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Knockout MiceLysosomal-Associated Membrane Protein-2Neutrophils Leads to Periodontitis in Impaired Phagosomal Maturation in

Eeva-Liisa Eskelinen and Paul SaftigAngelika Zirogianni, Rainer Podschun, Bernd Schröder, Wouter Beertsen, Marion Willenborg, Vincent Everts,

http://www.jimmunol.org/content/180/1/475doi: 10.4049/jimmunol.180.1.475

2008; 180:475-482; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/180/1/475.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2008 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Impaired Phagosomal Maturation in Neutrophils Leads toPeriodontitis in Lysosomal-Associated Membrane Protein-2Knockout Mice1

Wouter Beertsen,2§ Marion Willenborg,2* Vincent Everts,¶ Angelika Zirogianni,§

Rainer Podschun,† Bernd Schroder,* Eeva-Liisa Eskelinen,‡ and Paul Saftig3*

Inflammatory periodontal diseases constitute one of the most common infections in humans, resulting in the destruction ofthe supporting structures of the dentition. Circulating neutrophils are an essential component of the human innate immunesystem. We observed that mice deficient for the major lysosomal-associated membrane protein-2 (LAMP-2) developed severeperiodontitis early in life. This development was accompanied by a massive accumulation of bacterial plaque along the toothsurfaces, gingival inflammation, alveolar bone resorption, loss of connective tissue fiber attachment, apical migration ofjunctional epithelium, and pathological movement of the molars. The inflammatory lesions were dominated by polymor-phonuclear leukocytes (PMNs) apparently being unable to efficiently clear bacterial pathogens. Systemic treatment ofLAMP-2-deficient mice with antibiotics prevented the periodontal pathology. Isolated PMNs from LAMP-2-deficient miceshowed an accumulation of autophagic vacuoles and a reduced bacterial killing capacity. Oxidative burst response was notaltered in these cells. Latex bead and bacterial feeding experiments showed a reduced ability of the phagosomes to acquirean acidic pH and late endocytic markers, suggesting an impaired fusion of late endosomes-lysosomes with phagosomes. Thisstudy underlines the importance of LAMP-2 for the maturation of phagosomes in PMNs. It also underscores the requirementof lysosomal fusion events to provide sufficient antimicrobial activity in PMNs, which is needed to prevent periodontaldisease. The Journal of Immunology, 2008, 180: 475– 482.

P eriodontitis is an infectious disease that is one of the mostwidespread diseases worldwide (1). It is estimated to affectup to 15% of the adult dentate population (2). Periodontitis is

an inflammatory disease of the supporting tissues of the teeth leadingto resorption of alveolar bone and eventually tooth loss. The diseaseis characterized by a constant interaction between pathogenic bacteriaand the host defense mechanisms. In health, host immune responsesare sufficient to hold in check the pathogenic potential of both thenormal resident microbial flora and exogenous microbial pathogens.Complex inflammatory and immune reactions are involved in the pro-gression of periodontitis. Polymorphonuclear leukocytes (PMNs)4

and circulating neutrophils constitute the first defense barrier againstthe oral bacterial challenge in the periodontium (3). They are rapidly

recruited from the blood to the site at risk, and then phagocytose andkill the intruders. Neutrophils may release proinflammatory mediatorsthat amplify the local inflammatory reaction, further promotingleukocyte and platelet recruitment. Quantitative or qualitativeabnormalities of the PMNs may, therefore, have an effect on theaccumulation of plaque in the supra and subgingival regions.Malfunctioning of PMNs including a disturbed adhesion to theendothelium, chemotaxis, detoxification of bacterial products,phagocytosis, or degranulation have been associated with earlyonset periodontitis (4 –7).

Phagosome-lysosome fusion bestows on the phagocytic vacuolethe lytic properties for efficient removal of internalized pathogens.Lysosomes play a crucial role in the oxygen-independent killing ofbacteria, which is believed to be an important killing mechanism inthe oxygen-deprived periodontal pocket (8).

The limiting membrane of the lysosomal compartment isthought to be of importance for phagosome maturation (9).Lysosome-associated membrane protein (LAMP)-2 is a highlyglycosylated protein. It is an abundant and important constitu-ent of the lysosomal membrane involved in lysosomal biogen-esis and late steps of autophagy and phagocytosis (10 –14).LAMP-2-deficient mice exhibit elevated postnatal mortality,and the surviving mice are of reduced weight. Autophagic vacuolesaccumulate due to an impaired proteolysis of long-lived proteins in anumber of tissues, especially in myocytes, cardiomyocytes, and hepa-tocytes (10). In the latter cells, we observed an elevated secretion oflysosomal enzymes, impaired processing of cathepsin D, and an ab-normal retention of mannose-6-phosphate receptors in autophagicvacuoles (13).

We report that LAMP-2-deficient mice show an increased sus-ceptibility to periodontitis. PMNs isolated from these mice showan impaired fusion between phagosomes and lysosomes leading to

*Biochemical Institute, Christian-Albrechts-University Kiel and †Institute of Infec-tion Medicine, University Hospital Schleswig-Holstein, Kiel, Germany; ‡Departmentof Biological and Environmental Sciences, Division of Biochemistry, University ofHelsinki, Helsinki, Finland; and §Department of Periodontology and ¶Department ofOral Cell Biology, Academic Centre for Dentistry Amsterdam, Universiteit van Am-sterdam and Vrije Universiteit, Amsterdam, The Netherlands

Received for publication October 17, 2007. Accepted for publication October22, 2007.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by the Deutsche Forschungsgemeinschaft DFGSA683/6–1.2 W.B. and M.W. contributed equally to this work.3 Address correspondence and reprint requests to Dr. Paul Saftig, Biochemical Institute,Christian-Albrechts-University Kiel, Olshausenstrasse 40, D-24098 Kiel, Germany.E-mail address: psaftig@biochem.uni-kiel.de4 Abbreviations used in this paper: PMN, polymorphonuclear leukocyte; LAMP,lysosomal-associated membrane protein; CEJ, cemento-enamel junction.

Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00

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a decreased bacterial killing capacity. This impairment is the likelycause of the development of severe periodontitis.

Materials and MethodsMice, cell lines, and Abs

LAMP-2-deficient mice were generated as previously described (10). Themice were maintained in a conventional facility. For antibiotic treatmentselected mice received amoxicillin in the drinking water before birth (thepregnant mothers) and immediately after birth (in a concentration of 5 mg/L).

Mouse primary neutrophil granulocytes were isolated from 2- to 4-mo-old mice. Mice were injected i.p. with 1 ml of sterile 4% Brewer’s thio-glycolate solution (Difco/BD Biosciences). After 4 h the mice were killed,and cells were recovered by peritoneal lavage using 5–10 ml of ice-coldPBS/0.02% EDTA (w/v). The number of viable cells was checked by stain-ing the cells with 0.4% trypan blue (Invitrogen Life Technologies). Theneutrophil yield was determined by flow cytometry after staining with PE-conjugated anti-Gr-1 Ab (Miltenyi Biotec) and with FITC-conjugatedF4/80 Ab (Serotec) after three washes in PBS/0.5% BSA/0.02% NaN3.Before staining, nonspecific binding of Abs was blocked with the anti-Fcreceptor Ab 2.4G2 (BD Pharmingen). Flow cytometric measurements wereperformed using FACScan (BD Biosciences). Unless stated otherwise, thecells were cultured in RPMI 1640 containing 10% FCS and penicillin/streptomycin (Invitrogen Life Technologies) for 2 h to adhere to the sur-face. Before their use for the experiments nonadherent cells were washedaway, which resulted in �95% pure PMN cultures.

The following Abs were used in this study: rabbit antiserum againstmouse cathepsin D (15); rat anti-mouse LAMP-1 and rat anti-mouseLAMP-2 (Developmental Studies Hybridoma Bank); rabbit anti-LC3 fromI. Tanida and T. Ueno (Juntendo University, Tokyo, Japan); anti-lactoferrin(Upstate Biotechnology); anti-myeloperoxidase (Dianova), anti-�-tubulin(E7; Developmental Studies Hybridoma Bank); and rabbit anti-DNP (ICNBiomedicals). Alexa Fluor-conjugated secondary Abs were from Molecu-lar Probes (Invitrogen Life Technologies).

Tissue processing, measurements, and statistical analysis

Mice were killed between 7 wk and 12 mo after birth. Upper and lowerjaws were fixed in 4% paraformaldehyde and 1% glutaraldehyde in 0.1 Msodium cacodylate buffer (pH 7.4). Jaws were micrographed and visualizedby high resolution MicroCT. Following demineralization in EDTA, jawswere postfixed in 1% OsO4 and embedded in epoxy resin. Sections werecut parallel to the longitudinal axis of the molars and stained with meth-ylene blue. One midsagittal section of each molar block (four per animal)was subjected to image analysis using the Leica-Qwin-Pro software (ob-jectives �6.3 and �10). To this end, the area between the first and secondmolars, the standard interproximal area, was assessed for microbial plaque,infiltrated interdental epithelium, supraalveolar connective tissue, alveolarbone, and periodontal ligament. In addition, connective tissue attachmentand bone levels were determined distal to the first molar and mesial to thesecond molar in each molar block (four per animal). The connective tissueattachment level was defined as the distance from cemento-enamel junction(CEJ) to the apical termination of the junctional epithelium. Bone level wasdefined as the distance between CEJ and bone crest. Data were analyzed byStudent’s t test. Differences were considered significant at p � 0.05(two-tailed).

Analysis of periodontal bacteria

After dissection of the molar blocks but before fixation, supragingivalplaque samples were taken from the interdental areas (lingual aspect) of themolars using paper points. The samples were cultured aerobically andanaerobically according to routine laboratory procedures for detection ofperiodontal pathogens.

Latex bead phagocytosis and phagocytosis with Aggregatibacter(Actinobacillus) actinomycetemcomitans

Latex beads (3 �m; Sigma-Aldrich) were opsonized in RPMI 1640 me-dium containing 10% human serum for 30 min at 37°C and were washedthree times with medium. The 1–5 � 105 peritoneal PMNs per well werecultured in a 24-well plate with RPMI 1640 medium (without antibioticsand FCS) to adhere to the surface. Opsonized beads (0.05% solid) in se-rum-free RPMI 1640 were then added. Phagocytosis was synchronized byspinning at 300 � g for 1 min. To induce internalization, cells were incu-bated at 37°C. After different time periods the cells were fixed with 4%paraformaldehyde in PBS for 20–30 min at room temperature and used forimmunofluorescence. Before permeabilizing the cells external beads were

labeled with fluorophore-conjugated goat anti-human IgG (1:500) in PBSfor 1 h at room temperature.

A. actinomycetemcomitans bacteria were cocultured with peritonealneutrophils isolated from wild-type and LAMP-2-deficient mice (ratio1:50) at 37°C and 5% CO2 for 2 h. Extracellular bacteria were eliminatedby washing with PBS and gentamicin treatment. After different time pointsthe cells were fixed with 4% paraformaldehyde in PBS and examinedmicroscopically.

Immunofluorescence

PMNs were cultured on coverslips for 2 h and fixed with 4% paraformal-dehyde in PBS for 20–30 min at room temperature. Cells were permeabil-ized in PBS/0.2% saponin. Primary and secondary Abs were diluted in 3%BSA (Sigma-Aldrich) in PBS/0.2% saponin and added to the cells for 1 h.Goat anti-rabbit, anti-rat, or anti-mouse Abs conjugated to Alexa Fluor 488or 594 (Molecular Probes) were used. Nuclei were stained with DAPI(4�,6-diamidino-2-phenylindole; Sigma-Aldrich). Acidic compartmentswere labeled by incubating living cells in RPMI 1640 containing 20 mMHEPES (pH 7.4), and 0.1 mM DAMP (N-(3-((2,4-DNP)aminopropyl)-N-(3-aminopropyl)methylamine; Molecular Probes). The coverslips weremounted with Mowiol (Calbiochem) containing the anti-fading reagentDABCO (1,4 diazobicyclo-(2.2.2) octane; Sigma-Aldrich), and viewedwith an Axiovert 200M fluorescence microscope (Zeiss) with or without anApotome device for optical sectioning.

Electron microscopy

For electron microscopy, ultrathin sections were cut from the interdentalregion between the first and second mandibular and maxillary molars. Sec-tions were stained with uranyl acetate and lead citrate and examined in aPhilips EM 420. Isolated PMNs were fixed in 4% formaldehyde and 1%glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) and processedfor LX-112 embedding. For autophagic vacuole quantification, ultrathinsections of PMNs were scored under the microscope. The number of au-tophagic vacuole profiles was counted in at least 100 cell profiles perphenotype.

Killing assays

A total of 1 � 108 Escherichia coli cells were opsonized in RPMI 1640medium containing 10% human serum for 30 min at 37°C and werewashed three times with medium. Peritoneal PMNs (5 � 105 per well)were cultured in a 24-well plate with RPMI 1640 medium (without anti-biotics and FCS) to adhere to the surface before 5 � 106 E. coli wereadded. Phagocytosis was synchronized by spinning at 720 � g for 2 min.Cells were incubated at 37°C for 1 h. Phagocytosis was stopped by puttingthe cells on ice. Extracellular bacteria were eliminated by washing withPBS and incubation with 100 �g/ml gentamicin for 30 min. The PMNswere now cultured in RPMI 1640 containing 0.1% FCS. After various timeperiods the medium was removed, and 0.1% BSA in H2Odest was added tolyse the PMNs. The plates were frozen at �80°C before warm RPMI 1640(without antibiotics and FCS) was added and the plates were thawed fast byincubation at 37°C. This procedure did not affect the viability of E. coli(data not shown). The 100 �l of the lysate was plated on Luria-Bertani agarplates incubated at 37°C overnight, and CFUs were counted.

A. actinomycetemcomitans (American Type Culture Collection No.29522) was grown in tryptic soy broth (Sigma-Aldrich) with 0.1% yeastextract (BD Biosciences) and 2.5% glucose (Merck) or on Columbia bloodagar plates (Oxoid) at 37°C in 5% CO2. A total of 1 � 106 peritonealneutrophils were incubated with opsonized A. actinomycetemcomitans (ra-tio 1:50) in PBS at 37°C and 5% CO2 for 120 or 240 min. Phagocytosis wassynchronized by spinning the mixture at 720 � g for 2 min. Serial dilutionsof the supernatant were plated on blood agar plates and cultured for 48 h,and the number of CFU was counted. The percentage of viable bacteria ineach sample was then determined by comparing the number of CFU from thecontrol sample without added neutrophils (100% viability) to the number ofCFU obtained for A. actinomycetemcomitans incubated with neutrophils.

Hydrogen peroxide production by PMNs

The assay was performed according to Pick and Mizel (16). PeritonealPMNs (1 � 105 per well) were cultured in a 96-well plate with RPMI 1640medium (without antibiotics and FCS) for 2 h to allow adherence beforenonadherent cells were removed by shaking the plates and washing thewells three times with 0.1-ml volumes of warm phenol red-free HBSS. Atotal of 2 � 107 opsonized E. coli bacteria XL1 blue or 0.1 mg of zymosanwas added in 100 �l of phenol red solution (140 mM NaCl, 10 mMNaH2PO4, 5.5 mM sucrose, 0.56 mM phenol rot (pH 7.0), sterile filtered0.22 �m) containing 19 U/ml HRP. After 1 h at 37°C, the reaction was

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stopped by adding 10 �l of 1 M NaOH per well. The absorbance wasmeasured in a microtiter plate reader at 600 nm.

ResultsLAMP-2-deficient mice display periodontitis

We have reported earlier that LAMP-2 deficiency caused an in-creased postnatal lethality, a reduced weight development, and amassive accumulation of autophagic vacuoles in numerous tissues(10, 13). Because we needed to raise the LAMP-2-deficient miceafter the weaning period with liquid food, we wanted to study thereasons for this impaired feeding behavior. We observed that allLAMP-2 knockout animals, which were housed in a conventionalbreeding facility, exhibited early onset natural periodontitis withovert migration of molars (Fig. 1, B and C), alveolar bone loss andfurcation involvement (Fig. 1E). Increased mobility of molar teethwas diagnosed in several animals at the day of sacrifice.

Microscopically, loss of connective tissue attachment level andalveolar bone crest resorption was already evident at the age of 7wk after birth (Fig. 1G). In the knockout animals, all molar sur-faces exposed to the oral cavity proved to be covered with a layerof microbial plaque. Because the first molars erupt around day 14,this response implies that periodontitis had developed within atime period of 1 mo. Neither plaque nor signs of periodontitiscould be diagnosed in the knockout animals that were suppliedwith amoxicillin in the drinking water (Fig. 1M). In none of thewild-type or other transgenic animals kept in the same conven-tional breeding facility were signs of periodontitis and plaque de-velopment observed.

All interdental areas in the knockout mice exhibited massiveamounts of microbial plaque in the region occlusal to the level ofthe gingiva and within the sulcus area (Fig. 1, G and H). However,neither at the light microscopical level nor at the electron micro-scopical level were microorganisms observed within the gingivaltissues, except (very occasionally) within the very superficial lay-ers of the sulcular epithelium (Fig. 1I). In the older animals thebiofilm did not only occupy the crevicular domain but had grownout to cover the free dental surfaces up to the level of the occlusalplane. Microorganisms were sometimes found within the dentinaltubules of the molar cusp regions free of enamel (data not shown).Overt caries lesions, however, were not detected.

Upon culturing, it appeared that none of the classical periodontalpathogens that are commonly found in the human and are associ-ated with natural periodontitis in humans had nested. In particular,there was no colonization of A. actinomycetemcomitans, Porphy-romonas gingivalis, Prevotella intermedia, or Tannerella forsytia.The dominant flora in all animals consisted of facultative anaero-bic bacteria with relatively high percentage of Gram-negative rodswith a slightly increased number of Actinomyces species. In his-tological sections the bacterial morphotypes observed were clas-sified as cocci, rods, and filaments.

The dominant infiltrating cell type in the inflammatory lesionswas the PMN (Fig. 1H). Many of them had infiltrated the junc-tional and sulcular epithelia. Also within the gingival crevice (out-side the gingival tissue) numerous PMNs were observed, many ofthem loaded with phagocytosed bacteria (Fig. 1J). Signs of phago-cytosis by PMNs or by any other cell type (e.g., macrophage-likecells) were not found within the tissue, either at the light micro-scopical level or at the electron microscopical level. No plasmacells were noted within the connective tissue and only a few cellsbelonging to the lymphocytic lineage were identified. The epithe-lium in the interdental region had lost its normal appearance andwas characterized by extensive proliferation, widened intercellularspaces, and loss of keratinization (Fig. 1, G and H). Many smallblood vessels were observed throughout the interdental epithelium

and they showed fenestrations. PMNs (without showing signs ofbacterial phagocytosis) were abundantly present in contact withthe blood vessel wall, within the collagenous fiber framework ofthe gingiva, and within the epithelium. The area occupied by (in-filtrated) epithelium was significantly larger for LAMP-2 knockoutmice compared with wild-type mice ( p � 0.001) (Fig. 1K). Su-praalveolar connective tissue was significantly more extensive inknockout mice compared with wild-type animals ( p � 0.05) (Fig.1F). The surface area of alveolar bone in the interproximal area ofthe wild-type animals was larger compared with that of the knock-out mice ( p � 0.001) (Fig. 1K). The surface area of periodontalligament gave higher values for the knockout than the wild-typemice ( p � 0.05) (Fig. 1K).

Analysis of histomorphometric parameters showed considerableloss of connective tissue attachment (the distance between the CEJ tothe apical termination of junctional epithelium) in knockout animals(Fig. 1L). Also the alveolar bone crest was displaced in the apicaldirection (Fig. 1L), whereas the total molar root length (CEJ-apex)was about the same (Fig. 1L). Plaque growth and extent of connectivetissue attachment loss did not show statistically significant differencesbetween upper and lower molar regions (data not shown).

Reduced bacterial killing capacity of LAMP-2-deficient PMNs

The periodontal pathology may have been caused by PMNs able tophagocytose pathogens but unable to efficiently kill the microor-ganisms. To investigate this, we isolated PMNs from wild-typeand this LAMP-2-deficient mice. After attachment of these cellsthey were incubated for 1 h with E. coli cells. After 1 h phagocy-tosis, extracellular bacteria were killed by incubation for 30 minwith gentamicin. Cells were lysed and plated on bacterial plates toestimate CFUs after 0, 30, and 60 min of subsequent incubation(Fig. 2A). Wild-type PMNs were able to phagocytose and kill theingested bacteria as expected. LAMP-2-deficient PMNs were sig-nificantly less effective in killing bacteria. Even 60 min after in-cubation, the number of viable bacteria was comparable to thenumber of bacteria at 0 min of wild-type PMNs (Fig. 2A). To alsoanalyze whether periodontally relevant pathogens are susceptibleto phagocytotic killing, we isolated PMNs from wild-type andLAMP-2-deficient mice and incubated these cells with A. actino-mycetemcomitans (ratio 1:50) at 37°C for 120 or 240 min, respec-tively. We also observed that these bacteria were less efficientlykilled by LAMP-2-deficient PMNs (Fig. 2B), and the bacteria werepresent intracellularly in a higher number in LAMP-2-deficientPMNs (Fig. 2, C–E). These data suggest that the phagosomal kill-ing capacity is impaired in LAMP-2 lacking PMNs. To elucidatewhether oxidative or nonoxidative killing pathways were affectedwe analyzed the capacity of wild-type and LAMP-2 knockoutPMNs to produce oxygen radicals, which are known to be involvedin bacterial killing by these cells (17, 18). PMNs from both geno-types retained the capacity to react after addition of E. coli orzymosan by producing similar amounts of oxygen radicals mea-sured by the production of H2O2 (Fig. 3) and reactive oxygenspecies (data not shown). These data suggest that impairment ofthe nonoxidative, lysosomal killing pathway is mainly responsiblefor the reduced killing in LAMP-2-deficient PMNs.

Analysis of LAMP-2-deficient PMNs

Using electron microscopy, we observed that PMNs within thegingival crevice and PMNs isolated from LAMP-2-deficient micewere characterized by an accumulation of autophagic vacuoles(Figs. 1J and 4B), in contrast to wild-type cells (Fig. 4A). Fewearly autophagic vacuoles were detected in wild-type PMNs. Inagreement with our earlier results with PMNs in vivo (10), theLAMP-2-deficient PMNs showed a prominent accumulation of

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FIGURE 1. Periodontitis in LAMP-2 knockout mice. A, Mandibular molar block of 1-year-old wild-type (WT) mouse. Note that all three molars arein line with each other. B, Mandibular molars in 9-mo-old knockout animal. Note migration in buccal direction of second molar (between asterisks). C,Maxillary molars in 1-year-old knockout animal. Note severe migration in buccal direction of second molar (between asterisks). D and E, MicroCT scansof mandible of a wild-type (D) and a knockout (E) animal (4-mo-old). Note advanced bone loss around molars in E. F and G, Micrographs of interdentalregion between first and second molars in wild-type (F) and knockout (G) animal (7 wk). Note connective tissue attachment loss, epithelial proliferation(ig), interdental bone loss (rb), and plaque accumulation (arrow) in knockout animal (G). H and I, Micrographs showing interdental region between firstand second mandibular molars in knockout (7 wk). Note proliferation and infiltration by PMNs of the gingival epithelium (H). Accumulation of plaque isdenoted (arrow). I, Dental plaque is depicted at higher magnification. Only in the superficial layers of the gingival epithelium were signs of bacterialinvasion noted (arrowheads). J, Electron micrograph of neutrophil within sulcular area of knockout animal showing electron-lucent vacuoles (v) andinternalized bacteria (arrowheads). Scale bar represents 2 �m. K, Histomorphometric measurements of standard interproximal area (SIA) in wild-type (n �8) and knockout (n � 8) animals showing surface area (mean � SE) of plaque, epithelium, supraalveolar connective tissue (SACT), bone and periodontalligament (PDL). Note absence of plaque in wild-type animals and high values for plaque in knockout animals (p � 0.001). A highly statistically significantdifference (p � 0.001) was found for epithelium and supraalveolar connective tissue. L, Histomorphometric measurements shown mean � SE in interdentalregions in wild-type animals (n � 7: 7 wk, 4 mo, 9 mo, 1 year) and knockout animals (n � 5: 7 wk, 4 mo, 9 mo, 1 year). Although loss of attachmentlevel (the distance from CEJ to the apical termination of the junctional epithelium) (CEJ-ATJE) in wild-type animals was zero, in knockout groups meanloss was �200 �m (p � 0.001). Also with respect to crestal bone level (CEJ-B.crest), a significant difference was found between knockout and wild-typegroups (p � 0.005). Molar root length (CEJ-apex) was similar between the two animal groups. M, Effect of antibiotics on development of periodontitis inone wild-type and two knockout mice (4-mo-old; four standard interproximal areas per mouse). One knockout animal had received amoxicillin (KO � ab)in the drinking water immediately after weaning. Histomorphometric measurements show that amoxicillin did not only prevent plaque accumulation butalso proliferation of epithelium and loss of bone.

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late autophagic vacuoles, containing partially degraded cytoplas-mic material (Fig. 4C). Because LC3 was shown to be a specificmarker for early autophagic vacuoles (19), we performed an im-

FIGURE 2. Reduced killing capacity in LAMP-2-deficient PMNs. A,Scheme of the bacterial killing assay is boxed in upper right. Washingsteps are grayed boxes. A total of 5 � 105 primary peritoneal PMNs perwell were cultured in a 24-well plate with RPMI 1640 to allow adherence(A) to plastic surface. Nonadherent cells were removed by washing, and5 � 106 E. coli were added. After 1 h of phagocytosis (P), extracellularbacteria were washed away and subsequently killed by gentamicin (G)treatment for 30 min. Gentamicin was removed by extensive washing, andthe PMNs were cultured in RPMI 1640 medium containing 0.1% FCS.After various time points the PMNs were lysed and plated on Luria-Bertaniagar plates incubated at 37°C overnight. CFUs were counted. The exper-iment is a representative example of three independent assays performed.B, Killing of A. actinomycetemcomitans by PMNs. A total of 1 � 106

peritoneal neutrophils were mixed with opsonized A. actinomycetemcomi-tans (ratio 1:50) in PBS at 37°C and 5% CO2 for 120 or 240 min, and thenumber of CFU was counted. The percentage of viable bacteria in eachsample was then determined by comparing the number of CFU from thecontrol sample without added neutrophils (100% viability) to the numberof CFU obtained for A. actinomycetemcomitans incubated with neutro-phils. Data are presented as mean � SD. C, Quantitation of the number ofundigested A. actinomycetemcomitans cells inside neutrophils by micro-scopical examination. Data are presented as mean � SD. D and E, Phasecontrast pictures of representative images of wild-type (�/�) (D) andLAMP-2-deficient (�/�) (E) neutrophils with nondigested phagocytosedA. actinomycetmcomitans cells (arrowheads). Scale bar represents 1 �m.

FIGURE 3. Oxygen radical formation is unaltered in LAMP-2 knock-out PMNs. Hydrogen peroxide production by PMNs. After adherence tosurface, PMNs were incubated with opsonized E. coli bacteria or zymosanfor 1 h in phenol red solution containing HRP. H2O2 production was de-termined from the culture medium. Results represent the mean � SD ofthree experiments.

FIGURE 4. Microscopic examination of LAMP-2 knockout PMNs. Aand B, Electron microscopy of PMNs isolated from the peritoneum ofwild-type (�/�) and LAMP-2-deficient (�/�) mice 4 h after thioglycolateinjection. Neutrophil of a 3-mo-old wild-type mouse (A) and LAMP-2-deficient (B) mouse are shown. Early autophagic vacuoles (Avi) and lateautophagic vacuoles (Avd) were observed. Scale bar represents 500 nm. C,Number of autophagic vacuole profiles per cell profile, given as mean �SE. At least 100 cell profiles per phenotype were included in the analysis.D and E, Immunocytochemical analysis using an anti-LC3 Ab that detectsautophagic vacuoles in wild-type PMNs (D) and LAMP-2-deficient (E)PMNs. Scale bar represents 3 �m. F, LAMP-2 expression in wild-typePMNs. Scale bar represents 2 �m. Absence of expression in LAMP-2-deficient PMNs is shown (inset). Scale bar represents 3 �m. G and H,LAMP-1 (red) immunocytochemical staining of peritoneal PMNs. DAPI(blue) was used for counterstaining of the nuclei. G, Wild-type PMNs. H,LAMP-2-deficient PMNs display enlarged and clustered LAMP-1-positivecompartments. Scale bar represents 3 �m. I and J, Myeloperoxidase stain-ing (MPO) as a marker for primary granules in peritoneal PMNs does notreveal differences between the two genotypes. Inset, Costaining of MPO(red) with LAMP-1 (green) showed colocalization in some vesicles in bothwild-type (I) and LAMP-2-deficient (J) PMNs. DAPI was used for counter-staining of nuclei. Scale bar represents 3 �m including inset. K and L,Lactoferrin (LF) staining (green) as a marker for secondary granules. K, Inwild-type PMNs lactoferrin-positive vesicles are found in the center of thecell and at the membrane. L, LAMP-2-deficient PMNs show aggregatedvesicles and fewer signals at the membrane.

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munocytochemical analysis using Abs against this autophagosomemarker. The staining was diffuse in wild-type cells, whereas theLAMP-2-deficient cells showed a punctuate staining (Fig. 4, D andE). This result is consistent with autophagic accumulation inLAMP-2-deficient PMNs.

The localization of LAMP-2 in vesicular structures of wild-typePMNs was easily detectable (Fig. 4F). As expected no LAMP-2expression was observed in LAMP-2-deficient cells (Fig. 4F, in-set). We next analyzed the lysosomal compartment using Absagainst the related major lysosomal membrane protein LAMP-1. Inwild-type PMNs lysosomes were distributed within the entire cy-tosol and also close to the plasma membrane (Fig. 4G). Impor-tantly, in LAMP-2-deficient PMNs lysosomes appeared enlargedand clustered in the center of the cell (Fig. 4H). To monitor thedistribution and the presence of primary or secondary granules,which are essential to supply the phagosome with bactericidal sub-stances (20), we stained PMNs with Abs against myeloperoxidase(Fig. 4, I and J), a constituent of primary granules, and againstlactoferrin, a constituent of secondary granules (Fig. 4, K and L).We did not observe differences in the distribution, number and sizeof primary granules. Secondary, lactoferrin-containing granuleswere less frequently found close to the plasma membrane andshowed a reduced colocalization with LAMP-1 (Fig. 4L) inLAMP-2 knockout cells, which may suggest an impaired functionand/or transport of these vesicles.

FIGURE 5. Retarded maturation of latex bead phagosomes in LAMP-2-deficient PMNs. Peritoneal PMNs were incubated with opsonized latexbeads (3 �m) for 1 h and fixed with 4% paraformaldehyde in PBS. A andB, Electron microscopy of PMNs with a phagocytosed latex bead fromwild-type (A) and LAMP-2-deficient (B) neutrophil. Note the presence ofboth the latex bead phagosome (lb) and autophagic vacuoles (av) in theLAMP-2-deficient cells. The phagocytic index (PI, beads/cell) was deter-mined by counting at least 250 cells per genotype under the light micro-scope (phase contrast). The phagocytic index was 2.73 for wild-type and2.81 for knockout cells. Scale bar represent (A and B) 1 �m. C–F, Immu-nostaining of PMNs with phagocytosed latex beads. Phase contrast picturesare shown (inset). C and D, Immunohistochemical staining with theLAMP-1 Ab. DAPI showed a reduced recruitment of LAMP-1 to the latexbead phagosomal membranes in the LAMP-2-deficient PMNs. Magnifica-tion of one phagosome is shown (inset). E and F, Staining with cathepsinD Ab and DAPI. Scale bar represents 3 �m for staining and 5 �m for phasecontrast image (inset). Staining also revealed that cathepsin D was lessfrequently delivered to LAMP-2-deficient phagosomes. Magnification ofone phagosome is shown (inset). G and H, Recruitment of lysosomal mark-ers as shown in C–F was quantitated for 100 phagosomes in wild-type andLAMP-2-deficient PMNs in three independent experiments. G, Quantita-tion of LAMP-1-positive phagosomes. H, Quantitation of cathepsin D(CathD)-positive phagosomes. Results are shown as mean � SD.

FIGURE 6. Disturbed acidification in phagosomes in LAMP-2-defi-cient PMNs. A and B, Peritoneal PMNs were incubated with opsonizedlatex beads (3 �m) for 1 h, followed by 30 min incubation with 0.1 mMDAMP, which accumulates in acidified compartments. DAMP was de-tected with anti-DNP Abs. Phase contrast pictures are shown (inset). DAPIwas used for counter-staining of nuclei. A, Wild-type cells. DAMP-positivephagosomes are marked (arrowhead). B, LAMP-2-deficient cells. Pleasenote that a number of LAMP-2-deficient phagosomes are not acidified at all(arrowhead). Scale bar represents 3 �m for DAMP images and 5 �m forinset image. C, Quantitation of DAMP-positive phagosomes. Results rep-resent mean � SD of two experiments quantifying 100 phagosomes eachas depicted.

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Impaired maturation of phagosomes in LAMP-2-deficient PMNs

Our data suggested that impaired nonoxidative killing processesmay be the cause for the reduced bacterial killing capacity ofLAMP-2 knockout PMNs. To analyze this further, we made use ofthe ability of PMNs to spontaneously phagocytose latex beads inculture. The ingested latex bead-phagosomes can be easily moni-tored microscopically (Fig. 5). Electron microscopy confirmed thatLAMP-2-deficient PMNs were capable of phagocytosing suchbeads with similar efficiency as wild-type cells (Fig. 5, A and B).To analyze the maturation defects in more detail, we stained thecells after 1 h of phagocytosis with proteins localized in thelysosomal compartment. Whereas the majority of phagosomesin wild-type cells recruited LAMP-1 (Fig. 5C), and cathepsin D(Fig. 5E) there was a significant decrease in the number ofLAMP-2-deficient phagosomes positive for either LAMP-1(Fig. 5D) or cathepsin D (Fig. 5F). Under the given experimen-tal conditions, 60 –70% of wild-type latex bead phagosomeswere positive for lysosomal markers. In contrast, only 30% ofLAMP-2-deficient phagosomes contained lysosomal compo-nents (Fig. 5, G and H). In agreement with these results, we alsoobserved a decreased acidification in the LAMP-2 knockoutphagosomes using immunolabeling of the acidotropic com-pound DAMP (Fig. 6).

DiscussionLysosomes play a very important role in the oxygen-independentkilling of bacteria, which is believed to be an important mechanismin the oxygen-deprived periodontal pocket (8). Naturally occurringperiodontal disease normally does not happen in laboratory mice(21). We now demonstrate that in mice lacking LAMP-2 periodon-titis represents a very striking phenomenon. The early onset peri-odontal disease noted in the present study was clearly related to anaccumulation of plaque. No evidence was found (in any of thespecimens) for invasion of microorganisms into the deeper tissuesof the gingiva. Thus tissue damage and attachment loss were notlikely to be directly caused by bacterial invasion but more likelythe result of inflammation.

We recorded ample evidence of plaque formation in the inter-proximal areas of the knockout animals. In several cases plaquehad overgrown even the occlusal surfaces. Although our microbi-ological data were not very detailed, our observations suggest amixed flora in the oral cavity of the LAMP-2 knockout animals.Human periodontopathogens were not found.

Despite the fact that knockout mice were housed under the sameconditions as their wild-type littermates, in the latter animals bac-terial accumulations were never noticed. The prevention of plaqueaccumulation and tissue breakdown in the mice receiving antibi-otic treatment proves that periodontitis in LAMP-2 knockout miceis bacteria-related. Our observations also prove that LAMP-2 as-sociated cell functions are pivotal in self-cleaning properties of theoral cavity. Although we cannot exclude the possibility that theobserved periodontitis was, at least partially, due to systemic ef-fects arising from LAMP-2 deficiency, the most likely reason forthe huge outgrowth of dental plaque in the LAMP-2 knockoutmice is that bacterial killing by PMNs (analyzed in this study: E.coli and A. actinomycetemcomitans) was severely hindered.

In humans, disorders of neutrophil function are commonly as-sociated with severe periodontal destruction (7, 22, 23). Of courseother immune cells might have been affected as well by the defi-ciency. In this respect, dendritic cells are of interest, which captureexogenous Ags for eventual processing in endosomes-lysosomes(24). LAMP-2a facilitates MHC class II presentation of cytoplas-mic Ags. Decreased display of cytoplasmic epitopes via class II

molecules was observed in cells with diminished expression ofLAMP-2 (25).

An impaired function of macrophages in bacterial killing inLAMP-2-deficient mice is unlikely because these cells were onlyseldom observed within the affected tissue. We could also recentlyshow that phagosomal maturation is not affected in LAMP-1- orLAMP-2-deficient macrophages (14, 26).

PMNs are the most abundant immune cells in the inflammatorygingivial sites of patients with periodontitis, and their pathogenicrole in this setting has been suggested (27). The role of PMNs ininnate immunity and the specific role of the lysosomal compart-ment in these cells is underscored by congenital defects such asChediak-Higashi syndrome (4) and Papillon-Lefevre syndrome(28) in which lysosomal secretion events and lysosomal proteol-ysis is impaired, respectively. Our observations suggest thatLAMP-2-associated functions in PMNs are pivotal in the self-cleaning properties of the oral cavity. They help to orchestrate thenatural defense against oral biofilm formation.

Whereas in LAMP-2-deficient PMNs the distribution and fusionof primary granules with latex bead phagosomes is apparently un-affected (data not shown), the cellular localization of lactoferrin-positive granules was changed and these granules showed areduced colocalization with lysosomal markers, suggesting a dis-turbed biogenesis, traffic, or function of a subset of granules. Bothtypes of granules contribute to the killing of bacteria (29) and it islikely that LAMP-2 contributes to the fusion of granules withphagosomes.

Phagosome-lysosome fusion is essential for efficient degra-dation of internalized pathogens. Fusion with lysosomes resultsin delivery of an assortment of luminal and membrane proteinsto phagosomes. In a recent study we showed that in LAMP-1and LAMP-2 double-deficient fibroblasts, phagosomes acquiredthe early endosome markers Rab5 and PI3 phosphate, but failedto recruit Rab7 and did not fuse with lysosomes. We attributedthe deficiency to impaired organellar motility toward the cellcenter (14). We proposed that LAMPs might directly or indi-rectly assist the movement of phagosomes toward the cell cen-ter. Elimination of lysosome transport by disruption of micro-tubules (30) or by interference with RILP or dynein function(31) impairs contact and fusion between lysosomes and phago-somes. In contrast to fibroblasts, where either LAMP-1 orLAMP-2 is required for lysosomal motility and phagosomalmaturation, in PMNs the lack of LAMP-2 only seems to beenough to interfere with the maturation of phagosomes. As al-ready indicated by the accumulation of autophagic vacuoles inLAMP-2-, but not in LAMP-1-deficient PMNs (32), the lack ofLAMP-2 cannot be compensated by LAMP-1. Similar to thesuccessful maturation of autophagic vacuoles, the destruction ofphagocytosed bacteria requires the subsequent fusion with earlyand late endosomes and lysosomes. We show that the later mat-uration events are disturbed in LAMP-2-deficient PMNs, lead-ing to impaired bacterial clearance and development of peri-odontal disease in LAMP-2 knockout mice. Further analyses inPMNs and LAMP-deficient cells will be required to determinethe molecular mechanisms for the impaired maturation and dis-turbed lysosomal motility.

In conclusion, our data indicate that LAMP-2 is critically re-quired for the maturation process of phagosomes in PMNs. A lackof LAMP-2 leads to a reduced maturation and to a reduced bac-tericidal activity. This functional defect may be directly associatedwith the increased susceptibility of LAMP-2 knockout mice todevelop periodontal disease.

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AcknowledgmentsWe thank Martijn van Steenbergen (University of Amsterdam), Arja Strandell(University of Helsinki), and Marlies Rusch (University of Kiel) for assistancein the completion of this project.

DisclosuresThe authors have no financial conflict of interest.

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