Neutrophil dysfunction : a keystone for periodontal infection ? (I)

Introduction

The phagocyte system has two principal limbs, the mononuclear phagocytes and the neutrophilic polymorphonuclear phagocytes. Mononuclear phagocytes comprise monocytes and macrophages. Monocytes are circulating cells that spend a brief time in bloodstream and become macrophages after settling in tissues. Polymorphonuclear neutrophils, also called neutrophils, are rapidly moving phagocytic cells that constitute the first line of defense against bacteria. They circulate in bloodstream until they encounter appropriate chemotactic signals. Their principal task is to slow infection and to contain it until the mononuclear phagocytes and the specific immune system are mobilized to eradicate it.

Normal neutrophil function

Development/Neutrophil production

In adults, neutrophil development takes place within bone marrow. Like the other formed elements of blood, they arise from totipotent stem cells. Depending on the differentiation signals received, these stem cells may become myeloid pluripotent stem cells (CFU-GEMM or colony-forming unit-granulocyte, erythrocyte, monocyte, megacaryocyte), which will commit via a common granulocytic-monocytic progenitor (CFU-GM or colony-forming unit-granulocyte monocyte) to become a neutrophilic progenitor (CFU-G or colony-forming unit-granulocyte). Granulocytopoiesis occurs in two phases, a mitotic phase and a nonmitotic phase. Each phase last approximately one week. The mitotic phase comprises the myeloblast, promyelocyte and myelocyte stages (neutrophil proliferative pool). The nonmitotic phase includes metamyelocytes, band (or immature) and segmented (or mature) neutrophils (neutrophil storage pool) (^{Bainton, 1977}). During maturation, acquisition of deformability, motility, cell membrane receptors, as well as alteration in net surface charge helps neutrophils migrate into marrow sinusoids (^{Lichtman et al., 1977}). Band neutrophils are the first elements that enter bloodstream, they make up approximately 5 % of neutrophils in peripheral circulation. During infection, the transit time from myelocyte stage to migration into bloodstream may be shortened.

Morphologic and structural characteristics

Granules

Neutrophils are equipped with an abundance of discret granules classified according to their chronology of appearance during cell maturation, histochemical staining, contents and density (^{fig. 1}). The first granules to appear are observed during promyelocyte stage, they are called primary or azurophil granules. These granules, defined as peroxidase-positive granules, comprise one third of all granules in mature neutrophils (^{Bainton et al., 1971}). They are true lysosomes since they constitute the main neutrophil store of enzymatic or nonenzymatic bactericidal proteins. They also contain myeloperoxidase, an important element of the oxygen-dependent bactericidal mechanisms (^{Klebanoff, 1970}). The second granules to be seen arise during myelocyte and metamyelocyte stage, they are called secondary or specific granules (^{Bainton et al., 1971}). These peroxidase-negative granules are used as a stock of important plasma membrane proteins. They represent approximately two third of all granules in mature cells. Besides azurophil and specific granules, two additional organelles have been identified, the gelatinase or tertiary granules, and the secretory vesicles. Tertiary granules are peroxidase-negative granules containing gelatinase but no lactoferrin. They are formed later than specific granules (^{Kjeldsen et al., 1992} ; ^{Borregaard et al., 1993}). Secretory vesicles are formed in band and mature cells by endocytosis, they are alkaline phosphatase-positive and constitute an important stock of receptors (^{Borregaard et al., 1987} ; ¹⁹⁹³).

Other organelles

In mature neutrophils, the highly condensed nucleus is segmented into several lobes connected by thin strands of chromatin. Endoplasmic reticulum and Golgi apparatus are not prominent, and protein synthesis is limited. As a result, neutrophils are unable to renew their enzymatic stock. In addition, few mitochondria are present whereas glycogen granules are aboundant. This means that neutrophils draw most of their energy from anaerobic glycolysis.

Cytoskeleton

Microfilaments are located immediately below plasma membrane. They support membrane deformation and cell movement. In resting neutrophils, they form a cortical meshwork which prevents spontaneous fusion between granules and plasma membrane. Following stimulation, the enhanced turn over of microfilaments, accompanied by a decrease in viscosity of peripheral cytoplasm, could permit granules to have access to plasma membrane. As for microtubules, they appear necessary for initial orientation of neutrophils in a chemotactic gradient as well as for spatial organization of organelles during locomotion and degranulation (^{Oliver, 1978}).

Receptors

Mature neutrophils bear at least three different types of receptors for the Fc part of immunoglobulin G (IgG) subclasses, or FcγR (^{table I}). FcγRI (CD64) is not present on resting neutrophils ; but treatment with interferon gamma (IFN-g) induces its expression (^{Perussia et al., 1983}). This receptor binds monomeric IgG with high affinity. FcgRII (CD32) constitutes a family of receptors with high affinity for IgG aggregates. At least six isoforms of FcγRII have been identified ; they are encoded by three different genes, FcγRIIA, FcγRIIB, and FcγRIIC (^{Qiu et al., 1990}). One of these genes, FcγRIIA, is preferentially expressed in neutrophils. At this latter locus, two alleles encode allotypes which differ in their ability to support T cell proliferation induced by murine IgG1 antibodies (^{Tax et al., 1983}). The corresponding phenotypes, HR (high responder)/LR (low responder), are due to a difference of one amino acid (^{Warmerdam et al., 1991}) : at position 131, the high responder phenotype is characterized by an arginine (R), whereas the low responder is characterized by a histidine (H). This single amino acid difference appears to modulate the receptor affinity for IgG subclasses (^{Parren et al., 1992}). FcγRIII (CD16) form a family of receptors that preferentially bind immune complexes. Two FcγRIII isoforms are encoded by very homologous genes, FcγRIIIA and FcγRIIIB. The FcγRIIIB gene encodes the receptor found on neutrophils. At this latter locus, two alleles account for the NA system (^{Ory et al., 1989}). Mature neutrophils also bear receptors for the Fc part of immunoglobulin A (IgA) or FcaR (CD89) (^{table I}). This receptor is a heavily glycosylated protein related to FcgRII and FcgRIII ; it binds monomeric serum IgA as well as dimeric secretory IgA (^{van Dijk et al., 1996} ; ^{Hutchings and Kerr, 1997}). Its expression is constitutive but it is functionally up-regulated by cytokines such as tumor necrosis factor-alpha (TNF-α) or interleukin (IL)-8 (^{Russell et al., 1997}).

Five different complement receptors, C1qR, CR1, CR3, CR4, and C5aR, are present in neutrophils (^{table II}). At least four of these receptors, namely, C1qR, CR1, CR3, and CR4, are stored in secretory vesicles. The upregulation of their expression is the same irrespective of the activating stimulus. C1q receptor (C1qR), or collectin receptor, binds C1q and mannan-binding protein (MBP). Complement receptor 1 (CR1 or CD35) binds C3b, C4b and iC3b. Complement receptor 3 (CR3, Mac-1, or CD11b/CD18) and complement receptor 4 (CR4, p150,95, or CD11c/CD18) belong to the β₂-integrin family. CR3 binds lipopolysaccharides (LPS), fibrinogen and intercellular adhesion molecule-1 (ICAM-1, CD54) in addition to iC3b, C3b, and C3d. CR4 binds iC3b, C3b, and fibrinogen. As for C5a receptor (C5aR), it is constitutively present in high density on plasma membrane and binds C5a and C5a_desArg (C5a lacking the C-terminal Arg residue) (^{Sengeløv, 1995}).

Receptors for chemotactic factors, including N-formyl oligopeptides, IL-8, and platelet-activating factor (PAF) are present on mature neutrophils (^{Boulay et al., 1990} ; ^{Nakamura et al., 1991} ; ^{Thomas et al., 1991}).

Neutrophils also bear receptors and counterreceptors involved in leukocytes-endothelium interactions (^{table III}). The receptors expressed by neutrophils belong to two distinct families : the selectin and the integrin families. L-selectin (CD62L) is the only member of the selectin family to be present on neutrophils ; it is constitutively expressed and shed as a result of activation (^{Kishimoto et al., 1989}). This selectin binds carbohydrate-containing molecules such as glycosylation-dependent cell adhesion molecule-1 (GlyCAM-1) and mucosal addressin cell adhesion molecule (MAdCAM) (^{Nourshargh and Williams, 1995}). Neutrophils also express the β₂-integrins which are dimers having a unique α-chain noncovalently linked to an identical β-chain (b₂-chain, or CD18). This family of integrins consists of integrins α_Lβ₂ (lymphocyte functional antigen-1 [LFA-1] or CD11a/CD18), α_Mβ₂ (CR3 or CD11b/CD18), and α_Xβ₂ (CR4 or CD11c/CD18). Endothelial counterreceptors/ligands for the β₂-integrins include ICAM-1 (CD54) and ICAM-2 (CD102) belonging to the immunoglobulin-related receptor family. ICAM-1 expression is markedly increased following stimulation of endothelial cells by inflammatory mediators whereas ICAM-2 is constitutively expressed on these latter cells (^{Springer, 1990}). LFA-1 binds to both ICAM-1 and ICAM-2, whereas CR3 binds only to ICAM-1. No endothelial ligand for CR4 have been found (^{Bevilacqua, 1993}). The counterreceptors/ligands expressed by neutrophils include sialyl-LewisX (sLe^X)-containing surface glycoproteins and glycolipids which are able to bind to P- and E-selectins expressed by endothelial cells (^{Polley et al., 1991} ; ^{Springer and Lasky, 1991}). P-selectin (CD62P) is stored in Weibel-Palade bodies. Following cell activation, it translocates within minutes to the cell surface as a result of fusion of the Weibel-Palade bodies with plasma membrane ; then it is quickly shed. E-selectin (CD62E) is synthesized de novo by cytokine-activated endothelial cells, consequently its expression peaks a few hours following stimulation ; it is lost by 24 hours.

Neutrophil kinetics

The neutrophil intravascular pool is composed of two subpools : the freely circulating pool and the marginated pool. These two pools are approximately identical in size and their balance is constant. Freely circulating neutrophils comprise about 50 to 85 % of the total leukocyte population in normal adults ; their numbers are maintained at about 2,500-7,500 cells/ml. The marginated pool may consists of neutrophils which are loosely attached to endothelial cells by L-selectins and roll along the walls of small blood vessels (^{Lawrence and Springer, 1991}). Rolling slows down the neutrophils and enable them to sense activating or chemoattractant signals. If they encounter such signals, their L-selectins are shed and β₂-integrins concomitantly activated (^{Hynes, 1992}) while the expression of ICAM-1 on endothelial cells is upregulated (^{Bevilacqua, 1993}). Interactions between β₂-integrins and ICAM-1 permit the establishment of tight neutrophil binding to endothelial cells. Neutrophils flatten then onto endothelium, insert pseudopods between endothelial cells and migrate into tissues (diapedesis) (^{Butcher, 1991}) (^{fig. 2}). Neutrophils disappear from blood with a half-time of 6 to 8 hours. Once in tissues, they may function for 24 to 48 hours before they die or be lost from mucosal surfaces. In inflammatory foci, their life-span is very short.

Chemotaxis

Chemotaxis is an energy-dependent movement directed toward chemotactic factors that emanate from sites of inflammation or infection. These factors are generated either by invading bacteria (e.g. N-formyl oligopeptides) (^{Marasco et al., 1984}) and host cells present at sites of injury (e.g. leukotriene B₄ [LTB₄], PAF, IL-1, IL-8 and related chemokines) (^{Rola-Pleszczynski, 1991} ; ^{Baggiolini and Clark-Lewis, 1992}), or at the time of complement activation (^{Fernandez et al., 1978}). Interactions between chemoattractants and their receptors lead to a rapid polarization of the neutrophils (^{Snyderman, 1985}) and elicit activation of the motile apparatus, mobilization of different storage organelles, and rapid increase in oxygen consumption (respiratory burst). During locomotion toward the chemotactic source, neutrophils acquire a triangular shape with a lamellipodium (leading edge) in the front and a « hand mirror »-like tail (uropod) in the rear (^{Zigmond and Hirsch, 1973}). Neutrophils can perceive a 1 % difference in the concentration of a chemoattractant between their lamellipodium and their uropod (^{Zigmond, 1979}). Granules are mobilized and their fusion with plasma membrane at the leading edge increases the number of chemotactic receptors (^{Fletcher and Gallin, 1980}). Secretory vesicles, which are the lightest granules, are mobilized first, followed by tertiary granules and then by secondary granules which are denser (^{Sengeløv et al., 1993}). Following exposure to agonists, receptors are desensitized ; they become refractory to further stimulation despite the continuous presence of stimulus (^{Didsbury et al., 1991}). Interactions between leukocyte adherence molecules and extracellular matrix are also important for movement since migration can only take place by gliding or crawling along a surface (^{Sullivan and Mandell, 1983}).

Phagocytosis

Phagocytosis is a two-step process by which neutrophils isolate particles in membrane-bound compartments (phagosomes), where they are exposed to high concentrations of bactericidal substances. First, neutrophils must recognize the particle to ingest. This recognition (attachment) involves the coating of the particle with immunoglobulins (IgA or IgG) or complement fragments (C3b, iC3b, or C4b). This process is termed opsonization. Phagocytosis is most efficient when the particle is coated with both antibodies and complement fragments (^{Pereira and Hosking, 1984}). FcR cross-linking by immune complexes triggers the ingestion of the particle and the respiratory burst (^{Unkeless and Wright, 1984}) ; in neutrophils, only Fc±R and FcγRIIa trigger the respiratory burst (^{Lang et al., 1997}). Concomitant stimulation of phagocytosis and increase in oxygen consumption directly couples ingestion to killing. However, some micro-organisms may be ingested in the absence of serum factors (lectinophagocytosis) (^{Ofek and Sharon, 1988}). Two hypotheses have been proposed to explain ingestion : the trigger hypothesis and the zipper hypothesis (^{Swanson and Baer, 1995}). According to this latter hypothesis, neutrophil pseudopodia extend over the particle as long as FcR and CR, in their leading edge, encounter particle-bound ligands ; consequently, incompletely opsonized particles cannot be engulfed. This ingestion or engulfment require energy from anaerobic glycolysis. When pseudopodia meet, they fuse and isolate the particle in a phagosome. As phagosome is formed, granules fuse with it and release their contents (degranulation) to form a phagolysosome. The remaining specific granules fuse first, they are followed by primary granules that have been retained because of their high density. If granules fuse with the phagosome prior to complete closure, their contents may leak into the intercellular space (regurgitation during feeding). When neutrophils encounter particles attached to a nonphagocytosable surface, the granules that fuse with plasma membrane directly discharge their contents in the intercellular space (frustrated phagocytosis).

Respiratory burst

The respiratory burst is mediated by the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase the activity of which is not detected in resting neutrophils. When neutrophils are activated, the enzyme catalyzes the one-electron reduction of oxygen to superoxide anion (^¥O₂ ^-) at the expense of NADPH (^{Cross and Jones, 1991}) :

2O₂ + NADPH Æ 2^¥O₂ ^- + NADP⁺ + H⁺

The increased rate of NADP⁺ formation activates oxidation of glucose via the pentose phosphate pathway. Two enzymes in this cycle, glucose-6-phosphate dehydrogenase (G6PD) and phosphogluconate dehydrogenase (PGD), utilize NADP⁺ and regenerate NADPH (^{fig. 3}).

In activated neutrophils, the NADPH oxidase activity is associated with plasma membrane and therefore with the membrane of phagocytic vacuoles (^{Segal, 1989}). In resting neutrophils, the dormant enzyme complex is composed of two major cytosolic components (p47^phox and p67^phox) and two membrane components (p22^phox and gp91^phox) (^{table IV}). These specific NADPH oxidase components are named after their proteic (p) or glycoproteic (gp) nature, their molecular weight in kilodaltons, and phox, an abbreviation of phagocyte oxidase. The membrane components p22^phox (α-subunit) and gp91^phox (β-subunit) constitute the flavocytochrome b ₅₅₈ (where 558 indicates the absorption maximum in nm) or flavocytochrome b_-245 (where - 245 indicates the redox potential in mV) (^{Segal, 1987}). The β-subunit comprises a membrane-spaning domain with two heme prosthetic groups and docking sites for p47^phox and p67^phox, and a cytosolic domain that binds NADPH and FAD (flavin adenine dinucleotide). Upon stimulation, the cytosolic components translocate to plasma membrane permitting formation of the active NADPH oxidase (^{Heyworth et al., 1991}). The electrons are transferred from NADPH to FAD, then to hemes, and finally to molecular oxygen to form ^¥O₂ ^- in the phagocytic vacuoles.

Most of the ^¥O₂ ^- formed by NADPH oxidase rapidely undergoes dismutation to hydrogen peroxide (H₂O₂) in presence of cytosolic superoxide dismutase (SOD) (^{fig. 3}) :

2^¥O₂ ^- + 2H⁺ Æ H² O² + O²

The electron transfer causes the pH in the vacuole to rise to = 7.8 as protons are consumed when 2^.O₂ ^- dismutates to H₂O₂. This neutral or alkaline pH permits the granule contents to become active and lethal once released into the phagocytic vacuole. Therafter, pH slowly falls to 6.0-6.5 (^{Segal et al., 1981}).

Hydrogen peroxide is metabolized either by catalase (^{fig. 3}) :

2H₂O₂ Æ 2H₂O + O₂

or through the glutathione peroxidase-glutathione reductase system (^{fig. 3}) :

Glutathione peroxidase :

H₂O₂ + 2GSH Æ 2H₂O + GSSG

Glutathione reductase :

GSSG + H⁺ + NADPH Æ 2GSH + NADP⁺

Hydroxyl radical (OH^¥) may be produced by the Haber-Weiss reaction catalyzed by Fe⁺⁺⁺ according to the following scheme (^{fig. 3}) :

Haber-Weiss reaction

Bactericidal mechanisms

Nonoxidative mechanisms

The bactericidal arsenal of neutrophil granules issufficiently broad to permit relatively efficient killing in the absence of anoxidative burst (^{Vel et al., 1984} ; ^{Wetherall et al., 1984}). All the characterized microbicidal substances have been localized to azurophil (primary) or specific (secondary) granules, or both. In addition to enzymes, these granules contain nonenzymatic antibacterial proteins such as lactoferrin, defensins, and bactericidal/permeability-increasing protein (BPI). Lactoferrin is an iron-binding protein which exhibits bacteriostatic effects by depriving bacteria of the iron required for growth (^{Oram and Reiter, 1968}). Additionally, lactoferrin may play a role in hydroxyl radical formation (^{Ambruso and Johnston, 1981}). Defensins are arginine- and cystein-rich peptides which kill bacteria by disrupting their outer membrane (^{Selsted et al., 1985} ; ^{Viljanen et al., 1988}). BPI is a cationic protein which permeabilizes the envelope of Gram-negative bacteria and increases phospholipid turnover (^{Weiss et al., 1978}).

Oxidative mechanisms

Oxygen-dependent bactericidal mechanisms can be divided into myeloperoxide-dependent and -independent reactions (^{Klebanoff, 1975}). The myeloperoxidase-dependent reactions require myeloperoxidase, hydrogen peroxide and a halide ion. In neutrophils, chloride is the relevant halide (^{fig. 4}). It is oxidized by H₂O₂ in the presence of myeloperoxidase to form hypochlorous acid (HOCl) :

H₂O₂ + Cl^- + H⁺ -> HOCl + H₂O

myeloperoxydase

Hypochlorous acid is an extremely potent oxidant that rapidly attacks a wide range of molecules (potential targets include amines, amino acids, thiols, thioethers, nucleotides, hemoproteins) (^{Test and Weiss, 1986}). In tissues, HOCl can inactivate α-1-antitrypsin (^{Stolc, 1979}). This inactivation results in an enhanced proteolytic activity in vicinity of activated neutrophils. HOCl can also react with primary amines to form a complex family of nitrogen-chlorine (N-Cl) derivatives (^{Thomas, 1979}) (^{fig. 4}) :

RNH₂ + 2HOCl -> RNCl₂ + 2H₂O

These nitrogenous compounds are powerful oxidizing agents similar to hypochlorous acid in their ability to oxidize biological molecules.

The myeloperoxidase-independent reactions are based on the presence of oxygen metabolites, including hydrogen peroxide, superoxide anion and hydroxyl radical. The bactericidal effect of these reactive oxygen intermediates (ROIs) may be due to the initiation of a chain of peroxidation in bacterial cell wall :

OH^¥ + RH -> R^¥ + H₂O (1)

R^¥ + O₂ -> ^¥R₂ (2)

^¥RO₂ + RH -> R^¥ + ROOH (3)

Lipid peroxides (ROOH) can fragment to give a wide range of highly toxic products. However, if it is admitted that neutrophils produce hydroxyl radical, the release of myeloperoxidase limits the magnitude of its production and that of lactoferrin deprives the environment in iron.

Discussion

Polymorphonuclear neutrophils use a complex assortment of agents to kill pathogens : lytic enzymes or antimicrobial peptides stored in granules, reactive oxygen intermediates. Their efficacy in defending the periodontium against invading bacteria is highlighted by occurence of periodontal diseases when they manifest a dysfunction (^{Cottet, 1998}). However, as they may release their antimicrobial arsenal in the environment, they can amplify the destruction of normal cells and connective tissue especially when inflammation is chronic. Finally, it as recently been shown that these terminally differentiated cells may survive longer than currently believed in inflammatory foci and release cytokines that may influence the evolution of immune responses (^{Cassatella, 1995}).

Demande de tirés à part

Marie-Hélène COTTET, Faculté de Chirurgie dentaire Paris VI, 5, rue Garancière, 75006 PARIS - FRANCE.

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