761

The Neutrophil: Mechanisms of Controlling Periodontal Bacteria Kenneth T.

Miyasaki*

The control of potentially periodontopathic microorganisms by host neutrophils is crucial to periodontal health. Neutrophils may use oxidative or nonoxidative mechanisms and either kill bacteria, influence bacterial growth, or modify bacterial colonization in the periodontium. Delivery of antimicrobial substances by neutrophils involves respiratory burst activity, phagocytosis, secretion, or cytolysis/apoptosis. Neutrophils contain a number of antimicrobial components including calprotectin complex, lysozyme, defensins, cofactor-binding proteins, neutral serine proteases, bactericidal/ permeability increasing protein, myeloperoxidase, and a NADPH oxidase system. Many of these components are multifunctional and exhibit several mechanisms of antimicrobial activity. When comparisons are made among periodontal bacteria, differences in sensitivity to different components are observed. A hypothesis of specific defense is presented: That specific periodontal diseases can result from the failure of specific aspects of the host immune system (the neutrophil, in particular) in its interaction with specific periodontal pathogens. Failure may be due to phenotypic variation (pleomorphism) within the host or bacterial evasive strategies. J Periodontol 1991; 62:761-774.

Key Words: Neutrophils/therapeutic use; periodontal diseases/microbiology; periodontal

diseases/prevention

and control.

specific plaque hypothesis focused attention on the importance of certain bacteria in the pathogenesis of certain forms of periodontal disease.1 Predominantly Gram-negative bacteria have been cultured from clinically-defined disease lesions of gingivitis, adult Periodontitis (AP), localized juvenile Periodontitis (LJP), and generalized juvenile Periodontitis (GJP). The yeast, Candida albicans, also has been isolated from AP lesionai sites.2 Yet, despite the conceptual advance, the specific plaque hypothesis does not address the issue of the specific underlying host defense failures which enable specific pathogens to establish within the periodontal arena and produce disease. Neutrophils, monocytes, and other granulocytes form the myeloid arm of the immune system. The lymphoid arm includes T-cells, B-cells, and large granular lymphocytes. Bone marrow stem cells (in the presence of "stromal" cells) give rise to both cellular arms in a teeter-totter fashion, and the differentiation of stem cells toward the myeloid lineage is induced in vitro by lymphosuppressive steroids whereas increased lymphocyte (B-cell) production is observed in cyclic neutropenia.3 Cyclic neutropenia, but not lymphosuppressive therapy, is associated with destructive periodontal disease,4,5 suggesting that the myeloid arm, especially The

*Section of Oral Biology and Dental Research Institute, tistry, University of California, Los Angeles, CA.

School of Den-

neutrophils, is protective (or that the lymphoid arm is destructive) with respect to periodontal pathogenesis. Neutrophils are the predominant leukocytes in blood. It has become widely accepted that neutrophils defend periodontal tissues against infection by pathogens and subse-

quent tissue destruction.6 As evidence, individuals with defects in neutrophil function or biochemistry often exhibit severe forms of periodontal disease and, conversely, individuals with early onset or rapidly progressing forms of periodontal disease often exhibit relatively subtle neutrophil defects.7 One exciting area of periodontal research is the identification of the mechanisms host neutrophils may normally use to control specific periodontal pathogens. The aim of this review is to provide a perspective on the different mechanisms whereby host neutrophils normally control specific periodontopathic bacteria, to present evidence that different bacteria can be influenced differentially by individual mechanisms, and to consider the possibility of host pleomorphisms and some bacterial evasive strategies which permit circumvention of normal control systems. WHY ARE NEUTROPHILS IMPORTANT? The strategy of host defense against periodontal infection may be viewed as similar to that used in combatting any local infection (Fig. I).8,9 Initially, potential periodontal

pathogens

encounter

plasma factors,

such

as

complement,

762

J Periodontol December 1991

THE NEUTROPHIL MECHANISMS OF CONTROLLING PERIODONTAL BACTERIA

HOST DEFENSE STRATEGY

O

Serum

Complement

Microbes

Can serum factors control microbe?

Yes

LOCAL INFECTION

occurs

Can neutrophil control infection?

I

ACQUIRED IMMUNITY

MonocyteMacrophage

Neutrophil

9> No

7

6?

Figure

INNATE IMMUNITY

Does monocyte

completely digest antigen ?

Yes

Lymphocytes No

Can Ab, Ab + cells, orCMI control infection?

Yes

ACUTE INFLAMMATION RESOLUTION

CHRONIC INFLAMMATION RESOLUTION

1. A model sequence of immune system function in defense against periodontal and secondary systemic infection. if the host neutrophil is overwhelmed and chronic immune cells are activated.

within the crevicular or extracellular fluids. The result of this encounter is the initiation of inflammatory processes, as evidenced by the recovery of complement activation products from the gingival crevice.10 If complement (± antibody) is not successful in controlling the pathogen, the host defense turns to neutrophils.11 Neutrophils within the gingival crevice provide the first cellular host mechanism to control periodontal bacteria. They bring an astounding arsenal of antimicrobial weaponry packaged within almost all of their cellular compartments. If neutrophils are unsuccessful in controlling the pathogen (i.e., reducing bacterial antigen levels sufficiently), monocytes are recruited which infiltrate the connective tissue, develop into tissue macrophage, and either digest the antigen completely (and signal repair processes with cytokines such as transforming growth factor ß) or present partially digested antigen in association with major-histocompatibility complex (MHC)-encoded class II molecules to lymphocytes.12 Perhaps to avoid systemic infection, chronic periodontal inflammation may produce localized specific immune responses and a cytokine-orchestrated amputation of connective tissue which we call

"periodontal disease."13 Thus, neutrophils

are

important

because they control the periodontal microecology prior to the involvement of chronic inflammatory cells. In contrast, monocytes and lymphocytes dictate tissue responses to the periodontal microecology. Given the distinct tasks accorded to the acute and chronic immune elements, it may be proposed simplistically that either hypofunction of neutrophils or hyperfunction of monocytes/lymphocytes may result in increased susceptibility to periodontal diseases.7-13 NEUTROPHIL INTERFACE WITH PERIODONTAL MICROORGANISMS Half the leukocytes infiltrating the junctional epithelium and 90% of the leukocytes isolated from crevicular fluid

Yes

In this

SYSTEMIC INFECTION model, periodontal disease

neutrophils.14-15 Whereas neutrophils predominate the junctional epithelium and the gingival crevice, lymphocytes and monocytes/macrophage predominate within the subjacent connective tissue (Fig. 2.A).16 The concentration of neutrophils in the periodontal tissues exceeds the concentration of neutrophils in blood. In minimally-inflamed gin107 neutrophils/cm3 infiltrate the gingival givitis, 2.5 connective tissue and 1.7 x 10s neutrophils/cm3 are found in the junctional epithelium (blood levels normally range 106 neutrophils/cm3).17 Rather than from 1 x 106 to 4 a forming homogenous bacteria/leukocyte mixture in the gingival crevice, the neutrophils form a "leukocyte wall" interposed between the periodontal plaque mass and the junctional and sulcular (or pocket) epithelium (Fig. 2.B).9 The "leukocyte wall" may function both as a secretory and as a digestive organ, and crevicular neutrophils exhibit both partial degranulation and ingested bacteria.18"20 are

DELIVERY OF ANTIMICROBIAL SUBSTANCES Neutrophils are terminally-differentiated cells no longer concerned with growth or division. Their main function is to deliver antimicrobial substances to bacterial and fungal targets. Because neutrophils are not concerned with their own survival, they are free to use delivery modes which can be suicidal. Neutrophils deliver antimicrobial substances by 4 different mechanisms (Fig. 3).

Delivery of Oxygen Metabolites When phagocytes contact certain stimuli (such as opsonized bacteria or chemoattractants), they consume dioxygen if it is available (a process called the "respiratory burst") by transferring 1 or 2 electrons from cytosolic NADPH to the extracellular dioxygen via the NADPH oxidase system, forming potentially toxic Superoxide anion (02") and hy-

Volume 62 Number 12

MIYASAKI

763

specialized form of secretion. The phagolysosomal fusion process is dependent upon intracellular calcium and requires the activity of a mixture of 3 "synexin-like" cytosolic fusogenic proteins (molecular weights of 28 kdal, 47 kdal,

and 67 kdal).25 Antimicrobial substances are delivered at very high concentrations by intraphagolysosomal secretion and respiratory burst activity. In addition, phagocytosis isolates the ingested organism within an extremely stringent

environment.26

(Cytolysis and/or Apoptosis) Cytosolic and granule components can be delivered by apoptotic (programmed death) or cytolytic mechanisms.27-28 Apoptosis/cytolysis may be important in controlling mucocutaneous candidiasis.27'29 Death

OXIDATIVE AND NONOXIDATIVE MECHANISMS Neutrophils possess 2 pathways for controlling microorganisms, oxidative and nonoxidative.30-3' Components of both oxidative and nonoxidative pathways localize within the plasma membrane, cytosol, azurophil granule (sequestered by glycosaminoglycans), azurophil granule (sequestered within the membrane), specific granule, and possibly the tertiary granule (Fig. 4).29.32-34

2. A. Two immunologie zones can be distinguished in the periodontium. The neutrophil zone occupies the gingival crevice and junctional epithelium. The monocyte-lymphocyte zone forms within the subjacent connective tissue. B. Neutrophils in the gingival crevice form a leukocyte wall.

Figure

drogen peroxide (H202), respectively.21-22 The formation of chlorinating oxidants requires the secretion of myeloperoxidase (MPO) from the azurophil granule. Secretion Extracellular secretion involves the mobilization of cytoplasmic granules, fusion between the granule and plasma (or phagosomal) membranes, and discharge of granule contents into the external environment.23'24

Phagocytosis Phagocytosis is

the engulfment of particles within a membrane-bound structure called the "phagosome." Fusion between the cytoplasmic granules (lysosomes) and the phagosome form the "phagolysosome," and represents a

NONOXIDATIVE MECHANISMS OF CONTROLLING BACTERIA BY NEUTROPHILS Nonoxidative mechanisms of microbial control by neutrophils are those which do not involve the consumption of dioxygen and the formation of reduced oxygen metabolites. This does not mean that they are oxygen-independent and they may require the presence of dioxygen to exert antimicrobial effects.35 Nonoxidative mechanisms can include cytosolic components such as calprotectin and granule components such as 1) peptides, mainly the defensins; 2) lysozyme; 3) members of the neutral serine protease family (NSP) including cathepsin G, elastase, azurocidin, and p29b; 4) the bactericidal/permeability increasing protein (B/PI, 57 kdal CAP, and the related or identical molecule, BP); and 5) cofactor-binding proteins including apolactoferrin and possibly, cobalophilin (Table 1 and below). Other nongranule components, including histones, kill microorganisms and may also be important in host defense.35,36

Calprotectin Complex

The calprotectin complex is a mixture of 2 structurally related cytosolic proteins: the 10 kdal migration inhibition factor-related protein (MRP-8, cystic fibrosis antigen, the ß subunit) and the 14 kdal migration inhibition factor-related protein (MRP-14, the subunit). The calprotectin also has been called "LI complex protein and calgranulin," and it exerts zinc-reversible antimicrobial effects.37 Both subunits of calprotectin are members of the S-100 family (calcium-binding cytosolic proteins which are believed to be important in microtubule disassembly, cell growth, and

764

J Periodontol December 1991

THE NEUTROPHIL MECHANISMS OF CONTROLLING PERIODONTAL BACTERIA

secretionT

RESPIRATORY Bacterial factors Complement C5a

BURST| Bacterial factors Complement C5a

02" ,H202,HOCI Bacterial contact Complement iC3b

PHAGOCYTOSIS

Azurophil granule

Bacterial contact

Complement ¡C3b

components

|

LYSIS OR

Intraohagolysosomal

02"',H202,HOCI

APOPTOSISl

No oxygen metabolites

k Intraphagolysosomal degranulation

"

:

Complete cytoplasmic and granule discharge

Figure 3. The neutrophil has 4 ways of delivering antimicrobial substances to bacteria. Respiratory burst activity requires the presence of dioxygen and involves the delivery of toxic oxygen metabolites. Secretion involves the extracellular release of antimicrobial compounds. Phagocytosis is a complex process which results in the isolation of the microorganism in a stringent environment, and the delivery of virtually all antimicrobials except those of cytoplasmic and nuclear origin. Cytolysis and apoptosis, although different mechanisms, can be considered together as a means of delivering lysosomal, cytoplasmic, and nuclear substances.

OXIDATIVE

NEUTROPHIL

NADPH Oxidase

NONOXIDATIVE

Cytosol 47 kdal phosphoprotein 67 kdal protein

Cytosol Calprotectin

Membrane,Specific, Tertiary (?) Granule Cytochrome £> 65 kdal flavoprotein

Specific granule

5

+

02

o2-

HOCI

+ +

e ·

+

2e

H202

Lactoferrin

Cobalophilin (?) Lysozyme Azurophil granule

membrane B/PI

CT

Azurophil granule Myeloperoxidase

Azurophil granule Defensins Neutral serine proteases

Lysozyme

Figure 4. This diagram summarizes the oxidative and nonoxidative mechanisms neutrophils possess for controlling microbes. See text for briefdescriptions.

Volume 62 Number 12

MIYASAKI

Table 1. Characterized Nonoxidative Antimicrobial Substances Found in Substance

Weight (Kdal)

Calprotectin ( 2ß,)

36.5

Lysozyme

14.3

12

Lactoferrin Defensins Neutral Serine Proteases Cathepsin G Elastase Azurocidin

80 3-3.5

3

23-25 33-36 28-30 28-34 55-59

Molecular

p29b (AGP7) B/PI (BP?) *G

=

proteoglycan matrix;

Granule

Chromosome Localization

Neutrophils

Subcellular Localization*

GM

granule

Antimicrobial

Charge

Cationic

8

Specific and Azurophil G Specific G Azurophil G

Isomers* Mixture and ß subunits 1

Cationic Cationic

1 4

14 11 ND ND ND

Azurophil G Azurophil G Azurophil G Azurophil G Azurophil GM

Cationic Cationic Cationic Cationic Cationic

Characteristics Antonie

Cytosol

=

765

3-5C 3C 3C 3C

1(2?)

Spectrum*

References

P,N,F

37-39

P,N,F

40,41,43 44-49 50-55

P,N

P,N,F

59-64

P,N,F

59,74,81,82 75,76 75,76 83,88,89

P,N,F P,N,F

membrane.

Primary sequence isomers have no special designation; C carbohydrate-based glycoisomers, calprotectin isomers can result from a mixture of subunits. documented microbicidal activity (does not refer to other potential antimicrobial activities): Gram-negative bacteria, Gram-positive bacteria, F fungi (yeasts, mainly C. albicans). =

=

=

=

5Both subunits

are

encoded

on

chromosome 1.

and, significantly, calprotectin localizes neutrophils, monocytes, and normal epithelium of the

cell differentiation) to

muscosa, and esophagus.38,39 Calprotectin be microbiostatic at low concentrations and microbicidal at higher concentrations against yeasts such as C.

tongue, buccal can

albicans.29'37

Lysozyme Lysozyme is a multifunctional protein which is best known for its ability to hydrolyze the ßl-4 linkages between N-

acetylmuramic acid and N-acetylglucosamine in bacterial cell walls. Lysozyme possesses 2 mechanisms of antibacterial action: enzymatic (against Gram-positive organisms) and nonenzymatic (autolytic, against Gram-negative organisms). Many oral organisms are insensitive to lysozyme, but some are sensitive to the nonenzymatic bacteriolytic effects, especially in the presence of chaotropic ions.41'42 Lactoferrin and Cobalophilin The specific granule constituent,

apolactoferrin (or lactoiron-binding glycoprotein of the transfertransferrin), rin family.44"46 Apolactoferrin exerts iron-inhibitable bacteriostatic activity against many organisms and bactericidal activity against some.47'48 Bacteriostatic effects are attributed to iron deprivation, whereas bactericidal effects (against Gram-negatives, at least) may be due to a second mechanism involving the destabilization of the bacterial outer membrane by chelation of calcium, magnesium, and iron cations.49 Cobalophilin, a vitamin B12-binding protein, may exert antimicrobial effects against vitamin B12 auxotrophs. is

an

Defensins The defensins are multifunctional peptides with 29 to 34 amino acids which comprise 5 to 7% of the total neutrophil protein and 30 to 50% of the azurophil granule content.50'51 Three major defensins are found in human neutrophils (HNP-

1, HNP-2, and HNP-3).53 A fourth human leukocyte defensin, HNP-4, has been described which is extremely po-

in killing E. co//.54'55 Defensins exert antimicrobial effects against fungi, viruses, and bacteria under conditions of low ionic strength and mildly alkaline pH.50,53*56 The phagolysosomal concentration of the defensins can exceed 5 mg/ml, at which the defensins can exert a bactericidal effect under conditions of physiologic ionic strength.56 Besides their antimicrobial effects, defensins stimulate monocyte Chemotaxis and bind to adrenocorticotrophic hormone (ACTH) receptors and inhibit ACTH-stimulated corticosteroid production (HNP-4 has been referred to as a tent

"corticostatin").57-58 G G is

Cathepsin

an arginine-rich, cationic glycoprotein which Cathepsin exhibits chymotrypsin-like proteolytic activity and accounts for 1 to 2% of the total neutrophil protein.59 Cathepsin G shares primary sequence homology with all the members of the leukocyte NSP family, but is most closely related to mast cell chymases and T-cell granzymes.61'62 The microbicidal effect of cathepsin G against non-oral microorganisms does not depend upon enzymatic activity.63'64 Two small peptide fragments (IIGGR and HPQYNQR) from cathepsin G kill non-oral bacteria.65 Challenging the precept that nonoxidative peptides and proteins kill as cationic amphipathic molecules, the microbicidal activity is not dependent upon cationicity nor hydrophobicity.66 Plasma antiproteases such as -1-antitrypsin, -1-antichymotrypsin, and -2-macroglobulin inhibit the enzyme-independent microbicidal activity of cathepsin G.67 Similar to the defensins, the microbicidal activity of cathepsin G is impeded by increasing ionic strength and serum concentration.68 70 The biologic function of the enzymatic activity of cathepsin G is enigmatic, although it may enhance phagocytosis and promote complement-mediated

766

THE NEUTROPHIL MECHANISMS OF CONTROLLING PERIODONTAL BACTERIA

granulocyte chemotactic activity.7173 Cathepsin G potentiates the killing of bacteria by lysozyme, B/PI, and the myeloperoxidase-hydrogen peroxide-chloride system (MPO-H202-Cl- system).68"70'74 —

Azurocidin and the 37 kdalton Cationic Protein (CAP) Azurocidin lacks an active-site serine and is enzymatically inactive, yet highly bactericidal against, Escherichia coli. It is a major azurophil granule constituent, comprising 17% of the total azurophil granule protein.55,75'76 Azurocidin shares N-terminal sequence homology with the 37 kdal CAP ("Valley AB„ protein, Vab/').77 In contrast to azurocidin, 37 kdal CAP is not a major component of human neutrophil lysosomes. It kills Gram-negative bacteria by binding to the 4' phosphate of lipid A found in LPS.77,78 Increased substitution of 4'-amino-l-deoxyarabinose or O antigen decreases the susceptibility of Salmonella typhimurium to 37 kdal CAP,79 thus 37 kdal CAP selectively kills Gram-negative bacteria possessing the rough phenotype. The bactericidal activities of 37 kdal CAP and azurocidin are enhanced at low pH.75,80 Elastase

Elastase kills the Gram-negative Acinetobacter, and produces lysis of Staphylococcus aureus.14 Elastase, but not cathepsin G, produces marked proteolysis of the Acinetobacter outer membrane proteins. The microbicidal potential of purified elastase is generally low, leading some to believe that it has very little independent microbicidal activity.83 Elastase increases the sensitivity of Acinetobacter to the bactericidal effects of lysozyme.74 Elastase can potentiate the bactericidal effects of B/PI and the MPO-H202Cl~ system in an enzyme-independent manner.68"70 P29b P29b is a recently described cationic NSP which kills bacteria best in mildly acidic conditions.75 It appears to belong to a group of very closely related or identical elastolytic enzymes, which include proteinase 3, azurophil granule protein-7 (AGP-7), p29 (a possible antigenic target of antineutrophil cytoplasmic autoantibodies of Wegener's granulomatosis; also called the "ANCA-C" or "ACPA" antigen), and p29b.55,84-87 All have been distinguished by their N-terminal sequence, but not in side-by-side comparison, and it is therefore plausible that they are the same or allotypes of the same protein. Proteinase 3 and p29b are both azurophil granule components and both share substantial sequence homology with elastase.

Bactericidal/Permeability Increasing Protein Two relatively large, nonenzymatic, cationic proteins, B/ PI (57 kdal CAP) and BP, are similar if not identical molecules.77,88 91 B/PI is a 59 kdal intramembranous cationic protein (pi of 9.8), whereas BP is a 55 kdal glycoprotein (pi of 7.5).88,89 B/PI has a low proportion of arginine The

and

J Periodontol December 1991

cysteine and localizes exclusively to neutrophils and its

precursors.32,88,92 B/PI belongs to a class of proteins which transport or bind cholesterol esters and Hpopolysaccharides92,93

and enhance leukocyte activity in the presence of LPS.94 B/PI itself can "neutralize" endotoxin, suggesting other

potential biological functions.94 B/PI specializes in killing Gram-negative bacteria and does not kill Gram-positive bacteria or fungi.83 Killing by B/PI is reversed by both Mg++ which promotes B/PI desorption and serum albumin which does not promote desorption.96 In contrast, the bacteriostatic activity of B/PI is not reversed with Mg++ but is reversed by serum albumin. Bactericidal activity of B/PI resides entirely within a 25 kdal cationic, hydrophilic domain including the N-terminus.92,97 B/PI is more effective killing rough than smooth variants of certain enteric, Gram-negative bacteria.98 Modulating proteins

Two arginine-rich 15 kdal proteins, pl5A and pl5B, have been isolated from rabbit neutrophils which share sequence homology with no known protein but each other.99 Although they do not exert any microbicidal activity on their own, they potentiate the bactericidal activity of B/PI and inhibit that of azurocidin and the defensins. Modulatory proteins as well as interactions among all of the antimicrobial proteins discussed above add to the complexity of mechanisms neutrophils can use to control the oral

microecology.

OXIDATFVE MECHANISMS OF CONTROLLING BACTERIA BY NEUTROPHILS

The NADPH Oxidase System The NADPH oxidase system initiates oxygen reduction and consists of at least 3 membrane and 2 cytosolic proteins. The cytosolic proteins (a 47 kdal phosphoprotein and a 67 kdal protein) are involved in the binding of NADPH (with a redox potential of —320 mV) and electron shuttling.100-102 Facing the cytosol on the internal surface of the plasma membrane is a flavin (FAD)-binding protein (with a redox potential of 280 mV) which receives electrons from the cytosolic shuttles.102,103 This 65 kdal flavoprotein may be identical to the cytosolic 67 kdal NADPH-binding protein described above.104 Cytochrome b (called "cytochrome fr558" for its absorbance at 558 nm, and "cytochrome b_245" for its redox potential of -245 mV) is a heterodimeric structure consisting of a 22 kdal and 91 kdal subunit. Cytochrome b is found only in "professional phagocytes," including neutrophils, monocytes, and eosinophils.45 Cytochrome b adds electrons to external or intraphagolysosomal dioxygen (with 105 The 91 kdal subunit a redox couple of -160 mV).102 of cytochrome b is encoded on the X chromosome.26,34 About 80% of cytochrome b localizes to the specific granules and 20% to the plasma membrane.106 It has been -

Volume 62 Number 12

suggested that cytochrome b may localize to another organelle, the "tertiary" granules; as such, the subcellular distribution of cytochrome b may be complex.107 The translocation of cytochrome b558 from the specific granules to the plasma membrane is not required for respiratory burst activity, but is suspected to be involved in the enhanced respiratory activity observed after cytokine-priming.34,106 Reduced Oxygen Metabolites The reduced oxygen metabolites formed by the NADPH oxidase system, including 02~ and H202, may be toxic to many microorganisms. In contrast to 02~, H202 is capable of freely diffusing across the membranes of the neutrophil plasmalemma, phagolysosome, or target organism. An important "lethal hit" is the damage of large, vital targets of limited copy number, such as chromosomal DNA.108 H202 is incapable of producing damage to DNA unless in the presence of a reduced transition metal. The reduced transition metal provides an electron for the reduction of H202 to hydroxyl radical (OH-), which damages cellular DNA.109,110 Based on limited studies, it is generally felt that 02" itself is innocuous.109112

Myeloperoxidase-Mediated Bactericidal Activity

Myeloperoxidase (MPO) is a chlorin-containing enzyme found in neutrophils and blood monocytes which is encoded 113116 The 3 major glycoisoforms of on chromosome MPO (MPO I, II, and III) differ in charge, hydrophobicity, molecular mass of the heavy subunits, and subcellular localization.113,114,117 All 3 MPO forms catalyze the oxidation of chloride and reduction of H202 to form hypochlorous acid (HOCI). The hypochlorite anion, OC1", chlorinates amine groups. This produces peptide bond scission and the formation of low molecular weight chloramines

with bactericidal potential.118,119 It has been estimated that 28 to 72% of dioxygen consumed and up to 40% of the H202 formed during the respiratory burst is used to oxidize chloride.120,121 MPO is too large to freely cross membranes, therefore, chlorinating compounds are delivered first to the target surface. Opposite to killing by azurocidin and B/PI, smooth parent strains of enteric organisms are killed more efficiently by the MPO-H202-Cl~ system than their corresponding deep

rough mutants.78,98,121 Many periodontal Gram-negative organisms possess LPS with no carbohydrate chain structure analogous to the O-antigen of enteric microorganisms,122,123 suggesting that the MP0-H202-C1_ system may be subordinate to nonoxidative mechanisms in killing bacteria within the periodontium. NON OXIDATIVE KILLING OF ORAL BACTERIA Whereas serum alone is relatively ineffective in killing oral bacteria, serum plus neutrophils exhibit potent antimicrobial effects against all of the putative periodontal pathogens tested to date.124 Neutrophils kill Actinobacillus actinomy-

MIYASAKI ANAEROBIC

AEROBIC

F/G or

F/G or

ORGANISM

ABODE

767

HNP

ABODE

HNP

A.a. ATCC 29523 Y4

++

±

++

++

ND

++

ND

NCTC 9709

ND

Capno. ATCC 33123 ATCC 33124

+++ ++++

ATCC 27872

++ ++++

± -

+

+++ -

-

E.COli

ML-35

+++

ND

++

ND

++

+++

-

-

+++

Figure 5. Differential sensitivity of A. actinomycetemcomitans and Capnocytophaga Spp. to neutrophil granule components. Neutrophil granule components were separated by gel filtration chromatography. Seven fractions were generated, including A (containing myeloperoxidase and lactoferrin), (containing lactoferrin and NSP), C and D (containing NSP but enriched for elastase and cathepsin G, respectively), E (containing lysozyme), and F and G (containing defensins). The bactericidal activities at 100 ßg/ml protein under aerobic and anaerobic conditions was assessed. Each "+ represents 1-log order ofkilling over 2 hours. Fraction D (cathepsin G-containing) killed all bacteria tested. Fraction killed A. actinomycetemcomitans but not Capnocytophaga. Fraction C and the defensins killed Capnocytophaga. "

cetemcomitans by both oxidative and nonoxidative pathways under normoxic conditions.125 Although less than 50% of normoxic killing of A. actinomycetemcomitans can be ascribed to nonoxidative mechanisms, hypoxic conditions prevail within the gingival crevice.126 Three potential nonoxidative mechanisms are known to kill putative pathogens such as A. actinomycetemcomitans and/or Capnocytophaga Spp. in vitro. Lactoferrin kills both Streptococcus mutans and A. actinomycetemcomitans, both NSP and defensins kill Capnocytophaga Spp., and NSP kill A actinomycetemcomitans.48'127'130 As shown in Figure 5, differential killing potential of neutrophil granule components against oral bacteria was observed under aerobic and anaerobic conditions, thereby provide support for a specific defense hypothesis.131 Substantial cidal activity was observed among the defensin-containing and NSP-containing fractions.

Killing of Oral Bacteria by the Defensins A. actinomycetemcomitans, C. ochracea, C. sputigena, C. gingivalis, Eikenella corrodens, Haemophilus paraphrophilus, H. aphrophilus, and H. segnis are sensitive to the rabbit defensin, NP-1 (Fig. 6).128 The spectrum of activity of the human defensins, however, is more limited and they

killing Capnocytophaga but relatively weak against others, including A. actinomycetemcomitans and E. corrodens.129 Unlike the killing of non-oral organisms, the killing by defensins of Capnocytophaga under both aerobic are

active in

and anaerobic conditions occurred.

Killing of Oral Bacteria by the NSP Cathepsin G is particularly microbicidal against oral micro-

organisms,

and may

play a significant role in killing under

768

that the bactericidal activity of H202 against A. actinomycetemcomitans may be due to the iron-catalyzed reduction

KILLING OF PERIODONTAL BACTERIA BY THE RABBIT DEFENSIN, NP-1 H. s. HK498 H. s. HK316 H. a. 13252 H. a. 19415

S:

g

§ O

J Periodontol December 1991

THE NEUTROPHIL MECHANISMS OF CONTROLLING PERIODONTAL BACTERIA

of

H. p. 29242 H. p. 29241 H. p. 29240 E. c. E. c. A E. c. 23834

C. och. 27872 C. sput. 33123 C. ging. 33124 A. a. 67 A. a. 29523 A. a. Y4 A. a. 650

E. coll ML 35

-.—I-1-1— -1— -1-1— -I-1— -I

1

1

0

2

3

4

5

BACTERICIDAL ACTIVITY, 3LOG10

Figure 6. Sensitivity of oral bacteria to the rabbit defensin, NP-1. A.a. Eikenella corrodens; H.s. HaeA. actinomycetemcomitans; E.c. mophilus segnis; H.a. Haemophilus aphrophilus; Hp. Haemophilus paraphrophilus, C. ging., C. sput. and C. och. Capnocytophaga gingivalis, C. sputigena, and C. ochracea, respectively. The Uogw refers to the loglO order of killing observed after 1 hour. Wide variation in sensitivity of bacteria is observed. =

=

=

=

=

H202 to ·.133

Effects of Myeloperoxidase Against Oral Bacteria A. actinomycetemcomitans is killed rapidly by the isolated MP0-H202-C1- system.134 Although all 3 forms of MPO can kill oral bacteria, it has been shown that mainly MPO isoform III binds to oral bacteria.135 This finding suggests that MPO III, the most cationic of the MPO isomers, is also the most important in MPO-dependent bactericidal activity. The MPO system may have a number of other antimicrobial activities besides killing. For example, the MPOH202-C1~ system neutralizes the leukotoxin of A. actinomycetemcomitans136 and may modulate bacterial adherence to host tissues (below).

=

hypoxic or anaerobic conditions.130 It is more potent than other neutrophil granule antimicrobial substances, including the defensins, on a molar basis. Uniquely, an intact enzyme active site is crucial in the killing of Capnocytophaga Spp. but not E. coli}30 Therefore, cathepsin G appears to possess 2 mechanisms for killing bacteria, enzyme-dependent and enzyme-independent. In contrast, killing of A. actinomycetemcomitans is not enzyme-dependent. Thus, the chymotryptic activity of cathepsin G may have evolved to control organisms such as Capnocytophaga Spp. Intriguingly, we have observed that E. coli is sensitive to HPQYNQR but not IIGGR, and C. sputigena is sensitive to IIGGR but not HPQYNQR (unpublished data), suggesting that enzymatic activity may be required to expose IIGGR, which is normally buried.66 Unlike A. actinomycetemcomitans, the Capnocytophaga Spp. are also sensitive to killing by elastase.131 In summary, supportive of a specific defense hypothesis, not only do oral bacteria exhibit sensitivities to different molecules, but also, they are sensitive to different microbicidal domains and mechanisms of the same molecule. OXIDATrVE KILLING OF ORAL BACTERIA

Killing of Oral Metabolites

Bacteria

by Reduced Oxygen

Oxidative killing of A. actinomycetemcomitans by reagent H202 has been demonstrated, but the required concentration was generally 10 times higher than those anticipated within the phagolysosome.132 In contrast, Haemophilus aphrophilus is sensitive to "achievable" concentrations reagent H202. Killing of A. actinomycetemcomitans by the xanthine-xanthine oxidase system, which generates a mixture of H202 and 02", can be blocked partially by desferrioxamine, a

membrane-permeable iron-binding siderophore, suggesting

CONTROL OF ORAL BACTERIAL COLONIZATION Bacterial colonization is a complex process involving attachment, retention, survival, and growth of the microorganism on a host tissue.137 Periodontal neutrophils may play a key role in the modulation of the bacterial ecology of the root surface of teeth and preventing bacterial progression into the junctional epithelium. Short-term experimental neutropenia leads to a rapid apical extension of the subgingival plaque front into the junctional epithelium without the frank bacterial invasion of the subjacent connective

tissues.17'138

Nonoxidative systems may play an important role in influencing the oral microecology by mechanisms acting upon bacterial attachment. One lysosomal component which affects bacterial adherence to in vitro tooth models was lysozyme.139 It was believed that the effects of lysozyme on adherence could be attributed to its polycationic nature rather than enzymatic activity, although this was not demonstrated conclusively. Recently, Cimasoni et al.140 reported that neutrophil elastase or cathepsin G decreased the adherence of Streptococcus sanguis but increased the adherence of Porphyromonas gingivalis to saliva-coated hydroxyapatite. Although both neutral serine proteases bound to hydroxyapatite in active form, it was unclear as to whether enzymatic activity was responsible for the observed effects. Similarly, these lysosomal constituents affected the attachment of oral tréponèmes.141 The neutrophil may influence bacterial retention. Wilton142 found that neutrophils or neutrophil lysosomal enzymes were capable of detaching 5. mutans from glass. We have observed that intact neutrophils could detach Actinomyces viscosus from saliva-coated hydroxyapatite by an azide-

insensitive, energy-dependent mechanism.143

Oxidative antimicrobial systems may modulating bacterial colonization of the may be significant that Charon et al.144 plaque and gingivitis scores in patients

be important in tooth surface. It observed higher with CGD. We

Volume 62 Number 12

speculated that oxygen metabolism may be important in regulating bacterial colonization. The MP0-H202-C1~ sys-

MiYASAKI

NL

L

769

LNLNNLLN

tem blocked adherence of both A. viscosus and oral streptococci to saliva-coated hydroxyapatite.145'146 H202 can be derived from either host leukocytes or catalase-negative facultative bacteria such as streptococci.146 Enzyme activity was absolutely required for these effects to be observed.

NEUTROPHIL PLEOMORPHISM AND ABERRATION Chronic granulomatous disease (CGD) is an inherited disorder with at least 2 distinct inheritance modes: X-linked or autosomal recessive.103 The 91 kdal subunit of cytochrome b is absent in X-linked CGD and the 47 kdal cytosolic phosphoprotein is absent in an autosomal form of CGD.34 In CGD, neutrophils are incapable of mounting a respiratory burst. As a result, patients with CGD suffer recurrent, deep infection from catalase-positive organisms (which do not secrete H202), including S. aureus, E. coli, Serratia marcescens, C. albicans, Chromobacterium violaceum, Nocardia Spp., and Aspergillus Spp.147 Interestingly, these organisms can be killed by cationic proteins and peptides in vitro53,63 suggesting that the capacity for organisms to produce disease in CGD patients is dependent upon other evasive factors as well as catalase. Significantly, obligate anaerobes are not usually isolated from CGD lesions, suggesting that host defense mechanisms (presumably oxygen-independent, nonoxidative mechanisms) normally operating against such organisms are intact.26 Interestingly, CGD has been associated with severe gingivitis and oral ulcération but no strong association has been made with Periodontitis.144,148 MPO deficiency, the partial decrease or total lack of MPO from neutrophils, occurs in about 0.05% of the population.149 The relationship of partial MPO deficiency to the isoforms of MPO is unknown. In contrast to CGD, MPO deficiency is a relatively benign problem, infrequently associated with severe recurrent infections such as streptococcal cellulitis and conjunctivitis due to Haemophilus influenzae type b.150 Despite the relative insignificance of MPO from the medical standpoint, MPO-deficient neutrophils have difficulty killing certain microorganisms, including many fungi. Bacterial killing rates have also been reported to be retarded (including the killing of S. marcescens, E. coli, Lactobacillus acidophilus, Staphylococcus albus, and S. aureus).151 Nonoxidative mechanisms may be of greater importance in controlling the periodontal microflora. Individuals with "specific granule deficiency" (SGD) and Chediak-Higashi syndrome (CHS) often exhibit severe Periodontitis.144 Among other defects, neutrophils from individuals with SGD are deficient in defensins, lactoferrin, and cobalophilin; neutrophils from those with CHS are deficient in the neutral serine proteases.145,155 Can pleomorphic expression of neutrophil antimicrobial proteins lead to periodontal disease among otherwise healthy

Figure 7. Pleomorphic expression of the human neutrophil defensin, HNPnormal subjects; L UP subjects; LF 3. lactoferrin; NSP male. neutral serine proteases; HNP defensins; f female; m Electrophoretic separation of whole neutrophil lysates in 12.5% acrylamide gels containing acetic acid and urea. HNP-3 is virtually absent from =

=

=

=

2 individuals with UP and 1 normal. There is HNP-3 deficiency and UP in this sampling.

=

no

=

=

concordance between

individuals? At present, very little is known about the pleoof neutrophil antimicrobial substances among normal and periodontally diseased populations. However, peripheral blood neutrophils from individuáis with LIP exhibited decreased killing of A. actinomycetemcomitans despite normal phagocytosis, 02~ production, and secretion of specific granule components.154,155 These observations suggest that neutrophils from individuals with LJP may have a defect in their nonoxidative antimicrobial

morphic expression

systems.

We examined lysates from neutrophils of 6 LJP and 6 normal donors by acid-urea Polyacrylamide gel electrophoresis (AU-PAGE). Whereas all subjects possessed defensins HNP-1 and HNP-2, variable expressions of HNP-3 were observed: 1 normal and 1 LJP individual lacked HNP-3 completely, 3 normals and 2 LJP exhibited relatively low levels of HNP-3 (unpublished data; Fig. 7). No concordance between LJP and HNP-3 deficiency was observed. This is not totally surprising since HNP-3 is the least potent defensin against both oral and nonoral bacteria,53,129 and the actual biologic function (if any) of HNP-3 is unclear.

BACTERIAL EVASIVE STRATEGIES Although too numerous to detail in this review, bacteria possess a number of mechanisms to circumvent the antimicrobial effects of host defense systems. We examined several potential evasive mechanisms that bacteria may use against oxidative defense systems including catalase and bacterial mannans.132,156 Catalase, which catalyzes the disproportionation of H202 (reduction and oxidation of H202 to water and dioxygen, respectively) may be important in protecting bacteria against oxidative killing by reduced oxygen metabolites. Mannans are capable of interfering with the microbial activities of the MP0-H202-C1~ system, possibly by blocking the adsorption of the enzyme to the bacterial surface. Against nonoxidative defense, siderophores

770

THE NEUTROPHIL MECHANISMS OF CONTROLLING PERIODONTAL BACTERIA

may combat the bacteriostatic effects of lactoferrin by competing for iron.157 Extracellular polymers may also assist bacteria in evading host defense mechanisms; for example, sucrose-grown S. mutans are resistant to the bactericidal activity of lactoferrin.48 Any extracellular polyanion may also interfere with the activity of cationic proteins such as cathepsin G and B/PI.158,159 CONCLUSION Neutrophils contain numerous antimicrobial components. The antimicrobial compound may be multifunctional, exhibiting several mechanisms of antimicrobial activity. Multifunctionality can extend beyond interactions with microorganisms, and some antimicrobial components may interact with host systems; as such, neutrophils can also protect against periodontal disease by modulating chronic inflammation through the release of factors—perhaps identical to factors with antimicrobial roles—which suppress (and augment) lymphocyte and monocyte activity.57,160 Pleomorphism pervades the immune system, notably MHCencoded molecules (MHC class molecules I, MHC class II molecules, and complement factors B, C2, and C4) and in antigen receptors (Immunoglobulins and T-cell antigen receptors). The impact of host immune pleomorphism on periodontal diseases has never been studied in a comprehensive manner. Neutrophil antimicrobial components may exhibit pleomorphic characteristics or expression which may alter one or all of its functions. Theoretically, certain periodontal diseases may represent the failure of specific neutrophil immune mechanisms which control specific periodontopathic bacteria and specific subsequent chronic inflammatory responses. Evidence has been reviewed herein which suggests that the mechanisms whereby neutrophils control oral bacteria are selective. The MPOH202-C1~ system appears to be an important oxidative system, exerting both microbicidal activity against oral bacteria and modulating Gram-positive bacterial adherence to saliva-coated hydroxyapatite. Neutral serine proteases may be important nonoxidative mechanisms. These substances kill Gram-negative, facultative bacteria and have been shown to influence the adherence of Gram-negative bacteria to hydroxyapatite. Uniquely, certain oral bacteria are killed by the neutral serine protease, cathepsin G, in an apparently enzyme-dependent, oxygen-independent fashion. More research is required to 1) determine the impact of calprotectin complex on the oral ecology; 2) determine the synergistic interactions among neutrophil components in influencing the oral ecology; 3) expand upon our knowledge of the bacteriostatic and abhesive activities of neutrophil antimicrobial peptides and proteins; 4) explore evasive mechanisms of oral bacteria; and 5) relate host defense pleomorphism with periodontal health status.

Acknowledgments The author gratefully acknowledges the help and support of R.I. Lehrer, M.E. Selsted, T. Ganz, A.L. Bodeau, P.M.

J Periodontol December 1991

de Camargo, T.F. Flemmig, J.-C. Erard, L.E. Wolinsky, M.E. Wilson, W.M. Shafer, and A.R.K. Murthy. Supported by NIH-NIDR Research Grant DE 08161 and Research Career Development Award D.E. 00282. REFERENCES 1. Loesche WJ. Chemotherapy of dental plaque infections. Oral Sci Rev 1975; 9:65-107. 2. Slots J, Rams TE, Listgarten MA. Yeasts, enteric rods and pseudomonads in the subgingival flora of severe adult Periodontitis. Oral Microbiol Immunol 1988; 3:47-52. 3. Kincade PW, Lee G, Pietrangeli CE, Hayashi S-I, Gimble JM. Cells and molecules that regulate lymphopoiesis in bone marrow. Ann Rev Immunol 1989; 7:111-143. 4. Oshrain HI, Mender S, Mandel ID. Periodontal status of patients with reduced immunocapacity. J Periodontol 1979; 50:185-188. 5. Cohen DW, Morris AL. Periodontal manifestation of cyclic neutropenia. / Periodontol 1961; 32:159-168. 6. Genco RJ, Slots J. Host responses in periodontal diseases. / Dent Res 1984; 63:441-451. 7. Van Dyke TE, Hoop GA. Neutrophil function and oral disease. Crit Rev Oral Biol Med 1990; 1:117-133. 8. Wahl SM. Acute and chronic inflammation. In: Zembala M, Asherson GL, eds. Human Monocytes. New York: Academic Press; 1989:361-371. 9. Attström R, Lindhe J. Pathogenesis of plaque-associated periodontal disease. In: Lindhe J, ed. Textbook of Clinical Periodontology. Philadelphia: W.B. Saunders Company; 1985:154-187. 10. Shenkein HA, Genco RJ. Gingival fluid and serum in periodontal disease. II. Evidence for cleavage of complement components C3, C3 proactivator (Factor B) and C4 in gingival fluid. J Periodontol 1977; 48:778-784. 11. Miller DR, Lamster IB, Chasens AI. Role of the polymorphonuclear leukocyte in periodontal health and disease. J Clin Periodontol 1984; 11:1-15. 12. Unanue ER, Allen PM. The basis for the immunoregulatory role of macrophages and other accessory cells. Science 1987; 236:551-557. 13. Seymour GJ. Possible mechanisms involved in the immunoregulation of chronic inflammatory periodontal disease. / Dent Res 1987; 66:2-9. 14. Schroeder HE. Transmigration of leukocytes in human junctional epithelium. Helv Odontot Acta 1973; 17:6-18. 15. Attström R. Presence of leukocytes in crevices of healthy and chronically inflamed gingiva. / Periodont Res 1970; 5:42-47. 16. Page RC, Schroeder HE. Pathogenesis of inflammatory periodontal disease. A summary of current work. Lab Invest 1976; 33:235-249. 17. Kraal JH. Polymorphonuclear leukocytes in the periodontium. [Thesis]. Leiden, The Netherlands: Rijksuniversiteit te Leiden, 1979. 109 P18. Newman HN. Neutrophils and IgG at the host-plaque interface on children's teeth. J Periodontol 1980; 51:642-651. 19. Lange D, Schroeder HE. Cytochemistry and ultrastructure of gingival sulcus cells. Helv Odontol Acta 1971;15 (Suppl. VI): 65-86. 20. Saito I, Komiyama K, Moro I, Akachi K, Shiomi N, Ito , Seidai M, Umemura S. Ultrastructural and immunocytochemical characterization of polymorphonuclear leukocytes from gingival crevice in man. / Periodontol 1987; 58:493-497. 21. Klebanoff SJ. Phagocytic cells: products of oxygen metabolism. In: Gallin JI, Goldstein IM, Snyderman R, eds. Inflammation: Basic Principles and Clinical Correlates. New York: Raven Press Ltd;

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to: Dr. Kenneth T. Miyasaki, Section of Oral 63-050 CHS, UCLA School of Dentistry, Center for the Health Sciences, Los Angeles, CA 90024-1668. Accepted for publication July 15, 1991.

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