the endothelium in the area of an infection is mediated through interaction between surface adhesion glycoproteins on the neutrophil and adhesion molecules expressed by the endothelial cells. Cells in the peripheral blood may be moving, and the initial binding between the cells and the endothelium is not absolute but slows the cells, causing them to roll across the endothelium.

ICAM- intercellular cell adhesion molecule, LFA- leukocyte function associated antigen, VLA 4-very late antigen, VCAM vascular cell adhesion molecule

Chemotaxis

After traversing the endothelium, neutrophils enters the connective tissue to the site of bacterial challenge in response to chemoattractants expressed in a gradient. Chemoattractant agents liberated at the site include activated complement fragment C5a, an arachidonic acid metabolite leukotriene B, and formylated peptides (formylation is a unique characteristic of bacterial peptides). In addition, IL-8 can function as a chemoattractant specific for neutrophils.

3a

Fig. Leukocytes can "hone in" on various irritants, such as a microorganism, by chemotaxis. This requires a chemotaxin receptor (A). The chemotaxin receptors are members of the G-protein coupled family. Seven transmembranous domains, three extracellular loops (ELl-3), and three cytosolic loops (CL1-3) characterize this family of molecules. B, Neutrophils polarize, forming an anterior lamellipod and a posterior uropod. Cytoplasm appears to squirt through a contractile ring. Neutrophils exhibit strikingly sensitive chemotaxis and can detect a 1 % gradient over the length of its cell body at nanomolar concentrations.

PHAGOCYTOSIS

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Phagocytosis is the process by which cells ingest particles of a size visible to light microscope. Phagocytic cells: Neutrophils and monocytes / macrophages to be considered professional phagocytes Phagocytosis result in the evantual contaminent of a pathogen within a membrane-delimited structre the phagosome. The process of phagocytosis can be divided into the following steps: Recognition and attachment ( Opsonisation) Engulfment stage Secretion stage Digestion or degradation

Opsonisation

The immune system has evolved mechanisms of coating the pathogen with a few recognizable ligands or opsonin, which enable the phagocyte to bind and ingest the pathogen. This is referred to as opsonization

Fig. Routes of opsonization and phagocytosis. Bacterial control in the periodontal environment is achieved largely by opsonization, phagocytosis, and killing of the bacteria by neutrophils. Opsonization refers to the coating of the bacterial cell with host-derived proteins such as LPS binding protein (LBP), specific antibody, or the complement component iC3b. Opsonization with specific antibody of the Ig G subclass is required for phagocytosis of certain bacteria, such as Actinobacillus actinomycetemcomitans.

Engulfment stage
Opsonized particle bound to the surface of phagocyte

Formation of cytoplasmic pseudopodes around it due to activation of actin filament beneath cell wall

Phagocytic vacuole
+ lysosome of cell cytoplasm

Phagolysosome

Degranulation stage

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Preformed granules / stored products of PMNs are discharged or secreted into phagosome ( or extracellular environment). Neutrophils contain three types of granules: Specific or secondary granules: Both extracellulur and intra cellular secretion. Eg: lactoferrin, lysozyme, alkaline phosphatase and collagenase Azurophil granules: Intra phagolysosomal secretion. Eg: myeloperoxidase, acid hydrolases and neutral proteases such as elastase, collagenase and proteinase Tertiary granules: gelatinase , cathepsin

Killing and degradation stage

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Once microbe is ingested, it is killed by two broad categories of killing mechanism: Non-oxidative mechanism: Neutrophild do not require oxygen for energy can fusion under anaerobic condition. Oxidative mechanism: Based on the reduction of oxygen

NON- OXIDATIVE KILLING

Phagosome + lysosome phagolysosome Secretion of lysosomal components into the phagolysosome

In less than 30 seconds neutrophil secretes specific granules( lysosomes, lactoferrin, etc..) After secretion of specific granule it secrete azurophil granule among the microbiciadal compounds are small antimicrobial peptides compounds are: Defensins : Defensins are small cationic amphipathic, arginine- and cysteine-rich peptides composed of 29 to 38 amino acids. They make up 5-7% of the total protein and 30-50% of the azurophil granule content of human neutrophils.

Defensins are membrane-permeabilizing molecules that can kill a variety of gram negative bacteria, gram-positive bacteria, fungi and eukaryotic cells cathepsin G: Cathepsin G is a neutral serine protease found in the granules of neutrophils. Cathepsin G has proteolytic and esterolytic activities resembling that of chymotrypsin. have found that cathepsin G is the most potent nonoxidative antimicrobial agent against periodontal pathogens found in the human neutrophil. Calprotectin: Calprotectin exerts microbiostatic activity against fungi, including Candida albicans and bacteria, including Capnocytophuga sputigena serprocidins ( elastase, proteinase 3, azurocidin)

OXIDATIVE KILLING: Based on reduction of oxygen. Requires: 1) presence of oxygen 2) an oxidation-reduction potential In particular neutrophil exert intense microbicidal activity by forming toxic, reduced oxygen metabolites such as superoxide anion using NADPH oxidase system. Superoxide anion also contribute to formation of H2O2, which is capable diffusing inside the membrane. A phase of increased oxygen consumption(respiratory burst) by activated neutrophil requires the essential presence of NADPH oxidase.

Oxygen molecule
Superoxide ion
NADPH oxidase

NADPH

NADP + hydrogen ion

Bactericidal properties carried out by: 1) Myeloperoxidase(MPO) dependent pathway 2) Myeloperoxidase independent pathway

1) MPO Dependent killing

Hypochlorous acid

2) MPO Independent killing

Hydroxyl radical

Hydroxyl radical NADPH oxidase deficiency Chronic granulomatous disease Inconsistently associated with aggressive periodontal (AP) disease

Oxidative microbicidal mechanisms are of some importance in PDL infection

Role of Macrophages in Phagocytosis

The role of mononuclear phagocytes (monocytes in blood, macrophages in tissues) are closely related to neutrophils. Role of macrophages vary within and between the different tissue compartments. In general, macrophages follow two divergent pathways of functioning. Some macrophages function primarily as phagocytes, engulfing and destroying foreign substances including microorganisms and particulate insoluble agents Examples of these macrophages include alveolar macrophages in the lungs and peritoneal macrophages in the abdomen.

Other macrophages reside in the tissues, where they ingest foreign substances (antigens) and then present the antigens on the macrophage cell surface after processing. The combination of the antigen with appropriate histocompatibility molecules enables T-lymphocyte recognition, stimulating the release of cytokines that amplify the specific immune response.

In addition to processing antigens for activation of the specific immune system, macrophages within the tissues secrete a number of cytokines in response to antigens and other agents associated with microorganisms. These cytokines have several functions, including amplifying the specific immune system, inducing and amplifymg inflammation and stimulating tissue breakdown.

Tissue breakdown can occur either directly by macrophage-secreted enzymes or indirectly by stimulating enzymes in cells such as fibroblasts (collagenase). In addition, the macrophage produces cytokines such as interleukin (IL-1) and prostaglandin E2 that can promote breakdown of bone by stimulating osteoclasts. Hence, although macrophages have a critical role in the overall immune response, they play a somewhat secondary role in the acute response.

Fig. Mononuclear phagocytes (monocytes in blood, macrophages in tissues) are closely related to neutrophils. Their activities are similar, and they exhibit chemotaxis (A), phagocytosis (B), and killing (C), similar to neutrophils. Mononuclear phagocytes are particularly adept at processing and presenting antigen to T-cells (D), a process that may require more than 20 hours. That antigen is presented in association with MHC Class II molecules along with a costimulatory signal. Mononuclear phagocytes also release cytokines (E) that direct lymphocyte differentiation.

Major histocompatibility complex (MHC)/ human leukocyte antigen complex (HLA)

All cells of the body possess some form of glycoproteins unique to an individual. These glycoproteins are known as major histocompatibility complex (MHC) Proteins. The major histocompatibility complex (MHC) is a locus on the short arm of chromosome of that encodes a number of molecules, including MHC classes I, II and III molecules, which are involved with antigen uptake, processing and presentation. There are two major classes of MHC: MHC class I, MHC class II, MHC class I glycoproteins are found on the surfaces of all nucleated somatic cells. except (RBCs). MHC class II glycoproteins are found only on certain cells including Blymphocytes, T- lymphocytes, macrophages and macrophage like cells that present antigens to T-cells.

The processing and presenting of antigen by the macrophage to T cell require that both the cells possess surface determinants coded for the same major histocompatibility complex (MHC) genes. The T cell can accept the processed antigen only if it is presented by a macrophage carrying on its surface the self -MHC antigens. Cytotoxic T lymphocytes are able to kill and lysis virus infected target cells only when the T cells and target cells are of the MHC type, so that the T-cells can recognize the class I MHC antigens on the target cells. Helper T cells can accept antigen presented by macrophages only when the macrophages bear the same class II MHC molecules on the surface.

Conclusion

Phagocytosis is of primary importance in the ability of a host to resist or combat infection. The acute phase of the host response is critical in protecting the periodontium from the pathological sequelae of bacterial colonization and invasion. The role of the humoral and cellular components of the phagocytic cell system is important to emphasize the dynamic interplay between the infecting bacteria and the host. Deficiency of acute phase of host response or we can say defect of phagocytic cells enhanced susceptibility of the compromised host to periodontal breakdown which led's to periodontal disease.